Engineered gene effectors, compositions, and methods of use thereof

Abstract

The present disclosure provides one or more engineered gene effectors, as well as systems, compositions, and methods of use thereof, where the one or more engineered gene effectors can be used to effect modulation of a target gene in a cell (e.g., an endogenous target gene in a cell). The one or more engineered gene effector (s) may be operably coupled to a heterologous endonuclease, such as a CRISPR / Cas protein.

Description

Cross-references

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 489,873, filed on March 13, 2023, U.S. Provisional Application No. 63 / 381,255, filed on October 27, 2022, and U.S. Provisional Application No. 63 / 323,248, filed on March 24, 2022, each of which is incorporated herein by reference in its entirety. Sequence Listing Incorporated by Reference

[0002] This application is filed with a sequence listing in electronic format. The sequence listing is provided as a file named 55176-727_601_SL.xml, which was created on March 10, 2023 and is 132 kilobytes in size. The information in the electronic format of the sequence listing is incorporated by reference in its entirety. Background Art

[0003] Various effectors (e.g., transcriptional regulators) can be used to regulate the expression or activity of a target gene in a cell. For example, a heterologous gene effector can be introduced into a cell (e.g., delivered, expressed, etc.), and the heterologous gene effector can achieve such regulation of the target gene alone or together with additional reagents. In some examples, additional reagents may include clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated proteins (Cas) for specific binding to a target gene (e.g., a target deoxyribonucleic acid (DNA) sequence or ribonucleic acid (RNA) sequence of a target gene (e.g., an exogenous DNA sequence or RNA sequence)), and the heterologous gene effector can regulate the expression or activity level of the target gene. For example, such gene effectors can be used as gene therapy to treat or improve the condition (e.g., disease) of a subject. Summary of the invention

[0004] VP64-p65-Rta fusion polypeptide (VPR) is a benchmark gene effector that can activate a target gene in a cell. However, in some cases, VPR may not be optimal or sufficient for regulating all genes. Alternatively or additionally, the size of VPR (e.g., about 518 amino acid residues) may not be small enough to package them in a delivery method (e.g., a viral vector, such as an adeno-associated virus (AAV) vector) with at least one additional reagent (e.g., one or more guide RNAs, transgenics encoding therapeutic polynucleotides or proteins, various types of Cas enzymes, etc.). Therefore, for example, various aspects of the present disclosure provide engineered effectors, compositions thereof, and methods of use thereof, which are different from VPRs but have comparable effectiveness to the VPRs in activating the expression or activity levels of one or more target genes.

[0005] Krueppel-associated box (KRAB) is a domain (e.g., having about 75 amino acid residues or less) that can be found in eukaryotic Krueppel-type C2H2 zinc finger proteins (ZFPs). KRAB is a benchmark gene effector that is capable of repressing a target gene in a cell. However, in some cases, the KRAB may not be optimal or sufficient for regulating all genes. Thus, for example, various aspects of the present disclosure provide engineered effectors, compositions thereof, and methods of use thereof that are not identical to KRAB but have comparable effectiveness to the KRAB in reducing the expression or activity level of one or more target genes.

[0006] Disclosed herein are engineered gene effectors comprising a polypeptide, wherein: the polypeptide is heterologous to any one of the members selected from the group consisting of VP16, VP64, p65 and Rta; the size of the engineered gene effector is up to about 500 amino acid residues; and the engineered gene effector is capable of activating the expression level of a target gene in a cell, wherein the expression level of the target gene activated by the engineered gene effector is at least about 80% compared to the expression level activated by the VP64-p65-Rta fusion polypeptide (VPR) in a control cell.

[0007] Disclosed herein are engineered gene effectors comprising a polypeptide coupled to an additional polypeptide, wherein: the polypeptide comprises an amino acid sequence having at least about 70% sequence identity with 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; and the size of the engineered gene effector is less than or equal to about 250 amino acid residues.

[0008] Disclosed herein are engineered genetic effectors comprising a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least about 70% sequence identity with the polypeptide sequence of SEQ ID NO: 1, wherein the engineered genetic effector is not identical to any one of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

[0009] Disclosed herein are engineered gene effectors comprising a plurality of polypeptide domains, wherein each polypeptide domain of the plurality of polypeptide domains comprises a polypeptide comprising an amino acid sequence exhibiting at least about 70% sequence identity to the polypeptide sequence of SEQ ID NO: 1. A system comprising the engineered gene effector of any of the preceding claims.

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

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

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

[0013] Other aspects and advantages of the present disclosure will be readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. It will be appreciated that the present disclosure is susceptible of other different embodiments and that its several details may be modified in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative rather than restrictive. Incorporation by Reference

[0014] All publications, patents, and patent applications mentioned in this specification are incorporated herein 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. If publications and patents or patent applications incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The novel features of the present invention are particularly set forth in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description and the accompanying drawings (also referred to herein as "figures") which set forth exemplary embodiments utilizing the principles of the present invention, wherein:

[0016] Figure 1 Exemplary structures of engineered effectors are schematically illustrated.

[0017] Figure 2 An exemplary manipulation of a target gene modulated by an engineered effector in complex with a heterologous endonuclease and a guide nucleic acid molecule is schematically illustrated.

[0018] Figure 3 The correlation between two genes regulated by the engineered effector candidates is shown.

[0019] Figure 4 Predicted active polypeptide sequences used to generate engineered effectors are shown.

[0020] Figure 5 Examples of engineered effectors are schematically illustrated.

[0021] Figure 6 Additional examples of engineered effectors comprising flexible linkers are schematically illustrated.

[0022] Figure 7 Additional examples of engineered effectors comprising multiple active domains are schematically illustrated.

[0023] Figure 8 Different examples of engineered effectors containing multiple active domains are schematically illustrated.

[0024] Fig. 9 Additional examples of engineered effectors with different lengths are schematically illustrated.

[0025] Fig.10 Different examples of engineered effectors with different lengths are schematically illustrated.

[0026] Fig.11 Different examples of engineered effectors are schematically illustrated.

[0027] Fig.12 Activation of heterologous target genes by engineered effectors is shown.

[0028] Fig.13 Activation of endogenous genes encoding IFNs by engineered effectors is shown.

[0029] Fig.14 Activation of heterologous target genes by engineered effectors at different time points is shown.

[0030] Fig.15A and Fig. 15B Activation of the endogenous target gene encoding CXCR4 by the engineered effectors at different time points is shown.

[0031] Fig.16 Activation of the endogenous gene encoding CD2 by the engineered effectors is shown.

[0032] Fig.17 Activation of the endogenous gene encoding CD45 by the engineered effectors is shown.

[0033] Fig.18A Activation of a heterologous target gene by engineered genetic effectors is shown, each effector comprising an active domain at a different location.

[0034] Fig.18B Activation of endogenous target genes by engineered genetic effectors is shown, each effector containing an active domain at a different location.

[0035] Fig.19 Various expression levels of heterologous target genes after activation by engineered gene effectors are shown.

[0036] Fig. 20Shown are various expression levels of endogenous genes encoding IFNs after activation by engineered gene effectors.

[0037] Fig.21 Shown are various expression levels of the endogenous gene encoding CXCR4 after activation by the engineered gene effectors.

[0038] Fig. 22 The performance of engineered gene effectors in regulating heterologous and endogenous genes compared to control gene effectors is shown.

[0039] Fig.23 The performance of the engineered gene effectors in regulating endogenous genes compared to control gene effectors is shown.

[0040] Fig.24 Shown are the expression levels of CXCR4 3 days after transfection following activation by engineered gene effectors.

[0041] Fig.25 Shown are the expression levels of CXCR4 7 days after transfection following activation by engineered gene effectors.

[0042] Fig.26 Activation of endogenous and heterologous target genes by engineered genetic effectors is shown.

[0043] Fig. 27 Shown are the expression levels of CXCR4 28 days after transfection following knockdown of engineered gene effectors.

[0044] Fig.28 Shown are the expression levels of CXCR4 40 days after transfection following repression of the engineered gene effector.

[0045] Fig.29 JQ1 or GNE049-mediated reversal of gene expression by engineered genetic effectors is shown.

[0046] Fig.30 Durable and sustainable gene regulation by engineered gene effectors is shown.

[0047] Fig.31 Shown are the activation levels of CD45, IFNG (IFNγ), and CXCR4 by engineered gene effectors 3 days post transfection (dpt).

[0048] Fig.32 Persistent activation (eg, durable activation) of CD45, IFNG, CXCR4, and CD81 by engineered gene effectors at different time points (eg, 9-18 days post-transfection) is shown.

[0049] Fig.33Shown is the correlation between maximal activation (3 dpt) and activation (9-18 dpt) by engineered gene effectors.

[0050] Fig.34 Different sizes of VPR, p300, Rta, p65, VP64, and engineered gene effectors are shown.

[0051] Fig.35 Activation of CD45 by engineered gene effectors at 9, 12 and 18 days post-transfection is shown.

[0052] Fig.36 Persistent activation (eg, durable activation) of CD45, CXCR4, FIN-γ, and CD81 by engineered gene effectors is demonstrated.

[0053] Fig.37 The robustness and potency of activation of engineered genetic effectors for CD45, CXCR4, FIN-γ, and CD81 are demonstrated.

[0054] Fig.38 Activation of CD45 by engineered gene effectors at different time points after transfection is shown.

[0055] Fig.39 Activation of IFNG by engineered gene effectors at different time points after transfection is shown.

[0056] Fig.40 Activation of CXCR4 by engineered gene effectors at different time points after transfection is shown.

[0057] Fig.41 Activation of CD81 by engineered gene effectors at different time points after transfection is shown.

[0058] Fig.42 and Fig.43 Shown are changes in expression (in MFI) of engineered gene effectors (mCherry) and sgRNA (BFP) by engineered gene effectors at different time points after transfection.

[0059] Figure 44A-Figure 44C 3 days after transfection ( Fig.44A ), 6 days( Fig.44B ) and 9 days ( Fig.44C ) Activation of CD45 and IFNG by engineered gene effectors.

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

[0061] Fig.46The predicted structure of XV1.48 aligned with CC.2 is illustrated.

[0062] Fig.47 The predicted structure of CC.32 is illustrated.

[0063] Fig.48 The predicted structure of the Cas protein coupled to XV1.32 is illustrated.

[0064] Fig.49 Conserved sequence motifs from different human protein families are shown.

[0065] Fig.50 Conserved sequence motifs from different viral protein families are shown.

[0066] Fig.51 The process of amino acid screening using a regularized logistic regression model (ElasticNet) is illustrated.

[0067] Fig.52 The characteristic importance of the 20 amino acids identified is shown.

[0068] Fig.53 Peptide level motifs enriched by different properties (eg, charge) are shown.

[0069] Fig.54 The prediction structure of VPR is illustrated.

[0070] Fig.55 Illustrated predicted structure of XV1.1

[0071] Fig.56 A volcano plot illustrating the results of a screen for engineered gene effectors is shown.

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

[0073] Fig.58 A barrel view of a coiled coil variation is shown.

[0074] Fig.59 Activation of multiple target genes by engineered gene effectors is shown.

[0075] Fig.60 Activation of IFNG according to the engineered gene effector coding length (bp) is shown.

[0076] Fig.61 Activation of CD45 by engineered gene effectors at different time points is shown.

[0077] Fig.62 The potency and durability of CD45 activation by engineered gene effectors are shown.

[0078] Fig.63 Shown is the activation of IFNG by engineered gene effectors at different time points.

[0079] Fig.64 The potency and durability of IFNG activation by engineered gene effectors are shown.

[0080] Fig.65 Activation of CXCR4 by engineered gene effectors at different time points is shown.

[0081] Fig.66 The potency and durability of CXCR4 activation by engineered gene effectors are shown.

[0082] Fig.67 Activation of CD81 by engineered gene effectors at different time points is shown.

[0083] Fig.68 The potency and durability of CD81 activation by engineered gene effectors are shown.

[0084] Fig.69 Activation of CXCR4 mRNA levels by engineered gene effectors at day 40 is shown.

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

[0086] Fig.71 Schematic illustration of the epigenetic mechanism of persistent activation by engineered genetic effectors.

[0087] Fig.72 CD45 expression by engineered gene effectors at day 3 is shown.

[0088] Fig.73 CD45 expression by engineered gene effectors at day 9 is shown.

[0089] Fig.74 Shown is the expression of CD45 by the engineered gene effector 96 hours after inhibitor administration.

[0090] Fig.75 Mitotically durable gene activation by engineered gene effectors is shown. DETAILED DESCRIPTION

[0091] Although various embodiments of the present invention have been shown and described herein, it will be appreciated by those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art may conceive of many variations, changes and substitutions without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0092] Whenever the term "at least", "greater than", or "greater than or equal to" precedes the first value in a series of two or more values, the term "at least", "greater than", or "greater than or equal to" applies to each value in the series. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0093] Whenever the term "not greater than," "less than," or "less than or equal to" precedes the first value in a series of two or more values, the term "not greater than," "less than," or "less than or equal to" applies to every value in the series. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0094] The term "about" or "approximately" generally means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term can mean within an order of magnitude of a value, preferably within 5 times of a value, and more preferably within 2 times of a value. When describing specific values ​​in the application and claims, the term "about" should be assumed to mean within an acceptable error range for the specific value unless otherwise indicated.

[0095] The use of alternatives such as "or" should be understood to mean one, both, or any combination of the alternatives. The term "and / or" should be understood to mean one or both of the alternatives.

[0096] When used herein with respect to a polypeptide sequence or a nucleic acid sequence, the term "heterologous" means that the polypeptide sequence or nucleic acid sequence (1) is placed in a place (e.g., an environment, such as a cell, a virus, or a fusion polypeptide molecule or a fusion polynucleotide molecule) where it is not normally found (e.g., not normally found in nature); or (2) comprises two or more subsequences that are found in a different relationship to each other than that in which they are 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 polypeptide sequence and the second polypeptide sequence 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 polynucleotide sequence and the second polynucleotide sequence may be heterologous to each other.

[0097] The term "cell" generally refers to a biological cell. A cell can be the basic structural, functional and / or biological unit of a living organism. A cell can be derived from any organism having one or more cells. Some non-limiting examples include: prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotic organisms, protozoan cells, cells from plants (e.g., cells from plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugar cane, pumpkin, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, lycophytes, hornworts, liverworts, mosses), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. Agardh, etc.), algae (e.g., kelp), fungal cells (e.g., yeast cells, cells from mushrooms), animal cells, cells from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.), etc. Sometimes the cell is not derived from a natural organism (e.g., the cell can be synthetic, sometimes referred to as an artificial cell).

[0098] The term "nucleotide" as used herein generally refers to a base-sugar-phosphate combination. Nucleotide may include synthetic nucleotides. Nucleotide may include synthetic nucleotide analogs. Nucleotide may be a monomer unit of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside triphosphates, adenosine triphosphate (ATP), uridine triphosphate (UTP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphates, such as dATP, dCTP, dITP, dUTP, dGTP, dTTP or derivatives thereof. Such derivatives may include, for example, [αS] dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. The term nucleotide used herein may refer to dideoxyribonucleoside triphosphates (ddNTP) and derivatives thereof. Exemplary examples of dideoxyribonucleoside triphosphates may include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP and ddTTP. Nucleotide can be unlabeled, or detectably labeled by well-known technology.Labeling can also be carried out with quantum dots.Detectable labels can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels.The fluorescent labeling of nucleotides can include, but is 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-carboxyl-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 can 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, which are available from Perkin Elmer, Foster City, Calif.; FluoroLink deoxynucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP, which are available from Amersham, Arlington, Mass. Heights, Ill.; fluorescein-15-dATP, fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP and fluorescein-15-2′-dATP, which can be obtained from Boehringer Mannheim, Indianapolis, Ind.; and chromosome marker nucleotides, 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, which are available from Molecular Probes, Eugene, Oregon. Nucleotides can also be labeled or marked by chemical modification. Chemically modified single nucleotides can be biotin-dNTP.Some non-limiting examples of biotinylated dNTPs can include 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] The terms "polynucleotide," "oligonucleotide," or "nucleic acid" are used interchangeably herein and generally refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or analogs thereof, in single-stranded, double-stranded, or multi-stranded form. The polynucleotide may be exogenous or endogenous to the cell. The polynucleotide may be present in a cell-free environment. The polynucleotide may be a gene or a fragment thereof. The polynucleotide may be DNA. The polynucleotide may be RNA. The polynucleotide may have any three-dimensional structure and may perform any known or unknown function. The polynucleotide may contain one or more analogs (e.g., altered backbones, sugars, or nucleobases). If present, the nucleotide structure may be modified before or after polymer assembly. Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acids, xeno nucleic acids, morpholinos, locked nucleic acids, diol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to a sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine. Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, gene sites (locus) defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides (including cell-free DNA (cfDNA) and cell-free RNA (cfRNA)), nucleic acid probes and primers. The nucleotide sequence may be interrupted by non-nucleotide components.

[0100] The term "sequence identity" generally refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Typically, the techniques used to determine sequence identity include determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence encoded thereby, and comparing these sequences with a second nucleotide or amino acid sequence. Two or more sequences (polynucleotides or amino acids) can be compared by determining their "percent identity". The percentage identity of two sequences (whether nucleic acid sequences or amino acid sequences) is the number of exact matches between the two aligned sequences divided by the length of the longer sequence, then multiplied by 100. Percent identity can also be determined, for example, by comparing sequence information using an advanced BLAST computer program (including 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), and as 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 program can be used to determine the percent identity over the entire length of the compared proteins. Default parameters are provided to optimize searches using short query sequences, such as using the blastp program. The program also allows the use of SEG filters to mask fragments of the query sequence, as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). The desired degree of sequence identity ranges from about 50% to 100% and integer values ​​therebetween. In general, the disclosure encompasses 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 any of the sequences provided herein.

[0101] The term "gene" generally refers to nucleic acids (e.g., DNA, such as genomic DNA and cDNA) involved in encoding RNA transcripts and their corresponding nucleotide sequences. The term used herein for genomic DNA includes intervening non-coding regions and regulatory regions, and may include 5' and 3' ends. In some uses, the term encompasses transcribed sequences including 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons and introns. In some genes, the transcribed region will include an "open reading frame" encoding a polypeptide. In some uses of the term, a "gene" only includes coding sequences (e.g., "open reading frames" or "coding regions") necessary for encoding polypeptides. In some cases, a gene does not encode a polypeptide, such as a ribosomal RNA gene (rRNA) and a transfer RNA (tRNA) gene. In some cases, the term "gene" includes not only transcribed sequences, but also non-transcribed regions, including upstream and downstream regulatory regions, enhancers and promoters. A gene may refer to an "endogenous gene" or a natural gene located in its natural position in the genome of an organism. A gene may refer to an "exogenous gene" or a non-natural gene. Non-natural genes can refer to genes that are not normally present in the host organism but are introduced into the host organism by gene transfer. Non-natural genes can also refer to genes that are not located in their natural position in the genome of an organism. Non-natural genes can also refer to naturally occurring nucleic acids or polypeptide sequences (e.g., non-natural sequences) that comprise mutations, insertions, and / or deletions.

[0102] The term "expression" generally refers to one or more processes of polynucleotide transcription (e.g., transcription into mRNA or other RNA transcripts) from a DNA template and / or the process of transcribed mRNA being subsequently translated into peptides, polypeptides or proteins. Transcripts and encoded polypeptides can be collectively referred to as "gene products". If the polynucleotide is derived from genomic DNA, expression can include the splicing of mRNA in eukaryotic cells. "Upregulation" about expression generally refers to the expression level of polynucleotides (e.g., RNA, e.g., mRNA) and / or polypeptide sequences relative to their expression in the wild-type state, while "downregulation" generally refers to the expression level of polynucleotides (e.g., RNA, e.g., mRNA) and / or polypeptide sequences relative to their expression in the wild-type state. The expression of transfected genes can occur transiently or stably in cells. During "transient expression", transfected genes are not transferred to daughter cells during cell division. Since its expression is limited to transfected cells, the expression of the gene will disappear over time. On the contrary, when a gene is co-transfected with another gene that gives a selective advantage to transfected cells, stable expression of transfected genes can occur. Such a selective advantage might be resistance to a certain toxin presented by the cell.

[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., a cell). One or more genes can be expressed and determined in the form of nucleic acid molecules (e.g., mRNA or other RNA transcripts). Alternatively or in addition, one or more genes can be expressed and determined in the form of polypeptides (e.g., proteins measured by Western blot). The expression profile of a gene can be defined as the shape of the expression level of the gene over a period of time (e.g., at least or up to about 1 hour, at least or up to about 2 hours, at least or up to about 3 hours, at least or up to about 4 hours, at least or up to about 5 hours, at least or up to about 6 hours, at least or up to about 7 hours, at least or up to about 8 hours, at least or up to about 9 hours, at least or up to about 10 hours, at least or up to about 11 hours, at least or up to about 12 hours, at least or up to about 16 hours, at least or up to about 18 hours, at least or up to about 24 hours, at least or up to about 36 hours, at least or up to about 48 hours, at least up to about 3 days, at least up to about 4 days, at least or up to about 5 days, at least or up to about 6 days, at least or up to about 7 days, at least or up to about 8 days, at least or up to about 9 days, at least or up to about 10 days, at least or up to about 11 days, at least or up to about 12 days, at least or up to about 13 days, at least or up to about 14 days, etc.). Alternatively, the expression profile of a gene can be defined as the expression level of the gene at a time point of interest (e.g., at least or up to about 1 hour, at least or up to about 2 hours, at least or up to about 3 hours, at least or up to about 4 hours, at least or up to about 5 hours, at least or up to about 6 hours, at least or up to about 7 hours, at least or up to about 8 hours, at least or up to about 9 hours, at least or up to about 10 hours, at least or up to about 11 hours, at least or up to about 12 hours, at least or up to about 13 hours, at least or up to about 14 hours, at least or up to about 15 hours, at least or up to about 16 hours, at least or up to about 17 hours, at least or up to about 18 hours, at least or up to about 19 hours, at least or up to about 20 hours, at least or up to about 21 hours, at least or up to about 22 hours, at least or up to about 23 hours, at least or up to about 24 hours, at least or up to about 25 hours, at least or up to about 26 hours, at least or up to about 27 hours, at least or up to about 28 hours, at least or up to about 29 hours, at least or up to about 30 hours, at least or up to about 31 hours, at least or up to about 32 hours, at least or up to about 33 hours, at least or up to about 34 hours, at least or up to about 35 hours, at least or up to about 36 hours, at least or up to about 37 hours, at least or up to about 38 hours, at least or up to about 39 hours, at least or up to about or up to about 16 hours, at least or up to about 18 hours, at least or up to about 24 hours, at least or up to about 36 hours, at least or up to about 48 hours, at least or up to about 3 days, at least or up to about 4 days, at least or up to about 5 days, at least or up to about 6 days, at least or up to about 7 days, at least or up to about 8 days, at least or up to about 9 days, at least or up to about 10 days, at least or up to about 11 days, at least or up to about 12 days, at least or up to about 13 days, or at least or up to about 14 days).

[0104] The terms "peptide", "polypeptide" or "protein" are used interchangeably herein and generally refer to a polymer of at least two amino acid residues connected by a peptide bond. The term does not indicate a specific length of the polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant technology, chemical or enzymatic synthesis, or is naturally occurring. These terms apply to naturally occurring amino acid polymers and amino acid polymers comprising at least one modified amino acid. In some cases, the polymer may be interrupted by non-amino acids. These terms include amino acid chains of any length, including full-length proteins, and proteins with or without secondary and / or tertiary structures (e.g., domains). These terms also include amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other operation (e.g., conjugation with a labeling component). The term "amino acid" used herein generally refers to natural and non-natural amino acids, including but not limited to modified amino acids and amino acid analogs. Modified amino acids can include natural amino acids and non-natural amino acids that have been chemically modified to include non-naturally occurring groups or chemical moieties on amino acids. Amino acid analogs may refer to amino acid derivatives. The term "amino acid" includes both D-amino acids and L-amino acids.

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

[0106] The terms "engineered", "chimeric" or "recombinant" used herein with respect to polypeptide molecules (e.g., proteins) generally refer to polypeptide molecules having heterologous amino acid sequences or altered amino acid sequences due to the application of genetic engineering techniques to nucleic acids encoding polypeptide molecules and cells or organisms expressing the polypeptide molecules. The terms "engineered" or "recombinant" used herein with respect to polynucleotide molecules (e.g., DNA or RNA molecules) generally refer to polynucleotide molecules having heterologous nucleic acid sequences or altered nucleic acid sequences due to the application of genetic engineering techniques. 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, engineered or recombinant polynucleotides (e.g., genomic DNA sequences) can be modified or altered in part by gene editing. For example, the heterologous endonucleases disclosed herein (e.g., engineered Cas proteins) are not naturally occurring nucleases (e.g., not naturally occurring Cas proteins). In another example, the engineered gene effectors disclosed herein are not naturally occurring gene effectors.

[0107] The terms "engineered" and "modified" are used interchangeably herein. The terms "engineered" and "modified" are used interchangeably herein. The terms "engineered cells" or "modified cells" are used interchangeably herein. The terms "engineered properties" and "modified properties" are used interchangeably herein.

[0108] The terms "enhanced expression", "increased expression" or "up-regulated expression" generally refer to the production level of a portion of interest (e.g., a polynucleotide or polypeptide) in a host strain (e.g., a host cell) that is higher than the normal expression level of the portion of interest. The normal expression level can be substantially zero (or empty) or higher than zero. The portion of interest can include an endogenous gene or polypeptide construct of the host strain. The portion of interest can include a heterologous gene or polypeptide construct introduced into or entering the host strain. For example, a heterologous gene encoding a polypeptide of interest can be knocked-in (KI) into the genome of the host strain to enhance the expression of the polypeptide of interest in the host strain.

[0109] The terms "enhanced activity", "increased activity" or "up-regulated activity" generally refer to that the activity of a portion of interest (e.g., a polynucleotide or polypeptide) is modified to a level above the normal activity level of the portion of interest in a host strain (e.g., a host cell). The normal activity level can be substantially zero (or empty) or above zero. The portion of interest can include a polypeptide construct of the host strain. The portion of interest can include a heterologous polypeptide construct introduced into or into the host strain. For example, a heterologous gene encoding a polypeptide of interest can be knocked-in (KI) into the genome of a host strain to enhance the activity of the polypeptide of interest in the host strain.

[0110] The term "reduced expression", "lowered expression" or "down-regulated expression" generally refers to the production level of a portion of interest (e.g., a polynucleotide or polypeptide) in a host strain (e.g., a host cell) that is lower than the normal expression level of the portion of interest. The normal expression level is above zero. The portion of interest may include an endogenous gene or polypeptide construct of the host strain. In some cases, the portion of interest may be knocked out or knocked down in the host strain. In some instances, the reduced expression of the portion of interest may include complete inhibition of such expression in the host strain.

[0111] The term "reduced activity", "lowered activity" or "downregulated activity" generally refers to that the activity of a portion of interest (e.g., a polynucleotide or polypeptide) is modified to a level below the normal activity level of the portion of interest in a host strain (e.g., a host cell). The normal activity level is above zero. The portion of interest may include an endogenous gene or polypeptide construct of the host strain. In some cases, the portion of interest may be knocked out or knocked down in the host strain. In some instances, the reduced activity of the portion of interest may include complete inhibition of such activity in the host strain.

[0112] The terms "subject", "individual" or "patient" are used interchangeably herein and generally refer to vertebrates, preferably mammals, such as humans. Mammals include, but are not limited to, rodents, apes, humans, farm animals, sports animals, and pets. It also includes tissues, cells, and progeny of biological entities obtained in vivo or cultured in vitro.

[0113] The term "treatment" generally refers to a method of obtaining a beneficial or desired result, including 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 any treatment-related improvement or effect on one or more diseases, conditions, or symptoms being treated. For a prophylactic benefit, a composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even though the disease, condition, or symptom may not yet be manifested.

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

[0115] Overview

[0116] Various aspects of the present disclosure can provide engineered effectors (or engineered gene effectors, as used interchangeably herein) that can regulate (e.g., activate or reduce) the expression or activity level of a target gene (e.g., an endogenous target gene, a heterologous target gene, etc.) in a cell, compositions thereof, and methods of use thereof. Such engineered effectors can be used in combination with heterologous nucleases (e.g., engineered CRISPR / CAs nucleases, or inactivated variants thereof) to, for example, achieve manipulation of the expression or activity level of a target gene in a cell, for example, to treat or improve a condition (e.g., a disease) of a subject. Gene expression can support a variety of physiological and pathological effects in cells and tissues, leading to many diseases and disorders, and therefore compositions and methods utilizing the engineered gene effectors of the present disclosure can regulate the expression of specific genes in a desired manner, thereby obtaining therapeutic benefits.

[0117] Engineered gene effectors, compositions and methods thereof

[0118] In some aspects, the present disclosure provides engineered gene effectors (e.g., engineered gene activators, such as transcriptional activators), which are different from VP16 (e.g., comprising a polypeptide sequence of SEQ ID NO: 5), VP64 (e.g., comprising a polypeptide sequence of SEQ ID NO: 6), p65 (e.g., comprising a polypeptide sequence of SEQ ID NO: 7), Rta (e.g., comprising a polypeptide sequence of SEQ ID NO: 8), or VP64-p65-Rta fusion polypeptide (VPR) (e.g., comprising a polypeptide sequence of SEQ ID NO: 9). In some aspects, the present disclosure provides engineered gene effectors (e.g., engineered gene repressors, such as transcriptional repressors), which are different from Krueppel-associated box (KRAB) (e.g., comprising a polypeptide sequence of SEQ ID NO: 64).

[0119] In some embodiments, the size of an engineered genetic effector as disclosed herein can be at least or up to about 500 amino acid residues, at least or up to about 480 amino acid residues, at least or up to about 460 amino acid residues, at least or up to about 450 amino acid residues, at least or up to about 440 amino acid residues, at least or up to about 420 amino acid residues, at least or up to about 400 amino acid residues, at least or up to about 380 amino acid residues, at least or up to about 360 amino acid residues, at least or up to about 350 amino acid residues, at least or up to about 340 amino acid residues, at least or up to about 320 amino acid residues, at least or up to about 300 amino acid residues, at least or up to about 290 amino acid residues, at least or up to about 280 amino acid residues, at least or up to about 270 amino acid residues, at least or up to about 260 amino acid residues, at least or up to about 250 amino acid residues, at least or up to about 240 amino acid residues, at least or up to about 230 amino acid residues, at least or up to about 240 amino acid residues, or up to about 140 amino acid residues, at least or up to about 150 amino acid residues, at least or up to about 160 amino acid residues, at least or up to about 170 amino acid residues, at least or up to about 170 amino acid residues, at least or up to about 180 amino acid residues, at least or up to about 190 amino acid residues, at least or up to about 190 amino acid residues, at least or up to about 150 amino acid residues, at least or up to about 140 amino acid residues, at least or up to about 130 amino acid residues, at least or up to about 120 amino acid residues, at least or up to about 110 amino acid residues, at least or up to about 100 amino acid residues, at least or up to about 95 amino acid residues, at least or up to about 90 amino acid residues, at least or up to about 85 amino acid residues, at least or up to about 80 amino acid residues, at least or up to about 70 amino acid residues, at least or up to about 60 amino acid residues, at least or up to about 50 amino acid residues, at least or up to about 40 amino acid residues, at least or up to about 30 amino acid residues, or at least or up to about 20 amino acid residues.

[0120] In some embodiments, the size of the engineered genetic effector as disclosed herein can be between about 20 amino acid residues and about 200 amino acid residues, between about 40 amino acid residues and about 180 amino acid residues, between about 50 amino acid residues and about 150 amino acid residues, between about 60 amino acid residues and about 140 amino acid residues, between about 60 amino acid residues and about 130 amino acid residues, between about 60 amino acid residues and about 120 amino acid residues, between about 60 amino acid residues and about 110 amino acid residues, between about 60 amino acid residues and about 100 amino acid residues, between about 70 amino acid residues and about 120 amino acid residues, between about 70 amino acid residues and about 110 amino acid residues, between about 70 amino acid residues and about 100 amino acid residues, between about 80 amino acid residues and about 120 amino acid residues, between about 80 amino acid residues and about 110 amino acid residues, or between about 80 amino acid residues and about 100 amino acid residues.

[0121] In some embodiments, the size of an engineered genetic effector as disclosed herein may be less than about 680 amino acid residues, less than about 650 amino acid residues, less than about 600 amino acid residues, less than about 500 amino acid residues, less than about 400 amino acid residues, less than about 300 amino acid residues, less than about 200 amino acid residues, less than about 150 amino acid residues, less than about 140 amino acid residues, less than about 130 amino acid residues, less than about 120 amino acid residues, less than about 110 amino acid residues, or less than about 100 amino acid residues.

[0122] In some embodiments, the size of the engineered genetic effector as disclosed herein can be less than or equal to about 100 amino acid residues, less than or equal to about 95 amino acid residues, less than or equal to about 90 amino acid residues, less than or equal to about 85 amino acid residues, less than or equal to about 80 amino acid residues, less than or equal to about 75 amino acid residues, less than or equal to about 74 amino acid residues, less than or equal to about 73 amino acid residues, less than or equal to about 72 amino acid residues, less than or equal to about 71 amino acid residues, less than or equal to about 70 amino acid residues, less than or equal to about 69 amino acid residues, less than or equal to about 68 amino acid residues, less than or equal to about 67 amino acid residues, less than or equal to about 66 amino acid residues, less than or equal to about 65 amino acid residues. , less than or equal to about 64 amino acid residues, less than or equal to about 63 amino acid residues, less than or equal to about 62 amino acid residues, less than or equal to about 61 amino acid residues, less than or equal to about 60 amino acid residues, less than or equal to about 59 amino acid residues, less than or equal to about 58 amino acid residues, less than or equal to about 57 amino acid residues, less than or equal to about 56 amino acid residues, less than or equal to about 55 amino acid residues, less than or equal to about 54 amino acid residues, less than or equal to about 53 amino acid residues, less than or equal to about 52 amino acid residues, less than or equal to about 51 amino acid residues, less than or equal to about 50 amino acid residues, less than or equal to about 45 amino acid residues, less than or equal to about 40 amino acid residues, or less than or equal to about 35 amino acid residues.

[0123] In some embodiments, an engineered genetic effector as disclosed herein may comprise a polypeptide.

[0124] In some embodiments, a polypeptide of an engineered gene effector as disclosed herein may comprise an amino acid sequence, and the amino acid sequence of the polypeptide of the engineered gene effector may be at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 81%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 100%, at least or up to about 101%, at least or up to about 102%, at least or up to about 103%, at least or up to about 104%, at least or up to about 105%, at least or up to about 106%, at least or up to about 107%, at least or up to about 108%, at least or up to about 109%, at least or up to about 110%, at least or up to about 111 At least or up to about 82%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% identical. For example, the amino acid sequence of the engineered gene effector can be about 80% to about 100% identical to the polypeptide sequence of SEQ ID NO: 1.

[0125] In some embodiments, when aligned with the polypeptide sequence of SEQ ID No: 1, the amino acid sequence of the polypeptide may include C4. In some embodiments, when aligned with the polypeptide sequence of SEQ ID No: 1, the amino acid sequence of the polypeptide may include L5. In some embodiments, when aligned with the polypeptide sequence of SEQ ID No: 1, the amino acid sequence of the polypeptide may include M7. In some embodiments, when aligned with the polypeptide sequence of SEQ ID No: 1, the amino acid sequence of the polypeptide may include L19. In some embodiments, when aligned with the polypeptide sequence of SEQ ID NO: 1, the amino acid sequence of the polypeptide may include at least one, at least two, at least three or at least four members selected from the group consisting of C4, L5, M7 and L19. In some embodiments, when aligned with the polypeptide sequence of SEQ ID NO: 1, the amino acid sequence of the polypeptide may include one or more members selected from the group consisting of C4, L5, M7 and L19.

[0126] In some embodiments, the amino acid sequence of a polypeptide of an engineered gene effector as disclosed herein may be 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% identical to the polypeptide sequence of SEQ ID NO: 2. For example, the amino acid sequence of a polypeptide of an engineered gene effector may not be, and need not be, identical to the polypeptide sequence of SEQ ID NO: 2.

[0127] In some embodiments, the amino acid sequence of a polypeptide of an engineered gene effector as disclosed herein may be 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% identical to the polypeptide sequence of SEQ ID NO: 3. For example, the amino acid sequence of a polypeptide of an engineered gene effector may not be, and need not be, identical to the polypeptide sequence of SEQ ID NO: 3.

[0128] In some embodiments, the size of the polypeptide of the engineered gene effector as disclosed herein can be at least or up to about 250 amino acid residues, at least or up to about 240 amino acid residues, at least or up to about 230 amino acid residues, at least or up to about 220 amino acid residues, at least or up to about 210 amino acid residues, at least or up to about 200 amino acid residues, at least or up to about 190 amino acid residues, at least or up to about 180 amino acid residues, at least or up to about 170 amino acid residues, at least or up to about 160 amino acid residues, at least or up to about 150 amino acid residues, at least or up to about 140 amino acid residues, at least or up to about 130 amino acid residues, at least or up to about 120 amino acid residues, At least or up to about 110 amino acid residues, at least or up to about 100 amino acid residues, at least or up to about 95 amino acid residues, at least or up to about 90 amino acid residues, at least or up to about 85 amino acid residues, at least or up to about 80 amino acid residues, at least or up to about 70 amino acid residues, at least or up to about 60 amino acid residues, at least or up to about 50 amino acid residues, at least or up to about 45 amino acid residues, at least or up to about 40 amino acid residues, at least or up to about 35 amino acid residues, at least or up to about 30 amino acid residues, at least or up to about 25 amino acid residues, at least or up to about 20 amino acid residues, at least or up to about 15 amino acid residues, or at least or up to about 10 amino acid residues.

[0129] In some embodiments, the size of the polypeptide of the engineered gene effector as disclosed herein can be between about 10 amino acid residues and about 100 amino acid residues, between about 10 amino acid residues and about 90 amino acid residues, between about 10 amino acid residues and about 80 amino acid residues, between about 10 amino acid residues and about 70 amino acid residues, between about 10 amino acid residues and about 60 amino acid residues, between about 10 amino acid residues and about 50 amino acid residues, between about 10 amino acid residues and about 40 amino acid residues, between about 15 amino acid residues and about 100 amino acid residues, between about 15 amino acid residues and about 90 amino acid residues, between about 15 amino acid residues and about 80 amino acid residues, between about 15 amino acid residues and about 10 ... In some embodiments, the present invention relates to a polypeptide having a molecular weight of at least about 25 amino acid residues and about 35 amino acid residues, between about 15 amino acid residues and about 60 amino acid residues, between about 15 amino acid residues and about 50 amino acid residues, between about 15 amino acid residues and about 40 amino acid residues, between about 20 amino acid residues and about 100 amino acid residues, between about 20 amino acid residues and about 90 amino acid residues, between about 20 amino acid residues and about 80 amino acid residues, between about 20 amino acid residues and about 70 amino acid residues, between about 20 amino acid residues and about 60 amino acid residues, between about 20 amino acid residues and about 50 amino acid residues, between about 20 amino acid residues and about 40 amino acid residues, or between about 25 amino acid residues and about 35 amino acid residues.

[0130] In some embodiments, an engineered genetic effector may comprise only one of the polypeptides disclosed herein.

[0131] In some embodiments, the engineered gene effector may comprise a plurality (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) of such polypeptides disclosed herein. The plurality of such polypeptides may be substantially identical. Alternatively or additionally, the plurality of such polypeptides may be different from each other. In some embodiments, the plurality of such polypeptides may comprise a first polypeptide (e.g., exhibiting at least about 70% sequence identity to the polypeptide sequence of SEQ ID NO: 1) and a second polypeptide (e.g., exhibiting at least about 70% sequence identity to the polypeptide sequence of SEQ ID NO: 1). The first polypeptide and the second polypeptide may be coupled to each other, e.g., directly or indirectly (e.g., via a linker). For example, the first polypeptide and the second polypeptide may be fused to each other, e.g., directly or indirectly (e.g., via a linker). In another example, the first polypeptide and the second polypeptide may be non-covalently coupled to each other, e.g., via ionic bonds, hydrogen bonds, interactions mediated by oligomerization or dimerization domains, and the like.

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

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

[0134] In some embodiments, the polypeptide of the engineered effector (e.g., engineered gene activator) disclosed herein can be heterologous to one or more members (e.g., at least 1, 2, 3, 4, 5 or more members, or any one of the 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. Such one or more members can be referred to as transcriptional activators.

[0135] In some embodiments, the polypeptide of the engineered effector (e.g., engineered gene activator) disclosed herein can be heterologous to one or more members (e.g., 1, 2, 3, 4, or 5 members) selected from the group consisting of VP16, VP64, p65, Rta, and VPR. In some cases, the polypeptide can 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, the engineered gene effectors disclosed herein (e.g., engineered gene activators) may be able to activate the expression level of a target gene in a cell (e.g., alone or in combination with a heterologous endonuclease, such as a Cas or dCas protein, e.g., an inactivated variant of Un1Cas12f1 comprising the polypeptide sequence of SEQ ID NO: 11) as compared to a control. The control may be the expression level of the target gene in a control cell (e.g., basal expression level) in the absence of any external manipulation of the target gene (e.g., without an engineered gene effector and / or a heterologous endonuclease). The control may be the expression level of the target gene in a control cell that is treated with a heterologous endonuclease alone (e.g., dCas) without any gene effector operably coupled to the heterologous endonuclease. The control may be the expression level of the target gene in a control cell treated with a control gene effector (e.g., alone or in combination with a heterologous endonuclease). Non-limiting examples of control gene effectors may include VP16, VP64, p65, Rta, and VPR.

[0137] In some embodiments, the expression level of a target gene activated by an engineered gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) can be 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 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 an engineered gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) can be 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 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 an engineered gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) can be 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 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 an engineered gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) can be 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 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 an engineered gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) can be 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 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 an engineered gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) can be 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 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, 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%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, 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%, at least about 250%, at least about 300%, at least about 300%, at least about 300%, at least about 300%, at %, at least about 100%, 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 1.1 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 4.5 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, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, or more.

[0143] In some embodiments, the expression level of a target gene activated by an engineered gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) can be 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 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, 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 10 ...0%, at least about 120%, at least about 130%, at least about 141%, at least about 151%, at least about 162%, at least about 163%, at least about 164%, at least about 165%, at least about 166%, at least about 167%, at least about 168%, at least about 169%, at least about 171%, at least about 172%, at least about 173%, at least about 174%, at least about %, at least about 100%, 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 1.1 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 4.5 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, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, or more.

[0144] In some embodiments, the expression level of a target gene activated by an engineered gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) can be 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 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, 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 141%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, at least about 110%, at least about 120%, at least about 130%, at least about 141%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 250%, at least about 300%, at least about 300%, at least about 300%, at least about 300%, at %, at least about 100%, 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 1.1 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 4.5 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, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, or more.

[0145] In some embodiments, the expression level of a target gene activated by an engineered gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) can be 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 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, 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%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, 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%, at least about 250%, at least about 300%, at least about 300%, at least about 300%, at least about 300%, at %, at least about 100%, 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 1.1 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 4.5 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, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, or more.

[0146] In some embodiments, the expression level of a target gene activated by an engineered gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) can be 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 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, 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%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, 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%, at least about 250%, at least about 300%, at least about 300%, at least about 300%, at least about 300%, at %, at least about 100%, 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 1.1 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 4.5 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, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, or more.

[0147] In some embodiments, the expression level of a target gene activated by an engineered gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) can be substantially sustained (e.g., sustained or maintained 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 regulated level) for at least or up to about 1 hour, at least or up to about 2 hours, at least or up to about 6 hours, at least or up to about 12 hours, at least or up to about 18 hours. or up to about 14 days, at least or up to about 15 days, at least or up to about 16 days, at least or up to about 17 days, at least or up to about 18 days, at least or up to about 19 days, at least or up to about 20 days, at least or up to about 21 days, at least or up to about 22 days, at least or up to about 23 days, at least or up to about 24 days, at least or up to about 24 hours, at least or up to about 2 days, at least or up to about 3 days, at least or up to about 4 days, at least or up to about 5 days, at least or up to about 6 days, at least or up to about 7 days, at least or up to about 8 days, at least or up to about 9 days, at least or up to about 10 days, at least or up to about 11 days, at least or up to about 12 days, at least or up to about 13 days, at least or up to about 14 days, at least or up to about 3 weeks, at least or up to about 4 weeks, at least or up to about 2 months, at least or up to about 4 months, or at least or up to about 6 months.

[0148] In some embodiments, the expression level of a target gene activated by an engineered gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) can 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 the target gene in a control cell activated by a control gene effector (e.g., VPR, Rta, p65, VP64, VP16, P300, etc., in combination with a comparable heterologous endonuclease).

[0149] In some embodiments, the cell population contacted by the system provided herein (e.g., at least the engineered gene effector) can be such that a higher proportion of cells exhibit regulated expression levels (e.g., activation or inhibition) of the target gene compared to the cell population contacted by the system comprising a control gene effector (e.g., VPR, Rta, p65, VP64, VP16, P300, etc.). In some embodiments, the proportion of cells exhibiting regulated expression levels 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 100% of the cell population contacted by the system provided herein (e.g., at least the engineered gene effector). In some embodiments, a cell population contacted by a system provided herein (e.g., at least an engineered gene effector) can have a higher proportion of cells exhibiting modulated expression levels, which 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 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 higher than the proportion of cells in the population exhibiting modulated expression levels contacted by a system comprising a control gene effector (e.g., VPR, Rta, p65, VP64, VP16, P300, etc.).

[0150] In some embodiments, the polypeptide of the engineered effector (e.g., engineered gene repressor) disclosed herein can be heterologous to one or more members (e.g., at least 1, 2, 3, 4, 5 or more members, or any one of the members) selected from the group consisting of: Kruppel associated box (KRAB or SKD); KOX1 repression domain; Mad mSIN3 interaction domain (SID); ERF repressor domain (ERD), SRDX repression domain (e.g., for repression in plants), etc.; histone lysine methyltransferases, such as Pr-SET7 / 8, SUV4-20H1, RIZ1, etc.; histone lysine demethylases, such as JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARJD1A / RBP2, JARID1B / PLU-1, JARID 1C / SMCX, JARIDID / SMCY, etc.; histone lysine deacetylase, such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, etc.; DNA methylase, such as Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plant), ZMET2, CMT1, CMT2 (plant), etc.; and peripheral recruitment elements, such as lamin A, lamin B, etc. One or more members of this class can be referred to as transcriptional repressors.

[0151] In some embodiments, the polypeptide of the engineered effector (e.g., engineered gene repressor) disclosed herein can be heterologous to one or more members (e.g., 1, 2, or 3 members) selected from the group consisting of KRAB, DNMT3A, and DNMT3L.

[0152] In some embodiments, polypeptides of the engineered effectors (eg, engineered gene repressors) disclosed herein may not and need not include KRAB, DNMT3A, and / or DNMT3L.

[0153] In some embodiments, the engineered gene effectors disclosed herein (e.g., engineered gene repressors) can be capable of reducing the expression level of a target gene in a cell (e.g., alone or in combination with a heterologous endonuclease, such as a Cas or dCas protein, e.g., an inactivated variant of Un1Cas12f1 comprising the polypeptide sequence of SEQ ID NO: 11) compared to a control. The control can be the expression level of the target gene in a control cell (e.g., basal expression level) in the absence of any external manipulation of the target gene (e.g., without the engineered gene effector and / or the heterologous endonuclease). The control can be the expression level of the target gene in a control cell that is treated with a heterologous endonuclease alone (e.g., dCas) without any gene effector operably coupled to the heterologous endonuclease. The control can be the expression level of the target gene in a control cell treated with a control gene effector (e.g., alone or in combination with a heterologous endonuclease). Non-limiting examples of control gene effectors can include KRAB, DNMT3A, DNMT3L, and a fusion repressor comprising KRAB, DNMT3A, and DNMT3L (KAL).

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

[0155] In some embodiments, the expression level of a target gene reduced by an engineered genetic effector disclosed herein (e.g., in combination with a heterologous endonuclease) can be comparable (e.g., substantially the same) to the expression level of the target gene 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 reduced by an engineered gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) can be 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 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about At least about 40% less, at least about 50% less, at least about 60% less, at least about 70% less, at least about 80% less, at least about 90% less, 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 less.

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

[0158] In some embodiments, the expression level of a target gene reduced by an engineered genetic effector disclosed herein (e.g., in combination with a heterologous endonuclease) can be comparable (e.g., substantially the same) to the expression level of the target gene 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 reduced by an engineered gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) can be 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 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, 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 reduced by an engineered genetic effector disclosed herein (e.g., in combination with a heterologous endonuclease) can be at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 35%, at least or up to about 40%, at least or up to about 45%, at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, or at least or up to about 95%, as compared to the expression level of the target gene in a control cell reduced by DNMT3L (e.g., in combination with a heterologous endonuclease).

[0161] In some embodiments, the expression level of a target gene reduced by an engineered genetic effector disclosed herein (e.g., in combination with a heterologous endonuclease) can be comparable (e.g., substantially the same) to the expression level of the target gene 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 reduced by an engineered gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) can be 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 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, 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 reduced by an engineered gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) can be at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 35%, at least or up to about 40%, at least or up to about 45%, at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, or at least or up to about 95% as compared to the expression level of the target gene reduced by KAL (e.g., in combination with a heterologous endonuclease) in a control cell.

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

[0165] In some embodiments, the expression level of a target gene reduced by an engineered gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) can be 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 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 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 reduced by an engineered genetic effector disclosed herein (e.g., in combination with a heterologous endonuclease) can 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, as compared to the expression level of the target gene 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 reduced by an engineered gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) can 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, as compared to the expression level of the target gene 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 reduced by an engineered gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) can 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, as compared to the expression level of the target gene 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 reduced by an engineered gene effector disclosed herein (e.g., in combination with a heterologous endonuclease) can 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, as compared to the expression level of the target gene reduced by KAL (e.g., in combination with a heterologous endonuclease) in a control cell.

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

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

[0172] Without wishing to be bound by theory, in some embodiments, (1) the first engineered gene effector may comprise at least a portion of SEQ ID NO: 1, and (2) the second engineered gene effector may comprise at least a portion of the polypeptide sequence of SEQ ID NO: 1 and be different from the first engineered gene effector, and the first engineered gene effector and the second engineered gene effector may exhibit different gene regulatory activities. In some cases, even though the at least a portion of the polypeptide sequence of SEQ ID NO: 1 of (1) the first engineered gene effector may be substantially identical to the at least a portion of the polypeptide sequence of SEQ ID NO: 1 of (2) the second engineered gene effector, the first engineered gene effector and the second engineered gene effector may exhibit different (e.g., opposite) gene regulatory activities. For example, the first engineered gene effector may be a gene activator, and the second engineered gene effector may be a gene repressor. The different activities of the first and second engineered gene effectors may be due to one or more differences between the first and second engineered gene effectors, for example, (i) the position of the at least portion of the polypeptide sequence of SEQ ID NO: 1 in each engineered gene effector, (ii) the overall size / length of the engineered gene effector, (iii) the N-terminus of the at least portion of the polypeptide sequence of SEQ ID NO: 1, the presence, size or amino acid residue composition of additional polypeptide sequences, (iv) the C-terminus of the at least portion of the polypeptide sequence of SEQ ID NO: 1, the presence, size or amino acid residue composition of additional polypeptide sequences, and / or (v) the size or amino acid residue composition of the at least portion of the polypeptide sequence of SEQ ID NO: 1.

[0173] In some embodiments, the additional polypeptides of a gene effector disclosed herein may be disposed adjacent to the N-terminus of a polypeptide of a gene effector. In some embodiments, the additional polypeptides of a gene effector may be disposed adjacent to the C-terminus of a polypeptide of a gene effector. In some embodiments, a first type of additional polypeptide of a gene effector (e.g., a first additional polypeptide) may be disposed adjacent to the N-terminus of a polypeptide of a gene effector, and a second type of additional polypeptide of a gene effector (e.g., a second additional polypeptide) may be disposed adjacent to the C-terminus of a polypeptide of a gene effector (e.g., first additional polypeptide-polypeptide-second additional polypeptide). The polypeptide may be flanked by the first additional polypeptide and the second additional polypeptide. The first additional polypeptide and the second additional polypeptide may be substantially identical. Alternatively, the first additional polypeptide and the second additional polypeptide may be different. In some embodiments, a first type of polypeptide of a gene effector (e.g., a first polypeptide) may be disposed adjacent to the N-terminus of an additional polypeptide of a gene effector, and a second type of polypeptide of a gene effector (e.g., a second polypeptide) may be disposed adjacent to the C-terminus of an additional polypeptide of a gene effector (e.g., first polypeptide-additional polypeptide-second polypeptide). The additional polypeptide may be flanked by the first polypeptide and the second polypeptide. The first polypeptide and the second polypeptide may be substantially identical. Alternatively, the first polypeptide and the second polypeptide may be different.

[0174] In some embodiments, the additional polypeptides of the engineered gene effectors disclosed herein can be disposed within (e.g., inserted into) the polypeptides of the engineered gene effectors disclosed herein. The additional polypeptides can be inserted within the N-terminus of the polypeptide, or adjacent to the N-terminus of the polypeptide. Alternatively, the additional polypeptides can be inserted within the C-terminus of the polypeptide, or adjacent to the C-terminus of the polypeptide.

[0175] In some embodiments, the size of the polypeptide of the engineered genetic effector as disclosed herein and the size of the additional polypeptide can be substantially the same. In some embodiments, the size of the polypeptide of the engineered genetic effector and the size of the additional polypeptide can be different.

[0176] In some embodiments of the engineered gene effectors disclosed herein, the size of the polypeptide can be longer than the size of the additional polypeptide, for example, by at least or up to about 1 amino acid residue, at least or up to about 2 amino acid residues, at least or up to about 3 amino acid residues, at least or up to about 4 amino acid residues, at least or up to about 5 amino acid residues, at least or up to about 10 amino acid residues, at least or up to about 15 amino acid residues, at least or up to about 20 amino acid residues, at least or up to about 25 amino acid residues, at least or up to about 30 amino acid residues, at least or up to about 35 amino acid residues, at least or up to about 4 In some embodiments, the present invention comprises at least or up to about 100 amino acid residues, at least or up to about 45 amino acid residues, at least or up to about 50 amino acid residues, at least or up to about 60 amino acid residues, at least or up to about 70 amino acid residues, at least or up to about 80 amino acid residues, at least or up to about 90 amino acid residues, at least or up to about 100 amino acid residues, at least or up to about 110 amino acid residues, at least or up to about 120 amino acid residues, at least or up to about 130 amino acid residues, at least or up to about 140 amino acid residues, at least or up to about 150 amino acid residues, or at least or up to about 200 amino acid residues.

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

[0178] In some embodiments of the engineered genetic effectors disclosed herein, the size of the polypeptide can be at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 0%, 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%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, or more. In some embodiments of the engineered gene effectors disclosed herein, the size of the polypeptide can be at most about 1%, at most about 2%, at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, at most about 26%, at most about 27%, at most about 28%, at most about 29%, at most about 30%, at most about 31%, at most about 32%, at most about 33%, at most about 34%, at most about 35%, at most about 36%, at most about 37%, at most about 38%, at most about 39%, at most about 40%, at most about 41%, at most about 42%, at most about 43%, at most about 44%, at most about 45%, at most about 46%, at most about 47%, at most about 48%, at most about 49%, at most about 50%, at most about 51%, at most about 52%, at most about 53%, at most about 54%, at most about 55%, at most about 56%, at most about 57%, at most about 58%, at most about 59%, at most about 60%, at most about 61%, at most about 62%, at most about 63%, at most about 64%, at most about 65%, at most about 66%, at most about 67%, at most about 68%, at most about 69%, at most about In some embodiments, the present invention relates to an aqueous solution of at least about 40%, at most about 50%, at most about 60%, at most about 70%, at most about 80%, at most about 90%, at most about 100%, at most about 110%, at most about 120%, at most about 130%, at most about 140%, at most about 150%, at most about 160%, at most about 170%, at most about 180%, at most about 190%, at most about 200%, at most about 250%, at most about 300%, at most about 400%, or at most about 500%.

[0179] In some embodiments of the engineered genetic effectors disclosed herein, the size of the additional polypeptide can be at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 0%, 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%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, or more. In some embodiments of the engineered genetic effectors disclosed herein, the size of the additional polypeptide may be at most about 1%, at most about 2%, at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, at most about 26%, at most about 27%, at most about 28%, at most about 29%, at most about 30%, at most about 31%, at most about 32%, at most about 33%, at most about 34%, at most about 35%, at most about 36%, at most about 37%, at most about 38%, at most about 39%, at most about 40%, at most about 41%, at most about 42%, at most about 43%, at most about 44%, at most about 45%, at most about 46%, at most about 47%, at most about 48%, at most about 49%, at most about 50%, at most about 51%, at most about 52%, at most about 53%, at most about 54%, at most about 55%, at most about 56%, at most about 57%, at most about 58%, at most about 59%, at most about 60%, at most about 61%, at most about 62%, at most about 63%, at most about 64%, at most about 65%, at most about 66%, at most about 67%, at most about 68%, at most about 69%, at most about In some embodiments, the present invention relates to an aqueous solution of at least about 40%, at most about 50%, at most about 60%, at most about 70%, at most about 80%, at most about 90%, at most about 100%, at most about 110%, at most about 120%, at most about 130%, at most about 140%, at most about 150%, at most about 160%, at most about 170%, at most about 180%, at most about 190%, at most about 200%, at most about 250%, at most about 300%, at most about 400%, or at most about 500%.

[0180] In some embodiments, the size of the additional polypeptides of the engineered gene effectors disclosed herein can be at least or up to about 500 amino acid residues, at least or up to about 480 amino acid residues, at least or up to about 460 amino acid residues, at least or up to about 450 amino acid residues, at least or up to about 440 amino acid residues, at least or up to about 420 amino acid residues, at least or up to about 400 amino acid residues, at least or up to about 380 amino acid residues, at least or up to about 360 amino acid residues, at least or up to about 350 amino acid residues, at least or up to about 340 amino acid residues, at least or up to about 320 amino acid residues, at least or up to about 300 amino acid residues, at least or up to about 290 amino acid residues, at least or up to about 280 amino acid residues, at least or up to about 270 amino acid residues, at least or up to about 260 amino acid residues, at least or up to about 250 amino acid residues, at least or up to about 240 amino acid residues, at least or up to about 230 amino acid residues, at least or up to about 220 amino acid residues, at least or up to about 210 amino acid residues, at least or up to about 200 amino acid residues, at least or up to about 190 amino acid residues, at least or up to about 180 amino acid residues, at least or up to about 170 amino acid residues, at least or up to about 1 60 amino acid residues, at least or up to about 150 amino acid residues, at least or up to about 140 amino acid residues, at least or up to about 130 amino acid residues, at least or up to about 120 amino acid residues, at least or up to about 110 amino acid residues, at least or up to about 100 amino acid residues, at least or up to about 95 amino acid residues, at least or up to about 90 amino acid residues, at least or up to about 85 amino acid residues, at least or up to about 80 amino acid residues, at least or up to about 70 amino acid residues, at least or up to about 60 amino acid residues, at least or up to about 50 amino acid residues, at least or up to about 45 amino acid residues, at least or up to about 5 Up to about 40 amino acid residues, at least or up to about 35 amino acid residues, at least or up to about 30 amino acid residues, at least or up to about 25 amino acid residues, at least or up to about 20 amino acid residues, at least or up to about 15 amino acid residues, at least or up to about 10 amino acid residues, at least or up to about 9 amino acid residues, at least or up to about 8 amino acid residues, at least or up to about 7 amino acid residues, at least or up to about 6 amino acid residues, at least or up to about 5 amino acid residues, at least or up to about 4 amino acid residues, at least or up to about 3 amino acid residues, at least or up to about 2 amino acid residues, or at least or up to about 1 amino acid residue.

[0181] In some embodiments, the size of the additional polypeptides of the engineered gene effectors disclosed herein can be between about 2 and about 50, between about 2 and about 40, between about 2 and about 30, between about 2 and about 25, between about 2 and about 20, between about 2 and about 15, between about 2 and about 10, between about 2 and about 9, between about 2 and about 8, between about 2 and about 7, between about 2 and about 6, between about 2 and about 5, between about 2 and about 4, about 3 to about 15, about 3 to about 10, about 3 to about 9, about 3 to about 8, about 3 to about 7, about 3 to about 6, about 3 to about 5, about 1 to about 15, about 1 to about 10, about 1 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, or about 1 to about 3 amino acid residues.

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

[0183] In some embodiments, the additional polypeptides (of the engineered gene effectors disclosed herein) alone may be inert, for example, they do not induce any desired biological activity in the cell by themselves. In some embodiments, the additional polypeptides (of the engineered gene effectors disclosed herein) alone may not encode (or comprise) and need not encode (or comprise) any gene effectors. Thus, in the absence of the polypeptides of the engineered gene effectors disclosed herein, the additional polypeptides may not be able to modulate the expression and / or activity of the target gene in the cell.

[0184] In some embodiments, the additional polypeptides of the engineered gene effectors disclosed herein may include filler polypeptide sequences. The filler polypeptide sequence may be used in combination with another polypeptide sequence (e.g., a polypeptide of an engineered gene effector) to increase the size of the entire polypeptide (e.g., an engineered gene effector). A filler polypeptide sequence alone may not be able to exhibit the biological activity of interest. For example, in the absence of other parts of the engineered gene effector (e.g., in the absence of a polypeptide of an engineered gene effector), a filler polypeptide sequence alone may not be able to regulate (e.g., activate, repress, etc.) the expression or activity level of a target gene in a cell. The filler polypeptide sequence may not be found in natural proteins (e.g., natural mammalian proteins). In some embodiments, the filler polypeptide sequence may include 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, the filler polypeptide sequence may include multiple I residues (e.g., II, III, etc.). In some embodiments, the filler polypeptide sequence may include multiple L residues (e.g., LL, LLL, etc.). In some embodiments, the filler polypeptide sequence may comprise a plurality of V residues (e.g., VV, VVV, etc.). In some embodiments, the filler polypeptide sequence may comprise a plurality of A residues (e.g., AA, AAA, etc.). In some embodiments, the filler polypeptide sequence may comprise a plurality of G residues (e.g., GG, GGG, etc.).

[0185] In some embodiments, the filling polypeptide sequence disclosed herein can be a linker sequence. Any suitable linker can be used. A flexible linker can have a sequence of stretches containing glycine and serine residues. The small size of glycine and serine residues provides flexibility and allows the connected functional domains to move. The incorporation of serine or threonine can maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thereby reducing the adverse interactions between the linker and the protein portion. The flexible linker can also contain additional amino acids, such as threonine and alanine, to maintain flexibility, and polar amino acids, such as lysine and glutamine, to improve solubility. For example, a rigid connector can have an alpha helical structure. An alpha helical rigid linker can serve as a spacer between protein domains. Non-limiting examples of linkers include sequences in Table 4 and repetitions thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 repetitions. SEQ ID NO: 65-SEQ ID NO: 70 provide flexible linkers or their subunits. SEQ ID NO:71-SEQ ID NO:74 provide rigid linkers or subunits thereof.

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

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

[0188] In some embodiments, a non-peptide linker is used. For example, a non-peptide linker can be a chemical linker. The two parts of the complex disclosed herein can be connected by a chemical linker. Various chemical linkers disclosed herein can be alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene or heteroarylene, any of which is optionally substituted. In some embodiments, a chemical linker disclosed herein can be an ester, ether, amide, thioether or polyethylene glycol (PEG). In some embodiments, the linker can reverse the order of the amino acid sequence in the compound, for example, so that the amino acid sequence connected by the linker is head to head, rather than head to tail. The non-limiting examples of such linkers include the diesters of dicarboxylic acids, such as oxalyl diesters, malonyl diesters, succinyl diesters, glutaryl diesters, adipoyl diesters, pimetyl diesters, fumaryl diesters, maleoyl diesters, phthaloyl diesters, isophthaloyl diesters and terephthaloyl diesters. Non-limiting examples of such linkers include diamides of dicarboxylic acids, such as oxalyl diamide, malonyl diamide, succinyl diamide, glutaryl diamide, adipoyl diamide, pimetyl diamide, fumaryl diamide, maleoyl diamide, phthalyl diamide, isophthalyl diamide, and terephthalyl diamide. Non-limiting examples of such linkers include diamides of diamino linkers, such as ethylenediamine, 1,2-bis(methylamino)ethane, 1,3-diaminopropane, 1,3-bis(methylamino)propane, 1,4-bis(methylamino)butane, 1,5-bis(methylamino)pentane, 1,6-bis(methylamino)hexane, and pipyrizine. Non-limiting examples of optional substituents include hydroxyl groups, thiol groups, halogens, amino groups, nitro groups, nitroso groups, cyano groups, azide groups, sulfoxide groups, sulfone groups, sulfonamide groups, carboxyl groups, carboxaldehyde groups, imine groups, alkyl groups, halo-alkyl groups, alkenyl groups, halo-alkenyl groups, alkynyl groups, halo-alkynyl groups, halo-alkynyl groups, alkoxy groups, aryl groups, aryloxy groups, aralkyl groups, arylalkoxy groups, heterocyclyl groups, acyl groups, acyloxy groups, carbamate groups, amide groups, urea groups, epoxy groups, and ester groups.

[0189] In some embodiments, the additional polypeptides of the engineered gene effectors disclosed herein may comprise at most about 20, at most about 15, at most about 14, at most about 13, at most about 12, at most about 11, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, or about 1 glycine-serine (GS) linkers. In some embodiments, the additional polypeptides of the engineered gene effectors (e.g., engineered gene repressors) disclosed herein can comprise 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, 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 engineered gene effectors as disclosed herein can comprise at most about 20, at most about 15, at most about 14, at most about 13, at most about 12, at most about 11, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, or about 1 glycine (G) linker. In some embodiments, the additional polypeptides of the engineered gene effectors (e.g., engineered gene repressors) disclosed herein can comprise 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 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 engineered gene effector disclosed herein) may encode (or comprise) at least one gene effector alone. Thus, in the absence of the polypeptide of the engineered gene effector, the at least one gene effector of the additional polypeptide may be able to regulate the expression and / or activity of the target gene in the cell. In some cases, the at least one gene effector may include a plurality of identical gene effectors (e.g., at least 2, 3, 4, 5 or more gene effectors). Alternatively, the plurality of gene effectors may be different from each other. The plurality of gene effectors may include more than two gene activators. The plurality of gene effectors may include more than two gene repressors. The plurality of gene effectors may include gene activators and gene repressors.

[0192] In some embodiments, the additional polypeptides of the engineered gene effectors (e.g., engineered gene activators) 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 engineered gene effectors (e.g., engineered 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 polypeptides of the engineered gene effectors (e.g., engineered gene activators) 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 polypeptides of the engineered gene effectors (e.g., engineered gene activators) disclosed herein may include two or more members selected from the group consisting of VP16, VP64, p65, and Rta. In some embodiments, the additional polypeptides of the engineered gene effectors disclosed herein may include multiple VP16 domains. In some embodiments, the additional polypeptides of the engineered gene effectors disclosed herein may include 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 polypeptides of the engineered gene effectors (e.g., engineered gene activators) disclosed herein may not include two or more members selected from the group consisting of VP16, VP64, p65, and Rta. The additional polypeptide may include VP16, but not any one of VP64, p65, and Rta. The additional polypeptide may include VP64, but not any one of VP16, p65, and Rta. The additional polypeptide may include p65, but not any one of VP16, VP64, and Rta. The additional polypeptide may include Rta, but not any one of VP16, VP64, and p65. Alternatively, the additional polypeptide may not include any one of VP16, VP64, p65, Rta, and VPR.

[0196] In some embodiments, the additional polypeptides of the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise an amino acid sequence, and such amino acid sequence can be at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 81%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, at least or up to about 100%, at least or up to about 101%, at least or up to about 102%, at least or up to about 103%, at least or up to about 104%, at least or up to about 105%, at least or up to about 106%, at least or up to about 107%, at least or up to about 108%, at least or up to about 109%, at least or up to about 110%, at least or up to about 0%, at least or up to about 82%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% the same.

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

[0198] In some embodiments, the additional polypeptides of the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise an amino acid sequence, and such amino acid sequence can be at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 79%, at least or up to about 80%, at least or up to about 81%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, at least or up to about 100%, at least or up to about 101%, at least or up to about 102%, at least or up to about 103%, at least or up to about 104%, at least or up to about 105%, at least or up to about 106%, at least or up to about 107%, at least or up to about 108%, at least or up to about 109%, at least 0%, at least or up to about 82%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% the same.

[0199] In some embodiments, the engineered genetic effectors disclosed herein do not comprise Rta.

[0200] In some embodiments, the additional polypeptides of the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise an amino acid sequence, and such amino acid sequence can be at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 79%, at least or up to about 80%, at least or up to about 81%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, at least or up to about 100%, at least or up to about 101%, at least or up to about 102%, at least or up to about 103%, at least or up to about 104%, at least or up to about 105%, at least or up to about 106%, at least or up to about 107%, at least or up to about 108%, at least or up to about 109%, at least or up to about 0%, at least or up to about 82%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% the same.

[0201] In some embodiments, the engineered genetic effectors disclosed herein do not comprise p65.

[0202] In some embodiments, the additional polypeptides of the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise an amino acid sequence, and such amino acid sequence can be at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 81%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, at least or up to about 100%, at least or up to about 101%, at least or up to about 102%, at least or up to about 103%, at least or up to about 104%, at least or up to about 105%, at least or up to about 106%, at least or up to about 107%, at least or up to about 108%, at least or up to about 109%, at least or up to about 110%, at least or up to about 0%, at least or up to about 82%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% the same.

[0203] In some embodiments, the engineered genetic effectors disclosed herein do not comprise VP64.

[0204] In some embodiments, the additional polypeptides of the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise an amino acid sequence, and such amino acid sequence can be at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 81%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, at least or up to about 100%, at least or up to about 101%, at least or up to about 102%, at least or up to about 103%, at least or up to about 104%, at least or up to about 105%, at least or up to about 106%, at least or up to about 107%, at least or up to about 108%, at least or up to about 109%, at least or up to about 110%, at least or up to about 0%, at least or up to about 82%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% the same.

[0205] In some embodiments, the engineered gene effectors disclosed herein do not comprise VP 16. In some embodiments, the engineered gene effectors disclosed herein (e.g., engineered gene activators) may comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 81%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 100%, at least or up to about 101%, at least or up to about 102%, at least or up to about 103%, at least or up to about 104%, at least or up to about 105%, at least or up to about 106%, at least or up to about 107%, at least or up to about 108%, at least or up to about 109%, at least or up to about 110%, at least or up to about 1 or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0206] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise a polypeptide sequence that has at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 81%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, at least or up to about 100%, at least or up to about 101%, at least or up to about 102%, at least or up to about 103%, at least or up to about 104%, at least or up to about 105%, at least or up to about 106%, at least or up to about 107%, at least or up to about 108%, at least or up to about 109%, at least or up to about 110%, at least or up to about 111 or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0207] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein may comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 19. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0208] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein may comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 20. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0209] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein may comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 23. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0210] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein may comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 24. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0211] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein may comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 30. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0212] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein may comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 31. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0213] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80% similarity to 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). , at least or up to about 82%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0214] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein may comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 79. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0215] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise polypeptide sequences that have at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 80. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0216] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 81. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0217] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise polypeptide sequences that have at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 82. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0218] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 83. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0219] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise polypeptide sequences that have at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 84. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0220] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise polypeptide sequences that have at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 86. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0221] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 87. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0222] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 88. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0223] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 89. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0224] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein may comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 90. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0225] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 91. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0226] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise polypeptide sequences that have at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 92. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0227] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 94. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0228] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise polypeptide sequences that have at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 95. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0229] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise polypeptide sequences that have at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 96. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0230] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 97. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0231] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 98. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0232] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 99. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0233] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise a polypeptide sequence that has at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, at least or up to about 100%, at least or up to about 101%, at least or up to about 102%, at least or up to about 103%, at least or up to about 104%, at least or up to about 105%, at least or up to about 106%, at least or up to about 107%, at least or up to about 108%, at least or up to about 109 %, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0234] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise a polypeptide sequence that has at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, at least or up to about 100%, at least or up to about 101%, at least or up to about 102%, at least or up to about 103%, at least or up to about 104%, at least or up to about 105%, at least or up to about 106%, at least or up to about 107%, at least or up to about 108%, at least or up to about 109%, at least %, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0235] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise a polypeptide sequence that has at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, at least or up to about 100%, at least or up to about 101%, at least or up to about 102%, at least or up to about 103%, at least or up to about 104%, at least or up to about 105%, at least or up to about 106%, at least or up to about 107%, at least or up to about 108%, at least or up to about 109 %, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0236] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise a polypeptide sequence that has at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, at least or up to about 100%, at least or up to about 101%, at least or up to about 102%, at least or up to about 103. %, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0237] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise a polypeptide sequence that has at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, at least or up to about 100%, at least or up to about 101%, at least or up to about 102%, at least or up to about 103%, at least or up to about 104%, at least or up to about 105%, at least or up to about 106%, at least or up to about 107%, at least or up to about 108%, at least or up to about 109%, at least or up to about 110 %, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0238] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise a polypeptide sequence that has at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, at least or up to about 100%, at least or up to about 101%, at least or up to about 102%, at least or up to about 103%, at least or up to about 104%, at least or up to about 105%, at least or up to about 106%, at least or up to about 107%, at least or up to about 108%, at least or up to about 109%, at least or up to about 110 %, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0239] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise polypeptide sequences that have at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, at least or up to about 100%, at least or up to about 101%, at least or up to about 102%, at least or up to about 103%, at least or up to about 104%, at least or up to about 105%, at least or up to about 106%, at least or up to about 107%, at least or up to about 108%, at least or up to about 109%, at least or up to about 110%, at least or up to about 111 %, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0240] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise a polypeptide sequence that has at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, at least or up to about 100%, at least or up to about 101%, at least or up to about 102%, at least or up to about 103%, at least or up to about 104%, at least or up to about 105%, at least or up to about 106%, at least or up to about 107%, at least or up to about 108%, at least or up to about 109%, at least or up to about 110%, at least or up to about 111 %, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0241] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise a polypeptide sequence that has at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, at least or up to about 100%, at least or up to about 101%, at least or up to about 102%, at least or up to about 103%, at least or up to about 104%, at least or up to about 105%, at least or up to about 106%, at least or up to about 107%, at least or up to about 108%, at least or up to about 109%, at least or up to about 110%, at least or up to about 111 %, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0242] In some embodiments, the engineered gene effectors (e.g., engineered gene activators) disclosed herein can comprise a polypeptide sequence that has at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, at least or up to about 100%, at least or up to about 101%, at least or up to about 102%, at least or up to about 103%, at least or up to about 104%, at least or up to about 105%, at least or up to about 106%, at least or up to about 107%, at least or up to about 108%, at least or up to about 109%, at least or up to about 110%, at least or up to about 111 %, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

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

[0244] In some embodiments, the engineered gene effectors (e.g., engineered gene suppressors) disclosed herein can comprise a polypeptide sequence that has at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 81%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, at least or up to about 100%, at least or up to about 101%, at least or up to about 102%, at least or up to about 103%, at least or up to about 104%, at least or up to about 105%, at least or up to about 106%, at least or up to about 107%, at least or up to about 108%, at least or up to about 109%, at least or up to about 110%, at least or up to about 111 2%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity to an amino acid sequence (e.g., a contiguous polypeptide sequence).

[0245] In some embodiments, the engineered gene effectors (e.g., engineered gene suppressors) disclosed herein may comprise a polypeptide sequence that has at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 81%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 100%, at least or up to about 101%, at least or up to about 102%, at least or up to about 103%, at least or up to about 104%, at least or up to about 105%, at least or up to about 106%, at least or up to about 107%, at least or up to about 108%, at least or up to about 109%, at least or up to about 110%, at least or up to about 111 or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0246] In some embodiments, the engineered gene effectors (e.g., engineered gene suppressors) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 32. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0247] In some embodiments, the engineered gene effectors (e.g., engineered gene suppressors) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 33. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0248] In some embodiments, the engineered gene effectors (e.g., engineered gene suppressors) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 34. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0249] In some embodiments, the engineered gene effectors (e.g., engineered gene suppressors) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 35. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0250] In some embodiments, the engineered gene effectors (e.g., engineered gene suppressors) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 38. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0251] In some embodiments, the engineered gene effectors (e.g., engineered gene suppressors) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 39. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0252] In some embodiments, the engineered gene effectors (e.g., engineered gene suppressors) disclosed herein can comprise a polypeptide sequence that has at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 81%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, at least or up to about 100%, at least or up to about 101%, at least or up to about 102%, at least or up to about 103%, at least or up to about 104%, at least or up to about 105%, at least or up to about 106%, at least or up to about 107%, at least or up to about 108%, at least or up to about 109%, at least or up to about 110%, at least or up to about 111 2%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity to an amino acid sequence (e.g., a contiguous polypeptide sequence).

[0253] In some embodiments, the engineered gene effectors (e.g., engineered gene suppressors) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 44. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0254] In some embodiments, the engineered gene effectors (e.g., engineered gene suppressors) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 45. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0255] In some embodiments, the engineered gene effectors (e.g., engineered gene suppressors) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 46. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0256] In some embodiments, the engineered gene effectors (e.g., engineered gene suppressors) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 47. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0257] In some embodiments, the engineered gene effectors (e.g., engineered gene suppressors) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 48. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0258] In some embodiments, the engineered gene effectors (e.g., engineered gene suppressors) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 49. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0259] In some embodiments, the engineered gene effectors (e.g., engineered gene suppressors) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 50. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0260] In some embodiments, the engineered gene effectors (e.g., engineered gene suppressors) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 51. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0261] In some embodiments, the engineered gene effectors (e.g., engineered gene repressors) 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 engineered gene effectors (e.g., engineered gene suppressors) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 52. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0263] In some embodiments, the amino acid sequence of a polypeptide of an engineered gene effector disclosed herein may be 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% identical to the polypeptide sequence of SEQ ID NO: 53. For example, the amino acid sequence of a polypeptide of an engineered gene effector may not be, and need not be, identical to the polypeptide sequence of SEQ ID NO: 53.

[0264] In some embodiments, the amino acid sequence of a polypeptide of an engineered gene effector disclosed herein may be 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% identical to the polypeptide sequence of SEQ ID NO: 54. For example, the amino acid sequence of a polypeptide of an engineered gene effector may not be, and need not be, identical to the polypeptide sequence of SEQ ID NO: 54.

[0265] In some embodiments, the engineered gene effectors (e.g., engineered gene suppressors) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 55. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0266] In some embodiments, the amino acid sequence of a polypeptide of an engineered gene effector disclosed herein may be 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% identical to the polypeptide sequence of SEQ ID NO: 56. For example, the amino acid sequence of a polypeptide of an engineered gene effector may not be, and need not be, identical to the polypeptide sequence of SEQ ID NO: 56.

[0267] In some embodiments, the amino acid sequence of a polypeptide of an engineered gene effector disclosed herein may be 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% identical to the polypeptide sequence of SEQ ID NO: 57. For example, the amino acid sequence of a polypeptide of an engineered gene effector may not be, and need not be, identical to the polypeptide sequence of SEQ ID NO: 57.

[0268] In some embodiments, the engineered gene effectors (e.g., engineered gene suppressors) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 58. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0269] In some embodiments, the amino acid sequence of a polypeptide of an engineered gene effector disclosed herein may be 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% identical to the polypeptide sequence of SEQ ID NO: 59. For example, the amino acid sequence of a polypeptide of an engineered gene effector may not be, and need not be, identical to the polypeptide sequence of SEQ ID NO: 59.

[0270] In some embodiments, the amino acid sequence of a polypeptide of an engineered gene effector disclosed herein may be 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% identical to the polypeptide sequence of SEQ ID NO: 60. For example, the amino acid sequence of a polypeptide of an engineered gene effector may not be, and need not be, identical to the polypeptide sequence of SEQ ID NO: 60.

[0271] In some embodiments, the engineered gene effectors (e.g., engineered gene suppressors) disclosed herein can comprise a polypeptide sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 78%, at least or up to about 80%, at least or up to about 82% similarity to the polypeptide sequence of SEQ ID NO: 61. , at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity.

[0272] In some embodiments, the amino acid sequence of a polypeptide of an engineered gene effector disclosed herein may be 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% identical to the polypeptide sequence of SEQ ID NO: 62. For example, the amino acid sequence of a polypeptide of an engineered gene effector may not be, and need not be, identical to the polypeptide sequence of SEQ ID NO: 62.

[0273] In some embodiments, the amino acid sequence of a polypeptide of an engineered gene effector disclosed herein may be 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% identical to the polypeptide sequence of SEQ ID NO: 63. For example, the amino acid sequence of a polypeptide of an engineered gene effector may not be, and need not be, identical to the polypeptide sequence of SEQ ID NO: 63.

[0274] In some embodiments, the engineered gene effector can 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) compared 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 engineered gene effector can be enriched in one or more of aromatic amino acid residues, neutral amino acid residues and / or basic amino acid residues compared to hydrophobic amino acid residues and / or acidic amino acid residues.

[0275] In some embodiments, the polypeptide of the engineered gene effector can 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) compared 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 polypeptide of the engineered gene effector can be enriched in one or more of aromatic amino acid residues, neutral amino acid residues and / or basic amino acid residues compared to hydrophobic amino acid residues and / or acidic amino acid residues.

[0276] In some embodiments, the additional polypeptides of the engineered gene effector can 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) compared 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 engineered gene effector can be enriched in one or more of aromatic amino acid residues, neutral amino acid residues, and / or basic amino acid residues compared to hydrophobic amino acid residues and / or acidic amino acid residues.

[0277] sequence SEQ ID NO: 1 (vIRF2 domain 1) SEQ ID NO:2 (vIRF2 domain 2) SEQ ID NO:3 (vIRF2 domain 3) SEQ ID NO:4 (wild-type viral IRF2 or "vIRF2") SEQ ID NO:5 (VP16) SEQ ID NO:6 (VP64) SEQ ID NO:7(p65) SEQ ID NO:8 (Rta) SEQ ID NO:9 (VP64-p65-Rta or "VPR", linker sequence is underlined) SEQ ID NO: 10 (Un1Cas12f1 or "CasMini") SEQ ID NO: 11 (inactivated nuclease variant of Un1Cas12f1 or "dCasMini") SEQ ID NO:52 (Bel-1 domain 1) SEQ ID NO:53 (Bel-1 domain 2) SEQ ID NO:54 (wild type Bel-1) SEQ ID NO:55 (Chikungunya domain 1) SEQ ID NO:56 (Chikungunya domain 2) SEQ ID NO:57 (wild type Chikungunya) SEQ ID NO:58 (Eptv1 domain 1) SEQ ID NO:59 (Eptv1 domain 2) SEQ ID NO:60 (wild type Eptv1) SEQ ID NO:61 (Eptv2 active domain 1) SEQ ID NO:62 (Eptv2 active domain 2) SEQ ID NO:63 (wild-type Eptv2) SEQ ID NO:64 (KRAB)

[0278] Additional aspects of engineering genetic effectors

[0279] In some embodiments, the engineered gene effectors disclosed herein can be used to achieve regulation of the expression and / or activity level of a target gene (e.g., one or more target genes of Table 1). In some cases, a heterologous polypeptide comprising at least an engineered gene effector can be introduced into a cell (e.g., a mammalian cell) to achieve regulation of the expression and / or activity level of a target gene. In some embodiments, the engineered gene effectors disclosed herein, or proteins comprising engineered gene effectors (e.g., proteins comprising engineered gene effectors coupled to heterologous endonucleases) can be referred to as actuator motifs.

[0280] In some embodiments, the engineered gene effectors disclosed herein can be coupled to a heterologous endonuclease (e.g., an enzymatically active Cas protein, an enzymatically inactivated Cas protein, etc.). The engineered gene effector and the heterologous endonuclease can be coupled to each other, for example, directly or indirectly (e.g., through a linker). For example, the engineered gene effector and the heterologous endonuclease can be fused to each other, for example, directly or indirectly (e.g., through a linker). In another example, the engineered gene effector and the heterologous endonuclease can be non-covalently coupled to each other, for example, through ionic bonds, hydrogen bonds, interactions mediated by oligomerization or dimerization domains, etc. In some cases, the engineered gene effector and the heterologous endonuclease can be part of a single polypeptide molecule (e.g., a chimeric or fusion polypeptide).

[0281] Non-limiting examples of heterologous nucleases disclosed herein may include, but are not limited to, CRISPR-associated (Cas) proteins or Cas nucleases, including 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)); any derivatives thereof; any variants thereof, and any fragments thereof.

[0282] In some embodiments, the length of the heterologous endonucleases disclosed herein can be at most about 1000 amino acids, at most about 950 amino acids, at most about 900 amino acids, at most about 850 amino acids, at most about 800 amino acids, at most about 750 amino acids, at most about 700 amino acids, at most about 650 amino acids, at most about 640 amino acids, at most about 630 amino acids, at most about 620 amino acids, at most about 610 amino acids, at most about 600 amino acids, at most about 590 amino acids, at most about 580 amino acids, at most about 570 amino acids, at most about In some embodiments, the present invention relates to a polypeptide of the present invention to a polypeptide of the present invention. In some embodiments, the polypeptide of the present invention comprises at most about 550 amino acids, at most about 540 amino acids, at most about 530 amino acids, at most about 520 amino acids, at most about 510 amino acids, at most about 500 amino acids, at most about 490 amino acids, at most about 480 amino acids, at most about 470 amino acids, at most about 460 amino acids, at most about 450 amino acids, at most about 440 amino acids, at most about 430 amino acids, at most about 420 amino acids, at most about 410 amino acids, at most about 400 amino acids, at most about 350 amino acids, or at most about 300 amino acids.

[0283] In some embodiments, the heterologous endonucleases disclosed herein can be nuclease-deficient. In some embodiments, the heterologous endonucleases can be nuclease-null DNA binding proteins that do not induce transcriptional activation or repression of a target DNA sequence unless it is present in a complex with one or more heterologous gene effectors disclosed herein. In some embodiments, the heterologous endonucleases can be nuclease-null DNA binding proteins that can induce transcriptional activation or repression of a target DNA sequence (e.g., can be altered or enhanced by the presence of heterologous gene effectors provided herein).

[0284] In some embodiments, the heterologous endonucleases disclosed herein can be RNA nucleases, such as engineered (e.g., programmable or targetable) RNA nucleases. In some embodiments, the heterologous endonucleases disclosed herein can be nuclease-ineffective RNA binding proteins that do not induce transcriptional activation or repression of a target RNA sequence unless it is present in a complex with one or more heterologous gene effectors disclosed herein. In some embodiments, the heterologous endonucleases disclosed herein can be nuclease-ineffective RNA binding proteins that can induce transcriptional activation or repression of a target RNA sequence (e.g., can be altered or enhanced by the presence of heterologous gene effectors provided herein).

[0285] In some embodiments, the heterologous endonuclease can be a nucleic acid-guided targeting system. In some embodiments, the heterologous endonuclease can be a DNA-guided targeting system. In some embodiments, the heterologous endonuclease can be an RNA-guided targeting system. The nucleic acid-guided targeting system can include and utilize, for example, a guide amino acid sequence that promotes the specific binding of the CRISPR-Cas system (e.g., its nuclease-deficient form, such as dCas9 or dCas14) to a target gene (e.g., a target endogenous gene) or a target gene regulatory sequence. For example, the target gene can be any one of the genes listed in Table 1, and the target gene regulatory sequence can be operably coupled to any one of the genes listed in Table 1. Binding specificity can be determined by using a guide nucleic acid (e.g., a single guide RNA (sgRNA)) or a portion thereof. In some embodiments, the use of different sgRNAs enables the compositions and methods of the present disclosure to be used together with (e.g., targeting) different target genes (e.g., target endogenous genes) or target gene regulatory sequences.

[0286] In some embodiments, the heterologous endonuclease can comprise an amino acid sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity to the polypeptide sequence of SEQ ID NO: 10 (e.g., CasMini). In some embodiments, the heterologous endonuclease can comprise an amino acid sequence having at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% sequence identity to the polypeptide sequence of SEQ ID NO: 11 (e.g., dCasMini).

[0287] In some embodiments, the amino acid sequence of the heterologous endonuclease disclosed herein may be mutated and / or modified to generate a nuclease-deficient protein or a protein with reduced nuclease activity relative to a wild-type Cas protein. Nuclease-deficient proteins may retain the ability to bind to a target gene (e.g., DNA), but may lack or have reduced nucleic acid cleavage activity. In some embodiments, the heterologous endonuclease may exhibit reduced nuclease activity (e.g., nuclease deficiency or nuclease ineffectiveness) compared to wild-type Un1Cas12f1. The reduced nuclease activity may be at most about 95%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, at most about 5%, at most about 1%, at most about 0.5%, at most about 0.1% or less of wild-type Un1Cas12f1. In some cases, the heterologous endonuclease can include a substitution at D326 and / or D510 compared to the wild-type Un1Cas12f1 polypeptide sequence. For example, the D326 and / or D510 substitution can be an alanine substitution (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 include one or more substitutions in a native amino acid sequence, wherein the positions of at least some of these substitutions follow one or more specific rules that are determined to have unexpected advantages for the heterologous endonuclease. In some cases, these specific substitution rules are selected based on their ability to produce heterologous endonuclease variants (e.g., that can function in eukaryotic cells). According to these specific rules, one or more substitutions in a native amino acid sequence are all or partly (1) located within or no more than 30 amino acids downstream of a (D / E / K / N)X(R / F)(E / K)N motif of a native amino acid sequence, (2) located at position 241 of a native amino acid sequence or no more than 30 amino acids upstream or downstream thereof, (3) located at position 516 of a native amino acid sequence or no more than 30 amino acids upstream or downstream thereof, and / or (4) have a charged amino acid in the native amino acid sequence.

[0289] In some embodiments, for example, compared to the polypeptide sequence of wild-type Un1Cas12f1, the amino acid sequence of the heterologous nuclease (e.g., Cas protein) disclosed herein may include one or more substitutions at amino acid positions located within the (D / E / K / N)X(R / F)(E / K)N motif or upstream and / or downstream thereof not exceeding a threshold length (e.g., 30 amino acid residues). For example, at least one of the one or more substitutions may be located within or within the motif at no more than 28 amino acids, 26 amino acids, 24 amino acids, 22 amino acids, 20 amino acids, 18 amino acids, 16 amino acids, 14 amino acids, 12 amino acids, or 10 amino acids of the motif. In some cases, at least one of the one or more substitutions may be an R, A, S, or G amino acid residue. In some cases, the one or more substitutions may include substitutions located 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 substitutions 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 substitutions 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 disclosed herein comprising one or more substitutions upstream and / or downstream of a (D / E / K / N)X(R / F)(E / K)N motif can exhibit a cationic charge (e.g., a positive charge) that is higher than a wild-type variant of the heterologous endonuclease by at least or up to about 1 cationic charge, at least or up to about 2 cationic charges, at least or up to about 3 cationic charges, at least or up to about 4 cationic charges, at least or up to about 5 cationic charges, at least or up to about 6 cationic charges, at least or up to about 7 cationic charges, at least or up to about 8 cationic charges, at least or up to about 9 cationic charges, at least or up to about 10 cationic charges, at least or up to about 11 cationic charges, at least or up to about 12 cationic charges, at least or up to about 13 cationic charges, at least or up to about 14 cationic charges, at least or up to about 15 cationic charges, at least or up to about 16 cationic charges, at least or up to about 17 cationic charges, or at least or up to about 18 cationic charges.

[0291] In some embodiments, a heterologous endonuclease (e.g., and thus an engineered genetic effector operably coupled to a heterologous endonuclease) can form a complex with a guide nucleic acid (e.g., a guide RNA or a portion thereof). In some embodiments, a heterologous endonuclease can form a complex with a single guide nucleic acid (e.g., a single guide RNA (sgRNA)). In some embodiments, a heterologous endonuclease can be an RNA binding protein (RBP) optionally complexed with a guide nucleic acid (e.g., a guide RNA (e.g., sgRNA)), which can form a complex with a Cas protein. In some embodiments, a heterologous endonuclease can be a nuclease-ineffective DNA binding protein that can induce transcriptional activation or repression of a target DNA sequence. In some embodiments, a heterologous endonuclease can be a nuclease-ineffective RNA binding protein derived from RNA.

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

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

[0294] In some embodiments, the guide nucleic acid used in the compositions and methods of the present disclosure is about 8 to about 40 nucleotides, about 10 to about 40 nucleotides, about 11 to about 40 nucleotides, about 12 to about 40 nucleotides, about 13 to about 40 nucleotides, about 14 to about 40 nucleotides, about 15 to about 40 nucleotides, about 16 to about 40 nucleotides, about 17 to about 40 nucleotides, about 18 to about 40 nucleotides, about 19 to about 40 nucleotides, about 20 to about 40 nucleotides, about 22 to about 40 nucleotides, about 24 to about 40 nucleotides, about 26 to about 40 nucleotides, about 28 to about 40 nucleotides, about 30 to about 40 nucleotides, about 8 to about 30 nucleotides, about 10 to about 30 nucleotides, about 11 to about 30 nucleotides, about 12 to about 30 nucleotides, about 13 to about 30 nucleotides, about 14 to about 30 nucleotides, about 15 to about 30 nucleotides, about 16 to about 30 nucleotides, about 17 to about 30 nucleotides, about 18 to about 30 nucleotides, about 19 to about 30 nucleotides, about 20 to about 30 nucleotides, about 22 to about 30 nucleotides, about 24 to about 30 nucleotides, about 26 to about 30 nucleotides, about 28 to about 30 nucleotides, about 8 to about 25 nucleotides, about 10 to about 25 nucleotides, about 11 to about 30 nucleotides, about 25 nucleotides, about 12 to about 25 nucleotides, about 13 to about 25 nucleotides, about 14 to about 25 nucleotides, about 15 to about 25 nucleotides, about 16 to about 25 nucleotides, about 17 to about 25 nucleotides, about 18 to about 25 nucleotides, about 19 to about 25 nucleotides, about 20 to about 25 nucleotides, about 22 to about 25 nucleotides, about 24 to about 25 nucleotides, about 8 to about 20 nucleotides, about 10 to about 20 nucleotides, about 11 to about 20 nucleotides, about 12 to about 20 nucleotides, about 13 to about 20 nucleotides, about 14 to about 20 nucleotides, about 15 to about 20 nucleotides, about 1 In some embodiments, the guide nucleic acid can be a guide RNA or a portion thereof.

[0295] The heterologous endonucleases disclosed herein can be modified to enhance the regulation of gene expression by the compositions and methods disclosed herein (e.g., as part of the complex disclosed herein). The heterologous endonucleases can be modified to increase or decrease nucleic acid binding affinity, nucleic acid binding specificity, enzymatic activity and / or binding to other factors, such as heterodimerization or oligomerization domains and inducing ligands. The heterologous endonucleases can also be modified to change other activities or properties of the protein, such as stability. For example, one or more nuclease domains of the heterologous endonucleases can be modified, deleted or inactivated, or at least a portion of the heterologous endonucleases can be truncated to remove domains that are not necessary for the desired function of the protein or complex. The heterologous endonucleases can be modified to regulate (e.g., enhance or reduce) the activity of the heterologous endonucleases in regulating gene expression by the complex of the present disclosure comprising heterologous gene effectors.

[0296] For example, a heterologous endonuclease can be coupled (e.g., fused, covalently coupled, or non-covalently coupled) to a heterologous gene effector (e.g., an epigenetic modification domain, a transcriptional activation domain, and / or a transcriptional repression domain). A heterologous endonuclease can be coupled (e.g., fused, covalently coupled, or non-covalently coupled) to an oligomerization or dimerization domain disclosed herein (e.g., a heterologous dimerization domain). A heterologous endonuclease can be coupled (e.g., fused, covalently coupled, or non-covalently coupled) to a heterologous polypeptide that provides increased or decreased stability. A heterologous endonuclease can be coupled (e.g., fused, covalently coupled, or non-covalently coupled) to a sequence that can promote degradation of a heterologous endonuclease or a complex containing a heterologous endonuclease.

[0297] The heterologous endonuclease can be coupled (e.g., fused, covalently coupled, or non-covalently coupled) to any number of partners, e.g., at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight partners. In some embodiments, the heterologous endonuclease of the present disclosure is coupled (e.g., fused, covalently coupled, or non-covalently coupled) to at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, or at most ten partners. In some embodiments, the heterologous endonuclease disclosed herein is coupled (e.g., fused, covalently coupled, or non-covalently coupled) to 1-5, 1-4, 1-3, 1-2, 2-5, 2-4, 2-3, 3-5, 3-4, or 4-5 partners. In some embodiments, the heterologous endonuclease of the present disclosure is coupled (e.g., fused, covalently coupled, or non-covalently coupled) to one partner. In some embodiments, the heterologous endonucleases of the present disclosure are coupled (e.g., fused, covalently coupled, or non-covalently coupled) to two partners. In some embodiments, the heterologous endonucleases of the present disclosure are coupled (e.g., fused, covalently coupled, or non-covalently coupled) to three partners. In some embodiments, the heterologous endonucleases of the present disclosure are coupled (e.g., fused, covalently coupled, or non-covalently coupled) to four partners. In some embodiments, the heterologous endonucleases of the present disclosure are coupled (e.g., fused, covalently coupled, or non-covalently coupled) to five partners. In some embodiments, the heterologous endonucleases of the present disclosure are coupled (e.g., fused, covalently coupled, or non-covalently coupled) to six partners.

[0298] The heterologous endonucleases disclosed herein can be coupled (e.g., fused, covalently coupled, or non-covalently coupled) to one or more of the chaperones disclosed herein. The one or more chaperones may include: (i) any of the engineered gene effectors disclosed herein; and (ii) one or more additional chaperones provided by the present disclosure.

[0299] The heterologous endonucleases disclosed herein can be fusion proteins, for example, fusion proteins comprising a heterologous endonuclease and one or more of the chaperones disclosed herein. The fusion domain can be located at the N-terminus, C-terminus or internal of the heterologous endonuclease.

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

[0301] In some embodiments, the transcription effector can be a histone epigenetic modifier (or histone modifier). In some cases, histone epigenetic modifier can be methylated (for example, histone methylation modifier, such as amino acid methyltransferase, such as KRAB) to regulate histone. In some cases, histone epigenetic modifier can be acetylated to regulate histone. In some cases, histone epigenetic modifier can be phosphorylated to regulate histone. In some cases, histone epigenetic modifier can be ADP-ribosylation regulated histone. In some cases, histone epigenetic modifier can be glycosylated to regulate histone. In some cases, histone epigenetic modifier can be SUMOylation regulated histone. In some cases, histone epigenetic modifier can be ubiquitinated to regulate histone. In some cases, histone epigenetic modifier can be remodeled histone structure (for example, by ATP hydrolysis dependent process) to regulate histone.

[0302] In some embodiments, the transcription effector can be a gene epigenetic modifier (or gene modifier). In some cases, the gene modifier can regulate the gene by methylation (e.g., a gene methylation modifier such as DNA methyltransferase or DNMT). In some cases, the gene modifier can regulate the gene by acetylation.

[0303] In some embodiments, the transcriptional effector is from a family of related histone acetyltransferases. Non-limiting examples of histone acetyltransferases include 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 transcription effector may include an epigenetic modifier. In some embodiments, the transcription effector includes a histone epigenetic modifier (e.g., a histone lysine methyltransferase, a histone lysine demethylase, or a DNA methylase). Non-limiting examples of epigenetic modifiers may include 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, PRDM14, PRDM 15, 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, transcriptional activators, such as VP16, VP64, VP48, VP160, p65 subdomains (e.g., from NFkB), and the activation domains of EDLL and / or TAL activation domains (e.g., for activity in plants), SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, JHDM2a / b, UTX, JMJD3, GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZMYST3, MORFMYST4, SRC1, ACTR, PI 60, CLOCK, 10-11 translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, and the like.

[0306] Examples of proteins (or fragments thereof) that can be used as fusion partners to reduce transcription include, but are not limited to, transcriptional repressors, such as Kruppel-associated box (KRAB or SKD); KOX1 repression domain; Mad mSIN3 interaction domain (SID); ERF repressor domain (ERD), SRDX repression domain (e.g., for repression in plants), etc.; histone lysine methyltransferases, such as Pr-SET7 / 8, SUV4-20H1, RIZ1, etc.; histone lysine demethylases, such as JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARJD 1A / RBP2, JARID1B / PLU-1, JARID1C / SMCX, JARIDID / SMCY, etc.; histone lysine deacetylase, such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, etc.; DNA methylase, such as Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), etc.; and peripheral recruitment elements, such as lamin A, lamin B, etc.

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

[0308] The nucleic acid of at least heterologous endonuclease of coding disclosed herein, its fragment or derivative can be stably integrated into the genome of cell.The nucleic acid of at least heterologous endonuclease of coding can be operably connected to promoter, for example, promoter of constitutive or inducible activity in cell.The nucleic acid of at least heterologous endonuclease of coding can be operably connected to promoter in expression construct.Expression construct can include can instruct interested gene or other nucleotide sequence (for example, at least heterologous endonuclease) to express and can transfer interested nucleotide sequence to any nucleic acid construct in target cell.

[0309] In some embodiments, a heterologous endonuclease disclosed herein can be associated with a single guide RNA (sgRNA) to activate or repress transcription of a target gene (e.g., a target endogenous gene) (e.g., in combination with a heterologous gene effector disclosed herein). The sgRNA can be introduced into a cell expressing a heterologous endonuclease or a variant thereof as provided herein. In some cases, such cells can contain one or more different sgRNAs targeting the same target gene (e.g., a target endogenous gene) or target gene regulatory sequence. In other cases, the sgRNA targets different nucleic acids in the cell (e.g., different target genes, different target gene regulatory sequences, or different sequences within the same target gene or target gene regulatory sequence).

[0310] Enzymatic inactivation (e.g., nuclease deficiency) may refer to a nuclease that is able to bind to a nucleic acid sequence in a polynucleotide in a sequence-specific manner, but may not cut the target polynucleotide, or will cut it at a substantially reduced frequency. The guide portion of the enzymatic inactivation may include an enzymatically inactivated domain (e.g., a nuclease domain). Enzymatic inactivation may refer to inactivity. Enzymatic inactivation may refer to substantial inactivity. Enzymatic inactivation may refer to substantially inactivity. Enzymatic inactivation may refer to activity no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 6%, no more than 7%, no more than 8%, no more than 9% or no more than 10% activity compared to comparable wild-type activity (e.g., nucleic acid cleavage activity, wild-type Cas activity).

[0311] In some embodiments, the target nucleic acid of the heterologous endonucleases disclosed herein can be dsDNA. In such embodiments, the dsDNA targeting specificity is determined at least in part by two parameters: a gRNA spacer that targets a protospacer in the target dsDNA (a sequence in the target dsDNA that corresponds to the gRNA spacer on the non-complementary DNA strand) and a short sequence 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 can be RNA. In such embodiments, the RNA targeting specificity is determined at least in part by the original spacer-like sequence (sequence complementary to the gRNA spacer in the target RNA) gRNA spacer in the target RNA, and is independent of the sequence located immediately adjacent to the original spacer-like sequence 5' (upstream). In some embodiments, the heterologous endonuclease can also be able to target dsDNA molecules, wherein the gRNA spacer is selected to target the original spacer sequence in the target dsDNA molecule with a PAM selected from 5'-TTTG-3' and 5'-TTTA-3'. In other embodiments, the heterologous endonuclease cannot target dsDNA molecules, wherein the gRNA spacer is selected to make any original spacer sequence in the dsDNA molecule targeted by the gRNA spacer 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 an engineered gene effector and a heterologous endonuclease can regulate the expression and / or activity of a target gene (e.g., a target endogenous gene). In some embodiments, a heterologous polypeptide and / or a complex thereof can edit the sequence of a nucleic acid (e.g., a gene and / or a gene product). Nuclease-active variants of a heterologous endonuclease can edit a nucleic acid sequence by generating double-strand breaks or single-strand breaks in a target polynucleotide.

[0314] In some embodiments, a heterologous polypeptide (e.g., and / or a complex comprising a heterologous polypeptide) comprising an engineered gene effector and a heterologous endonuclease can generate a double-strand break in a target polynucleotide (e.g., DNA). Double-strand breaks in DNA can lead to DNA break repair, thereby allowing the introduction of genetic modifications (e.g., nucleic acid editing). In some embodiments, nucleases induce site-specific single-stranded DNA breaks or nicks, resulting in HDR.

[0315] Double-strand breaks in DNA can lead to DNA break repair, thereby allowing the introduction of genetic modifications (e.g., nucleic acid editing). DNA break repair can occur by non-homologous end joining (NHEJ) or homology-mediated repair (HDR). In HDR, a donor DNA repair template or template polynucleotide comprising homology arms flanking the target DNA site can be provided.

[0316] In some embodiments, a heterologous polypeptide (e.g., and / or a complex comprising a heterologous polypeptide) comprising an engineered gene effector and a heterologous endonuclease does not generate a double-strand break in a target polynucleotide (e.g., DNA). Binding of a heterologous polypeptide or a complex comprising a heterologous polypeptide (e.g., a complex comprising a nuclease-deficient variant of a heterologous endonuclease and a guide RNA) in the absence of nucleic acid breakage can be sufficient to modulate (e.g., enhance or repress) the 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 gene can be one or more heterologous target genes. The target gene can be one or more endogenous target genes, such as (i) pathogenic alleles, such as mutant alleles, and / or (ii) non-pathogenic alleles, such as wild-type alleles. For example, disclosed herein is a complex comprising a guide portion and one or more heterologous polypeptides, the heterologous polypeptide comprising an engineered gene effector and a heterologous endonuclease, which can regulate (e.g., increase or decrease) the activity or expression level 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 cell genome), or endogenous to the subject (e.g., present in the subject's genome). 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, for example, a pathogen target gene or an exogenous gene expressed due to a 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) the expression of a target gene (e.g., after introducing a complex comprising an engineered gene effector and a heterologous polypeptide into a cell or cell population). In some embodiments, the expression level is an RNA expression level, which can be measured by, for example, RNAseq, qPCR, microarrays, gene arrays, FISH, etc. In some embodiments, the expression level is a protein expression level, which can be measured by, for example, protein immunoblotting, 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) the expression of a target gene (e.g., after introducing a complex comprising an engineered gene effector and a heterologous polypeptide into a cell or cell population) 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 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 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times, at least about 150 times, at least about 200 times, at least about 250 times, at least about 300 times, at least about 350 times, at least about 400 times, at least about 500 times, at least about 600 times, at least about 700 times, at least about 800 times, at least about 900 times, at least about 1000 times, at least about 1500 times, at least about 2000 times, or at least about 3000 times.

[0323] In some embodiments, the systems and methods disclosed herein can modulate (e.g., increase or decrease) the expression of a target gene (e.g., after introducing a complex comprising an engineered gene effector and a heterologous polypeptide into a cell or cell population) by 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 13-fold, up to about 14-fold, up to about 15-fold, up to about 16-fold, up to about 17-fold, up to about 18-fold, up to about 19-fold, up to about 20-fold, up to about 21-fold, up to about 22-fold, up to about 23-fold, up to about 24-fold, up to about 25-fold, up to about 26-fold, up to about 27-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 In some embodiments, the present invention relates to an increase in the amount of at least one molecule of the present invention, wherein the amount of the present invention is at least about 20 times, at most about 30 times, at most about 40 times, at most about 50 times, at most about 60 times, at most about 70 times, at most about 80 times, at most about 90 times, at most about 100 times, at most about 150 times, at most about 200 times, at most about 250 times, at most about 300 times, at most about 350 times, at most about 400 times, at most about 500 times, at most about 600 times, at most about 700 times, at most about 800 times, at most about 900 times, at most about 1000 times, at most about 1500 times, at most about 2000 times, at most about 3000 times, at most about 5000 times, or at most about 10000 times.

[0324] In some embodiments, the systems and methods disclosed herein can modulate (e.g., increase or decrease) the expression of a target gene (e.g., after introducing a complex comprising an engineered gene effector and a heterologous polypeptide into a cell or cell population) by 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 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, about 20-fold, about 21-fold, about 22-fold, about 23-fold, about 24-fold, about 25-fold, about 26-fold, about 27-fold, about 28-fold, about 29-fold, about 30-fold, about 31-fold, about 32-fold, about 33-fold, about 34-fold, about 35-fold, about 36-fold, about 37-fold, about 38-fold, about 39-fold, about 40-fold, about 41-fold, about 42-fold, about 43-fold, about 44-fold, about 45-fold, about 46-fold, about 47-fold, about 48-fold, about 49-fold, about 50-fold, about 51-fold, about 52-fold, about 53-fold, about 54-fold, about 55-fold About 13 times, about 14 times, about 15 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, about 100 times, about 150 times, about 200 times, about 250 times, about 300 times, about 350 times, about 400 times, about 500 times, about 600 times, about 700 times, about 800 times, about 900 times, about 1000 times, about 1500 times, about 2000 times, about 3000 times, about 5000 times, or about 10000 times.

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

[0326] In some embodiments, the extent of expression change is relative to before the system of the present disclosure (e.g., a complex comprising an engineered gene effector and a heterologous polypeptide) is introduced into a cell or cell population. In some embodiments, the extent of expression change is relative to a corresponding control cell or cell population that has not been treated with the system of the present disclosure. In some embodiments, the extent of expression change is relative to a corresponding control cell or cell population treated with an alternative approach to the 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., after introducing a complex into a cell or cell population, the complex comprising an engineered gene effector and a heterologous polypeptide comprising a heterologous endonuclease disclosed herein). Depending on the functional properties of the target gene, the activity level can be determined by a suitable functional assay for the target gene in question. For example, the activity level of a target gene that is a mitogen can be determined by measuring cell proliferation; the activity level of a target gene that induces apoptosis can be measured by an Annexin V assay or other suitable cell death assay; the activity level of an anti-inflammatory cytokine can be measured by an LPS-induced cytokine release assay.

[0328] The systems and methods of the present disclosure can in some cases cause changes in the expression and / or activity level of a target gene (e.g., a target endogenous gene) that last longer than what can be achieved with alternative compositions and methods (e.g., suppression by RNAi, such as using siRNA). In some embodiments, persistent regulation of gene expression (e.g., durable gene activation or durable gene repression) has advantages over transient regulation.

[0329] In some embodiments, the systems and methods of the present disclosure can modulate (e.g., increase or decrease) the expression and / or activity level of a target gene for 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, at least about 4 days, 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, at least about 5 days, 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, at least about 4 ... 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, or 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., a target endogenous gene) to exceed a certain threshold value for at most about 1 hour, at most about 2 hours, at most about 3 hours, at most about 4 hours, at most about 5 hours, at most about 6 hours, at most about 7 hours, at most about 8 hours, at most about 9 hours, at most about 10 hours, at most about 12 hours, at most about 14 hours, at most about 18 hours, at most about 20 hours, at most about 1 day ... hour, at most about 2 hours, at most about 3 hours, at most about 4 hours, at most about 5 hours, at most about 6 hours, at most about 7 hours, at most about 8 hours, at most about 9 hours, at most about 10 hours, at most about 12 hours, at most about 14 hours, at most about 18 hours, at most about 20 hours In some embodiments, the present invention may be performed for at most about 2 days, at most about 3 days, at most about 4 days, at most about 5 days, at most about 6 days, at most about 7 days, at most about 8 days, at most about 9 days, at most about 10 days, at most about 14 days, at most about 21 days, at most about 28 days, at most about 5 weeks, at most about 6 weeks, at most about 7 weeks, at most about 8 weeks, at most about 9 weeks, at most about 10 weeks, at most about 12 weeks, at most about 14 weeks, at most about 18 weeks, at most about 20 weeks, at most about 26 weeks, or at most about 5 months, at most about 6 months, at most about 9 months, at most 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., a target endogenous gene) above a certain threshold for 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 (for example, endogenous target gene) can be a pathogenic allele, such as a mutant variant of a wild-type allele. The disease can be a genetic disease, such as a hereditary disorder. Non-limiting examples of genetic diseases can include Duchenne muscular dystrophy (DMD), hemophilia, cystic fibrosis, Huntington's chorea, familial hypercholesterolemia (LDL receptor defect), hepatoblastoma, Wilson's disease, congenital hepatic porphyria, hereditary liver metabolic disorders, Lesch-Nyhan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, telangiectasia ataxia, Bloom's syndrome, retinoblastoma and Tay-Sachs disease. In some cases, the target gene can be a gene encoding a protein. In some cases, the target gene can be a gene regulatory sequence (e.g., a promoter, an enhancer, a repressor, a silencer, an insulator, a cis-regulatory element, a trans-regulatory element, an epigenetic modification (e.g., DNA methylation) site, etc.) that can affect the expression of a gene encoding a protein of interest provided herein. For example, a target gene regulatory sequence can be physically located outside the transcription unit or open reading frame encoding the target gene product.

[0333] In some embodiments, the target gene regulatory sequence does not include a nucleotide sequence that is foreign to the subject or host cell. In some embodiments, the target gene regulatory sequence does not include an engineered or artificially generated or introduced nucleotide sequence.

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

[0335] In some embodiments, the target gene (e.g., target endogenous gene) is a gene that is overexpressed or underexpressed in cancer, such as acute leukemia, astrocytoma, biliary cancer (cholangiocarcinoma), bone cancer, breast cancer, brain stem glioma, bronchoalveolar cell lung cancer, adrenal cancer, anal region cancer, bladder cancer, endocrine system cancer, esophageal cancer, head and neck cancer, kidney cancer, parathyroid cancer, penis 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, skin melanoma, ependymoma, epidermoid tumor, Ewing's sarcoma, gastric cancer, glioblastoma The invention relates to a type of cancer comprising: a neoplasm of the lung, a glioma of the ovarian, a prostate, a colorectal, a glioma, a hematological malignancy, a hepatocellular (liver) carcinoma, a hepatoma, a Hodgkin's disease, an intraocular melanoma, a Kaposi's sarcoma, a lung cancer, a lymphoma, a medulloblastoma, a melanoma, a meningioma, a mesothelioma, a multiple myeloma, a muscle cancer, a central nervous system (CNS) neoplasm, a neuronal cancer, a small cell lung cancer, a non-small cell lung cancer, an osteosarcoma, an ovarian cancer, a pancreatic cancer, a childhood malignancy, a pituitary adenoma, a prostate cancer, a colorectal cancer, a renal cell carcinoma, a soft tissue sarcoma, a schwannoma, a skin cancer, a spinal tumor, a squamous cell carcinoma, a stomach cancer, a synovial sarcoma, a testicular cancer, a uterine cancer, or a tumor and its metastasis, including a refractory version of any of the foregoing cancers, or a combination thereof.

[0336] As disclosed herein, non-limiting examples of target genes or genes encoding proteins of interest are included in Table 1. Table 1 List of target gene examples (eg, encoding a protein of interest)

[0337] Heterologous polynucleotide

[0338] In some embodiments, the systems and compositions of the present disclosure further comprise a heterologous polynucleotide (e.g., encoding a gene of interest, such as one or more genes selected from Table 1), which is introduced into the cell without entering the genome of the cell through the action of a heterologous endonuclease of the present disclosure. In some cases, such heterologous polynucleotides encoding a gene of interest can enter the genome of the cell by other means (e.g., via an adeno-associated viral vector (such as AAV2 or AAV8)). Alternatively, such heterologous polynucleotides encoding a gene of interest can be introduced into the intracellular portion of the cell and remain non-chromosomal (e.g., as a non-chromosomal plasmid).

[0339] Thus, the systems and compositions may include a non-pathogenic wild type or variant of a target gene as described above. Alternatively or additionally, the systems and compositions may include a heterologous polynucleotide sequence encoding (or comprising) at least a non-pathogenic wild type or variant of a target gene (e.g., an endogenous target gene) disclosed herein.

[0340] System and composition

[0341] In some aspects, the disclosure provides a system or composition for any method disclosed herein, comprising: (i) an engineered gene effector disclosed herein (e.g., an engineered gene activator, an engineered gene repressor), and optionally one or more of the following: (ii) a heterologous endonuclease disclosed herein, (iii) a guide nucleic acid disclosed herein, and / or (iv) a heterologous polynucleotide (e.g., encoding one or more genes from Table 1). The system or composition may comprise one or more polynucleotides encoding any of the above members (i)-(iv). The subject composition can be used to modify cells in vitro, in vitro, or in vivo. The subject composition can be used to treat or enhance the condition of a subject, as described herein.

[0342] The compositions disclosed herein may include active ingredients (e.g., engineered gene effectors, heterologous endonucleases, guide nucleic acids, etc.) and optional additional ingredients (e.g., excipients). If necessary and / or desired, the compositions may be divided, molded, and / or packaged into desired single-dose units or multiple-dose units, or single or multiple implant units.

[0343] In some embodiments, the composition may include one or more heterologous polynucleotides encoding active ingredients disclosed herein. When different members are included in the active ingredient, each member can be encoded by a different heterologous polynucleotide. Alternatively, two or more (e.g., all) components can be encoded by a single heterologous polynucleotide. In some cases, a heterologous polynucleotide can encode an engineered gene effector disclosed herein. In some cases, a single heterologous polynucleotide can encode (i) an engineered gene effector, (ii) a heterologous nuclease, and (iii) one or more guide nucleic acids (e.g., at least 1, at least 2, at least 3, at least 4, at least 5 or more guide nucleic acids) for targeting a specific region or sequence of a target gene.

[0344] One or more heterologous polynucleotides may also include one or more promoters (or one or more transcription control elements, as used interchangeably herein). Different active ingredients encoded by one or more heterologous polynucleotides may be under the control of the same promoter or different promoters. The promoter disclosed herein may be active in eukaryotic cells, mammalian cells, non-human mammalian cells, or human cells. The promoter may be an inducible or constitutively active promoter. Alternatively or in addition, the promoter may be tissue-specific or cell-specific. Non-limiting examples of suitable eukaryotic promoters (i.e., promoters functional in eukaryotic cells) may include long terminal repeats (LTRs) from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, retroviruses, human elongation factor-1 promoter (EF1), hybrid constructs containing cytomegalovirus (CMV) enhancers fused to chicken beta actin promoter (CAG), mouse stem cell virus promoter (MSCV), phosphoglycerate kinase-1 locus promoter (PGK), and mouse metallothionein-I. The promoter may be a fungal promoter. The promoter can be a plant promoter. A database of plant promoters can be found (e.g., PlantProm). The expression vector can also include a ribosome binding site and a transcription terminator for translation initiation. The expression vector can also include an appropriate sequence for amplifying expression. In some cases, the promoter disclosed herein can be any tissue-specific promoter provided herein, or any cell type-specific promoter provided herein.

[0345] The size of the heterologous polynucleotide in the one or more heterologous polynucleotides (e.g., a single heterologous polynucleotide) can be at least or up to about 2.5 kilobases, at least or up to about 2.6 kilobases, at least or up to about 2.7 kilobases, at least or up to about 2.8 kilobases, at least or up to about 2.9 kilobases, at least or up to about 3.0 kilobases, at least or up to about 3.1 kilobases, at least or up to about 3.2 kilobases, at least or up to about 3.3 kilobases, at least or up to about 3.4 kilobases, at least or up to about 3.5 kilobases, at least or up to about 3.6 kilobases, at least or up to about 3.7 kilobases, at least or up to about 3.8 kilobases, at least or up to about 3.9 kilobases, at least or up to about 4. .0 kilobases, at least or up to about 4.1 kilobases, at least or up to about 4.2 kilobases, at least or up to about 4.3 kilobases, at least or up to about 4.4 kilobases, at least or up to about 4.5 kilobases, at least or up to about 4.6 kilobases, at least or up to about 4.7 kilobases, at least or up to about 4.8 kilobases, at least or up to about 4.9 kilobases, at least or up to about 5.0 kilobases, at least or up to about 5.5 kilobases, at least or up to about 6.0 kilobases, at least or up to about 6.5 kilobases, at least or up to about 7.0 kilobases, at least or up to about 7.5 kilobases, at least or up to about 8.0 kilobases, at least or up to about 9.0 kilobases, or at least or up to about 10 kilobases. In some cases, the size of the heterologous polynucleotide in one or more heterologous polynucleotides (e.g., a single heterologous polynucleotide) can be about 3 kilobases to about 5 kilobases, about 3 kilobases to about 4.8 kilobases, about 3 kilobases to about 4.6 kilobases, about 3 kilobases to about 4.4 kilobases, about 3 kilobases to about 4.2 kilobases, about 3 kilobases to about 4.0 kilobases, about 3 kilobases to about 3.5 kilobases, about 3.5 kilobases to about 5 kilobases, about 3.5 kilobases to about 4.8 kilobases, about 3.5 kilobases to about 4.6 kilobases. , about 3.5 kilobases to about 4.4 kilobases, about 3.5 kilobases to about 4.2 kilobases, about 3.5 kilobases to about 4 kilobases, about 4 kilobases to about 5 kilobases, about 4 kilobases to about 4.9 kilobases, about 4 kilobases to about 4.8 kilobases, about 4 kilobases to about 4.7 kilobases, about 4 kilobases to about 4.6 kilobases, about 4 kilobases to about 4.5 kilobases, about 4 kilobases to about 4.4 kilobases, about 4 kilobases to about 4.3 kilobases, about 4 kilobases to about 4.2 kilobases, or about 4 kilobases to about 4.1 kilobases.

[0346] The method for delivering one or more heterologous polynucleotides provided herein to a cell can be related to a viral delivery method or a non-viral delivery method. Therefore, one or more heterologous polynucleotides can be one or more viral vectors (e.g., one or more AAV vectors). Alternatively, one or more heterologous polynucleotides can be a non-viral vector that is compounded with a non-viral delivery portion (e.g., a cationic lipid and / or a lipid particle (e.g., a lipid nanoparticle (LNP))) or is wrapped by a non-viral delivery portion.

[0347] Non-viral delivery methods for nucleic acids can include lipofection, nuclear transfection, microinjection, gene guns, viral particles, liposomes, immunoliposomes, polycations or lipids: nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced DNA uptake. Cationic and neutral lipids suitable for effective receptor recognition lipofection of polynucleotides can be used. Can be delivered to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration).

[0348] In some embodiments, viral vectors can be used to deliver the compositions and systems provided herein to a subject. In some cases, the viral vector is an adeno-associated virus (AAV) vector. The term "AAV" is an abbreviation for adeno-associated virus, and can be used to refer to the virus itself or its derivatives. Unless otherwise required, the term encompasses all serotypes, subtypes, and both naturally occurring forms and recombinant forms. The abbreviation "rAAV" refers to recombinant adeno-associated virus, also referred to as recombinant AAV vectors (or "rAAV vectors"). The term "AAV" includes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh10 and hybrids thereof, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. The genomic sequences of various serotypes of AAV, as well as the sequences of natural terminal repeats (TR), Rep proteins, and capsid subunits are known in the art. Such sequences can be found in literature or public databases (such as GenBank). "rAAV vector" used herein refers to an AAV vector comprising a polynucleotide sequence (i.e., a polynucleotide heterologous to AAV) from a non-AAV source, usually a sequence of interest for cell gene transformation. Typically, the heterologous polynucleotide is flanked by at least one, usually two AAV inverted terminal repeats (ITRs). The term "rAAV vector" includes both rAAV vector particles and rAAV vector plasmids. The rAAV vector can be single-stranded (ssAAV) or self-complementary (scAAV). The term "AAV virus" or "AAV virus particle" or "rAAV vector particle" ref...

Claims

1. An engineered gene effector, the engineered gene effector comprising a polypeptide, wherein: The polypeptide is heterologous to any member selected from the group consisting of VP16, VP64, p65 and Rta; The engineered gene effector has a size of up to about 500 amino acid residues; and The engineered gene effector is capable of activating the expression level of a target gene in a cell, wherein the expression level of the target gene activated by the engineered gene effector is at least about 80% compared to the expression level of the target gene activated by the VP64-p65-Rta fusion polypeptide (VPR) in a control cell.

2. The engineered gene effector according to claim 1, wherein: The expression level of the target gene activated by the engineered gene effector is at least about 90% of the expression level of the target gene activated by the VPR in the control cells.

3. The engineered gene effector according to claim 1, wherein: The expression level of the target gene activated by the engineered gene effector is at least about 95% of the expression level of the target gene activated by the VPR in the control cells.

4. The engineered gene effector according to claim 1, wherein: The expression level of the target gene activated by the engineered gene effector is at least about 100% of the expression level of the target gene activated by the VPR in the control cells.

5. The engineered gene effector according to claim 1, wherein: The expression level of the target gene activated by the engineered gene effector is at least about 5% higher than the expression level of the target gene activated by the VPR in the control cells.

6. The engineered gene effector according to claim 1, wherein: The expression level of the target gene activated by the engineered gene effector is at least about 10% higher than the expression level of the target gene activated by the VPR in the control cells.

7. The engineered gene effector according to any one of claims 1 to 6, wherein: The expression level of the target gene activated by the engineered gene effector persists for a duration of at least about 12 days.

8. The engineered gene effector according to any one of claims 1 to 6, wherein: The expression level of the target gene activated by the engineered gene effector persists for a duration of at least about 18 days.

9. The engineered gene effector according to any one of claims 1 to 6, wherein: The expression level of the target gene activated by the engineered gene effector is at least about 80% of the expression level of the target gene activated by the VPR in the control cells, and the expression level persists for a duration of at least about 9 days.

10. The engineered gene effector according to any one of claims 1 to 6, wherein: The expression level of the target gene activated by the engineered gene effector is at least about 90% of the expression level of the target gene activated by the VPR in the control cells, and the expression level persists for a duration of at least about 12 days.

11. The engineered gene effector according to any one of claims 1 to 10, wherein: The engineered gene effector has a size of less than or equal to about 400 amino acid residues.

12. The engineered gene effector according to any one of claims 1 to 10, wherein: The engineered gene effector has a size of less than or equal to about 300 amino acid residues.

13. The engineered gene effector according to any one of claims 1 to 10, wherein: The engineered gene effector has a size of less than or equal to about 250 amino acid residues.

14. The engineered gene effector according to any one of claims 1 to 13, wherein: The target gene is endogenous to the cell.

15. The engineered gene effector according to any one of claims 1 to 14, wherein: The polypeptide comprises an amino acid sequence that exhibits at least about 70% sequence identity to the polypeptide sequence of SEQ ID NO:

1.

16. An engineered genetic effector, comprising a polypeptide coupled to an additional polypeptide, wherein: 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; and The engineered gene effector has a size of less than or equal to about 250 amino acid residues.

17. The engineered gene effector according to claim 16, wherein: The additional polypeptides include VP16.

18. The engineered gene effector according to claim 16, wherein: The additional polypeptides include VP64.

19. The engineered gene effector according to any one of claims 16 to 18, wherein: The engineered gene effector can activate the expression level of the target gene in the cell.

20. The engineered gene effector according to any one of claims 16 to 19, wherein: The polypeptide is fused to the additional polypeptide.

21. The engineered gene effector according to any one of claims 16 to 20, wherein: The polypeptide is not identical to any of SEQ ID NO:2 and SEQ ID NO:

3.

22. The engineered gene effector according to any one of claims 16 to 21, wherein: The at least portion comprises an amino acid sequence having at least about 70% sequence identity to the polypeptide sequence of SEQ ID NO:

5.

23. The engineered gene effector according to any one of claims 16 to 21, wherein: The at least portion comprises an amino acid sequence having at least about 70% sequence identity to the polypeptide sequence of SEQ ID NO:

6.

24. The engineered gene effector according to any one of claims 16 to 23, wherein: The additional polypeptide is located adjacent to the C-terminus of the polypeptide.

25. The engineered gene effector according to any one of claims 16-23, wherein: The additional polypeptide is located adjacent to the N-terminus of the polypeptide.

26. The engineered gene effector according to any one of claims 16-25, wherein: The additional polypeptide comprises a stuffer polypeptide sequence.

27. The engineered gene effector according to any one of claims 16 to 26, wherein: The additional polypeptide comprises a linker sequence.

28. An engineered gene effector, the engineered gene effector comprising a polypeptide, wherein: The polypeptide comprises an amino acid sequence having at least about 70% sequence identity with the polypeptide sequence of SEQ ID NO: 1, Wherein, the engineered gene effector is different from any one of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:

4.

29. The engineered gene effector according to claim 28, wherein: The engineered gene effector can activate the expression level of the target gene in the cell.

30. An engineered gene effector, comprising a plurality of polypeptide domains, wherein: Each polypeptide domain of the plurality of polypeptide domains comprises a polypeptide comprising an amino acid sequence exhibiting at least about 70% sequence identity to the polypeptide sequence of SEQ ID NO:

1.

31. The engineered gene effector according to claim 30, wherein: The engineered gene effector can activate the expression level of the target gene in the cell.

32. The engineered genetic effector according to claim 30 or 31, wherein: The plurality of polypeptide domains comprises three or more polypeptides.

33. The engineered genetic effector according to any one of claims 30 to 32, wherein: The plurality of polypeptide domains include a first polypeptide domain and a second polypeptide domain coupled to each other.

34. The engineered genetic effector of claim 33, wherein: The first polypeptide domain is fused to the second polypeptide domain.

35. The engineered genetic effector according to any one of the preceding claims, wherein: The amino acid sequence of the polypeptide exhibits at least about 80% sequence identity with the polypeptide sequence of SEQ ID NO:

1.

36. The engineered genetic effector according to any one of the preceding claims, wherein: The amino acid sequence of the polypeptide exhibits at least about 90% sequence identity with the polypeptide sequence of SEQ ID NO:

1.

37. The engineered genetic effector of any one of the preceding claims, wherein: The amino acid sequence of the polypeptide exhibits at least about 95% sequence identity to the polypeptide sequence of SEQ ID NO:

1.

38. The engineered genetic effector of any one of the preceding claims, wherein: The size of the amino acid sequence of the polypeptide is at most about 50 amino acid residues, at most about 40 amino acid residues, or at most about 35 amino acid residues.

39. The engineered genetic effector according to any one of the preceding claims, wherein: The size of the amino acid sequence of the polypeptide is at least about 20 amino acid residues, at least about 25 amino acid residues, or at least about 30 amino acid residues.

40. The engineered genetic effector of any one of the preceding claims, wherein: The engineered gene effector has a size of less than or equal to about 150 amino acid residues.

41. The engineered genetic effector of any one of the preceding claims, wherein: The engineered gene effector has a size of less than or equal to about 100 amino acid residues.

42. The engineered genetic effector of any one of the preceding claims, wherein: When aligned with the polypeptide sequence of SEQ ID NO: 1, the amino acid sequence of the polypeptide comprises one or more members selected from the group consisting of C4, L5, M7 and L19.

43. The engineered genetic effector of any one of the preceding claims, wherein: The engineered gene effector is capable of activating the expression level of the target gene in the cell for at least about 9 days.

44. A system comprising the engineered genetic effector of any one of the preceding claims.

45. The system of claim 44, wherein: The system comprises the engineered gene effector coupled to a heterologous endonuclease.

46. ​​A system according to claim 44 or 45, wherein: The heterologous endonuclease is a Cas protein.

47. The system of any of the preceding claims, further comprising a guide nucleic acid capable of forming a complex comprising the engineered gene effector and the heterologous endonuclease, wherein The complex exhibits specific binding to the target gene.

48. One or more polynucleotides encoding the system of any of the preceding claims.

49. A cell comprising the system of any preceding claim.

50. A method of controlling a target gene in a cell, the method comprising contacting the cell with the system of any preceding claim.

51. The method of claim 50, wherein: The controlling includes activating the expression level of the target gene.

52. The method of claim 50, wherein: The target gene is endogenous to the cell.

Images