Recombinant nucleases and compositions and methods of use thereof

Abstract

The present disclosure provides one or more recombinant nucleases that can be used to bind, cleave and / or edit a target polynucleotide sequence, as well as systems, compositions and methods thereof. The one or more recombinant nucleases of the present invention may be recombinant variants of small CRISPR / Cas proteins.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 315,159, filed March 1, 2022; U.S. Provisional Patent Application No. 63 / 380,178, filed October 19, 2022; and U.S. Provisional Patent Application No. 63 / 385,171, filed November 28, 2022, which applications are incorporated by reference herein in their entireties.

[0002] INCORPORATION BY REFERENCE OF SEQUENCE LISTING This application has been filed with an electronic Sequence Listing, which has been provided as a 426 kilobyte file with the file name 55176-726_601_SL.XML, created on February 15, 2023. The information set forth in this electronic Sequence Listing is incorporated herein by reference in its entirety. [Background technology]

[0003] Nucleases (e.g., endonucleases) can be used to edit target sequences in cells or to regulate the expression or activity of target genes in cells. For example, heterologous nucleases can be introduced (e.g., delivered, expressed, etc.) into cells, and the heterologous nucleases can be used alone or in combination with additional agents to edit or regulate target genes. For example, CRISPR (clustered regularly interspaced short palindromic repeats) / CRISPR-associated proteins (Cas) are a family of nucleases involved in the specific binding, cleavage, and / or editing of target deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences (e.g., foreign DNA or RNA sequences). The programmable nature of these nucleases has led to their use as versatile technologies to revolutionize the field of targeted gene manipulation, for example, as gene therapy for the treatment or alleviation of a condition (e.g., disease) of interest. Summary of the Invention [Means for solving the problem]

[0004] Various endonucleases, such as CRISPR / Cas proteins (e.g., the currently utilized Cas12f protein), may be smaller in size than Cas9, Cas12a, and the like. However, the size of the endonucleases may be too large to be packaged with at least one additional agent (e.g., a transgene encoding a therapeutic polynucleotide or therapeutic protein, a guide RNA, etc.) in certain delivery methods (e.g., viral vectors, e.g., adeno-associated virus (AAV) vectors). Thus, various embodiments of the present disclosure provide, for example, recombinant nucleases that are smaller than conventional endonucleases yet are effective in binding, cleaving, and / or editing target polynucleotide sequences, as well as compositions and methods of use thereof.

[0005] In one aspect of the present disclosure, there is provided a recombinant polypeptide comprising a recombinant nuclease, wherein the recombinant nuclease comprises an amino acid sequence having at least 80% identity to the polypeptide sequence of SEQ ID NO: 1, and the amino acid sequence comprises at least one deletion from amino acid residues 2 to 100 compared to the polypeptide sequence of SEQ ID NO: 1.

[0006] Another aspect of the present disclosure provides a recombinant polypeptide comprising a recombinant nuclease, wherein the recombinant nuclease comprises an amino acid sequence having greater than 92% identity to the polypeptide sequence of SEQ ID NO: 12.

[0007] In another aspect of the present disclosure, there is provided a recombinant polypeptide comprising a recombinant nuclease variant, the recombinant nuclease variant comprises an amino acid sequence having at least 70% identity to the polypeptide sequence of SEQ ID NO: 12; the amino acid sequence comprises modifications compared to the polypeptide sequence of SEQ ID NO: 1, the modification comprises one or more members selected from the group consisting of A21Q, V23I, N32E, D29E, N33R, E35K, K36Q, I37A, A38G, E40D, K73G, A74T, R75G, K76E, Q83K, G87K, E151A, A340S, H353K, A374K, I387E, N423D, K473Q, T474L, T474R, H497K, L515R, N519T, K521D, K521N and L522I, and at least one deletion from amino acid residues 400 to 529 of SEQ ID NO: 1; Recombinant polypeptides are provided.

[0008] In another aspect of the present disclosure, there is provided a recombinant polypeptide comprising a recombinant nuclease variant, The recombinant nuclease variant is a chimeric polypeptide, the chimeric polypeptide comprising: a first polypeptide sequence comprising at least three consecutive amino acid residues in common with a first Cas protein; a second polypeptide sequence that includes at least three consecutive amino acid residues in common with a second Cas protein that is distinct from the first Cas protein; and Including, the first Cas protein comprises an amino acid sequence having at least 80% identity to the polypeptide sequence of SEQ ID NO: 1; Recombinant polypeptides are provided.

[0009] In another aspect of the present disclosure, there is provided a recombinant polypeptide comprising a recombinant nuclease variant, The recombinant nuclease is a chimeric polypeptide, the chimeric polypeptide comprising: a first polypeptide sequence (CP1) comprising at least three consecutive amino acid residues in common with a portion of a first Cas protein; a second polypeptide sequence (CP2) that includes at least three consecutive amino acid residues in common with a portion of a second Cas protein that is distinct from the first Cas protein; a third polypeptide sequence (CPx); Including, the third polypeptide sequence (CPx) is (i) a portion of a first Cas protein that is not immediately adjacent to the portion of the first Cas protein; (ii) a second Cas protein, a portion of the second Cas protein that is not immediately adjacent to the portion of the second Cas protein; or (iii) a portion of a third Cas protein that is distinct from the first Cas protein and the second Cas protein; and comprising at least three consecutive amino acid residues in common with the length of the chimeric polypeptide is about 1,000 amino acids or less; Recombinant polypeptides are provided.

[0010] In another aspect of the present disclosure, there is provided a recombinant polypeptide comprising a recombinant nuclease variant, The recombinant nuclease variant is a chimeric polypeptide, the chimeric polypeptide comprising: a first polypeptide comprising at least three consecutive amino acid residues in common with a first Cas protein; a second polypeptide that includes at least three consecutive amino acid residues in common with a second Cas protein that is distinct from the first Cas protein; and Including, the length of the second polypeptide sequence is less than about 20% of the length of the first polypeptide sequence; Recombinant polypeptides are provided.

[0011] In another aspect of the present disclosure, there is provided a recombinant polypeptide comprising a recombinant nuclease variant, The recombinant nuclease variant comprises: (i) comprises an amino acid sequence having at least 80% identity to the polypeptide sequence of a member selected from Table 3B, Table 4B, or Table 5C; (ii) is not any of SEQ ID NOs: 1 to 3, 10, and 13 to 19; (iii) having a length of about 800 amino acids or less; Recombinant polypeptides are provided.

[0012] In another aspect of the disclosure, there is provided a recombinant polypeptide comprising a recombinant nuclease variant operably linked to a gene modulator, The recombinant nuclease variant comprises: (i) comprises an amino acid sequence having at least 70% identity to the polypeptide sequence of SEQ ID NO:1; (ii) when operably linked to a gene modulator, induces enhanced regulation of a target gene in a cell compared to a control recombinant polypeptide comprising SEQ ID NO: 10 operably linked to said gene modulator; Recombinant polypeptides are provided.

[0013] In another aspect of the disclosure, there is provided a method for regulating a target gene in a cell, comprising: Methods are provided that include contacting a cell with any one of the recombinant polypeptides disclosed herein.

[0014] In another aspect of the disclosure, there is provided a method for modulating a target gene in a cell, comprising: contacting the cell with a recombinant polypeptide comprising a recombinant nuclease variant operably linked to a gene modulator; the recombinant nuclease variant comprises an amino acid sequence having at least 70% identity to the polypeptide sequence of SEQ ID NO: 1; the contacting step induces enhanced regulation of a target gene in the cell compared to a control recombinant polypeptide comprising SEQ ID NO: 10 operably linked to the gene modulator. A method is provided.

[0015] In another aspect of the present disclosure, a composition is provided comprising a guide nucleic acid molecule configured to form a complex with a Cas protein.

[0016] In another aspect of the disclosure, there is provided a composition comprising a guide nucleic acid molecule configured to form a complex with a Cas protein, the guide nucleic acid molecule a spacer sequence that exhibits specific binding to a target polynucleotide sequence; Scaffold sequence for forming a complex with Cas protein Including, The scaffold sequence is (i) having a contiguous polynucleotide sequence having at least 96% sequence identity to the polynucleotide sequence of SEQ ID NO: 555, or having a contiguous polynucleotide sequence having at least 84% sequence identity to the polynucleotide sequence of SEQ ID NO: 597; (ii) having a contiguous polynucleotide sequence having at least 97% sequence identity to the polynucleotide sequence of SEQ ID NO: 557 or having a contiguous polynucleotide sequence having at least 88% sequence identity to the polynucleotide sequence of SEQ ID NO: 598; (iii) having a contiguous polynucleotide sequence having at least 90% sequence identity to the polynucleotide sequence of SEQ ID NO: 578, or having a contiguous polynucleotide sequence having at least 84% sequence identity to the polynucleotide sequence of SEQ ID NO: 597, or having a contiguous polynucleotide sequence having at least 81% sequence identity to the polynucleotide sequence of SEQ ID NO: 599; (iv) having a contiguous polynucleotide sequence having at least 93% sequence identity to the polynucleotide sequence of SEQ ID NO: 568, or having a contiguous polynucleotide sequence having at least 84% sequence identity to the polynucleotide sequence of SEQ ID NO: 597, or having a contiguous polynucleotide sequence having at least 67% sequence identity to the polynucleotide sequence of SEQ ID NO: 600; or (v) having a contiguous polynucleotide sequence having at least 95% sequence identity to the polynucleotide sequence of SEQ ID NO: 569, or having a contiguous polynucleotide sequence having at least 84% sequence identity to the polynucleotide sequence of SEQ ID NO: 597, or having a contiguous polynucleotide sequence having at least 71% sequence identity to the polynucleotide sequence of SEQ ID NO: 601. A composition is provided, characterized in that:

[0017] In another aspect of the disclosure, there is provided a composition comprising a guide nucleic acid molecule configured to form a complex with a Cas protein, the guide nucleic acid molecule a spacer sequence that exhibits specific binding to a target polynucleotide sequence operably linked to a target gene; Scaffold sequence for forming a complex with Cas protein Including, the scaffold sequence exhibits at least 80% sequence identity with a polynucleotide sequence of a member selected from Table 6B, Table 7B and Table 8B, and is not identical to SEQ ID NO: 500; When the complex binds to the target polynucleotide sequence in a cell, the expression level of the target gene in the cell is regulated, and (A1) the expression level of the target gene regulated by the complex becomes similar to or higher than (A2) the expression level regulated by a control complex comprising a control guide nucleic acid molecule comprising the polynucleotide sequence of SEQ ID NO: 500 and the Cas protein. A composition is provided.

[0018] In another aspect of the disclosure, there is provided a composition comprising a guide nucleic acid molecule configured to form a complex with a Cas protein, the guide nucleic acid molecule a spacer sequence that exhibits specific binding to a target polynucleotide sequence operably linked to a target gene; Scaffold sequence for forming a complex with Cas protein Including, the length of the scaffold sequence is a maximum of about 158 ​​nucleotides in length; When the complex binds to the target polynucleotide sequence in a cell, the expression level of the target gene in the cell is regulated, and (A1) the expression level of the target gene regulated by the complex becomes similar to or higher than (A2) the expression level regulated by a control complex comprising a control guide nucleic acid molecule comprising the polynucleotide sequence of SEQ ID NO: 500 and the Cas protein. A composition is provided.

[0019] In another aspect of the disclosure, there is provided a composition comprising a vector encoding a Cas protein and a guide nucleic acid molecule configured to form a complex with the Cas protein, The vector is a first polynucleotide sequence encoding the Cas protein; and a second polynucleotide sequence encoding a scaffold sequence of the guide nucleic acid molecule for forming a complex with the Cas protein; Including, the total length of the first polynucleotide sequence plus the length of the second polynucleotide sequence is at most about 1700 nucleotides; A composition is provided.

[0020] In another aspect of the disclosure, there is provided a method for regulating a target gene in a cell, comprising: Methods are provided that include contacting a cell with any one of the compositions disclosed herein.

[0021] In another aspect of the disclosure, there is provided a method for modulating a target gene in a cell, comprising: contacting a cell with a complex comprising a guide nucleic acid molecule and a Cas protein; the complex exhibits specific binding to a target polynucleotide sequence operably linked to a target gene; When the complex binds to the target polynucleotide sequence in the cell, the expression level of the target gene in the cell is regulated, and (A1) the expression level of the target gene regulated by the complex becomes similar to or higher than (A2) the expression level regulated by a control complex comprising a control guide nucleic acid molecule comprising the polynucleotide sequence of SEQ ID NO: 500 and the Cas protein. A method is provided.

[0022] Those skilled in the art will readily appreciate further aspects and advantages of the present disclosure from the following detailed description, which shows and describes only illustrative embodiments of the disclosure. It will also be readily apparent that the present disclosure is susceptible to other and various embodiments, the details of which can be modified in various obvious respects without departing from the present disclosure. Accordingly, the drawings and detailed description are to be regarded as illustrative in nature and not restrictive.

[0023] Incorporation by Reference All publications, patents, and patent applications mentioned herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event that an incorporated-by-reference publication, patent, or patent application conflicts with the disclosure of this specification, the disclosure of this specification will govern and / or take precedence over such conflicting matter. [Brief explanation of the drawings]

[0024] The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "figures").

[0025] [Figure 1]Schematic representation of a structural alignment between Un1Cas12f1 and AsCas12f to identify one or more domains in Un1Cas12f1 that may not be conserved in one or more additional Cas12f homologous structures.

[0026] [Figure 2] Schematic representation of a structural alignment between Un1Cas12f1 and Un2Cas12f1 to identify one or more domains in Un1Cas12f1 that may not be conserved in one or more additional Cas12f homologous structures.

[0027] [Figure 3] Figure 3A shows a schematic representation of a selection of different domains of Un1Cas12f1 that can be at least partially deleted to generate one or more recombinant nucleases, and Figure 3B shows a schematic representation of a deletion landscape approach to generating recombinant nuclease variants of Un1Cas12f1.

[0028] [Figure 4] Schematic representation of an example deletion landscape of a Cas protein (e.g., dCas9) to identify one or more domains that may be deleted in the Cas protein with minimal or no substantial effect on the activity of the Cas protein (e.g., the ability to induce transcriptional expression when an engineered variant of dCas9 is operably linked to a gene repressor).

[0029] [Figure 5] Figure 1 shows the enhanced expression of endogenous IFNγ (top plot) and endogenous CD2 (bottom plot) induced in cells by various recombinant nuclease variants disclosed herein that were engineered to exhibit reduced nuclease activity (e.g., dCas variants) and then fused to a gene activation modulator.

[0030] [Figure 6] Figure 1 shows the reduction in target gene (e.g., GFP) expression induced in cells by various recombinant nuclease variants disclosed herein. These recombinant nuclease variants were engineered to exhibit reduced nuclease activity (e.g., dCas variants) and then fused to gene repression modulators. Two gene repression modulators were used: gene repressor A (top plot) and gene repressor B (bottom plot).

[0031] [Figure 7] Figure 1 shows the enhanced expression of endogenous CD2 (top plot), endogenous IFNγ (middle plot), and endogenous CXCR4 (bottom plot) induced in cells by various recombinant nuclease variants disclosed herein that were engineered to exhibit reduced nuclease activity (e.g., dCas variants) and then fused to gene activation modulators.

[0032] [Figure 8] Schematic representation of a guide nucleic acid molecule configured to form a complex with a Cas protein.

[0033] [Figure 9] Schematically shown are regions of the scaffold region that can be modified (e.g., mutated or deleted) in the guide nucleic acid molecule to recombine the guide nucleic acid molecule.

[0034] [Figure 10] 1 shows the reduction in expression of target genes (e.g., GFP) induced in cells by multiple recombinant guide RNA variants disclosed herein. These multiple recombinant guide RNA variants have at least modified scaffold regions compared to control guide RNA sequences ("SQ"). These gene expression reductions were observed using the same dCas protein linked to a gene-silencing modulator.

[0035] [Figure 11]

[0023] Figure 1 shows the reduction in target gene (e.g., GFP) expression induced in cells by additional recombinant guide RNA variants disclosed herein, which had altered at least the scaffold region compared to a control guide RNA sequence ("SQ"). These gene expression reductions were observed using the same dCas protein linked to a gene-silencing modulator.

[0036] [Figure 12] This figure shows the enhanced expression of endogenous CD2 induced in cells by various recombinant guide RNA variants disclosed herein. The various recombinant guide RNA variants have at least modified scaffold regions compared to the control guide RNA sequence ("SQ"). These enhanced gene expression were observed using the same dCas protein linked to a gene activation modulator.

[0037] [Figure 13] 1 shows a schematic diagram of an example of a recombination guide nucleic acid molecule disclosed herein.

[0038] [Figure 14] A comparison of the sizes of various DNA vectors encoding the Cas protein and each single-guide nucleic acid molecule is shown.

[0039] [Figure 15] 1 shows the increase in endogenous IFNγ expression induced in cells by the truncated nuclease variant t1 disclosed herein, which recombinant nuclease variant t1 was fused to the gene activation modulator VPR.

[0040] [Figure 16]1 shows the reduction in endogenous CXCR4 expression induced in cells by the chimeric nuclease variant disclosed herein. The recombinant nuclease variant t1 was fused to a gene repression modulator (ZNF10-KRAB-hDNMT3L). DETAILED DESCRIPTION OF THE INVENTION

[0041] While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art will also recognize that various variations, changes, and substitutions are possible without departing from the invention. Various alternative aspects of the embodiments of the invention described herein may also be employed.

[0042] When the terms "at least," "greater than," or "greater than or equal to" appear before or after the first or last number in a series of two or more numbers, the terms "at least," "greater than," or "greater than or equal to" always apply to each number in the series. For example, "1, 2, or 3 or more" is the same as "1 or more, 2 or more, or 3 or more."

[0043] When the term "within," "less than," or "less than or equal to" appears after the last number in a series of two or more numbers, the term "within," "less than," or "less than or equal to" always applies to each number in the series. For example, "less than or equal to 1, 2, or 3" means the same as "less than or equal to 1, 2, or 3."

[0044] The term "about" typically refers to a range within an acceptable error of a particular numerical value, as determined by one of ordinary skill in the art; this acceptable error range depends in part on how the numerical value is measured or determined, i.e., in part on the limitations of the measurement system. For example, "about" may mean within one standard deviation per measurement or more than one standard deviation. Alternatively, "about" may refer to a range of up to 20%, up to 10%, up to 5%, or up to 1% of a numerical value. Alternatively, particularly with respect to biological systems or processes, the term "about" may mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a numerical value. Where specific numerical values ​​are described in this application and claims, unless otherwise specified, the term "about" is intended to mean within an acceptable error range of the particular numerical value.

[0045] The use of alternatives (e.g., those indicated by "or") means one, both, or a combination of the alternatives. The term "and / or" means one or both of the alternatives.

[0046] The term "cell" generally refers to a biological cell. A cell may be the basic structural, functional, and / or biological unit of a living organism. A cell may be from any organism having one or more cells. Some examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of single-celled eukaryotes, protozoan cells, cells obtained from plants (e.g., cells obtained from cereal plants, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, club mosses, bryophytes, liverworts, or mosses), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, etc.), seaweed (e.g., kelp), fungal cells (e.g., yeast cells, cells obtained from mushrooms), animal cells, cells obtained from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells obtained from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells obtained from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.), etc. In some cases, the cells may not be derived from a naturally occurring organism (e.g., the cells may be synthetically produced, sometimes referred to as "artificial cells").

[0047] As used herein, the term "nucleotide" generally refers to a combination of a base, a sugar, and a phosphate. A nucleotide may be a synthetic nucleotide. A nucleotide may be a synthetic nucleotide analog. A nucleotide may be a monomeric unit of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)). The term "nucleotide" may include the ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), and guanosine triphosphate (GTP), as well as deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives include, for example, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. As used herein, "nucleotide" may also refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Specific examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may be unlabeled or may be detectably labeled by known techniques. Labeling may also be performed using quantum dots. Detectable labels include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels for nucleotides include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, cyanine, and 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP, available from PerkinElmer (Foster City, CA); and FluoroLink deoxynucleotides FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink available from Amersham (Arlington Heights, IL). Cy5-dUTP; fluorescein-15-dATP, fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2'-dATP, available from Boehringer Mannheim (Indianapolis, IN); and Molecular Available chromosome-labeled nucleotides include BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, Fluorescein-12-UTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, Tetramethylrhodamine-6-UTP, Tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP, all available from Probes, Inc. (Eugene, Oregon). Nucleotides can also be labeled by chemical modification. The chemically modified single nucleotide may be a biotin-dNTP.Some examples of biotinylated dNTPs include, but are not limited to, biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

[0048] As used herein, the terms "polynucleotide," "oligonucleotide," or "nucleic acid" are used interchangeably and generally refer to a polymeric form of nucleotides of a certain length, which may be deoxyribonucleotides or ribonucleotides, or analogs thereof, and which may be single-stranded, double-stranded, or multi-stranded. A polynucleotide may be exogenous to a cell or endogenous to a cell. A polynucleotide may be in a cell-free environment. A polynucleotide may be a gene or a fragment thereof. A polynucleotide may be DNA. A polynucleotide may be RNA. A polynucleotide may have any three-dimensional structure and may perform any function, known or unknown. A polynucleotide may contain one or more analogs (e.g., modified backbones, sugars, or nucleobases). If a modification is included in the nucleotide structure, the modification can be imparted before or after assembly of the nucleotide. Some examples of analogs include, but are not limited to, 5-bromouracil, peptide nucleic acids, xenonucleic acids, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorescent dyes (e.g., rhodamine or fluorescein linked to the sugar), thiol-containing nucleotides, biotin-labeled nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queusine, and wyosine.Examples of polynucleotides include, but are not limited to, coding or non-coding regions of a gene or gene fragment, loci defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, DNA isolated from a sequence, RNA isolated from a sequence, cell-free polynucleotides such as cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. Non-nucleotide components can be inserted into the nucleotide sequence.

[0049] The term "gene" generally refers to a nucleic acid (e.g., DNA, such as genomic DNA or cDNA) and the corresponding nucleotide sequence involved in encoding an RNA transcript. As used herein with respect to genomic DNA, the term also includes intervening non-coding and regulatory regions, including the 5' and 3' ends. In some uses, the term "gene" encompasses transcribed sequences, such as 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcribed region contains an "open reading frame" that encodes a polypeptide. In some uses of the term "gene," the term "gene" includes only the coding sequence (e.g., "open reading frame" or "coding region") required to encode a polypeptide. In some cases, a gene does not encode a polypeptide, such as, for example, a ribosomal RNA gene (rRNA) or a transfer RNA (tRNA) gene. In some cases, the term "gene" includes not only the transcribed sequence but also non-transcribed regions, including upstream and downstream regulatory regions, such as enhancers and promoters. "Gene" may refer to an "endogenous gene" or a native gene that is present in its natural location in the genome of an organism. "Gene" may also refer to a "foreign gene" or a non-native gene. A "non-native gene" may refer to a gene that is not normally found in the host organism, but that has been introduced into the host organism by gene transfer. Furthermore, a "non-native gene" may refer to a gene that is not present in its natural location in the genome of an organism. A "non-native gene" may refer to a naturally occurring nucleic acid sequence or polypeptide sequence that contains mutations, insertions, and / or deletions (e.g., a non-native sequence).

[0050] The term "deletion" typically refers to the removal (or loss) of one or more (or a specified number) amino acids (e.g., contiguous or non-contiguous amino acids) from a polypeptide sequence, or the removal (or loss) of one or more (or a specified number) nucleobases (e.g., contiguous or non-contiguous nucleobases) from a polynucleotide sequence (e.g., a polynucleotide sequence encoding a polypeptide sequence). An "internal deletion" typically refers to a deletion that does not include the N- or C-terminus of a polypeptide or the 5' or 3' end of a polynucleotide. Deletions (e.g., internal deletions) can be identified by comparison to a reference sequence, e.g., by determining the start and end positions of the deletion relative to the reference sequence. Deletions (e.g., internal deletions) are distinct from and are distinguished from substitutions. For example, compared to the reference polypeptide sequence, at least one different amino acid is not inserted at the position where at least one amino acid has been deleted, and as a result, the size (e.g., number of amino acid residues) of the modified (or recombined) polypeptide sequence containing the deletion of at least one amino acid is smaller than the reference polypeptide sequence by the size of the at least one deleted amino acid.

[0051] The term "sequence identity" typically refers to a perfect nucleotide-to-nucleotide correspondence between two polynucleotide sequences or a perfect amino acid-to-amino acid correspondence between two polypeptide sequences. Techniques for determining sequence identity generally involve determining the nucleotide sequence of a polynucleotide and / or the amino acid sequence encoded by that nucleotide sequence and then comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid sequences) can be compared by determining their "percent identity." The percent identity of two sequences, whether nucleic acid or amino acid, is determined by dividing the number of perfectly matched residues between the two aligned sequences by the length of the longer sequence and multiplying by 100. For example, percent identity may be determined by comparing sequence information using the advanced BLAST computer program (e.g., version 2.2.9) available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87:2264-2268 (1990), which is discussed in Altschul, et al., J. Mol. Biol., 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997). The BLAST program may be used to determine percent identity over the entire length of the proteins being compared. Default parameters are set to optimize searches with short query sequences, for example, using the blastp program.The BLAST program can further employ a SEG filter to mask specific segments of the query sequence as determined by the SEG program described in Wootton and Federhen, Computers and Chemistry 17:149-163 (1993). The desired sequence identity range is between about 50% and 100%, including integer values ​​within that range. Typically, the present disclosure includes sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the sequences provided herein.

[0052] The term "expression" typically refers to one or more processes by which a polynucleotide (e.g., mRNA or other RNA transcript) is transcribed from a DNA template and / or the process by which the transcribed mRNA is translated into a peptide, polypeptide, or protein. A transcript and the encoded polypeptide can be collectively referred to as a "gene product." When a polynucleotide is derived from genomic DNA, "expression" can also include splicing of mRNA in eukaryotic cells. "Up-regulation," with respect to expression, typically refers to an increase in the expression level of a polynucleotide sequence (e.g., RNA such as mRNA) and / or polypeptide sequence compared to the wild-type expression level, while "down-regulation" typically refers to a decrease in the expression level of a polynucleotide sequence (e.g., RNA such as mRNA) and / or polypeptide sequence compared to the wild-type expression level. Expression of a transfected gene can occur transiently or stably in a cell. In "transient expression," the transfected gene is not transferred to daughter cells during cell division. In transient expression, expression occurs only in transfected cells, and gene expression is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selectable advantage to the transfected cells. Such a selectable advantage may be resistance to a particular toxin to which the cells are exposed.

[0053] The term "expression profile" generally refers to the quantitative (e.g., abundance) and qualitative expression of one or more genes in a sample (e.g., cells). The one or more genes may be expressed and identified in the form of nucleic acid molecules (e.g., mRNA or other RNA transcripts). Additionally or alternatively, the one or more genes may be expressed and identified in the form of polypeptides (e.g., proteins as measured by Western blot). The expression profile of a gene may be defined as the amount of expression of the gene over a certain time frame (e.g., a time frame of at least about 1 hour or less, at least about 2 hours or less, at least about 3 hours or less, at least about 4 hours or less, at least about 5 hours or less, at least about 6 hours or less, at least about 7 hours or less, at least about 8 hours or less, at least about 9 hours or less, at least about 10 hours or less, at least about 11 hours or less, at least about 12 hours or less, 2 hours or less, at least about or about 16 hours or less, at least about or about 18 hours or less, at least about or about 24 hours or less, at least about or about 36 hours or less, at least about or about 48 hours or less, at least up to about 3 days, at least up to about 4 days, at least up to about 5 days, at least up to about 6 days, at least up to about 7 days, at least up to about 8 days, at least up to about 9 days, at least up to about 10 days, at least up to about 11 days, at least up to about 12 days, at least up to about 13 days, at least up to about 14 days, etc.).Alternatively, a gene expression profile may be defined as the amount of expression of a gene at a time point of interest (e.g., at least about 1 hour or at most about 1 hour, at least about 2 hours or at most about 2 hours, at least about 3 hours or at most about 3 hours, at least about 4 hours or at most about 4 hours, at least about 5 hours or at most about 5 hours, at least about 6 hours or at most about 6 hours, at least about 7 hours or at most about 7 hours, at least about 8 hours or at most about 8 hours, at least about 9 hours or at most about 9 hours, at least about 10 hours or at most about 10 hours, at least about 11 hours or at most about 11 hours, The expression level of the gene measured may be at least about 12 hours or at most about 12 hours, at least about 16 hours or at most about 16 hours, at least about 18 hours or at most about 18 hours, at least about 24 hours or at most about 24 hours, at least about 36 hours or at most about 36 hours, at least about 48 hours or at most about 48 hours, at least at most about 3 days, at least at most about 4 days, at least at most about 5 days, at least at most about 6 days, at least at most about 7 days, at least at most about 8 days, at least at most about 9 days, at least at most about 10 days, at least at most about 11 days, at least at most about 12 days, at least at most about 13 days, or at least at most about 14 days).

[0054] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and generally refer to a polymer of at least two amino acid residues linked by a peptide bond. These terms do not imply a specific length of the polymer, nor do they refer to or distinguish between peptides produced using recombinant technology, chemical synthesis, enzymatic synthesis, or natural origin. These terms also apply to naturally occurring amino acid polymers as well as amino acid polymers containing at least one modified amino acid. In some cases, non-amino acids can be inserted into these polymers. These terms also include amino acid chains of any length, including full-length proteins, proteins with secondary and / or tertiary structure (e.g., domains), or proteins lacking secondary and / or tertiary structure. These terms also include modified amino acid polymers, such as those modified by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, or other manipulations, such as conjugation with a labeling component. As used herein, the term "amino acid" generally refers to natural and unnatural amino acids, including, but not limited to, modified amino acids and amino acid analogs. Modified amino acids include natural and unnatural amino acids that have been chemically modified to contain groups or chemical moieties not found in naturally occurring amino acids. Amino acid analogs may refer to amino acid derivatives. The term "amino acid" includes both D- and L-amino acids.

[0055] As used herein, the terms "derivative," "variant," or "fragment" when used with respect to a polypeptide generally refer to a polypeptide that is related to a wild-type polypeptide, e.g., a polypeptide that is related in amino acid sequence, structure (e.g., secondary and / or tertiary structure), activity (e.g., enzymatic activity), and / or function to the wild-type polypeptide. Polypeptide derivatives, variants, and fragments may include one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof, compared to the wild-type polypeptide.

[0056] As used herein, the term "recombined," "chimeric," or "recombinant" in reference to a polypeptide molecule (e.g., a protein) generally refers to a polypeptide molecule having a heterologous or modified amino acid sequence, as well as a cell or organism expressing the polypeptide molecule, resulting from the application of genetic engineering techniques to the nucleic acid encoding the polypeptide molecule. As used herein, the term "recombined" or "recombinant" in reference to a polynucleotide molecule (e.g., a DNA molecule or an RNA molecule) generally refers to a polynucleotide molecule having a heterologous or modified nucleic acid sequence, resulting from 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-specific mutagenesis; and gene fusion. In some cases, a recombined polynucleotide or recombinant polynucleotide (e.g., a genomic DNA sequence) can be modified or changed by a gene editing moiety.

[0057] For example, the recombinant nucleases (e.g., recombinant Cas proteins) disclosed herein are not naturally occurring nucleases (e.g., are not naturally occurring Cas proteins). As used herein, the terms "recombinant nuclease" and "recombinant nuclease variant" may be used interchangeably.

[0058] As used herein, the terms "recombinant" and "modified" are used interchangeably. As used herein, the terms "recombining" and "modifying" are used interchangeably. As used herein, the terms "recombinant cell" and "modified cell" are used interchangeably. As used herein, the terms "recombinant characteristic" and "modified characteristic" are used interchangeably.

[0059] The terms "enhanced expression," "increased expression," or "upregulated expression" generally refer to the production of a moiety of interest (e.g., a polynucleotide or polypeptide) at an expression level that exceeds the normal expression level of the moiety of interest in a host strain (e.g., a host cell). The normal expression level may be substantially zero (or null) or may be an expression level greater than zero. The moiety of interest may comprise an endogenous gene or endogenous polypeptide construct of the host strain. The moiety of interest may comprise a heterologous gene or heterologous polypeptide construct introduced into the host strain. For example, to enhance expression of a polypeptide of interest in a host strain, a heterologous gene encoding the polypeptide of interest can be knocked in (KI) into the genome of the host strain.

[0060] The terms "enhanced activity," "increased activity," or "upregulated activity" generally refer to the activity of a moiety of interest (e.g., a polynucleotide or polypeptide) that has been modified to exceed the normal activity level of the moiety of interest in a host strain (e.g., a host cell). The normal activity level may be substantially zero (or null) or an activity level greater than zero. The moiety of interest may include a polypeptide construct of the host strain. The moiety of interest may include a heterologous polypeptide construct introduced into the host strain. For example, to enhance the activity of a polypeptide of interest in a host strain, a heterologous gene encoding the polypeptide of interest can be knocked in (KI) into the genome of the host strain.

[0061] The terms "suppression of expression," "reduced expression," or "downregulated expression" generally refer to the production of a moiety of interest (e.g., a polynucleotide or polypeptide) at an expression level that is below the normal expression level of the moiety of interest in a host strain (e.g., a host cell). Normal expression is an expression level that is greater than zero. The moiety of interest may comprise an endogenous gene or endogenous polypeptide construct of the host strain. In some cases, the moiety of interest can be knocked out or knocked down in the host strain. In some instances, suppression of expression of the moiety of interest may include complete inhibition of such expression in the host strain.

[0062] The terms "suppressed activity," "reduced activity," or "downregulated activity" generally refer to the activity of a moiety of interest (e.g., a polynucleotide or polypeptide) that has been altered to be below the normal activity level of the moiety of interest in a host strain (e.g., a host cell). A normal activity level is an activity that is greater than zero. The moiety of interest may comprise an endogenous gene or endogenous polypeptide construct of the host strain. In some cases, the moiety of interest can be knocked out or knocked down in the host strain. In some instances, reducing the activity of the moiety of interest may include complete inhibition of such activity in the host strain.

[0063] As used herein, the terms "subject," "individual," or "patient" are used interchangeably and generally refer to a vertebrate, preferably a mammal such as a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Also included are tissues, cells, and their progeny of biological matter obtained in vivo or in vitro culture.

[0064] The terms "treatment" or "treating" generally refer to an approach for obtaining a beneficial or desired result, such as, but not limited to, a therapeutic benefit and / or a prophylactic benefit. For example, "treatment" can include administering a system or cell population disclosed herein. A "therapeutic benefit" refers to a treatment-related improvement in, or an effect on, one or more diseases, conditions, or symptoms being treated. To obtain a prophylactic benefit, a composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or a subject experiencing one or more physiological symptoms of a disease, even before the disease, condition, or symptom appears.

[0065] The term "effective amount" or "therapeutically effective amount" generally refers to the amount of a composition sufficient to achieve a desired activity when administered to a subject in need thereof, such as a composition comprising a heterologous polypeptide, a heterologous polynucleotide, and / or a recombinant cell (e.g., a modified stem cell). In the context of the present disclosure, the term "therapeutically effective" generally refers to the amount of a composition sufficient to delay the onset of, arrest the progression of, or alleviate or relieve at least one symptom of, a disease treated by the methods of the present disclosure.

[0066] overview In various embodiments of the present disclosure, recombinant nucleases that are smaller than conventional nucleases but are effective in binding, cleaving, and / or editing target polynucleotide sequences, as well as compositions and methods for use thereof, can be provided.Such recombinant nucleases (e.g., recombinant CRISPR / Cas nucleases) can, for example, treat or alleviate a subject's condition (e.g., disease) by manipulating the expression or activity of a target gene (e.g., endogenous target gene) in a cell.Gene expression underpins various physiological and pathological effects in cells and tissues, thereby contributing to numerous diseases and conditions.Therefore, the compositions and methods utilizing recombinant nucleases of the present disclosure have therapeutic benefits because they can regulate the expression of specific genes in a desired manner.

[0067] Recombinant nucleases, compositions and methods thereof In some embodiments, the present disclosure provides a recombinant nuclease comprising an amino acid sequence having at least 50% identity to the polypeptide sequence of SEQ ID NO: 1. The amino acid sequence of the recombinant nuclease may contain at least one deletion compared to (e.g., when aligned with) the polypeptide sequence of SEQ ID NO: 1 (or SEQ ID NO: 10). This at least one deletion can be selectively removed according to the present disclosure. As disclosed herein, SEQ ID NO: 1 encodes the polypeptide sequence of Un1Cas12f1 (i.e., Cas14a1). Also disclosed herein, SEQ ID NO: 10 encodes a recombinant variant of Un1Cas12f1 with reduced nuclease activity. Thus, the amino acid sequence of the recombinant nuclease disclosed herein may be a mutant sequence (or mutant variant) of Un1Cas12f1 (or an inactivated variant thereof).

[0068] Without wishing to be bound by any theory, as disclosed herein, at least one deletion in the amino acid sequence of the recombinant nuclease may be present in one or more regions of the native Un1Cas12f1 nuclease that do not structurally align with the control CRISPR / Cas protein. The control CRISPR / Cas protein may be derived from a Class 1 CRISPR system or a Class 2 CRISPR system (e.g., as a wild-type CRISPR / Cas protein). Class 1 CRISPR systems can be classified into types I, III, and IV, while Class 2 CRISPR systems can be classified into types II, V, and VI. In some cases, the control CRISPR / Cas protein may be a Type V Cas protein, such as a Type VA Cas protein, a Type VB Cas protein, a Type VC Cas protein, a Type VD Cas protein, a Type VE Cas protein, a Type VF Cas protein, a Type VG Cas protein, a Type VH Cas protein, a Type VI Cas protein, a Type VJ Cas protein, a Type VK Cas protein, or a Type VU Cas protein. In some cases, the control CRISPR / Cas protein may be a VJ-type protein, such as a wild-type CasΦ (Cas12J) protein. In some cases, the control CRISPR / Cas protein may be Un2Cas12f1 (SEQ ID NO: 2) or AsCas12f (SEQ ID NO: 3).

[0069] Without wishing to be bound by any theory, as disclosed herein, the deletion of at least one amino acid sequence of the recombinant nuclease can be determined by performing a deletion landscape test (e.g., recursive deletion and / or global deletion) of the Cas nuclease encoded by the polypeptide sequence of SEQ ID NO: 1 (or SEQ ID NO: 10).

[0070] Without wishing to be bound by any theory, as disclosed herein, different variants of a recombinant nuclease may exhibit different activities (e.g., different binding affinities for a control sgRNA, different binding affinities for a control target gene, different levels of cleavage of the target gene, different levels of activation of the target gene, different levels of repression of the target gene, etc.). In some embodiments, a first variant of the different variants may contain at least one deletion in amino acid residues 1-100 (e.g., when aligned with the polypeptide sequence of SEQ ID NO: 1), and a second variant of the different variants may contain at least one deletion in amino acid residues 101-529 (e.g., when aligned with the polypeptide sequence of SEQ ID NO: 1), and the first and second variants may exhibit different activities (e.g., the first variant may enhance activation and / or expression of a target gene compared to the second variant, or vice versa). Alternatively, the first variant and the second variant may exhibit comparable activities. In some embodiments, the first variant of the plurality of different variants may contain a single deletion in the amino acid residues 1-100 (e.g., when aligned with the polypeptide sequence of SEQ ID NO: 1), and the second variant of the plurality of different variants may contain multiple deletions in the amino acid residues 1-100 (e.g., when aligned with the polypeptide sequence of SEQ ID NO: 1), and the first variant and the second variant may exhibit different activities (e.g., the second variant may enhance target gene activation and / or expression compared to the first variant, or vice versa). Alternatively, the first variant and the second variant may exhibit comparable activities.

[0071] SEQ ID NO: 1 (Un1Cas12f1) JPEG2025507822000002.jpg71146

[0072] SEQ ID NO: 10 (inactive nuclease variant of Un1Cas12f1, i.e., dCasMINI) JPEG2025507822000003.jpg73147

[0073] SEQ ID NO: 2 (Un2Cas12f1) JPEG2025507822000004.jpg66146

[0074] SEQ ID NO: 3 (AsCas12f) JPEG2025507822000005.jpg59144

[0075] Throughout this disclosure, (i) a sequence comparison between the amino acid sequence of a recombinant nuclease disclosed herein and the polypeptide sequence of SEQ ID NO: 1 may be equivalent (e.g., substantially identical) to (ii) a sequence comparison between the amino acid sequence of a recombinant nuclease disclosed herein and the polypeptide sequence of SEQ ID NO: 10.

[0076] In some embodiments, the amino acid sequence of a recombinant nuclease disclosed herein has a similar affinity to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) any one of the polypeptide sequences of the Cas proteins selected from Table 2 by at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 62% or less, at least about 64% or less, at least about 65% or less, at least about 66% or less, at least about 68% or less, at least about 70% or less, at least about 72% or less, at least about 74% or less, at least about 75% or less, at least about 76% or more. the sequence of the amino acid sequence of the present invention may have an identity of at least about 78% or less, at least about 80% or less, at least about 82% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 88% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, or at least about 99% or less. For example, the amino acid sequence of a recombinant nuclease disclosed herein may have about 80% to about 100% identity to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from Table 2.

[0077] In some embodiments, the amino acid sequence of a recombinant nuclease disclosed herein does not share identity with (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from Table 2.

[0078] In some embodiments, the at least one deletion in the amino acid sequence of a recombinant nuclease disclosed herein can include an N-terminal deletion, a C-terminal deletion, and / or an internal deletion. For example, the at least one deletion can be an internal deletion and / or a C-terminal deletion, but can also be an N-terminal deletion.

[0079] In some embodiments, at least one deletion in the amino acid sequence of a recombinant nuclease disclosed herein may be a deletion from amino acid residues 1-100 (e.g., amino acid residues 2-100), amino acid residues 101-429, amino acid residues 101-200, amino acid residues 201-300, amino acid residues 301-400, amino acid residues 401-500, amino acid residues 500-529, and / or amino acid residues 430-529, relative to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from Table 2 (if applicable).

[0080] In some embodiments, the at least one deletion in the amino acid sequence of a recombinant nuclease disclosed herein may be at least one deletion from amino acid residues 2-100 compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from Table 2 (as applicable).

[0081] In some embodiments, at least one deletion in the amino acid sequence of a recombinant nuclease disclosed herein is at least one deletion of amino acid residues 2-98, amino acid residues 2-96, amino acid residues 2-95, amino acid residues 2-94, amino acid residues 2-92, amino acid residues 2-90, amino acid residues 2-88, amino acid residues 2-86, or amino acid residues 2-98, compared to the polypeptide sequence of (i) SEQ ID NO: 1 or (ii) any one of the polypeptide sequences of the Cas proteins selected from Table 2 (if applicable). Amino acid residues 2 to 85, amino acid residues 2 to 84, amino acid residues 2 to 82, amino acid residues 2 to 80, amino acid residues 2 to 78, amino acid residues 2 to 76, amino acid residues 2 to 75, amino acid residues 2 to 74, amino acid residues 2 to 72, amino acid residues 2 to 70, amino acid residues 2 to 68, amino acid residues 2 to 66, amino acid residues 2 to 65, amino acid residues 2 to 64, amino acid residues 2 to 62, amino acid residues 2 to 60, amino acid residues 2 to 58 , amino acid residues 2 to 56, amino acid residues 2 to 55, amino acid residues 2 to 54, amino acid residues 2 to 52, amino acid residues 2 to 50, amino acid residues 2 to 48, amino acid residues 2 to 46, amino acid residues 2 to 45, amino acid residues 2 to 44, amino acid residues 2 to 42, amino acid residues 2 to 40, amino acid residues 2 to 38, amino acid residues 2 to 36, amino acid residues 2 to 35, amino acid residues 2 to 34, amino acid residues 2 to 32, amino acid residues 2 to 30 The deletion may be from the 2nd to 28th amino acid residues, the 2nd to 26th amino acid residues, the 2nd to 25th amino acid residues, the 2nd to 24th amino acid residues, the 2nd to 22nd amino acid residues, the 2nd to 20th amino acid residues, the 2nd to 18th amino acid residues, the 2nd to 16th amino acid residues, the 2nd to 15th amino acid residues, the 2nd to 14th amino acid residues, the 2nd to 12th amino acid residues, the 2nd to 10th amino acid residues, the 2nd to 8th amino acid residues, the 2nd to 6th amino acid residues, the 2nd to 5th amino acid residues, or the 2nd to 4th amino acid residues.

[0082] In some embodiments, at least one deletion in the amino acid sequence of a recombinant nuclease disclosed herein is at least one deletion of amino acid residues 2-100, 4-100, 5-100, 6-100, 8-100, 10-100, 12-100, 14-100, 15-100, 16-100, 17-100, 18-100, 19-200, 21-210, 22-220, 23-230, 24-240, 25-260, 27-280, 28-290, 29-300, 30-310, 31-320, 32-330, 33-340, 34-350, 35-360, 37-380, 38-400, 39-410, 40-420, 41-430, 42-440, 43-450, 44-460, 45-470, 46-480, 47-480, 48-490, 49-500, 51-520, 53-540, 55-560, 57-580, 58-590, 59-600, 60-610, 61-620, 62-630, 63-640, 64-650, 65-700, 70-710, 72-730, 74-750, 75-760, 76-770, 77-780, 78-790, 79-800, 80- ~100th amino acid residues, 16th to 100th amino acid residues, 18th to 100th amino acid residues, 20th to 100th amino acid residues, 22nd to 100th amino acid residues, 24th to 100th amino acid residues, 25th to 100th amino acid residues, 26th to 100th amino acid residues, 28th to 100th amino acid residues, 30th to 100th amino acid residues, 32nd to 100th amino acid residues, 34th to 100th amino acid residues, 35th to 100th amino acid residues, 36th to 100th amino acid residues, 38th to 100th amino acid residues, 40th to 100th amino acid residues amino acid residues, 42-100th amino acid residues, 44-100th amino acid residues, 45-100th amino acid residues, 46-100th amino acid residues, 48-100th amino acid residues, 50-100th amino acid residues, 52-100th amino acid residues, 54-100th amino acid residues, 55-100th amino acid residues, 56-100th amino acid residues, 58-100th amino acid residues, 60-100th amino acid residues, 62-100th amino acid residues, 64-100th amino acid residues, 65-100th amino acid residues, 66-100th amino acid residues amino acid residues, 68 to 100 amino acid residues, 70 to 100 amino acid residues, 72 to 100 amino acid residues, 75 to 100 amino acid residues, 76 to 100 amino acid residues, 78 to 100 amino acid residues, 80 to 100 amino acid residues, 82 to 100 amino acid residues, 84 to 100 amino acid residues, 85 to 100 amino acid residues, 86 to 100 amino acid residues, 88 to 100 amino acid residues, 90 to 100 amino acid residues, 92 to 100 amino acid residues, 94 to 100 amino acid residues,The deletion may be from amino acid residues 95 to 100, amino acid residues 96 to 100, or amino acid residues 98 to 100.

[0083] In some embodiments, at least one deletion in the amino acid sequence of a recombinant nuclease disclosed herein is at least one deletion of amino acid residues 30-100, 30-98, 30-96, 30-95, 30-94, 30-92, 30-90, 30-88, 30-86, 30-85, 30-84, 30-82, 30-80, 30-78, 30-76, 30-75, 30-74, or 30-86, as compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from Table 2 (if applicable). The deletion may be from amino acid residues at positions 30 to 72, amino acid residues at positions 30 to 70, amino acid residues at positions 30 to 68, amino acid residues at positions 30 to 66, amino acid residues at positions 30 to 65, amino acid residues at positions 30 to 64, amino acid residues at positions 30 to 62, amino acid residues at positions 30 to 60, amino acid residues at positions 30 to 58, amino acid residues at positions 30 to 56, amino acid residues at positions 30 to 55, amino acid residues at positions 30 to 54, amino acid residues at positions 30 to 52, amino acid residues at positions 30 to 50, amino acid residues at positions 30 to 48, amino acid residues at positions 30 to 46, amino acid residues at positions 30 to 45, amino acid residues at positions 30 to 44, amino acid residues at positions 30 to 42, amino acid residues at positions 30 to 40, amino acid residues at positions 30 to 38, amino acid residues at positions 30 to 36, amino acid residues at positions 30 to 34, or amino acid residues at positions 30 to 32.

[0084] In some embodiments, at least one deletion in the amino acid sequence of a recombinant nuclease disclosed herein is a deletion of amino acid residues 55-56, 54-57, 54-58, 53-59, 52-60, 51-61, 50-62, 49-63, 48-64, or 59-65, as compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from Table 2 (if applicable). The amino acid residues may include one or more deletions from the amino acid residues at positions 47 to 65, the amino acid residues at positions 46 to 66, the amino acid residues at positions 45 to 67, the amino acid residues at positions 44 to 68, the amino acid residues at positions 43 to 69, the amino acid residues at positions 42 to 70, the amino acid residues at positions 41 to 71, the amino acid residues at positions 40 to 72, the amino acid residues at positions 39 to 73, the amino acid residues at positions 38 to 74, the amino acid residues at positions 37 to 73, the amino acid residues at positions 36 to 74, or the amino acid residues at positions 35 to 75 (for example, substantially all of these amino acid residues may be deleted).

[0085] In some embodiments, at least one deletion in the amino acid sequence of a recombinant nuclease disclosed herein may be a deletion from one or more members (e.g., one member, at least two members, at least three members, at least four members, at least five members, or more members) selected from the group consisting of amino acid residues 2 to 10, amino acid residues 11 to 20, amino acid residues 21 to 30, amino acid residues 31 to 40, amino acid residues 41 to 50, amino acid residues 51 to 60, amino acid residues 61 to 70, and amino acid residues 71 to 80, compared to the polypeptide sequence of SEQ ID NO: 1. In some embodiments, the at least one deletion in the amino acid sequence of a recombinant nuclease disclosed herein may be a deletion from one or more members (e.g., 1 member, at least 2 members, at least 3 members, at least 4 members, at least 5 members, or more members) selected from the group consisting of amino acid residues 2-5, amino acid residues 6-10, amino acid residues 11-15, amino acid residues 16-20, amino acid residues 21-25, amino acid residues 26-30, amino acid residues 31-35, amino acid residues 36-40, amino acid residues 41-45, amino acid residues 46-50, amino acid residues 51-55, amino acid residues 56-60, amino acid residues 61-65, amino acid residues 66-70, amino acid residues 71-75, and amino acid residues 76-80, relative to the polypeptide sequence of (i) SEQ ID NO: 1 or (ii) any one of the Cas proteins selected from Table 2 (if applicable).

[0086] In some embodiments, at least one deletion in the amino acid sequence of a recombinant nuclease disclosed herein may be a deletion from one or more members (e.g., two or more members, three or more members, four or more members, five or more members, etc.) selected from the group consisting of amino acid residues 10-90, amino acid residues 15-85, amino acid residues 20-80, amino acid residues 25-75, amino acid residues 30-70, amino acid residues 35-75, amino acid residues 40-70, amino acid residues 45-65, and amino acid residues 50-60, relative to the polypeptide sequence of (i) SEQ ID NO: 1 or (ii) any one of the Cas proteins selected from Table 2 (if applicable).

[0087] In some embodiments, at least one deletion in the amino acid sequence of a recombinant nuclease disclosed herein may be a deletion from one or more members (e.g., two or more members, three or more members, four or more members, five or more members, etc.) selected from the group consisting of amino acid residues 20-30, amino acid residues 25-35, amino acid residues 30-40, amino acid residues 35-45, amino acid residues 40-50, amino acid residues 45-55, amino acid residues 50-60, amino acid residues 55-65, amino acid residues 60-70, amino acid residues 65-75, amino acid residues 70-80, amino acid residues 75-85, and amino acid residues 80-90, relative to the polypeptide sequence of (i) SEQ ID NO: 1 or (ii) any one of the Cas proteins selected from Table 2 (if applicable).

[0088] In some embodiments, at least one deletion in the amino acid sequence of a recombinant nuclease disclosed herein may be a deletion from one or more members (e.g., two or more members, three or more members, four or more members, five or more members, etc.) selected from the group consisting of amino acid residues 20-25, amino acid residues 25-30, amino acid residues 30-35, amino acid residues 35-40, amino acid residues 40-45, amino acid residues 45-50, amino acid residues 50-55, amino acid residues 55-60, amino acid residues 60-65, amino acid residues 65-70, amino acid residues 70-75, amino acid residues 75-80, and amino acid residues 85-90, relative to the polypeptide sequence of (i) SEQ ID NO: 1 or (ii) any one of the Cas proteins selected from Table 2 (if applicable).

[0089] In some embodiments, at least one deletion in the amino acid sequence of a recombinant nuclease disclosed herein may be a deletion from amino acid residues 430 to 529 relative to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from Table 2 (if applicable).

[0090] In some embodiments, at least one deletion in the amino acid sequence of a recombinant nuclease disclosed herein is at least one deletion of amino acid residues 430-528, 430-526, 430-525, 430-524, 430-522, 430-520, 430-518, 430-529, 430-530, 430-531, 430-532, 430-533, 430-534, 430-535, 430-536, 430-537, 430-538, 430-539, 430-540, 430-541, 430-542, 430-543, 430-544, 430-545, 430-546, 430-547, 430-548, 430-549, 430-550, 430-551, 430-552, 430-553, 430-554, 430-555, 430-556, 430-557, 430-558, 430-559, 430-560, 430-561, 430-562, 430-563, 430-564, 430-565, 430-566, 430-567, 430-568, 430-569, 430-570, 430-571, 430-572, 430-573, 430-574, 4 Amino acid residues at positions 516, 430 to 515, 430 to 514, 430 to 512, 430 to 510, 430 to 508, 430 to 506, 430 to 505, 430 to 504, 430 to 502, 430 to 500, 430 to 498, 430 to 496, 430 to 495, 430 to 494 residues, 430-492 amino acid residues, 430-490 amino acid residues, 430-488 amino acid residues, 430-486 amino acid residues, 430-485 amino acid residues, 430-484 amino acid residues, 430-482 amino acid residues, 430-480 amino acid residues, 430-478 amino acid residues, 430-476 amino acid residues, 430-475 amino acid residues, 430-474 amino acid residues, 430-472 amino acid residues, 430-470 amino acid residues, 430-468 amino acid residues, 430 to 466 amino acid residues, 430 to 465 amino acid residues, 430 to 464 amino acid residues, 430 to 462 amino acid residues, 430 to 460 amino acid residues, 430 to 458 amino acid residues, 430 to 456 amino acid residues, 430 to 455 amino acid residues, 430 to 454 amino acid residues, 430 to 452 amino acid residues, 430 to 450 amino acid residues, 430 to 448 amino acid residues, 430 to 446 amino acid residues, 430 to 445 amino acid residues,The deletion may be from amino acid residues 430 to 444, amino acid residues 430 to 442, amino acid residues 430 to 440, amino acid residues 430 to 438, amino acid residues 430 to 436, amino acid residues 430 to 435, amino acid residues 430 to 434, or amino acid residues 430 to 432.

[0091] In some embodiments, at least one deletion in the amino acid sequence of a recombinant nuclease disclosed herein is at least one of the following amino acid residues: 430-529, 432-529, 434-529, 435-529, 436-529, 438-529, 440-529, 442-529, 443-529, 444-529, 445-529, 446-529, 447-529, 448-529, 449-529, 450-529, 451-529, 452-529, 453-529, 454-529, 455-529, 456-529, 457-529, 458-529, 459-529, 460-529, 461-529, 462-529, 463-529, 464-529, 465-529, 466-529, 467-529, 468-529, 469-529, 470-529, 471-529, 472-529, 473-529, 474-529, 475-529, 476-529, 477-529, 478-529, 479-529, 480-529, 481-529, 482-529, 483-529, 484-529, 485-529, 486-529, 487-529, Amino acid residues at positions 529, 444 to 529, 445 to 529, 446 to 529, 448 to 529, 450 to 529, 452 to 529, 454 to 529, 455 to 529, 456 to 529, 458 to 529, 460 to 529, 462 to 529, 464 to 529, 465 to 529 residues, 466 to 529 amino acid residues, 468 to 529 amino acid residues, 470 to 529 amino acid residues, 472 to 529 amino acid residues, 474 to 529 amino acid residues, 475 to 529 amino acid residues, 476 to 529 amino acid residues, 478 to 529 amino acid residues, 480 to 529 amino acid residues, 482 to 529 amino acid residues, 484 to 529 amino acid residues, 485 to 529 amino acid residues, 486 to 529 amino acid residues, 488 to 529 amino acid residues, 490 to 529 amino acid residues, 492 to 529 amino acid residues, 494 to 529 amino acid residues, 495 to 529 amino acid residues, 496 to 529 amino acid residues, 498 to 529 amino acid residues, 500 to 529 amino acid residues, 502 to 529 amino acid residues, 504 to 529 amino acid residues, 505 to 529 amino acid residues, 506 to 529 amino acid residues, 508 to 529 amino acid residues, 510 to 529 amino acid residues, 512 to 529 amino acid residues, 514 to 529 amino acid residues,The deletion may be from amino acid residues 515 to 529, amino acid residues 516 to 529, amino acid residues 518 to 529, amino acid residues 520 to 529, amino acid residues 522 to 529, amino acid residues 524 to 529, amino acid residues 525 to 529, amino acid residues 526 to 529, or amino acid residues 528 to 529.

[0092] In some embodiments, the at least one deletion in the amino acid sequence of a recombinant nuclease disclosed herein may be a deletion from one or more members (e.g., 1 member, at least 2 members, at least 3 members, at least 4 members, at least 5 members, or more members) selected from the group consisting of amino acid residues 450-459, amino acid residues 460-469, amino acid residues 470-479, amino acid residues 480-489, amino acid residues 490-499, amino acid residues 500-509, amino acid residues 510-519, and amino acid residues 520-529, relative to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from Table 2 (if applicable). In some embodiments, at least one deletion in the amino acid sequence of a recombinant nuclease disclosed herein is at least one of the following amino acid residues: 450-459, 460-465, 466-469, 470-475, 476-479, 480-485, 486-489, 490-495, 491-492, 492-493, 493-494, 494-495, 495-500, 501-502, 502-503, 503-504, 505-506, 507-508, 509-510, 511-512, 513-514, 515-516, 517-518, 519-520, 521-522, 522-523, 523-524, 524-525, 525-526, 526-527, 527-528, 528-529, 530-531, 532-533, 534-535, 536-537, 538-539, 540-541, 542-543, 544-545, 546-547, 548-549, 549-550, 551-552, 552-553, 553-554, 554-555, 555-556, 557-558, 559-560, 561-562, 562-563, 563-564, The deletion may be from one or more members (e.g., one member, at least two members, at least three members, at least four members, at least five members, or more members) selected from the group consisting of amino acid residue 495, amino acid residues 496 to 499, amino acid residues 500 to 505, amino acid residues 506 to 509, amino acid residues 510 to 515, amino acid residues 516 to 519, amino acid residues 520 to 525, and amino acid residues 526 to 529.

[0093] In some embodiments, at least one deletion in the amino acid sequence of a recombinant nuclease disclosed herein may comprise a deletion of a single amino acid residue compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from Table 2 (if applicable). In some embodiments, at least one deletion in the amino acid sequence of a recombinant nuclease disclosed herein may comprise a deletion of multiple amino acid residues compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from Table 2 (if applicable). The deleted amino acid residues may be adjacent to each other (e.g., contiguous) when aligned with (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from Table 2 (if applicable). Additionally or alternatively, the deleted amino acid residues may not be adjacent to each other when aligned with (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from Table 2 (if applicable). The number of amino acid residues that may be included in this plurality of amino acid residues is at least about 2 or less amino acid residues, at least about 3 or less amino acid residues, at least about 4 or less amino acid residues, at least about 5 or less amino acid residues, at least about 6 or less amino acid residues, at least about 7 or less amino acid residues, at least about 8 or less amino acid residues, at least about 9 or less amino acid residues, at least about 10 or less amino acid residues, at least about 11 or less amino acid residues, at least about 12 or less amino acid residues, at least about 13 or less amino acid residues, at least about 14 or less amino acid residues, at least about 15 or less amino acid residues, at least about 16 or less amino acid residues, at least about 17 or less amino acid residues,At least about 18 or less amino acid residues, at least about 19 or less amino acid residues, at least about 20 or less amino acid residues, at least about 22 or less amino acid residues, at least about 24 or less amino acid residues, at least about 25 or less amino acid residues, at least about 26 or less amino acid residues, at least about 28 or less amino acid residues, at least about 30 or less amino acid residues, at least about 32 or less amino acid residues, at least about 34 or less amino acid residues, at least about 35 or less amino acid residues, at least about 36 or less amino acid residues, at least about 38 or less amino acid residues, at least about 40 or less amino acid residues, at least about 42 or less amino acid residues, at least about 44 or less amino acid residues, at least about 45 or less amino acid residues, at least about 46 or about 46 or less amino acid residues, at least about 48 or less amino acid residues, at least about 50 or less amino acid residues, at least about 52 or less amino acid residues, at least about 54 or less amino acid residues, at least about 55 or less amino acid residues, at least about 56 or less amino acid residues, at least about 58 or less amino acid residues, at least about 60 or less amino acid residues, at least about 62 or less amino acid residues, at least about 64 or less amino acid residues, at least about 65 or less amino acid residues, at least about 66 or less amino acid residues, at least about 68 or less amino acid residues, at least about 70 or less amino acid residues, at least about 72 or less amino acid residues, at least about 74 or less amino acid residues, at least about 75 or less amino acid residues, at least about 76 or less amino acid residues,It may be at least about 78 or less amino acid residues, at least about 80 or less amino acid residues, at least about 82 or less amino acid residues, at least about 84 or less amino acid residues, at least about 85 or less amino acid residues, at least about 86 or less amino acid residues, at least about 88 or less amino acid residues, at least about 90 or less amino acid residues, at least about 92 or less amino acid residues, at least about 94 or less amino acid residues, at least about 95 or less amino acid residues, at least about 96 or less amino acid residues, at least about 98 or less amino acid residues, or at least about 100 or less amino acid residues.

[0094] In some embodiments, the deletion of multiple amino acid residues to generate the amino acid sequence of a recombinant nuclease disclosed herein may comprise the deletion of multiple, non-contiguous amino acid residues, for example, compared to the polypeptide sequence of (i) SEQ ID NO: 1 or (ii) any one of the Cas proteins selected from Table 2 (if applicable). In some cases, the deletion of multiple, non-contiguous amino acid residues in the amino acid sequence of a recombinant nuclease disclosed herein may comprise the deletion of a first amino acid residue (e.g., a first single amino acid residue, or a first set of multiple amino acid residues, e.g., the first set of contiguous amino acid residues) and the deletion of a second amino acid residue (e.g., a second single amino acid residue, or a second set of multiple amino acid residues, e.g., the second set of contiguous amino acid residues) when aligned with the polypeptide sequence of (i) SEQ ID NO: 1 or (ii) any one of the Cas proteins selected from Table 2 (if applicable), where the deletion of the first amino acid residue and the deletion of the second amino acid residue are not contiguous with each other.For example, the number of amino acid residues separating a first amino acid residue position from a second amino acid residue position (e.g., when aligned with the polypeptide sequence of SEQ ID NO:1) can be at least about 1 or less, at least about 2 or less, at least about 3 or less, at least about 4 or less, at least about 5 or less, at least about 10 or less, at least about 15 or less, at least about 20 or less, at least about 30 or less, at least about 40 or less, at least about 50 or less, at least about 60 or less, at least about 70 or less, at least about or less, at least about 80 or less, at least about 90 or less, at least about 100 or less, at least about 120 or less, at least about 150 or less, at least about 200 or less, at least about 250 or less, at least about 300 or less, at least about 350 or less, at least about 400 or less, at least about 450 or less, at least about 500 or less, at least about 510 or less, at least about 520 or less, or at least about 525 or less.

[0095] In some embodiments, the deletion of multiple amino acid residues to generate the amino acid sequence of a recombinant nuclease disclosed herein may comprise the deletion of multiple consecutive amino acid residues, for example, compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from Table 2 (if applicable). In some cases, the deletion of multiple contiguous amino acid residues can include, for example, a deletion of at least about 2 or no more than about 2 contiguous amino acid residues, a deletion of at least about 3 or no more than about 3 contiguous amino acid residues, a deletion of at least about 4 or no more than about 4 contiguous amino acid residues, a deletion of at least about 5 or no more than about 5 contiguous amino acid residues, a deletion of at least about 6 or no more than about 6 contiguous amino acid residues, a deletion of at least about 7 or no more than about 7 contiguous amino acid residues, a deletion of at least about 8 or no more than about 8 contiguous amino acid residues, a deletion of at least about 9 or no more than about 9 contiguous amino acid residues, a deletion of at least about 10 or no more than about 10 contiguous amino acid residues, a deletion of at least about 11 or no more than about 11 contiguous amino acid residues, or a deletion of at least about 11 or no more than about 11 contiguous amino acid residues, compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from Table 2 (if applicable). deletion of at least about 12 or less consecutive amino acid residues, deletion of at least about 13 or less consecutive amino acid residues, deletion of at least about 14 or less consecutive amino acid residues, deletion of at least about 15 or less consecutive amino acid residues, deletion of at least about 16 or less consecutive amino acid residues, deletion of at least about 17 or less consecutive amino acid residues, deletion of at least about 18 or less consecutive amino acid residues, deletion of at least about 19 or less consecutive amino acid residues, deletion of at least about 20 or less consecutive amino acid residues, deletion of at least about 21 or less consecutive amino acid residues, deletion of at least about 22 or less consecutive amino acid residues, deletion of at least about 23 or less consecutive amino acid residues,Deletion of at least about 24 or less contiguous amino acid residues, deletion of at least about 25 or less contiguous amino acid residues, deletion of at least about 26 or less contiguous amino acid residues, deletion of at least about 27 or less contiguous amino acid residues, deletion of at least about 28 or less contiguous amino acid residues, deletion of at least about 29 or less contiguous amino acid residues, deletion of at least about 30 or less contiguous amino acid residues, deletion of at least about 31 or less contiguous amino acid residues The deletion of at least about 31 or less contiguous amino acid residues is selected from the group consisting of deletions of at least about 32 or less contiguous amino acid residues, deletions of at least about 34 or less contiguous amino acid residues, deletions of at least about 35 or less contiguous amino acid residues, deletions of at least about 36 or less contiguous amino acid residues, deletions of at least about 37 or less contiguous amino acid residues, deletions of at least about 38 or less contiguous amino acid residues, deletions of at least about 39 or less contiguous amino acid residues, Deletion of amino acid residues, deletion of at least about 40 or less contiguous amino acid residues, deletion of at least about 45 or less contiguous amino acid residues, deletion of at least about 50 or less contiguous amino acid residues, deletion of at least about 55 or less contiguous amino acid residues, deletion of at least about 60 or less contiguous amino acid residues, deletion of at least about 65 or less contiguous amino acid residues, deletion of at least about 70 or less contiguous amino acid residues, deletion of at least The deletion may include a deletion of at least about 75 or less contiguous amino acid residues, a deletion of at least about 80 or less contiguous amino acid residues, a deletion of at least about 85 or less contiguous amino acid residues, a deletion of at least about 80 or less contiguous amino acid residues, a deletion of at least about 90 or less contiguous amino acid residues, a deletion of at least about 95 or less contiguous amino acid residues, or a deletion of at least about 100 or less contiguous amino acid residues.

[0096] For example, the deletion may be a single deletion of multiple contiguous amino acid residues. In another example, the deletion may include a first deletion of a first plurality of contiguous amino acid residues and a second deletion of a second plurality of contiguous amino acid residues when aligned with (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from Table 2 (if applicable), where the deletion of the first plurality of contiguous amino acid residues and the deletion of the second plurality of contiguous amino acid residues are not necessarily contiguous (e.g., not adjacent to each other).

[0097] In some embodiments, the amino acid sequence of a recombinant nuclease disclosed herein may comprise the addition of one or more heterologous amino acid residues (e.g., one or more polypeptide sequences) compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from Table 2 (if applicable). The one or more heterologous amino acid residues may be present at the position of the at least one deletion in the recombinant nuclease. Additionally or alternatively, the one or more heterologous amino acid residues may not be present at the position of the at least one deletion in the recombinant nuclease. For example, the one or more heterologous amino acid residues may be present upstream and / or downstream of the position of the at least one deletion in the recombinant nuclease.

[0098] In some embodiments, the one or more heterologous amino acid residues of the recombinant nucleases disclosed herein may be a single amino acid residue. In some embodiments, the one or more heterologous amino acid residues of the recombinant nucleases disclosed herein can be a plurality of amino acid residues, for example, at least about 2 or less amino acid residues, at least about 3 or less amino acid residues, at least about 4 or less amino acid residues, at least about 5 or less amino acid residues, at least about 6 or less amino acid residues, at least about 7 or less amino acid residues, at least about 8 or less amino acid residues, at least about 9 or less amino acid residues, at least about 10 or less amino acid residues, at least about 11 or less amino acid residues, at least about 12 or less amino acid residues, at least about 13 or less amino acid residues, at least about 14 or less amino acid residues, at least about 15 or less amino acid residues, or at least about 16 or less amino acid residues. a group having at least about 17 or less amino acid residues, at least about 18 or less amino acid residues, at least about 19 or less amino acid residues, at least about 20 or less amino acid residues, at least about 22 or less amino acid residues, at least about 24 or less amino acid residues, at least about 25 or less amino acid residues, at least about 26 or less amino acid residues, at least about 28 or less amino acid residues, at least about 30 or less amino acid residues, at least about 32 or less amino acid residues, at least about 34 or less amino acid residues, at least about 35 or less amino acid residues, at least about 36 or less amino acid residues, at least about 38 or less amino acid residues, at least about 40 or less amino acid residues, at least about 42 or less amino acid residues,At least about 44 or less amino acid residues, at least about 45 or less amino acid residues, at least about 46 or less amino acid residues, at least about 48 or less amino acid residues, at least about 50 or less amino acid residues, at least about 52 or less amino acid residues, at least about 54 or less amino acid residues, at least about 55 or less amino acid residues, at least about 56 or less amino acid residues acid residues, at least about 58 or less amino acid residues, at least about 60 or less amino acid residues, at least about 62 or less amino acid residues, at least about 64 or less amino acid residues, at least about 65 or less amino acid residues, at least about 66 or less amino acid residues, at least about 68 or less amino acid residues, at least about 70 or less amino acid residues, at least about 72 or less amino acid residues, at least about 74 or less amino acid residues, at least about 75 or less amino acid residues, at least about 76 or less amino acid residues, at least about 78 or less amino acid residues, at least about 80 or less amino acid residues, at least about 82 or less amino acid residues, at least about 84 or less amino acid residues, at least about 85 or less amino acid residues, at least about 86 or less amino acid residues It may be 6 or fewer amino acid residues, at least about 88 or fewer amino acid residues, at least about 90 or fewer amino acid residues, at least about 92 or fewer amino acid residues, at least about 94 or fewer amino acid residues, at least about 95 or fewer amino acid residues, at least about 96 or fewer amino acid residues, at least about 98 or fewer amino acid residues, or at least about 100 or fewer amino acid residues.

[0099] In some embodiments, the one or more heterologous amino acid residues may comprise a plurality of contiguous amino acid residues, or may comprise a plurality of non-contiguous amino acid residues.

[0100] In some embodiments, the one or more heterologous amino acid residues of a recombinant nuclease disclosed herein can be a heterologous polypeptide sequence (e.g., a polypeptide sequence heterologous to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one of the Cas proteins selected from Table 2, if applicable). The heterologous polypeptide sequence can exhibit a positive overall charge (e.g., a total charge of +1, +2, +3, +4, +5, etc., when measured in a buffer at a pH of about 7.4). The heterologous polypeptide sequence can exhibit a negative overall charge (e.g., a total charge of −1, −2, −3, −4, −5, etc., when measured in a buffer at a pH of about 7.4). The heterologous polypeptide sequence can exhibit a neutral charge (e.g., a total charge of −1, −2, −3, −4, −5, etc., when measured in a buffer at a pH of about 7.4). In some cases, the heterologous polypeptide sequence may comprise an amino acid sequence having at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% identity to the polypeptide sequence of SEQ ID NO:11.

[0101] SEQ ID NO: 11 (heterologous polypeptide) JPEG2025507822000006.jpg7145

[0102] In some embodiments, the amino acid sequence of a recombinant nuclease disclosed herein may include (i) a deletion of one or more amino acid residues from amino acid residues 2 to 100 relative to the polypeptide sequence of SEQ ID NO:1, and / or (ii) a deletion of one or more amino acid residues from amino acid residues 430 to 529 relative to the polypeptide sequence of SEQ ID NO:1, and may further include a deletion of one or more additional amino acid residues from amino acid residues 101 to 429 disclosed herein relative to the polypeptide sequence of SEQ ID NO:1 (e.g., a deletion of at least 1, 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, at least 15, at least 20, or more additional amino acid residues).

[0103] In some embodiments, the amino acid sequence of a recombinant nuclease disclosed herein may include (i) a deletion of one or more amino acid residues from amino acid residues 2 to 100 relative to the polypeptide sequence of any one of the Cas proteins selected from Table 2, and / or (ii) a deletion of one or more amino acid residues from the last 100 C-terminal amino acid residues relative to the polypeptide sequence of any one of the Cas proteins selected from Table 2, and may further include a deletion of one or more additional amino acid residues (e.g., a deletion of at least 1, 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, at least 15, at least 20, or more additional amino acid residues) between the deletions in (i) and (ii) relative to the polypeptide sequence of any one of the Cas proteins selected from Table 2.

[0104] In some embodiments, the recombinant polypeptides disclosed herein can include one or more heterologous amino acid residues (e.g., other than mutations) compared to the native Un1Cas12fl nuclease provided in SEQ ID NO: 1. The presence of one or more heterologous amino acid residues can, for example, enhance the activity, stability, expression, binding to a corresponding guide nucleic acid molecule, etc. of the recombinant polypeptide.

[0105] In some embodiments, the recombinant polypeptides disclosed herein (e.g., inactive Cas nuclease variants) have a sequence identity that is at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 65% or less, at least about 70% or less, at least about 75% or less, at least about 80% or less, or at least about 85% or more identical to the polypeptide sequence of SEQ ID NO: 12. The amino acid sequence may comprise an amino acid sequence having at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% identity.

[0106] SEQ ID NO: 12 (example of a recombinant nuclease polypeptide) JPEG2025507822000007.jpg67145

[0107] In some embodiments, the recombinant polypeptides disclosed herein are at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 65% or less, at least about 70% or less, at least about 75% or less, to the polypeptide sequence of a recombinant nuclease variant selected from Table 3B (e.g., any one or more of SEQ ID NOs:20-27), Table 4B (e.g., any one or more of SEQ ID NOs:28-111), and / or Table 5C (e.g., any one or more of SEQ ID NOs:112-201). % or less, at least about 80% or less, at least about 85% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or about 100% identity to the amino acid sequence.

[0108] In some embodiments, the recombinant polypeptides disclosed herein may not be identical to any one of the Cas proteins listed in SEQ ID NO: 1 and Table 2 (SEQ ID NOs: 2, 3, and 13-19).

[0109] In some embodiments, the recombinant polypeptides disclosed herein may contain at least one amino acid modification compared to the polypeptide sequence of SEQ ID NO:1 or SEQ ID NO:10. The at least one amino acid modification may include one or more members selected from the group consisting of A21Q, V23I, N32E, D29E, N33R, E35K, K36Q, I37A, A38G, E40D, K73G, A74T, R75G, K76E, Q83K, G87K, E151A, A340S, H353K, A374K, I387E, N423D, K473Q, T474L, T474R, H497K, L515R, N519T, K521D, K521N, and L522I, as compared to the polypeptide sequence of SEQ ID NO:1 or SEQ ID NO:10 disclosed herein, and one or more amino acid residue deletions (e.g., at least one deletion from amino acid residues 400 to 529 of SEQ ID NO:1 or SEQ ID NO:10).The one or more members may include any of the following amino acids as compared to the polypeptide sequence of SEQ ID NO:1 or SEQ ID NO:10: A21Q, V23I, N32E, D29E, N33R, E35K, K36Q, I37A, A38G, E40D, K73G, A74T, R75G, K76E, Q83K, G87K, E151A, A340S, H353K, A374K, I387E, N423D, K473Q, T474L, T474R at least about one or less amino acid modifications selected from the group consisting of H497K, L515R, N519T, K521D, K521N, and L522I; at least about two or less amino acid modifications; at least about three or less amino acid modifications; at least about four or less amino acid modifications; at least about five or less amino acid modifications; The amino acid sequence may include at least about 6 or less amino acid modifications, at least about 7 or less amino acid modifications, at least about 8 or less amino acid modifications, at least about 9 or less amino acid modifications, at least about 10 or less amino acid modifications, at least about 11 or less amino acid modifications, at least about 12 or less amino acid modifications, at least about 13 or less amino acid modifications, at least about 14 or less amino acid modifications, at least about 15 or less amino acid modifications, at least about 20 or less amino acid modifications, at least about 25 or less amino acid modifications, or at least about 30 or less amino acid modifications. Optionally, the at least one amino acid modification may include at least one set of modifications selected from Table 5A. Optionally, the at least one amino acid modification may include at least one set of modifications selected from Table 5B. For example, at least one set of alterations selected from Table 5B may not be cA2.55 or cA2.84.

[0110] In some embodiments, a recombinant polypeptide disclosed herein may comprise at least one amino acid modification compared to the polypeptide sequence of SEQ ID NO: 1 or SEQ ID NO: 10. The at least one amino acid modification may comprise one or more members (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more members) selected from the group consisting of A21Q, V23I, D29E, N33R, E40D, Q83K, G87K, E151A, A340S, H353K, A374K, I387E, N423D, K473Q, T474L, T474R, H497K, L515R, N519T, K521D, K521N, and L522I.

[0111] In some embodiments, a recombinant polypeptide disclosed herein may comprise at least one amino acid modification compared to the polypeptide sequence of SEQ ID NO: 1 or SEQ ID NO: 10. The at least one amino acid modification may comprise one or more members (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 members) selected from the group consisting of N32E, N33R, E35K, K36Q, I37A, A38G, K73G, A74T, R75G, and K76E.

[0112] In some embodiments, a recombinant nuclease variant of a recombinant polypeptide disclosed herein may be a chimeric polypeptide comprising multiple polynucleotide sequence domains of different types derived from multiple Cas proteins of different types. The chimeric polypeptide may comprise a first polypeptide sequence comprising at least three consecutive amino acid residues in common with a first Cas protein and a second polypeptide sequence comprising at least three consecutive amino acid residues in common with a second Cas protein, where the second Cas protein is distinct from the first Cas protein. The first and second Cas proteins may be naturally occurring Cas proteins of different types. The first and second Cas proteins may differ in size (i.e., length of amino acid sequence) from one another, and the difference may be 500 amino acid residues or less, 400 amino acid residues or less, 350 amino acid residues or less, 300 amino acid residues or less, 250 amino acid residues or less, 200 amino acid residues or less, 150 amino acid residues or less, 140 amino acid residues or less, 130 amino acid residues or less, 120 amino acid residues or less, 110 amino acid residues or less, 100 amino acid residues or less, 90 amino acid residues or less, 80 amino acid residues or less, 70 amino acid residues or less, 60 amino acid residues or less, 50 amino acid residues or less, 40 amino acid residues or less, 30 amino acid residues or less, or 20 amino acid residues or less.

[0113] In some cases, the length of the first polypeptide sequence may be substantially the same as the length of the second polypeptide sequence. Alternatively, the length of the first polypeptide sequence may be different from the length of the second polypeptide sequence. The length of the second polypeptide sequence may be about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 18% or less, about 16% or less, about 15% or less, about 14% or less, about 12% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less of the length of the first polypeptide sequence. The length of the second polypeptide sequence is at least about 1% or less, at least about 2% or less, at least about 3% or less, at least about 4% or less, at least about 5% or less, at least about 6% or less, at least about 7% or less, at least about 8% or less, at least about 9% or less, at least about 10% or less, at least about 12% or less, at least about 14% or less, at least about 15% or less, at least about 16% or less of the length of the first polypeptide sequence. or less, at least about 18% or less, at least about 20% or less, at least about 25% or less, at least about 30% or less, at least about 35% or less, at least about 40% or less, at least about 45% or less, at least about 50% or less, at least about 55% or less, at least about 60% or less, at least about 70% or less, at least about 80% or less, at least about 90% or less, or at least about 95% or less.

[0114] In some cases, the first Cas protein and the second Cas protein may be two members selected from the Cas proteins set forth in SEQ ID NO: 1 and Table 2 (SEQ ID NOs: 2, 3, and 13-19). The first Cas protein or the second Cas protein may have at least about 5% or less identity, at least about 10% or less identity, at least about 15% or less identity, at least about 20% or less identity, at least about 30% or less identity, at least about 40% or less identity, at least about 50% or less identity, at least about 60% or less identity, at least about 70% or less identity, or at least about 80% or less identity to the polypeptide sequence of a member selected from the Cas proteins set forth in SEQ ID NO: 1 and Table 2 (SEQ ID NOs: 2, 3, and 13-19). or may include an amino acid sequence having at least about 70% identity, at least about 75% or less identity, at least about 80% or less identity, at least about 85% or less identity, at least about 90% or less identity, at least about 92% or less identity, at least about 95% or less identity, at least about 96% or less identity, at least about 97% or less identity, at least about 98% or less identity, at least about 99% or less identity, or substantially about 100% identity. For example, such a member may be SEQ ID NO: 1 or SEQ ID NO: 2.

[0115] In some cases, the first polypeptide of the chimeric polypeptide of the recombinant nuclease variant disclosed herein shares at least about 4 or no more than about 4 contiguous amino acid residues, at least about 5 or no more than about 5 contiguous amino acid residues, at least about 6 or no more than about 6 contiguous amino acid residues, at least about 7 or no more than about 7 contiguous amino acid residues, at least about 8 or no more than about 8 contiguous amino acid residues, at least about 9 or no more than about 9 contiguous amino acid residues, at least about 10 or no more than about 10 contiguous amino acid residues, at least about 11 or no more than about 11 contiguous amino acid residues, at least about 12 or no more than about 12 contiguous amino acid residues, at least about 13 or no more than about 13 contiguous amino acid residues, at least about 14 or no more than about 14 contiguous amino acid residues, at least about 15 or no more than about 15 contiguous amino acid residues, at least about 16 or no more than about 16 contiguous amino acid residues, at least about 17 or no more than about 17 contiguous amino acid residues, at least about 18 or no more than about 18 contiguous amino acid residues, at least about 19 or no more than about 20 contiguous amino acid residues, at least about 21 or no more than about 22 contiguous amino acid residues, at least about 23 or no more than about 24 contiguous amino acid residues, at least about 25 or no more than about 26 contiguous amino acid residues, at least about 26 or no more than about 27 contiguous amino acid residues, at least about 27 or no more than about 28 contiguous amino acid residues, at least about 28 or no more than about 29 contig It may contain amino acid residues, at least about 12 or less contiguous amino acid residues, at least about 13 or less contiguous amino acid residues, at least about 14 or less contiguous amino acid residues, at least about 15 or less contiguous amino acid residues, at least about 18 or less contiguous amino acid residues, at least about 20 or less contiguous amino acid residues, at least about 30 or less contiguous amino acid residues, at least about 40 or less contiguous amino acid residues, or at least about 50 or less contiguous amino acid residues.

[0116] In some cases, the second polypeptide of the recombinant nuclease variant chimeric polypeptide disclosed herein has at least about 4 or no more than about 4 contiguous amino acid residues, at least about 5 or no more than about 5 contiguous amino acid residues, at least about 6 or no more than about 6 contiguous amino acid residues, at least about 7 or no more than about 7 contiguous amino acid residues, at least about 8 or no more than about 8 contiguous amino acid residues, at least about 9 or no more than about 9 contiguous amino acid residues, at least about 10 or no more than about 10 contiguous amino acid residues, at least about 11 or no more than about 1 ... It may contain amino acid residues, at least about 12 or less contiguous amino acid residues, at least about 13 or less contiguous amino acid residues, at least about 14 or less contiguous amino acid residues, at least about 15 or less contiguous amino acid residues, at least about 18 or less contiguous amino acid residues, at least about 20 or less contiguous amino acid residues, at least about 30 or less contiguous amino acid residues, at least about 40 or less contiguous amino acid residues, or at least about 50 or less contiguous amino acid residues.

[0117] In some cases, in the second polypeptide of the recombinant nuclease variant chimeric polypeptide disclosed herein, the length of the first polypeptide may be longer than the length of the second polypeptide, the difference being at least or not more than about 1 amino acid, at least or not more than about 2 amino acids, at least or not more than about 5 amino acids, at least or not more than about 10 amino acids, at least or not more than about 15 amino acids, at least or not more than about 20 amino acids, at least or not more than about 25 amino acids, at least or not more than about 30 amino acids, at least or not more than about 35 amino acids, at least or not more than about 40 amino acids, at least or not more than about 45 amino acids, at least or not more than about 50 amino acids, at least or not more than about 55 amino acids, at least or not more than about 60 amino acids, at least about 70 amino acids or less in length, at least about 80 amino acids or less in length, at least about 90 amino acids or less in length, at least about 100 amino acids or less in length, at least about 110 amino acids or less in length, at least about 120 amino acids or less in length, at least about 130 amino acids or less in length, at least about 140 amino acids or less in length, at least about 15 The length may be 0 or less than about 150 amino acids, at least or less than about 200 amino acids, at least or less than about 250 amino acids, at least or less than about 300 amino acids, at least or less than about 350 amino acids, at least or less than about 400 amino acids, at least or less than about 450 amino acids, or at least or less than about 500 amino acids.

[0118] In some cases, the first polypeptide may be derived from the N-terminal 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the first Cas protein. Additionally or alternatively, the first polypeptide may be derived from the C-terminal 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the first Cas protein. In some cases, the second polypeptide may be derived from the N-terminal 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the second Cas protein. Additionally or alternatively, the second polypeptide may be derived from the C-terminal 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the second Cas protein.

[0119] In some cases, the first polypeptide may be derived from the first 5 amino acid residues, the first 10 amino acid residues, the first 15 amino acid residues, the first 20 amino acid residues, the first 30 amino acid residues, the first 40 amino acid residues, the first 50 amino acid residues, the first 60 amino acid residues, the first 70 amino acid residues, the first 80 amino acid residues, the first 90 amino acid residues, the first 100 amino acid residues, the first 150 amino acid residues, the first 200 amino acid residues, the first 250 amino acid residues, or the first 300 amino acid residues from the N-terminus of the first Cas protein. Additionally or alternatively, the first polypeptide may be derived from the first 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or 300 amino acid residues of the C-terminus of the first Cas protein.

[0120] In some cases, the second polypeptide may be derived from the first 5 amino acid residues, the first 10 amino acid residues, the first 15 amino acid residues, the first 20 amino acid residues, the first 30 amino acid residues, the first 40 amino acid residues, the first 50 amino acid residues, the first 60 amino acid residues, the first 70 amino acid residues, the first 80 amino acid residues, the first 90 amino acid residues, the first 100 amino acid residues, the first 150 amino acid residues, the first 200 amino acid residues, the first 250 amino acid residues, or the first 300 amino acid residues of the N-terminus of the second Cas protein. Additionally or alternatively, the second polypeptide may be derived from the first 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or 300 amino acid residues of the C-terminus of the second Cas protein.

[0121] In some cases, a recombinant nuclease variant of a recombinant polypeptide disclosed herein can include a third polypeptide sequence that includes at least three consecutive amino acid residues (or more consecutive amino acid residues, as disclosed herein) in common with the first Cas protein. In such cases, the first and third polypeptide sequences can be contiguous or non-contiguous within the chimeric polypeptide. Additionally or alternatively, the third polypeptide sequence can include at least three consecutive amino acid residues (or more consecutive amino acid residues, as disclosed herein) in common with the second Cas protein. The first and third polypeptide sequences can be non-contiguous within the chimeric polypeptide. Alternatively, the first and third polypeptide sequences can be contiguous within the chimeric polypeptide. The second and third polypeptide sequences can be non-contiguous within the chimeric polypeptide. Alternatively, the second and third polypeptide sequences can be contiguous within the chimeric polypeptide. In yet another embodiment, or in addition, the third polypeptide sequence can include at least three consecutive amino acid residues in common with a third Cas protein that is different from the first Cas protein and the second Cas protein. For example, the first Cas protein, the second Cas protein, and the third Cas protein can be three members selected from the Cas proteins set forth in SEQ ID NO: 1 and Table 2.

[0122] In some cases, the third polypeptide of the recombinant nuclease variant chimeric polypeptide shares at least about 4 or less contiguous amino acid residues, at least about 5 or less contiguous amino acid residues, at least about 6 or less contiguous amino acid residues, at least about 7 or less contiguous amino acid residues, at least about 8 or less contiguous amino acid residues, at least about 9 or less contiguous amino acid residues, at least about 10 or less contiguous amino acid residues, or at least about 11 or less contiguous amino acid residues with the third Cas protein. The group may comprise at least about 12 or less contiguous amino acid residues, at least about 13 or less contiguous amino acid residues, at least about 14 or less contiguous amino acid residues, at least about 15 or less contiguous amino acid residues, at least about 18 or less contiguous amino acid residues, at least about 20 or less contiguous amino acid residues, at least about 30 or less contiguous amino acid residues, at least about 40 or less contiguous amino acid residues, or at least about 50 or less contiguous amino acid residues.

[0123] In some cases, the third polypeptide may be derived from the first 5 amino acid residues, the first 10 amino acid residues, the first 15 amino acid residues, the first 20 amino acid residues, the first 30 amino acid residues, the first 40 amino acid residues, the first 50 amino acid residues, the first 60 amino acid residues, the first 70 amino acid residues, the first 80 amino acid residues, the first 90 amino acid residues, the first 100 amino acid residues, the first 150 amino acid residues, the first 200 amino acid residues, the first 250 amino acid residues, or the first 300 amino acid residues of the N-terminus of the third Cas protein. Additionally or alternatively, the third polypeptide may be derived from the first 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or 300 amino acid residues of the C-terminus of the third Cas protein.

[0124] In some cases, within the chimeric polypeptide of a recombinant nuclease variant disclosed herein, the first polypeptide sequence, the second polypeptide sequence, and the third polypeptide sequence can be arranged from the N-terminus to the C-terminus of the chimeric polypeptide in a structure designated CP1-CP2-CPx(I), CP1-CPx-CP2(II), CP2-CP1-CPx(III), CP2-CPx-CP1(IV), CPx-CP1-CP2(V), or CPx-CP2-CP1(VI), where "-" can be an amino acid linker (e.g., a linker comprising one or more amino acid sequences) or a direct covalent bond. The amino acid linkers disclosed herein may comprise one amino acid, at least about two or no more than two amino acids, at least about three or no more than three amino acids, at least about four or no more than four amino acids, at least about five or no more than five amino acids, at least about eight or no more than eight amino acids, at least about ten or no more than ten amino acids, at least about 12 or no more than twelve amino acids, at least about 15 or no more than fifteen amino acids, at least about 16 or no more than sixteen amino acids, or at least about 20 or no more than twenty amino acids. The amino acid linker may comprise at least one glycine, at least one serine, or at least one glycine-serine dipeptide.

[0125] In some embodiments, the amino acid sequence of a recombinant nuclease disclosed herein (e.g., a chimeric polypeptide disclosed herein) is up to 528 amino acids in length, up to 527 amino acids in length, up to 526 amino acids in length, up to 525 amino acids in length, up to 524 amino acids in length, up to 523 amino acids in length, up to 522 amino acids in length, up to 521 amino acids in length, up to 520 amino acids in length, up to 519 amino acids in length, up to 518 amino acids in length, up to 517 amino acids in length, up to 516 amino acids in length, up to 515 amino acids in length, up to 518 ... 514 amino acids long, up to 513 amino acids long, up to 512 amino acids long, up to 511 amino acids long, up to 510 amino acids long, up to 509 amino acids long, up to 508 amino acids long, up to 507 amino acids long, up to 506 amino acids long, up to 505 amino acids long, up to 504 amino acids long, up to 503 amino acids long, up to 502 amino acids long, up to 501 amino acids long, up to about 500 amino acids long, up to about 495 amino acids long, up to about 490 amino acids long, up to about 485 amino acids long, up to about 480 amino acids long, up to about 475 Amino acid length, up to about 470 amino acids, up to about 465 amino acids, up to about 460 amino acids, up to about 455 amino acids, up to about 450 amino acids, up to about 445 amino acids, up to about 440 amino acids, up to about 435 amino acids, up to about 430 amino acids, up to about 425 amino acids, up to about 420 amino acids, up to about 415 amino acids, up to about 410 amino acids, up to about 405 amino acids, up to about 400 amino acids, up to about 395 amino acids, up to about 390 amino acids, up to about 385 amino acids The amino acid sequence may be up to about 380 amino acids in length, up to about 375 amino acids in length, up to about 370 amino acids in length, up to about 365 amino acids in length, up to about 360 amino acids in length, up to about 355 amino acids in length, up to about 350 amino acids in length, up to about 345 amino acids in length, up to about 340 amino acids in length, up to about 335 amino acids in length, up to about 330 amino acids in length, up to about 325 amino acids in length, up to about 320 amino acids in length, up to about 315 amino acids in length, up to about 310 amino acids in length, up to about 305 amino acids in length, or up to about 300 amino acids in length.

[0126] In some embodiments, the recombinant nuclease comprising an amino acid sequence disclosed herein is up to about 1000 amino acids in length, up to about 950 amino acids in length, up to about 900 amino acids in length, up to about 850 amino acids in length, up to about 800 amino acids in length, up to about 750 amino acids in length, up to about 700 amino acids in length, up to about 650 amino acids in length, up to about 640 amino acids in length, up to about 630 amino acids in length, up to about 620 amino acids in length, up to about 610 amino acids in length, up to about 600 amino acids in length, up to about 590 amino acids in length, up to about 580 amino acids in length, up to about 570 amino acids in length, up to about It may have a length of 560 amino acids, up to about 550 amino acids, up to about 540 amino acids, up to about 530 amino acids, up to about 520 amino acids, up to about 510 amino acids, up to about 500 amino acids, up to about 490 amino acids, up to about 480 amino acids, up to about 470 amino acids, up to about 460 amino acids, up to about 450 amino acids, up to about 440 amino acids, up to about 430 amino acids, up to about 420 amino acids, up to about 410 amino acids, up to about 400 amino acids, up to about 350 amino acids, or up to about 300 amino acids.

[0127] In some embodiments, at least a portion of the recombinant nuclease variants disclosed herein may be derived from (e.g., obtained by recombining) a naturally occurring Cas protein (e.g., a first, second, or third Cas protein described herein). In some cases, the naturally occurring Cas protein may have a length of up to about 800 amino acids, up to about 750 amino acids, up to about 700 amino acids, up to about 650 amino acids, up to about 600 amino acids, up to about 550 amino acids, up to about 540 amino acids, up to about 530 amino acids, up to about 510 amino acids, up to about 500 amino acids, up to about 490 amino acids, up to about 480 amino acids, up to about 470 amino acids, up to about 460 amino acids, up to about 450 amino acids, or up to about 400 amino acids. The naturally occurring Cas protein may be, for example, a member selected from the Cas proteins set forth in SEQ ID NO: 1 and Table 2.

[0128] In some embodiments, the first polypeptide sequence, the second polypeptide sequence, and / or the third polypeptide sequence of the recombinant nuclease variant chimeric polypeptides disclosed herein may not be derived from Cas12a. In some embodiments, the recombinant nuclease variant chimeric polypeptides disclosed herein may not be derived from Cas12a. In some embodiments, the recombinant nuclease variant chimeric polypeptides disclosed herein may be derived entirely from one or more Cas12f-type orthologs (e.g., a Cas protein selected from Table 2 or SEQ ID NO: 1).

[0129] In some embodiments, recombinant nucleases comprising the amino acid sequences disclosed herein can be mutated and / or modified to provide proteins deficient in nuclease activity or proteins with reduced nuclease activity compared to wild-type Cas proteins. Nuclease-defective proteins can retain the ability to bind to target genes (e.g., DNA) but may lack or have reduced nucleic acid cleavage activity. In some embodiments, recombinant nucleases comprising the amino acid sequences disclosed herein can have reduced nuclease activity (e.g., nuclease-defective or nuclease null) compared to the Cas nuclease encoded by SEQ ID NO: 1 or a Cas protein selected from Table 2. The reduced nuclease activity may be up to about 95% nuclease activity, up to about 90% nuclease activity, up to about 80% nuclease activity, up to about 70% nuclease activity, up to about 60% nuclease activity, up to about 50% nuclease activity, up to about 40% nuclease activity, up to about 30% nuclease activity, up to about 20% nuclease activity, up to about 10% nuclease activity, up to about 5% nuclease activity, up to about 1% nuclease activity, up to about 0.5% nuclease activity, up to about 0.1% nuclease activity, or less nuclease activity relative to the nuclease activity of the Cas nuclease encoded by SEQ ID NO:1 or the nuclease activity of a Cas protein selected from Table 2. In some cases, the recombinant nuclease may include a substitution at D326 and / or D510 compared to the polypeptide sequence of SEQ ID NO: 1. For example, the substitution at D326 and / or D510 may be a substitution to alanine (e.g., D326A and / or D510A).

[0130] In some embodiments, the amino acid sequence of a recombinant nuclease disclosed herein may contain one or more substitutions in a native amino acid sequence, at least some of which follow one or more specific rules determined to provide surprising advantages to the recombinant nuclease. In some cases, the specific substitution rules are selected to, for example, generate recombinant nuclease variants capable of functioning in eukaryotic cells. According to these specific rules, all or part of the one or more substitutions in the native amino acid sequence may be: (1) substitutions within or not exceeding 30 amino acid residues downstream of the (D / E / K / N)X(R / F)(E / K)N motif of the native amino acid sequence; (2) substitutions within or not exceeding 30 amino acid residues upstream or downstream of position 241 of the native amino acid sequence; (3) substitutions within or not exceeding 30 amino acid residues upstream or downstream of position 516 of the native amino acid sequence; and / or (4) substitutions of charged amino acids in the native amino acid sequence.

[0131] In some embodiments, the amino acid sequence of a recombinant nuclease disclosed herein may contain one or more substitutions at amino acid positions within or below a threshold length (e.g., 30 amino acid residues in length) upstream and / or downstream of the (D / E / K / N)X(R / F)(E / K)N motif compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the amino acid sequence of a recombinant nuclease disclosed herein that does not have one or more substitutions. Optionally, at least one of the one or more substitutions may be, for example, within 28 or less, 26 or less, 24 or less, 22 or less, 20 or less, 18 or less, 16 or less, 14 or less, 12 or less, or 10 or less amino acid residues upstream and / or downstream of the motif. Optionally, at least one of the one or more substitutions may be with an R, A, S, or G amino acid residue. In some cases, the one or more substitutions may include a substitution at one or more positions selected from the group consisting of D143, T147, E151, and K154 (e.g., D143R, T147R, E151R, and / or K154R). In some cases, the one or more substitutions may include a substitution at one or more positions selected from the group consisting of N504, E507, N516, N519, E527, and E528 (e.g., N504R, E507R, N516R, N519R, E527R, and / or E528R).In some cases, the one or more substitutions may include a substitution at one or more positions selected from the group consisting of K11, K73, D143, T147, E151, K154, E241, D318, K330, K457, E425, E462, N504, E507, N516, N519, E527 and E528 (e.g., K11R, K73R, D143R, T147R, E151R, K154R, E241R, D318R, K330R, E425N, K457R, E462R, N504R, E507R, N516R, N519R, E527R and / or E528R).

[0132] In some embodiments, the amino acid sequence of a recombinant nuclease comprising one or more substitutions upstream and / or downstream of a (D / E / K / N)X(R / F)(E / K)N motif disclosed herein may exhibit a higher cationic charge (e.g., positive charge) than a control amino acid sequence of a recombinant nuclease that does not have said one or more substitutions, the difference being at least about monovalent or less than monovalent cationic charge, at least about divalent or less than divalent cationic charge, at least about trivalent or less than trivalent cationic charge, at least about tetravalent or less than divalent cationic charge, at least about pentavalent or less than divalent cationic charge, at least about hexavalent or less than divalent cationic charge, at least about heptavalent or less than divalent cationic charge, at least about hexavalent or less than divalent cationic charge, at least about 7 or less than divalent cationic charge, at least about 8 or less than divalent cationic charge, at least about 9 or less than divalent cationic charge, at least about 10 or less than divalent cationic charge, at least about 11 or less than divalent cationic charge, at least about 12 or less than divalent cationic charge, at least about 13 or less than divalent cationic charge, at least about 14 or less than divalent cationic charge, at least about 15 or less than divalent cationic charge, at least about 16 or less than divalent cationic charge, at least about 17 or less than divalent cationic charge, at least about 18 or less than divalent cationic charge, at least about 19 or less than divalent cationic charge, at least about 20 or less than divalent cationic charge, at least about 21 or less than divalent cationic charge, at least about 22 or less than divalent cationic charge, at least about 23 or less than divalent cationic charge, at least about 24 or less than divalent cationic charge, at least about 25 or less than divalent cationic charge, at least about 26 or less than divalent cationic charge may be a cationic charge of at least about 7 or less, a cationic charge of at least about 8 or less, a cationic charge of at least about 9 or less, a cationic charge of at least about 10 or less, a cationic charge of at least about 11 or less, a cationic charge of at least about 12 or less, a cationic charge of at least about 13 or less, a cationic charge of at least about 14 or less, a cationic charge of at least about 15 or less, a cationic charge of at least about 16 or less, a cationic charge of at least about 17 or less, or a cationic charge of at least about 18 or less.

[0133] Without wishing to be bound by any theory, the amino acid sequence of a recombinant nuclease comprising one or more substitutions upstream and / or downstream of the (D / E / K / N)X(R / F)(E / K)N motif disclosed herein may exhibit enhanced binding affinity (e.g., higher binding affinity) to (i) a guide nucleic acid sequence (e.g., a guide RNA sequence) and / or (ii) a target polynucleotide sequence of the Cas / guide nucleic acid complex (e.g., a target gene in a cell, e.g., an endogenous target gene) as determined by surface plasmon resonance (SPR) or isothermal titration calorimetry (IRC) assays, compared to (A) a control amino acid sequence of a recombinant nuclease not having the one or more substitutions, and / or (B) a CRISPR / Cas protein encoded by SEQ ID NO: 1, where the difference in binding affinity is at or about 1% or less, at or about 5% or less, at or about 10% or less, at or about 15% or less, at or about 2% or less, or at least about 2% or less. 0% or less than about 20%, at least about 25% or less than about 25%, at least about 30% or less than about 30%, at least about 40% or less than about 40%, at least about 50% or less than about 50%, at least about 60% or less than about 60%, at least about 70% or less than about 70%, at least about 80% or less than about 80%, at least about 90% or less than about 90%, at least about 100% or less than about 100%, at least about 110% or less than about It may be at or below 120%, at least at or below about 125%, at least at or below about 150%, at or below about 150%, at least at or below about 200%, at or below about 250%, at or below about 250%, at or below about 300%, at or below about 350%, at or below about 400%, at or below about 450%, or at or below about 500%.

[0134] Without wishing to be bound by any theory, the amino acid sequence of a recombinant nuclease comprising one or more substitutions upstream and / or downstream of the (D / E / K / N)X(R / F)(E / K)N motif disclosed herein may exhibit enhanced binding affinity (e.g., higher binding affinity) to (i) a guide nucleic acid sequence (e.g., a guide RNA sequence) and / or (ii) a target polynucleotide sequence of the Cas / guide nucleic acid complex (e.g., a target gene in a cell, e.g., an endogenous target gene) as determined by surface plasmon resonance (SPR) or isothermal titration calorimetry (IRC) assays, compared to (A) a control amino acid sequence of a recombinant nuclease not comprising the one or more substitutions, and / or (B) a CRISPR / Cas protein encoded by SEQ ID NO: 1, where the difference in binding affinity is at least about 0. The ratio may be at least about 0.1-fold or less, at least about 0.2-fold or less, at least about 0.5-fold or less, at least about 1-fold or less, at least about 2-fold or less, at least about 3-fold or less, at least about 4-fold or less, at least about 5-fold or less, at least about 6-fold or less, at least about 7-fold or less, at least about 8-fold or less, at least about 9-fold or less, at least about 10-fold or less, at least about 15-fold or less, at least about 20-fold or less, at least about 25-fold or less, at least about 30-fold or less, at least about 35-fold or less, or at least about 40-fold or less.

[0135] In some embodiments, the present disclosure provides a system comprising a recombinant nuclease disclosed herein. In some embodiments, the system may comprise a recombinantly expressed form of the recombinant nuclease (or a generated recombinant nuclease). In some embodiments, the system may comprise one or more polynucleotides encoding at least the recombinant nuclease. In some embodiments, the system may comprise a cell (or population of cells) comprising at least the recombinant nuclease (e.g., a cell engineered to contain the recombinant nuclease, e.g., a cell transfected or transduced to express the recombinant nuclease).

[0136] How to use recombinant nucleases In some embodiments, the recombinant nucleases disclosed herein can be used to bind, cleave, and / or edit a target polynucleotide sequence, e.g., to regulate the expression level and / or activity level of a target polynucleotide sequence of a polypeptide (e.g., protein) encoded by the target polynucleotide sequence or a polypeptide (e.g., protein) operably linked to the target polynucleotide sequence. In some cases, a heterologous polypeptide comprising a recombinant nuclease disclosed herein can be introduced into a cell (e.g., a mammalian cell) to bind, cleave, and / or edit a target polynucleotide sequence in the cell (e.g., an endogenous gene or a heterologous gene in the cell). In some embodiments, the recombinant nucleases disclosed herein, or proteins comprising the recombinant nucleases, can be referred to as "actuator moieties."

[0137] In some embodiments, the recombinant nucleases disclosed herein can retain at least a portion (e.g., substantially full nuclease activity) of the nuclease encoded by the polypeptide sequence of SEQ ID NO: 1 or the nuclease activity of a Cas protein selected from Table 2.

[0138] In some embodiments, the recombinant nucleases disclosed herein may be deficient in nuclease activity. In some embodiments, the recombinant nucleases disclosed herein may be nuclease-null DNA-binding proteins that are incapable of inducing transcriptional activation or repression of a target DNA sequence when complexed with one or more heterologous gene effectors of the present disclosure. In some embodiments, the recombinant nucleases disclosed herein may be nuclease-null DNA-binding proteins that are capable of inducing transcriptional activation or repression of a target DNA sequence (e.g., transcriptional activation or repression of a target DNA sequence can be altered or enhanced by the presence of a heterologous gene effector provided herein). As used herein, the terms "gene effector" and "gene modulator" may be used interchangeably. Also, as used herein, the terms "gene effector polypeptide" and "gene modulator polypeptide" may be used interchangeably.

[0139] In some embodiments, the recombinant nucleases disclosed herein can be RNA nucleases, such as recombinant RNA nucleases (e.g., programmable RNA nucleases or targetable RNA nucleases). In some embodiments, the recombinant nucleases disclosed herein can be RNA binding proteins in a nuclease-null form that are incapable of inducing transcriptional activation or repression of a target RNA sequence when complexed with one or more heterologous effectors of the present disclosure. In some embodiments, the recombinant nucleases disclosed herein can be RNA binding proteins in a nuclease-null form that are capable of inducing transcriptional activation or repression of a target RNA sequence (e.g., transcriptional activation or repression of a target RNA sequence can be altered or enhanced by the presence of a heterologous effector provided herein).

[0140] In some embodiments, the recombinant nucleases disclosed herein may be nucleic acid-guided targeting systems. In some embodiments, the recombinant nucleases disclosed herein may be DNA-guided targeting systems. In some embodiments, the recombinant nucleases disclosed herein may be RNA-guided targeting systems. The nucleic acid-guided targeting systems may include, for example, a guide nucleic acid sequence that facilitates specific binding of a CRISPR-Cas system (e.g., a nuclease-deficient version thereof, e.g., dCas9 or dCas14) to a target gene (e.g., an endogenous target gene) or a regulatory sequence of the target gene, and this guide nucleic acid sequence can be utilized. For example, the target gene may be any one of the genes listed in Table 1, and the regulatory sequence of the target gene may be operably linked to any one of the genes listed in Table 1. Binding specificity can be determined using a guide nucleic acid, such as a single guide RNA (sgRNA) or a portion thereof. In some embodiments, the compositions and methods of the present disclosure can be used against different target genes (e.g., endogenous target genes) or regulatory sequences of target genes (e.g., to target different target genes or their regulatory sequences) by using different types of sgRNAs.

[0141] In some embodiments, the recombinant nuclease can form a complex with a guide nucleic acid, such as a guide RNA or a portion thereof. In some embodiments, the recombinant nuclease can form a complex with a single guide nucleic acid, such as a single guide RNA (sgRNA). In some embodiments, the recombinant nuclease can be an RNA-binding protein (RBP) that can form a complex with a guide nucleic acid, such as a guide RNA (e.g., sgRNA), which can form a complex with a Cas protein. In some embodiments, the recombinant nuclease can be a DNA-binding protein in a nuclease-null form that can induce activation or repression of transcription of a target DNA sequence. In some embodiments, the recombinant nuclease can be an RNA-binding protein in a nuclease-null form derived from RNA.

[0142] Guide nucleic acids used in the compositions and methods of the disclosure may be, for example, at least 8 nucleotides in length, at least 9 nucleotides in length, at least 10 nucleotides in length, at least 11 nucleotides in length, at least 12 nucleotides in length, at least 13 nucleotides in length, at least 14 nucleotides in length, at least 15 nucleotides in length, at least 16 nucleotides in length, at least 17 nucleotides in length, at least 18 nucleotides in length, at least 19 nucleotides in length, at least 20 nucleotides in length, at least 21 nucleotides in length, at least 22 nucleotides in length, at least 23 nucleotides in length, at least 24 nucleotides in length, at least 25 nucleotides in length, at least 26 nucleotides in length, at least 27 nucleotides in length, at least 28 nucleotides in length, at least 29 nucleotides in length, at least 30 nucleotides in length, at least 31 nucleotides in length, at least 32 nucleotides in length, at least 33 nucleotides in length, at least 34 nucleotides in length, at least 35 nucleotides in length, at least 36 nucleotides in length, at least 37 nucleotides in length, at least 38 nucleotides in length, at least 39 nucleotides in length, or at least 40 nucleotides in length.

[0143] In some embodiments, a guide nucleic acid used in the compositions and methods of the disclosure is up to 10 nucleotides in length, up to 11 nucleotides in length, up to 12 nucleotides in length, up to 13 nucleotides in length, up to 14 nucleotides in length, up to 15 nucleotides in length, up to 16 nucleotides in length, up to 17 nucleotides in length, up to 18 nucleotides in length, up to 19 nucleotides in length, up to 20 nucleotides in length, up to 21 nucleotides in length, up to 22 nucleotides in length, up to 23 nucleotides in length, up to 24 nucleotides in length, up to 25 nucleotides in length, up to 26 nucleotides in length, up to 27 nucleotides in length, up to 28 nucleotides in length, up to 29 nucleotides in length, up to 30 nucleotides in length, up to 31 nucleotides in length, up to 32 nucleotides in length, up to 33 nucleotides in length, up to 34 nucleotides in length, up to 35 nucleotides in length, up to 36 nucleotides in length, up to 37 nucleotides in length, up to 38 nucleotides in length, up to 39 nucleotides in length, or up to 40 nucleotides in length.

[0144] In some embodiments, the guide nucleic acid used in the compositions and methods of the present disclosure is between about 8 nucleotides and about 40 nucleotides in length, between about 10 nucleotides and about 40 nucleotides in length, between about 11 nucleotides and about 40 nucleotides in length, between about 12 nucleotides and about 40 nucleotides in length, between about 13 nucleotides and about 40 nucleotides in length, between about 14 nucleotides and about 40 nucleotides in length, between about 15 nucleotides and about 40 nucleotides in length, between about 16 nucleotides and about 40 nucleotides in length, between about 17 nucleotides and about 40 nucleotides in length, between about 18 nucleotides in length, between about 20 nucleotides in length, between about 22 nucleotides in length, between about 24 nucleotides in length, between about 26 nucleotides in length, between about 28 nucleotides in length, between about 29 nucleotides in length, between about 30 nucleotides in length, between about 31 nucleotides in length, between about 32 nucleotides in length, between about 33 nucleotides in length, between about 34 nucleotides in length, between about 35 nucleotides in length, between about 36 nucleotides in length, between about 37 nucleotides in length, between about 38 nucleotides in length, between about 39 nucleotides in length, between about 40 nucleotides in length, between about 41 nucleotides in length, between about 42 nucleotides in length, between about 43 nucleotides in length, between about 44 nucleotides in length, between about 45 nucleotides in length, between about 46 nucleotides in length, between about 47 nucleotides in length, between about 48 nucleotides in length, between about 49 nucleotides in length, between about 50 nucleotides in length, between about 51 nucleotides in length, between about nucleotide length to about 40 nucleotides, about 19 nucleotides to about 40 nucleotides, about 20 nucleotides to about 40 nucleotides, about 22 nucleotides to about 40 nucleotides, about 24 nucleotides to about 40 nucleotides, about 26 nucleotides to about 40 nucleotides, about 28 nucleotides to about 40 nucleotides, about 30 nucleotides to about 40 nucleotides, about 8 nucleotides to about 30 nucleotides, about 10 nucleotides to about 30 nucleotides, about 11 nucleotides to about 30 nucleotides, about 12 nucleotides to about 30 nucleotides long, about 13 nucleotides to about 30 nucleotides long, about 14 nucleotides to about 30 nucleotides long, about 15 nucleotides to about 30 nucleotides long, about 16 nucleotides to about 30 nucleotides long, about 17 nucleotides to about 30 nucleotides long, about 18 nucleotides to about 30 nucleotides long, about 19 nucleotides to about 30 nucleotides long, about 20 nucleotides to about 30 nucleotides long, about 22 nucleotides to about 30 nucleotides long, about 24 nucleotides to about 30 nucleotides long, about 26 nucleotides to about 30 nucleotides long nucleotide length, about 28 nucleotides to about 30 nucleotides, about 8 nucleotides to about 25 nucleotides, about 10 nucleotides to about 25 nucleotides, about 11 nucleotides to about 25 nucleotides, about 12 nucleotides to about 25 nucleotides, about 13 nucleotides to about 25 nucleotides, about 14 nucleotides to about 25 nucleotides, about 15 nucleotides to about 25 nucleotides, about 16 nucleotides to about 25 nucleotides, about 17 nucleotides to about 25 nucleotides, about 18 nucleotides to about 25 nucleotides,about 19 nucleotides to about 25 nucleotides in length, about 20 nucleotides to about 25 nucleotides in length, about 22 nucleotides to about 25 nucleotides in length, about 24 nucleotides to about 25 nucleotides in length, about 8 nucleotides to about 20 nucleotides in length, about 10 nucleotides to about 20 nucleotides in length, about 11 nucleotides to about 20 nucleotides in length, about 12 nucleotides to about 20 nucleotides in length, about 13 nucleotides to about 20 nucleotides in length, about 14 nucleotides to about 20 nucleotides in length, about 15 nucleotides to about 20 nucleotides in length, about 16 nucleotides to about 20 nucleotides in length, about 17 nucleotides to about 20 nucleotides in length, about 18 nucleotides to about 20 nucleotides in length, about 19 nucleotides to about 20 nucleotides in length, about 8 nucleotides to about 20 nucleotides in length, about 19 nucleotides to about 20 nucleotides in length, about 20 ... The length of the guide nucleic acid is about 1 to about 18 nucleotides, about 10 to about 18 nucleotides, about 11 to about 18 nucleotides, about 12 to about 18 nucleotides, about 13 to about 18 nucleotides, about 14 to about 18 nucleotides, about 15 to about 18 nucleotides, about 16 to about 18 nucleotides, about 8 to about 16 nucleotides, about 10 to about 16 nucleotides, about 11 to about 16 nucleotides, about 12 to about 16 nucleotides, about 13 to about 16 nucleotides, about 14 to about 16 nucleotides, or about 15 to about 16 nucleotides. In some embodiments, the guide nucleic acid may be a guide RNA or a portion thereof.

[0145] The recombinant nucleases disclosed herein can be modified to enhance regulation of gene expression using the compositions and methods of the present disclosure, for example, as part of a complex disclosed herein. The recombinant nucleases of the present disclosure can be modified to increase or decrease nucleic acid binding affinity, increase or decrease nucleic acid binding specificity, increase or decrease enzymatic activity, and / or increase or decrease binding to and ligand attraction of other factors, such as heterodimerization or oligomerization domains. The recombinant nucleases of the present disclosure can also be modified to alter other activities or properties (e.g., stability). For example, one or more nuclease domains of the recombinant nucleases of the present disclosure can be modified, deleted, or inactivated, or at least a portion of the recombinant nuclease can be truncated to remove domains that are not essential for the desired function of the recombinant nuclease or complex. Furthermore, the recombinant nucleases of the present disclosure can be modified to modulate (e.g., increase or decrease) the activity of the recombinant nuclease to regulate gene expression using a complex of the present disclosure that includes a heterologous gene effector.

[0146] For example, a recombinant nuclease of the present disclosure can be linked (e.g., fused, covalently, or non-covalently) to a heterologous gene effector (e.g., an epigenetic modification domain, a transcriptional activation domain, and / or a transcriptional repression domain). A recombinant nuclease of the present disclosure can be linked (e.g., fused, covalently, or non-covalently) to an oligomerization or dimerization domain (e.g., a heterodimerization domain) disclosed herein. A recombinant nuclease of the present disclosure can be linked (e.g., fused, covalently, or non-covalently) to a heterologous polypeptide that increases or decreases stability. A recombinant nuclease of the present disclosure can be linked (e.g., fused, covalently, or non-covalently) to a sequence that can promote degradation of the recombinant nuclease or a complex comprising the recombinant nuclease. For example, a recombinant nuclease of the present disclosure can be linked (e.g., fused, covalently linked, or non-covalently linked) to a gene editing moiety (e.g., a heterologous protein, or a domain or functional fragment thereof) that edits, mutates, or modifies (directly or indirectly) a target polynucleotide sequence.

[0147] A recombinant nuclease of the disclosure can be linked (e.g., fused, covalently linked, or non-covalently linked) to any suitable number of partners, for example, at least 1 partner, at least 2 partners, at least 3 partners, at least 4 partners, at least 5 partners, at least 6 partners, at least 7 partners, or at least 8 partners. In some embodiments, a recombinant nuclease of the disclosure is linked (e.g., fused, covalently linked, or non-covalently linked) to up to 2 partners, up to 3 partners, up to 4 partners, up to 5 partners, up to 6 partners, up to 7 partners, up to 8 partners, or up to 10 partners. In some embodiments, a recombinant nuclease of the disclosure is linked to 1 to 5 partners, 1 to 4 partners, 1 to 3 partners, 1 to 2 partners, 2 to 5 partners, 2 to 4 partners, 2 to 3 partners, 3 to 5 partners, 3 to 4 partners, or 4 to 5 partners (e.g., fused, covalently linked, or non-covalently linked). In some embodiments, a recombinant nuclease of the disclosure is linked to one partner (e.g., fused, covalently linked, or non-covalently linked). In some embodiments, a recombinant nuclease of the disclosure is linked to two partners (e.g., fused, covalently linked, or non-covalently linked). In some embodiments, a recombinant nuclease of the disclosure is linked to three partners (e.g., fused, covalently linked, or non-covalently linked). In some embodiments, the recombinant nuclease of the present disclosure is linked (eg, fused, covalently linked, or non-covalently linked) to four partners.In some embodiments, the recombinant nucleases of the disclosure are linked to five partners (e.g., fused, covalently linked, or non-covalently linked), hi some embodiments, the recombinant nucleases of the disclosure are linked to six partners (e.g., fused, covalently linked, or non-covalently linked).

[0148] The recombinant nucleases disclosed herein may be fusion proteins, for example, fusion proteins comprising a recombinant nuclease disclosed herein and one or more partners, where the fused domain or heterologous polypeptide may be located at the N-terminus, C-terminus, or internal sequence of the recombinant nuclease.

[0149] The partner of the recombinant nuclease disclosed herein (e.g., a partner covalently or non-covalently linked to a nuclease activity-deficient or nuclease null variant of the recombinant nuclease disclosed herein) may be a transcriptional effector (e.g., a transcriptional activator or transcriptional repressor). The transcriptional effector may be heterologous to the cells provided herein.

[0150] In some embodiments, the transcriptional effector may be an epigenetic histone modifier (or histone modifier). In some cases, the epigenetic histone modifier may regulate histones by methylation (e.g., a histone methylation modifier, e.g., an amino acid methyltransferase (e.g., KRAB)). In some cases, the epigenetic histone modifier may regulate histones by acetylation. In some cases, the epigenetic histone modifier may regulate histones by phosphorylation. In some cases, the epigenetic histone modifier may regulate histones by ADP-ribosylation. In some cases, the epigenetic histone modifier may regulate histones by glycosylation. In some cases, the epigenetic histone modifier may regulate histones by sumoylation. In some cases, the epigenetic histone modifier may regulate histones by ubiquitination. In some cases, epigenetic histone modifiers can regulate histones by remodeling histone structure, such as through ATP hydrolysis-dependent processes.

[0151] In some embodiments, the transcriptional effector may be an epigenetic gene modifier (or gene modifier). In some cases, the gene modifier may regulate genes by methylation (e.g., a gene methylation modifier, e.g., a DNA methyltransferase, or DNMT). In some cases, the gene modifier may regulate genes by acetylation.

[0152] In some embodiments, the transcriptional effector is selected from a family of related histone acetyltransferases, including, but not limited to, the GNAT subfamily, the MYST subfamily, the p300 / CBP subfamily, the HAT1 subfamily, GCN5, PCAF, Tip60, MOZ, MORF, MOF, HBO1, p300, CBP, HAT1, ATF-2, SRC1, and TAFII250.

[0153] In some embodiments, the transcriptional effector may comprise an epigenetic modifier. In some embodiments, the transcriptional effector comprises an epigenetic histone modifier (e.g., histone lysine methyltransferase, histone lysine demethylase, or DNA methylase). Examples of epigenetic modifiers include the EZH subfamily, non-SET subfamily, other SET subfamily, PRDM subfamily, SET1 subfamily, SET2 subfamily, SUV39 subfamily, SYMD subfamily, ASH1L, EHMT1, EHMT2, EZH1, EZH2, MLL, MLL2, MLL3, MLL4, MLL5, NSD1, NSD2, NSD3, PRDM1, PRDM10, PRDM11, PRDM12, PRDM13, and PRDM14. 14, PRDM15, PRDM16, PRDM2, PRDM4, PRDM5, PRDM6, PRDM7, PRDM8, PRDM9, SET1, SET1L, SET2L, SETD2, SETD3, SETD4, SETD5, SETD6, SETD7, SETD8, SETDB1, SETDB2, SETMAR, SUV39H1, SUV39H2, SUV420H1, SUV420H2, SYMD1, SYMD2, SYMD3, SYMD4, and SYMD5.

[0154] Examples of proteins (or fragments thereof) that can be used as fusion partners to increase transcription include, but are not limited to, the VP16 subdomain, VP64 subdomain, VP48 subdomain, VP160 subdomain, and p65 subdomain (e.g., from NFkB); the activation domain of EDLL; and / or the TAL activation domain (e.g., for activity in plants); and transcriptional activators such as SET1A, SET1B, MLL1-5, ASH1, SYMD2, NSD1, JHDM2a / b, UTX, JMJD3, GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZMYST3, MORFMYST4, SRC1, ACTR, PI 60, CLOCK, Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, etc. Another example of such a gene activation modulator is the VP64-p65-Rta fusion polypeptide (VPR).Examples of proteins (or fragments thereof) that can be used as fusion partners to reduce transcription include Kruppel-associated boxes (KRAB or SKD); KOX1 repression domains; Mad mSIN3-interacting domains (SIDs); ERF repressor domains (ERDs) (e.g., for repression in plants), SRDX repression domains, etc.; histone lysine methyltransferases such as Pr-SET7 / 8, SUV4-20H1, and RIZ1; histone lysine demethylases such as JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARJD 1 A / RBP2, JARIDlB / PLU-1, JARID 1C / SMCX, and JARIDID / SMCY; histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, and HDAC11; and Hhal DNA These include, but are not limited to, DNA methylases such as m5c methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants); and transcriptional repressors such as peripheral recruitment factors such as lamin A and lamin B.

[0155] In various aspects, the recombinant nucleases provided herein may induce editing or mutation of a target polynucleotide as described herein. In some embodiments, editing or mutation of a target polynucleotide sequence includes changing one or more nucleotides in the target polynucleotide to one or more different nucleotides. In some embodiments, editing or mutation of a target polynucleotide includes changing a guanine (G) to another nucleotide. In some cases, a guanine (G) may be changed to an adenine (A). In some cases, a guanine (G) may be changed to a thymine (T). In some cases, a guanine (G) may be changed to a cytosine (C). In some cases, a guanine (G) may be changed to a uracil (U). In some cases, a guanine (G) may be changed to an inosine (I). In some embodiments, editing or mutation of a target polynucleotide includes changing a cytosine (C) to another nucleotide. In some cases, a cytosine (C) may be changed to a guanine (G). In some cases, a cytosine (C) may be changed to an adenine (A). In some embodiments, editing or mutating a target polynucleotide includes changing a thymine (T) to another nucleotide. In some embodiments, changing a thymine (T) to a cytosine (T). In some embodiments, changing a cytosine (C) to a uracil (U). In some embodiments, changing a cytosine (C) to an inosine (I). In some embodiments, editing or mutating a target polynucleotide includes changing a thymine (T) to another nucleotide. In some embodiments, changing a thymine (T) to a cytosine (C). In some embodiments, changing a thymine (T) to a guanine (G). In some embodiments, changing a thymine (T) to an adenine (A). In some embodiments, changing a thymine (T) to a uracil (U). In some embodiments, changing a thymine (T) to an inosine (I). In some embodiments, editing or mutating a target polynucleotide includes changing adenine (A) to another nucleotide. In some embodiments, changing adenine (A) to a guanine (G). In some embodiments, changing adenine (A) to a cytosine (C). In some cases, adenine (A) may be changed to thymine (T).In some embodiments, editing or mutating a target polynucleotide includes changing uracil (U) to another nucleotide. In some embodiments, changing uracil (U) to guanine (G). In some embodiments, changing uracil (U) to cytosine (C). In some embodiments, changing uracil (U) to thymine (T). In some embodiments, changing uracil (U) to adenine (A). In some embodiments, changing uracil (U) to inosine (I). In some embodiments, editing or mutating a target polynucleotide includes changing inosine (I) to another nucleotide. In some embodiments, changing inosine (I) to guanine (G). In some embodiments, changing inosine (I) to cytosine (C). In some embodiments, changing inosine (I) to thymine (T). In some cases, inosine (I) may be changed to adenine (A). In some cases, inosine (I) may be changed to uracil (U).

[0156] In some embodiments, editing or mutating the target polynucleotide includes introducing one or more point mutations into the target polynucleotide. In some embodiments, editing or mutating the target polynucleotide includes introducing one or more deletions (e.g., deletion of one or more nucleotides) into the target polynucleotide. In some embodiments, editing or mutating the target polynucleotide includes introducing one or more insertions (e.g., insertion of one or more nucleotides) into the target polynucleotide. In some embodiments, editing or mutating the target polynucleotide includes introducing one or more substitutions (e.g., substitutions of two or more nucleotides) into the target polynucleotide. In some embodiments, editing or mutating the target polynucleotide includes introducing one or more translocations (e.g., translocations of one or more nucleotides) into the target polynucleotide. In some embodiments, editing or mutating the target polynucleotide includes introducing one or more transitions into the target polynucleotide.

[0157] In some cases, the recombinant nuclease described herein may be linked to a partner (e.g., a gene editing moiety) that induces editing or mutation of a target polynucleotide, as described herein. In some cases, the nuclease activity-deficient recombinant nuclease or nuclease-null recombinant nuclease provided herein is linked to a gene editing moiety (e.g., a protein or functional domain or functional fragment thereof) that induces editing or mutation of a target polynucleotide via a covalent or non-covalent bond. In some embodiments, the gene editing moiety (e.g., a protein or functional domain or functional fragment thereof) that induces editing or mutation of a target polynucleotide is a gene editing moiety that changes one or more nucleotides to another nucleotide. In some embodiments, the gene editing moiety (e.g., a protein or functional domain or functional fragment thereof) that induces editing or mutation of a target polynucleotide is a gene editing moiety that changes a guanine (G) to another nucleotide. In some cases, the gene editing moiety that induces editing or mutation of a target polynucleotide changes a guanine (G) to a cytosine (C). In some cases, the gene editing portion that edits or induces mutation of the target polynucleotide changes a guanine (G) to a thymine (T). In some cases, the gene editing portion that edits or induces mutation of the target polynucleotide changes a guanine (G) to an adenine (A). In some cases, the gene editing portion that edits or induces mutation of the target polynucleotide changes a guanine (G) to a uracil (U). In some cases, the gene editing portion that edits or induces mutation of the target polynucleotide changes a guanine (G) to an inosine (I). In some embodiments, the gene editing portion (e.g., a protein or functional domain or functional fragment thereof) that edits or induces mutation of the target polynucleotide sequence is a gene editing portion that changes a cytosine (C) to another nucleotide. In some cases, the gene editing portion that edits or induces mutation of the target polynucleotide changes a cytosine (C) to a guanine (G). In some cases, the gene editing portion that edits or induces mutation of the target polynucleotide changes a cytosine (C) to a thymine (T).In some cases, the gene editing portion that edits or induces mutation of the target polynucleotide changes a cytosine (C) to an adenine (A). In some cases, the gene editing portion that edits or induces mutation of the target polynucleotide changes a cytosine (C) to an uracil (U). In some cases, the gene editing portion that edits or induces mutation of the target polynucleotide changes a cytosine (C) to an inosine (I). In some embodiments, the gene editing portion (e.g., a protein or functional domain or functional fragment thereof) that edits or induces mutation of the target polynucleotide sequence is a gene editing portion that changes a thymine (T) to another nucleotide. In some cases, the gene editing portion that edits or induces mutation of the target polynucleotide changes a thymine (T) to a cytosine (C). In some cases, the gene editing portion that edits or induces mutation of the target polynucleotide changes a thymine (T) to a guanine (G). In some cases, the gene editing portion that edits or induces mutation of the target polynucleotide changes a thymine (T) to an adenine (A). In some cases, the gene editing portion that induces editing or mutation of the target polynucleotide changes thymine (T) to uracil (U). In some cases, the gene editing portion that induces editing or mutation of the target polynucleotide changes thymine (T) to inosine (I). In some embodiments, the gene editing portion (e.g., a protein or functional domain or functional fragment thereof) that induces editing or mutation of the target polynucleotide sequence is a gene editing portion that changes adenine (A) to another nucleotide. In some cases, the gene editing portion that induces editing or mutation of the target polynucleotide changes adenine (A) to cytosine (C). In some cases, the gene editing portion that induces editing or mutation of the target polynucleotide changes adenine (A) to thymine (T). In some cases, the gene editing portion that induces editing or mutation of the target polynucleotide changes adenine (A) to guanine (G). In some cases, the gene editing portion that induces editing or mutation of the target polynucleotide changes adenine (A) to uracil (U).In some cases, the gene editing portion that induces editing or mutation of the target polynucleotide changes an adenine (A) to an inosine (I). In some embodiments, the gene editing portion (e.g., a protein or functional domain or functional fragment thereof) that induces editing or mutation of the target polynucleotide sequence is a gene editing portion that changes an uracil (U) to another nucleotide. In some cases, the gene editing portion that induces editing or mutation of the target polynucleotide changes an uracil (U) to a cytosine (C). In some cases, the gene editing portion that induces editing or mutation of the target polynucleotide changes an uracil (U) to a thymine (T). In some cases, the gene editing portion that induces editing or mutation of the target polynucleotide changes an uracil (U) to an adenine (A). In some cases, the gene editing portion that induces editing or mutation of the target polynucleotide changes an uracil (U) to a guanine (G). In some cases, the gene editing portion that induces editing or mutation of the target polynucleotide changes an uracil (U) to an inosine (I). In some embodiments, the gene editing moiety (e.g., a protein or functional domain or functional fragment thereof) that induces editing or mutation of a target polynucleotide sequence is a gene editing moiety that changes inosine (I) to another nucleotide. In some cases, the gene editing moiety that induces editing or mutation of a target polynucleotide changes inosine (I) to cytosine (C). In some cases, the gene editing moiety that induces editing or mutation of a target polynucleotide changes inosine (I) to thymine (T). In some cases, the gene editing moiety that induces editing or mutation of a target polynucleotide changes inosine (I) to adenine (A). In some cases, the gene editing moiety that induces editing or mutation of a target polynucleotide changes inosine (I) to uracil (U). In some cases, the gene editing moiety that induces editing or mutation of a target polynucleotide changes inosine (I) to guanine (G).

[0158] In some embodiments, a recombinant nuclease described herein (e.g., a nuclease activity-deficient recombinant nuclease described herein or a nuclease-null recombinant nuclease) may be linked (e.g., via a covalent or non-covalent bond) to a partner (e.g., a gene editing moiety) that introduces one or more point mutations into a target polynucleotide. In some embodiments, a recombinant nuclease described herein (e.g., a nuclease activity-deficient recombinant nuclease described herein or a nuclease-null recombinant nuclease) may be linked (e.g., via a covalent or non-covalent bond) to a partner (e.g., a gene editing moiety) that introduces one or more deletions (e.g., deletions of one or more nucleotides) into a target polynucleotide. In some embodiments, a recombinant nuclease described herein (e.g., a nuclease activity-deficient recombinant nuclease described herein or a nuclease-null recombinant nuclease) may be linked (e.g., via a covalent or non-covalent bond) to a partner (e.g., a gene editing moiety) that introduces one or more insertions (e.g., insertions of one or more nucleotides) into a target polynucleotide. In some embodiments, a recombinant nuclease described herein (e.g., a nuclease activity-deficient recombinant nuclease described herein or a nuclease-null recombinant nuclease) may be linked (e.g., via a covalent or non-covalent bond) to a partner (e.g., a gene editing moiety) that introduces one or more replacements (e.g., replacements of two or more nucleotides) into a target polynucleotide. In some embodiments, a recombinant nuclease described herein (e.g., a nuclease activity-deficient recombinant nuclease described herein or a nuclease-null recombinant nuclease) may be linked (e.g., via a covalent or non-covalent bond) to a partner (e.g., a gene editing moiety) that introduces one or more translocations (e.g., translocations of one or more nucleotides) into a target polynucleotide.In some embodiments, a recombinant nuclease described herein (e.g., a nuclease activity-deficient recombinant nuclease described herein or a nuclease-null recombinant nuclease) may be linked (e.g., via a covalent or non-covalent bond) to a partner (e.g., a gene editing moiety) that introduces one or more transitions into a target polynucleotide.

[0159] In some embodiments, the gene editing portion may be a base-editing protein or a base-editing enzyme. In some embodiments, the base-editing protein or base-editing enzyme is a deaminase. In some cases, the deaminase is a cytidine deaminase. In some cases, the cytidine deaminase catalyzes a reaction that converts cytosine (C) to uracil (U), which can form base pairs with thymine. In some embodiments, for example, when a polynucleotide is double-stranded (e.g., double-stranded DNA), a uridine base may be replaced with a thymidine base (e.g., by a cellular repair mechanism), resulting in a C / G to T / A transition. In some embodiments, the deaminase is an adenine deaminase. In some cases, the adenine deaminase catalyzes a reaction that converts adenosine (A) to inosine (I). Examples of deaminases suitable for use herein include, but are not limited to, APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3E deaminase, APOBEC3F deaminase, APOBEC3G deaminase, APOBEC3H deaminase, APOBEC4 deaminase, activation-induced cytidine deaminase (AID), adenosine deaminase 1 (ADAR1), adenosine deaminase 2 (ADAR2), adenosine deaminase 3 (ADAR3), or TadA.

[0160] In some embodiments, the recombinant nucleases described herein may be used for prime editing. For example, the recombinant nucleases described herein may be linked to a reverse transcriptase enzyme (e.g., recombinant M-MLV reverse transcriptase) and a prime-editing RNA (pegRNA). In such cases, the recombinant nuclease may have nickase activity. In some embodiments, prime editing may be used to perform targeted insertions, deletions, or base-to-base conversions.

[0161] In some embodiments, the recombinant nucleases described herein may be used to write genes.

[0162] In some embodiments, the recombinant nuclease described herein may be linked to a partner (e.g., a protein, or a functional domain or fragment thereof) that methylates a target substrate. In some cases, the partner (e.g., a protein, or a functional domain or fragment thereof) that methylates a target substrate is a methyltransferase. In some cases, the methyltransferase is a DNA methyltransferase, a histone methyltransferase, or an RNA methyltransferase. In some cases, the DNA methyltransferase is DNMT1 or DNMT3.

[0163] In some embodiments, the recombinant nucleases described herein may be linked to a partner (e.g., a protein or functional domain or fragment thereof) that has demethylase activity (e.g., capable of removing methyl groups from nucleic acids, proteins, or other molecules). In some cases, the partner (e.g., a protein or functional domain or fragment thereof) that has demethylase activity is a histone lysine demethylase, including, but not limited to, KDM1, KDM2, KDM3, KDM4, KDM5, and KDM6.

[0164] In some embodiments, the recombinant nucleases described herein may be linked to a partner (e.g., a protein or functional domain or fragment thereof) having dismutase activity. In some cases, the partner (e.g., a protein or functional domain or fragment thereof) having dismutase activity is superoxide dismutase, formaldehyde dismutase, or chlorite dismutase.

[0165] In some embodiments, the recombinant nucleases described herein may be linked to a partner having alkylating activity (e.g., a protein or functional domain or functional fragment thereof). In some cases, the partner having alkylating activity (e.g., a protein or functional domain or functional fragment thereof) is a prenyltransferase, a terpene cyclase, or a terpene synthase.

[0166] In some embodiments, the recombinant nucleases described herein may be linked to a partner (e.g., a protein or functional domain or fragment thereof) having depurination activity. In some cases, the partner (e.g., a protein or functional domain or fragment thereof) having depurination activity is a DNA glycosylase.

[0167] In some embodiments, the recombinant nucleases described herein may be linked to a partner having oxidative activity (e.g., a protein or functional domain or fragment thereof). In some cases, the partner having oxidative activity (e.g., a protein or functional domain or fragment thereof) is a peroxidase or an oxidase.

[0168] In some embodiments, the recombinant nucleases described herein may be linked to a partner (eg, a protein or functional domain or functional fragment thereof) that has pyrimidine dimer-forming activity.

[0169] In some embodiments, the recombinant nucleases described herein may be linked to a partner having integrase activity (e.g., a protein or a functional domain or fragment thereof). In some cases, the partner having integrase activity (e.g., a protein or a functional domain or fragment thereof) is a retroviral integrase or an HIV integrase.

[0170] In some embodiments, the recombinant nucleases described herein may be linked to a partner having transposase activity (e.g., a protein or a functional domain or fragment thereof). In some cases, the partner having transposase activity (e.g., a protein or a functional domain or fragment thereof) is ty1, Mariner transposase, Tn3, transposase (Tnp), Tn5, or Tn7 transposon.

[0171] In some embodiments, the recombinant nucleases described herein may be linked to a partner having recombinase activity (e.g., a protein or functional domain or fragment thereof). In some cases, the partner having recombinase activity (e.g., a protein or functional domain or fragment thereof) is a tyrosine recombinase, Rad51 recombinase, RecA recombinase, or Dmc1 recombinase.

[0172] In some embodiments, the recombinant nucleases described herein may be linked to a partner having polymerase activity (e.g., a protein or functional domain or fragment thereof). In some cases, the partner having polymerase activity (e.g., a protein or functional domain or fragment thereof) is a DNA polymerase, an RNA polymerase, a reverse transcriptase, or an RdRp replicase.

[0173] In some embodiments, the recombinant nucleases described herein may be linked to a partner (e.g., a protein or functional domain or fragment thereof) having ligase activity. In some cases, the partner (e.g., a protein or functional domain or fragment thereof) having ligase activity is a ubiquitin ligase, a glutamate-cysteine ​​ligase, an aminoacyl-tRNA synthetase, a succinyl-coenzyme A synthetase, an acetyl-CoA synthetase, a pyruvate carboxylase, an acetyl-CoA carboxylase, a propionyl-CoA carboxylase, a methylcrotonyl-CoA carboxylase, a DNA ligase, a magnesium chelatase, a cobalt chelatase, or a DNA synthetase.

[0174] In some embodiments, the recombinant nucleases described herein may be linked to a partner having helicase activity (e.g., a protein or a functional domain or fragment thereof). In some cases, the partner having helicase activity (e.g., a protein or a functional domain or fragment thereof) is a DNA helicase, an RNA helicase, a chromodomain helicase, or a DEAD box / DEAD / DEAH box helicase.

[0175] In some embodiments, the recombinant nucleases described herein may be linked to a partner (e.g., a protein or functional domain or fragment thereof) having photolyase activity. In some cases, the partner (e.g., a protein or functional domain or fragment thereof) having photolyase activity is photoreactivatable enzyme, DNA photolyase, DNA photoreactivatable enzyme, DNA cyclobutane dipyrimidine photolyase, DNA photolyase, deoxyribonucleic acid photolyase, deoxyribodipyrimidine photolyase, photolyase, PRE, PhrB photolyase, deoxyribonucleic acid cyclobutane dipyrimidine photolyase, phrA photolyase, dipyrimidine photolyase (photosensitive), or deoxyribopyrimidine dimer lyase (photosensitive).

[0176] In some embodiments, the recombinant nucleases described herein may be linked to a partner having glycosylase activity (e.g., a protein or a functional domain or fragment thereof). In some cases, the partner having glycosylase activity (e.g., a protein or a functional domain or fragment thereof) is N-methylpurine DNA glycosylase, UNG, hOGG1, hNTH1, hNEIL1, hMYH, hSMUG1, TDG, MBD4, Mag1, Ung1, Ogg1, Ntg1, AlkE, Ntg2, hNEIL2, hNEIL3, AlkC, AlkD, MutY, Nei, Nth, Fpg, or UDG.

[0177] In some embodiments, the recombinant nucleases described herein may be linked to a partner (e.g., a protein or functional domain or fragment thereof) having acetyltransferase activity. In some cases, the partner (e.g., a protein or functional domain or fragment thereof) having acetyltransferase activity is CBP histone acetyltransferase, choline acetyltransferase, chloramphenicol acetyltransferase, serotonin N-acetyltransferase, NatA acetyltransferase, or NatB acetyltransferase.

[0178] In some embodiments, the recombinant nucleases described herein may be linked to a partner (e.g., a protein or functional domain or fragment thereof) having deacetylase activity. In some cases, the partner (e.g., a protein or functional domain or fragment thereof) having deacetylase activity is HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-6, HDAC-7, or HDAC-8.

[0179] In some embodiments, the recombinant nucleases described herein may be linked to a partner having kinase activity (e.g., a protein or a functional domain or fragment thereof). In some cases, the partner having kinase activity (e.g., a protein or a functional domain or fragment thereof) is linked to a Ca 2+ / calmodulin-dependent protein kinase, cyclin-dependent kinase, nucleoside diphosphate kinase, phosphatidylinositol phosphate kinase, thymidine kinase, thymidylate kinase, or cell wall-bound kinase.

[0180] In some embodiments, the recombinant nucleases described herein may be linked to a partner having phosphatase activity (e.g., a protein or functional domain or fragment thereof). In some cases, the partner having phosphatase activity (e.g., a protein or functional domain or fragment thereof) is an acid phosphatase, alkaline phosphatase, an endonuclease / exonuclease / phosphatase family, a kinase, a phosphatome, a phosphotransferase, a protein phosphatase, or protein phosphatase 2.

[0181] In some embodiments, the recombinant nucleases described herein may be linked to a partner (e.g., a protein or functional domain or fragment thereof) having ubiquitin ligase activity. In some cases, the partner (e.g., a protein or functional domain or fragment thereof) having ubiquitin ligase activity is selected from the group consisting of E3A, mdm2, anaphase-promoting complex (APC), UBR5 (EDD1), SOCS / BC-box / eloBC / CUL5 / RING, LNXp80, CBX4, CBLL1, HACE1, HECTD1, HECTD2, HECTD3, HECTD4, HECW1, HECW2, HERC1, HERC2, HERC3, HE, RC4, HERC5, HERC6, HUWE1, ITCH, NEDD4, NEDD4L, PPIL2, PRPF19, PIAS1, PIAS2, PIAS3, PIAS4, RANBP2, RNF4, RBX1, SMURF1, SMURF2, STUB1, TOPORS, TRIP12, UBE3A, UBE3B, UBE3C, UBE3D, UBE4A, UBE4B, UBOX5, UBR5, VHL, WWP1, WWP2, Parkin, or MKRN1.

[0182] In some embodiments, the recombinant nuclease described herein may be linked to a partner (e.g., a protein or functional domain or fragment thereof) having deubiquitinating activity. In some cases, the partner (e.g., a protein or functional domain or fragment thereof) having deubiquitinating activity is a deubiquitinating peptidase, a deubiquitinating isopeptidase, a deubiquitinating enzyme, a ubiquitin protease, a ubiquitin hydrolase, or a ubiquitin isopeptidase.

[0183] In some embodiments, the recombinant nuclease described herein may be linked to a partner (e.g., a protein or functional domain or fragment thereof) having adenylation activity. In some cases, the partner (e.g., a protein or functional domain or fragment thereof) having adenylation activity is a carboxylic acid reductase.

[0184] In some embodiments, the recombinant nucleases described herein may be linked to a partner (e.g., a protein or functional domain or fragment thereof) having deadenylation activity. In some cases, the partner (e.g., a protein or functional domain or fragment thereof) having deadenylation activity is 5'-deadenylation, CNOT6 deadenylase, CNOT6L deadenylase, or CCR4-NOT deadenylase.

[0185] In some embodiments, the recombinant nucleases described herein may be linked to a partner (e.g., a protein or functional domain or fragment thereof) having sumoylation activity. In some cases, the partner (e.g., a protein or functional domain or fragment thereof) having sumoylation activity is a small ubiquitin-like modifier (SUMO-1), SUMO-2, or SUMO-3.

[0186] In some embodiments, a recombinant nuclease described herein may be linked to a partner (e.g., a protein or functional domain or fragment thereof) having desumoylating activity. In some cases, the partner (e.g., a protein or functional domain or fragment thereof) having desumoylating activity is SENP1, SENP2, SENP3, or SENP5.

[0187] In some embodiments, the recombinant nucleases described herein may be linked to a partner (e.g., a protein, or a functional domain or fragment thereof) having ribosylating activity. In some cases, the partner (e.g., a protein, or a functional domain or fragment thereof) having ribosylating activity is a mono(ADP-ribosyl)transferase, a poly(ADP-ribose)polymerase, or a histone ribosylating enzyme.

[0188] In some embodiments, the recombinant nucleases described herein may be linked to a partner (e.g., a protein, or a functional domain or fragment thereof) having deribosylating activity. In some cases, the partner (e.g., a protein, or a functional domain or fragment thereof) having deribosylating activity is a histone lysine deribosylase or an ADP-ribose deribosylase.

[0189] In some embodiments, the recombinant nuclease described herein may be linked to a partner (e.g., a protein or functional domain or fragment thereof) having myristoylation activity. In some cases, the partner (e.g., a protein or functional domain or fragment thereof) having myristoylation activity is N-myristoyltransferase (NMT) 1, N-myristoyltransferase (NMT) 2, or glycylpeptide N-tetradecanoyltransferase.

[0190] In some embodiments, the recombinant nucleases described herein may be linked to a partner (e.g., a protein or functional domain or fragment thereof) having remodeling activity. In some cases, the partner (e.g., a protein or functional domain or fragment thereof) having remodeling activity is a histone acetyltransferase (HAT), deacetylase, or methyltransferase.

[0191] In some embodiments, the recombinant nucleases described herein may be linked to a partner having protease activity (e.g., a protein or functional domain or fragment thereof). In some cases, the partner having protease activity (e.g., a protein or functional domain or fragment thereof) is trypsin, chymotrypsin, elastase, papain, bromelain, serine protease, cysteine ​​protease, threonine protease, aspartic acid protease, glutamic acid protease, metalloprotease, or asparagine peptide lyase.

[0192] In some embodiments, a recombinant nuclease described herein may be linked to a partner having oxidoreductase activity (e.g., a protein or functional domain or fragment thereof). In some cases, the partner having oxidoreductase activity (e.g., a protein or functional domain or fragment thereof) is a donor dehydrogenase, peroxidase, reductase, dehydrogenase, oxidase, oxygenase, hydroxylase, luciferase, DMSO reductase, glucose oxidase, L-gulonolactone oxidase, thiamine oxidase, xanthine oxidase, acetaldehyde dehydrogenase, pyruvate dehydrogenase, oxoglutarate dehydrogenase, monoamine oxidase, biliverdin reductase, dihydrofolate reductase, methylenetetrahydrofolate reductase, sarcosine oxidase, or dihydrobenzophenanthridine oxidase.

[0193] In some embodiments, the recombinant nucleases described herein may be linked to a partner having transferase activity (e.g., a protein or functional domain or fragment thereof). In some cases, the partner having transferase activity (e.g., a protein or functional domain or fragment thereof) is a coenzyme A transferase, an acyltransferase, a peptidyl transferase, an N-acetyltransferase, or a pyruvate dehydrogenase.

[0194] In some embodiments, the recombinant nucleases described herein may be linked to a partner having hydrolase activity (e.g., a protein or functional domain or fragment thereof). In some cases, the partner having hydrolase activity (e.g., a protein or functional domain or fragment thereof) is an esterase, protease, glycosidase, or lipase.

[0195] In some embodiments, the recombinant nucleases described herein may be linked to a partner having lyase activity (e.g., a protein or functional domain or fragment thereof). In some cases, the partner having lyase activity (e.g., a protein or functional domain or fragment thereof) is phenylalanine ammonia lyase, citrate lyase, isocitrate lyase, hydroxynitrile, pectate lyase, argininosuccinate lyase, pyruvate formate lyase, alginate lyase, or pectin lyase.

[0196] In some embodiments, the recombinant nucleases described herein may be linked to a partner (e.g., a protein or functional domain or fragment thereof) having isomerase activity. In some cases, the partner (e.g., a protein or functional domain or fragment thereof) having isomerase activity is a ribose phosphate isomerase, a bisphosphoglycerate mutase, or a photoisomerase enzyme.

[0197] In some embodiments, the recombinant nucleases described herein may be linked to a partner having synthase activity (e.g., a protein or functional domain or fragment thereof). In some cases, the partner having synthase activity (e.g., a protein or functional domain or fragment thereof) is ATP synthase, citrate synthase, tryptophan synthase, pseudouridine synthase, or fatty acid synthase.

[0198] In some embodiments, the recombinant nucleases described herein may be linked to a partner (e.g., a protein or functional domain or fragment thereof) having demyristoylation activity. In some cases, the partner (e.g., a protein or functional domain or fragment thereof) having demyristoylation activity is a T3SS effector protein.

[0199] In some embodiments, the recombinant nucleases described herein may be linked to a partner (e.g., a protein or functional domain or fragment thereof) having transposition activity. In some cases, the partner (e.g., a protein or functional domain or fragment thereof) having transposition activity is transposase Tn5 or Sleeping Beauty transposase.

[0200] The recombinant nucleases disclosed herein may be provided in any form. For example, the recombinant nucleases disclosed herein can be provided in the form of a protein, e.g., the recombinant nuclease alone or complexed with a guide nucleic acid as a ribonucleoprotein. The recombinant nuclease can be provided in the form of a complex, e.g., complexed with a guide nucleic acid and / or one or more heterologous gene effectors of the present disclosure. The recombinant nuclease can be provided in the form of a nucleic acid encoding at least the recombinant nuclease, e.g., RNA (e.g., messenger RNA (mRNA)) or DNA. The nucleic acid encoding at least the recombinant nuclease can be codon-optimized (e.g., optimized for human codons) for efficient protein translation in a particular cell or organism.

[0201] The nucleic acid encoding at least the recombinant nuclease disclosed herein, or a fragment or derivative thereof, can be stably integrated into the genome of a cell. The nucleic acid encoding at least the recombinant nuclease can be operably linked to a promoter, such as a promoter that is constitutively active in the cell or a promoter that is activated upon induction. The nucleic acid encoding at least the recombinant nuclease can be operably linked to a promoter in an expression construct. The expression construct can include a nucleic acid construct capable of inducing expression of a gene or other nucleic acid sequence of interest (e.g., at least the recombinant nuclease), and such a nucleic acid sequence of interest can be transferred into a target cell.

[0202] In some embodiments, the recombinant nucleases disclosed herein can bind to a single guide RNA (sgRNA) to activate or repress transcription of a target gene (e.g., an endogenous target gene), for example, by cooperating with a heterologous gene effector disclosed herein. The sgRNA can also be introduced into cells expressing the recombinant nucleases provided herein or variants thereof. In some cases, such cells may contain one or more sgRNAs that target the same target gene (e.g., an endogenous target gene) or the same regulatory sequence of a target gene. In other cases, the sgRNAs target different nucleic acids within the cell (e.g., targeting different target genes, different regulatory sequences of a target gene, or different sequences within the same target gene or the same regulatory sequence of a target gene).

[0203] "Enzymatically inactive" (e.g., as used with respect to a defective nuclease) refers to a nuclease that can bind sequence-specifically to a nucleic acid sequence in a polynucleotide but that may not cleave the target polynucleotide or that cleaves the target polynucleotide significantly less frequently. An enzymatically inactive guide portion may comprise an enzymatically inactive domain (e.g., an enzymatically inactive nuclease domain). "Enzymatically inactive" may mean no activity. "Enzymatically inactive" may mean substantially no activity. "Enzymatically inactive" may mean essentially no activity. "Enzymatically inactive" may mean 1% or less, 2% or less, 3% or less, 4% or less, 5% or less, 6% or less, 7% or less, 8% or less, 9% or less, or 10% or less of the activity of a comparable wild-type activity (e.g., nucleic acid cleavage activity or wild-type Cas activity).

[0204] In some embodiments, the target nucleic acid of the recombinant nuclease disclosed herein may be dsDNA. In such embodiments, target specificity for dsDNA is determined at least in part by two parameters: the gRNA spacer that targets the protospacer in the target dsDNA (the sequence in the target dsDNA that corresponds to the gRNA spacer on the non-complementary DNA strand) and a short sequence called a 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', 5'-TTTA-3', or 5'-TTTR-3'. In some embodiments, the PAM is 5'-TTTG-3'. In some embodiments, the PAM is 5'-TTTA-3'. In some embodiments, the PAM is 5'-TTTR-3'.

[0205] In some embodiments, the target nucleic acid of the recombinant nuclease disclosed herein may be RNA. In such embodiments, target specificity for RNA is determined at least in part by the gRNA spacer targeting a protospacer-like sequence (a sequence in the target RNA that is complementary to the gRNA spacer) in the target RNA, and is independent of the sequence located immediately 5' (upstream) of the protospacer-like sequence. In some embodiments, the recombinant nuclease can also target a dsDNA molecule, and the gRNA spacer is selected to target a protospacer in a target dsDNA molecule having a PAM selected from 5'-TTTG-3', 5'-TTTA-3', and 5'-TTTR-3'. In another embodiment, the recombinant nuclease does not target a dsDNA molecule, and the gRNA spacer is selected to target a protospacer in a dsDNA molecule without a PAM selected from 5'-TTTG-3', 5'-TTTA-3', and 5'-TTTR-3'.

[0206] In some embodiments, a heterologous polypeptide comprising a recombinant nuclease (e.g., and / or a complex comprising a heterologous polypeptide) can regulate the expression and / or activity of a target gene (e.g., an endogenous target gene). In some embodiments, the heterologous polypeptide and / or complex thereof can edit the sequence of a nucleic acid (e.g., a gene and / or gene product). Nuclease-active variants of the recombinant nuclease can edit a nucleic acid sequence by introducing a double-stranded or single-stranded break in a target polynucleotide.

[0207] In some embodiments, a heterologous polypeptide (e.g., and / or a complex comprising a heterologous polypeptide) comprising a recombinant nuclease can introduce double-strand breaks into a target polynucleotide, such as DNA. By introducing double-strand breaks into DNA, the DNA breaks can be repaired, allowing for the introduction of genetic modifications (e.g., nucleic acid editing). In some embodiments, the nuclease induces site-specific single-strand DNA breaks or nicks, allowing homologous recombination repair to occur.

[0208] By introducing double-strand breaks into DNA, DNA breaks can be repaired and genetic modifications (e.g., nucleic acid editing) can be introduced. DNA break repair can occur by non-homologous end joining (NHEJ) or homology-directed repair (HDR). In homology-directed repair, a donor DNA repair template or template polynucleotide can be provided that includes homologous arms that flank the target DNA site.

[0209] In some embodiments, a heterologous polypeptide comprising a recombinant nuclease (e.g., and / or a complex comprising a heterologous polypeptide) does not introduce a double-strand break into a target polynucleotide such as DNA. The heterologous polypeptide or a complex comprising the heterologous polypeptide (e.g., a complex comprising a nuclease-deficient variant of a recombinant nuclease and a guide RNA) can bind to a target polynucleotide without cleaving the nucleic acid, thereby sufficiently regulating (e.g., enhancing or suppressing) the expression of a target gene (e.g., an endogenous target gene).

[0210] Target gene The present disclosure provides compositions, methods, and systems for modulating the expression of one or more target genes. The target genes may be one or more endogenous target genes, for example, (i) disease-causing alleles (e.g., mutant alleles) and / or (ii) non-disease-causing alleles (e.g., wild-type alleles). For example, disclosed herein are one or more complexes comprising one or more heterologous polypeptides, including recombinant polypeptides disclosed herein (e.g., recombinant polypeptides comprising recombinant nucleases), that can modulate (e.g., increase or decrease) the activity level or expression amount of a target gene (e.g., in a cell), and a guide moiety. Such complexes comprising a guide moiety (e.g., a short guide RNA) and a recombinant nuclease can modulate the expression of the target gene by cleaving the target gene. Additionally or alternatively, such complexes may comprise a gene modulator operably linked (e.g., fused) to the recombinant nuclease, thereby modulating the expression of the target gene without cleaving the target gene. In some cases, increased expression of the target gene may be achieved by the gene modulator. Also, in some cases, a gene modulator may achieve a decrease in expression of a target gene. In some cases, a gene modulator (e.g., a gene editing moiety described herein) may edit a target gene, for example, to correct an undesired mutation in the target gene, thereby decreasing expression of the mutated gene and increasing expression of the corrected gene.

[0211] In some embodiments, the target gene or its regulatory sequence is endogenous to the cell, e.g., present in the genome of the cell, or endogenous to the subject, e.g., present in the genome of the subject. In some embodiments, the target gene or its regulatory sequence is not part of an engineered reporter system.

[0212] In some embodiments, the target gene is exogenous to the host subject, e.g., a pathogen-targeting gene or foreign gene is expressed as a result of therapeutic intervention, such as gene therapy and / or cell therapy. In some embodiments, the target gene is an exogenous reporter gene. In some embodiments, the target gene is an exogenous synthetic gene.

[0213] In some embodiments, the systems and methods disclosed herein can modulate (e.g., increase or decrease) the expression or duration of a target gene (e.g., by introducing a complex comprising a heterologous polypeptide into a cell or cell population). In some embodiments, the expression level is RNA expression level, which can be measured, for example, by RNAseq, qPCR, microarray, gene array, FISH, etc. In some embodiments, the expression level is protein expression level, which can be measured, for example, by Western blot, ELISA, multiplex immunoassay, mass spectrometry, NMR, proteomics, flow cytometry, mass cytometry, etc.

[0214] In some embodiments, the systems and methods disclosed herein can modulate (e.g., increase or decrease) the expression or duration of a target gene (e.g., by introducing a complex comprising 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, or at least about 11-fold. , at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, at least about 350-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1000-fold, at least about 1500-fold, at least about 2000-fold, or at least about 3000-fold.

[0215] In some embodiments, the systems and methods disclosed herein can modulate (e.g., increase or decrease) the expression or duration of a target gene (e.g., by introducing into a cell or cell population a complex comprising a heterologous polypeptide), the degree of modulation being 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 20-fold, up to about 22-fold, up to about 24-fold, up to about 26-fold, up to about 28-fold, up to about 29-fold, up to about 30-fold, up to about 31-fold, up to about 32-fold, up to about 33-fold, up to about 34-fold, up to about 35-fold, up to about 36-fold, up to about 37-fold, up to about 38-fold, up to about 39-fold, up to about 40-fold, up to about 41-fold, up to about 42-fold, up to about 43-fold, up to about 44-fold, up to about 45-fold, up to about 46-fold, up to about 47-fold, up to about 48-fold, up to about 49-fold, up to about 50-fold, up to about 51-fold, up to about 52-fold, up to about 53-fold, up to The amplification may be up to about 14 times, up to about 15 times, up to about 20 times, up to about 30 times, up to about 40 times, up to about 50 times, up to about 60 times, up to about 70 times, up to about 80 times, up to about 90 times, up to about 100 times, up to about 150 times, up to about 200 times, up to about 250 times, up to about 300 times, up to about 350 times, up to about 400 times, up to about 500 times, up to about 600 times, up to about 700 times, up to about 800 times, up to about 900 times, up to about 1000 times, up to about 1500 times, up to about 2000 times, up to about 3000 times, up to about 5000 times, or up to about 10000 times.

[0216] In some embodiments, the systems and methods disclosed herein can modulate (e.g., increase or decrease) the expression or duration of a target gene (e.g., by introducing a complex comprising a heterologous polypeptide into a cell or cell population), and the degree of modulation can be about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10 ... fold, about 9 fold, about 10 fold, about 11 fold, about 12 fold, about 13 fold, about 14 fold, about 15 fold, about 20 fold, about 30 fold, about 40 fold, about 50 fold, about 60 fold, about 70 fold, about 80 fold, about 90 fold, about 100 fold, about 150 fold, about 200 fold, about 250 fold, about 300 fold, about 350 fold, about 400 fold, about 500 fold, about 600 fold, about 700 fold, about 800 fold, about 900 fold, about 1000 fold, about 1500 fold, about 2000 fold, about 3000 fold, about 5000 fold, or about 10000 fold.

[0217] In some embodiments, the systems and methods disclosed herein can modulate (e.g., increase or decrease) the expression or duration of a target gene from below the limit of detection to detectable levels (e.g., by introducing a complex comprising a heterologous polypeptide into a cell or cell population).

[0218] In some embodiments, the degree of change in expression or its duration is relative to the state before introduction of a system of the present disclosure (e.g., a complex comprising a heterologous polypeptide) into the cell or cell population. In some embodiments, the degree of change in expression or its duration is relative to the state before introduction of a system of the present disclosure into the cell or cell population. In some embodiments, the degree of change in expression or its duration is relative to the state before introduction of a system of the present disclosure into the cell or cell population. In some embodiments, the degree of change in expression or its duration is relative to the state before introduction of a system of the present disclosure into the cell or cell population.

[0219] In some embodiments, the degree of change in expression or its duration is relative to a control nuclease. The control nuclease may comprise a native nuclease (e.g., (i) the polypeptide sequence of SEQ ID NO: 1, or (ii) the polypeptide sequence of any Cas protein selected from Table 2) or a variant thereof (e.g., a variant thereof having reduced nuclease activity operably linked to a gene modulator). For example, the control nuclease may be dCasMINI linked to (e.g., fused to) the same gene modulator as the recombinant nuclease disclosed herein.

[0220] In some embodiments, the systems and methods disclosed herein can modulate (e.g., increase or decrease) the activity level or duration of a target gene (e.g., by introducing into a cell or cell population a complex comprising a heterologous polypeptide comprising a recombinant nuclease disclosed herein). Depending on the functional nature of the target gene of interest, the activity level can be measured by a functional assay appropriate for the target gene. For example, the activity level of a mitogenic target gene can be measured by measuring cell proliferation; the activity level of a target gene that induces apoptosis can be measured by an Annexin V assay or other appropriate cell death assay; and the activity level of an anti-inflammatory cytokine can be measured by an LPS-induced cytokine release assay.

[0221] In some cases, the disclosed systems and methods can induce changes in the expression and / or activity levels of a target gene (e.g., an endogenous target gene) for a longer duration than can be achieved by alternative compositions and methods (e.g., RNAi-mediated suppression, e.g., suppression using siRNA). In some embodiments, sustained modulation of gene expression is advantageous compared to transient modulation.

[0222] In some embodiments, the systems and methods disclosed herein can modulate (e.g., increase or decrease) the expression and / or activity levels of a target gene over a period of time, wherein the duration of modulation is at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 18 hours, at least about 20 hours, at least about 1 day, at least about 2 days, at least about 3 days, The period may be at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 28 days, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 12 weeks, at least about 14 weeks, at least about 18 weeks, at least about 20 weeks, at least about 26 weeks, at least about 5 months, at least about 6 months, at least about 9 months, at least about 12 months or longer.

[0223] In some embodiments, the systems and methods disclosed herein can modulate (e.g., increase or decrease) the expression and / or activity level of a target gene (e.g., an endogenous target gene) above a particular threshold over a period of time, the duration of modulation being at or below about 1 hour, at or below about 2 hours, at or below about 3 hours, at or below about 4 hours, at or below about 5 hours, at or below about 6 hours, at or below about 7 hours, at or below about 8 hours, at or below about 9 hours, at or below about 10 hours, at or below about 12 hours, at or below about 14 hours, at or below about 18 hours, at or below about 20 hours, at or below about 1 day, at or below about 2 days, at or below about 3 days, at or below about 4 days, or at least about 4 days or less, at least about 5 days or less, at least about 6 days or less, at least about 7 days or less, at least about 8 days or less, at least about 9 days or less, at least about 10 days or less, at least about 14 days or less, at least about 21 days or less, at least about 28 days or less, at least about 5 weeks or less, at least about 6 weeks or less, at least about 7 weeks or less weeks or less, at least about 8 weeks or less, at least about 9 weeks or less, at least about 10 weeks or less, at least about 12 weeks or less, at least about 14 weeks or less, at least about 18 weeks or less, at least about 20 weeks or less, at least about 26 weeks or less, at least about 5 months or less, at least about 6 months or less, at least about 9 months or less,Or it may be at least about 12 months or less than about 12 months.

[0224] In some embodiments, the systems and methods disclosed herein can modulate (e.g., increase or decrease) the expression and / or activity level of a target gene (e.g., an endogenous target gene) above a particular threshold over a period of time, where the duration of modulation can be about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 14 hours, about 18 hours, about 20 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 14 days, about 21 days, about 28 days, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 12 weeks, about 14 weeks, about 18 weeks, about 20 weeks, about 26 weeks, about 5 months, about 6 months, about 9 months, or about 12 months.

[0225] In some embodiments, a recombinant polypeptide disclosed herein (e.g., a recombinant nuclease operably linked to a gene modulator) is capable of or can enhance regulation of a target gene compared to regulation of the target gene by a control polypeptide (e.g., a control nuclease operably linked to the same gene modulator). The control nuclease may be a naturally occurring nuclease (e.g., (i) the polypeptide sequence of SEQ ID NO: 1, or (ii) the polypeptide sequence of any Cas protein selected from Table 2), or a variant thereof (e.g., dCasMINI disclosed herein).

[0226] In some cases, enhanced target gene regulation may be characterized by a change in expression of the target gene that is greater than regulation by a control polypeptide. In some instances, such a change may result in an increase in expression of the target gene. The increased expression of the target gene by a recombinant polypeptide disclosed herein may be greater than the increased expression by a control polypeptide by at least about 1% or less, at least about 2% or less, at least about 3% or less, at least about 4% or less, at least about 5% or less, at least about 10% or less, at least about 15% or less, at least about 20% or less, at least about 30% or less, at least about 40% or less, at least about 50% or less. may be about 50% or less, at least at or about 60% or less, at least at or about 70% or less, at least at or about 80% or less, at least at or about 90% or less, at least at or about 100% or less, at least at or about 110% or less, at least at or about 120% or less, at least at or about 150% or less, at least at or about 200% or less, at least at or about 300% or less, at least at or about 400% or less, or at least at or about 500% or less.The expression level of a target gene increased by a recombinant polypeptide disclosed herein may be greater than the expression level increased by a control polypeptide, and the difference may be at least about 0.1 fold or less, at least about 0.2 fold or less, at least about 0.5 fold or less, at least about 1 fold or less, at least about 1.5 fold or less, at least about 2 fold or less, at least about 3 fold or less, or at least about 4 fold or less. The increase may be 4-fold or less, at least about 5-fold or less, at least about 6-fold or less, at least about 7-fold or less, at least about 8-fold or less, at least about 9-fold or less, at least about 10-fold or less, at least about 15-fold or less, at least about 20-fold or less, at least about 30-fold or less, at least about 40-fold or less, or at least about 100-fold or less. In some examples, such an alteration may result in a decrease (reduction) in the expression level of the target gene.The expression level of a target gene reduced by a recombinant polypeptide disclosed herein may be less than the expression level reduced by a control polypeptide, and the difference may be at least about 1% or less, at least about 2% or less, at least about 3% or less, at least about 4% or less, at least about 5% or less, at least about 10% or less, at least about 15% or less, at least about 20% or less, at least about 30% or less, at least about 40% or less, at least about 50% or less. may be about 50% or less, at least at or about 60% or less, at least at or about 70% or less, at least at or about 80% or less, at least at or about 90% or less, at least at or about 100% or less, at least at or about 110% or less, at least at or about 120% or less, at least at or about 150% or less, at least at or about 200% or less, at least at or about 300% or less, at least at or about 400% or less, or at least at or about 500% or less.The expression level of a target gene decreased (reduced) by a recombinant polypeptide disclosed herein may be less than the expression level decreased by a control polypeptide, and the difference may be at least about 0.1-fold or less, at least about 0.2-fold or less, at least about 0.5-fold or less, at least about 1-fold or less, at least about 1.5-fold or less, at least about 2-fold or less, at least about 3-fold or less, at least about 4-fold or less. may be about 4-fold or less, at least about 5-fold or less, at least about 6-fold or less, at least about 7-fold or less, at least about 8-fold or less, at least about 9-fold or less, at least about 10-fold or less, at least about 15-fold or less, at least about 20-fold or less, at least about 30-fold or less, at least about 40-fold or less, or at least about 100-fold or less.

[0227] In some cases, enhanced regulation of a target gene may be characterized by a change in expression level of the target gene (e.g., an increase or decrease in expression level above a particular threshold as disclosed herein) that lasts longer than the change in expression level caused by a control polypeptide. The change in expression level of the target gene caused by a recombinant polypeptide disclosed herein may last longer than the change in expression level caused by a control polypeptide, and the difference may be at least about 1% or less, at least about 2% or less, at least about 3% or less, at least about 4% or less, at least about 5% or less, at least about 10% or less, at least about 15% or less, at least about 20% or less, at least about 30% or less, at least about 40% or less, at least about 50% or less. may be about 50% or less, at least at or about 60% or less, at least at or about 70% or less, at least at or about 80% or less, at least at or about 90% or less, at least at or about 100% or less, at least at or about 110% or less, at least at or about 120% or less, at least at or about 150% or less, at least at or about 200% or less, at least at or about 300% or less, at least at or about 400% or less, or at least at or about 500% or less.The change in expression level of a target gene caused by a recombinant polypeptide disclosed herein may last longer than the change in expression level caused by a control polypeptide, and the difference may be at least about 0.1 fold or less, at least about 0.2 fold or less, at least about 0.5 fold or less, at least about 1 fold or less, at least about 1.5 fold or less, at least about 2 fold or less, at least about 3 fold or less, at least about 4 fold or less The increase may be 4-fold or less, at least about 5-fold or less, at least about 6-fold or less, at least about 7-fold or less, at least about 8-fold or less, at least about 9-fold or less, at least about 10-fold or less, at least about 15-fold or less, at least about 20-fold or less, at least about 30-fold or less, at least about 40-fold or less, or at least about 100-fold or less. For example, the threshold may be (i) a threshold for the expression level of the target gene before regulation is enhanced, (ii) a threshold for the maximum increase in the expression level of the target gene for activating the target gene or a portion thereof (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the maximum increase), or (iii) a maximum decrease in the expression level of the target gene for suppressing the target gene or a portion thereof (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the maximum decrease). In some embodiments, the target gene (e.g., an endogenous target gene) may be a disease-causing allele, e.g., a mutant variant of a wild-type allele. The disease may be a genetic disease (e.g., an inherited disease).Examples of genetic diseases include, but are not limited to, Duchenne muscular dystrophy (DMD), hemophilia, cystic fibrosis, Huntington's disease, familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, congenital hepatic porphyria, inherited liver metabolic disorders, Lesch-Nyhan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia-telangiectasia, Bloom's syndrome, retinoblastoma and Tay-Sachs disease.In some cases, the target gene may be a gene encoding a protein.In some cases, the target gene may be a gene regulatory sequence (e.g., promoter, enhancer, repressor, silencer, insulator, cis-regulatory element, trans-regulatory element, epigenetic modification site (e.g., DNA methylation site) etc.) that can affect the expression of the gene encoding the protein of interest provided herein. For example, the regulatory sequences of a target gene can be positioned physically outside the transcription unit or open reading frame that encodes the target gene product.

[0228] In some embodiments, the target gene regulatory sequence does not comprise a nucleotide sequence exogenous to the subject or host cell, hi some embodiments, the target gene regulatory sequence does not comprise a recombinant, artificially created, or introduced nucleotide sequence.

[0229] In some embodiments, the target gene (e.g., an endogenous target gene) is a gene that is overexpressed or underexpressed in a disease or condition. In some embodiments, the target gene is a gene that is overexpressed or underexpressed in an inherited genetic disease.

[0230] In some embodiments, the target gene (e.g., endogenous target gene) is a gene that is overexpressed or underexpressed in cancer, such as acute leukemia, astrocytoma, biliary tract cancer (cholangiocarcinoma), bone cancer, breast cancer, brainstem glioma, bronchioloalveolar cell lung cancer, adrenal gland cancer, anal region cancer, bladder cancer, endocrine system cancer, esophageal cancer, head and neck cancer, kidney cancer, parathyroid cancer, penile cancer, pleural / peritoneal cancer, salivary gland cancer, small intestine cancer, thyroid cancer, ureter cancer, urethral cancer, cervical cancer, endometrial cancer, fallopian tube cancer, renal pelvis cancer, vaginal cancer, vulvar cancer, cervical cancer, chronic leukemia, colon cancer, colorectal cancer, cutaneous melanoma, ependymoma, epidermoid tumor, Ewing's disease, sarcoma, gastric cancer, glioblastoma, glioblastoma multiforme, glioma, hematologic malignancies, hepatocellular (liver) carcinoma, hepatoma, Hodgkin's disease, intraocular melanoma, Kaposi's sarcoma, lung cancer, lymphoma, medulloblastoma, malignant melanoma, meningioma, mesothelioma, multiple myeloma, muscle cancer, central nervous system (CNS) neoplasms, neuronal carcinoma, small cell lung cancer, non-small cell lung cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pediatric malignancies, pituitary adenoma, prostate cancer, rectal cancer, renal cell carcinoma, soft tissue sarcoma, schwannoma, skin cancer, spinal axis tumor, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, uterine cancer, and tumors, as well as metastases and refractory forms of said cancers, and combinations thereof.

[0231] Non-limiting examples of target genes or genes encoding proteins of interest disclosed herein are shown in Table 1.

[0232] Guide nucleic acid molecule In some embodiments, the present disclosure provides a guide nucleic acid molecule (e.g., a recombinant guide nucleic acid molecule) configured to form a complex with a Cas protein. The Cas protein may be a naturally occurring protein. The Cas protein may be a recombinant nuclease variant provided herein. The guide nucleic acid molecule may include a spacer sequence that exhibits specific binding to a target polynucleotide sequence operably linked to a target gene (e.g., in a cell). The target polynucleotide sequence may be a portion of the target gene. Alternatively, the target polynucleotide sequence may be located upstream of the target gene (e.g., a portion of the promoter sequence of the target gene or adjacent to the promoter sequence of the target gene) or downstream of the target gene (e.g., a portion of the termination sequence of the target gene or adjacent to the termination sequence of the target gene). The guide nucleic acid molecule may include a scaffold sequence for forming a complex with the Cas protein. The spacer sequence and the scaffold sequence may be part of a single polynucleotide sequence (e.g., a single guide nucleic acid molecule such as an sgRNA). Alternatively, the spacer sequence and scaffold sequence may be separate molecules that can hybridize to form a complex with the Cas protein.

[0233] Without wishing to be bound by any theory, the guide nucleic acid molecules disclosed herein can be operably linked to (e.g., can form a functional complex with) one or more Cas proteins, including, but not limited to, Un1Cas12f1, a Cas protein selected from Table 2, and recombinant nuclease variants provided throughout this disclosure (e.g., the polypeptide of SEQ ID NO: 12).

[0234] In some embodiments, the scaffold sequence disclosed herein is not identical to the polynucleotide sequence of SEQ ID NO: 500. The scaffold sequence may comprise at least one deletion when compared to (e.g., when aligned with) the polynucleotide sequence of SEQ ID NO: 500. Without wishing to be bound by any theory, the at least one deletion in the scaffold sequence can be determined by performing a deletion landscape examination (e.g., recursive deletions and / or comprehensive deletions) of a control scaffold sequence of SEQ ID NO: 500. The scaffold sequence may comprise at least one mutation when compared to (e.g., when aligned with) the polynucleotide sequence of SEQ ID NO: 500. Without wishing to be bound by any theory, the at least one mutation in the scaffold sequence can be determined by performing a mutation landscape examination (e.g., recursive mutations and / or comprehensive mutations) of a control scaffold sequence of SEQ ID NO: 500. The at least one deletion disclosed herein may be a removal of a nucleotide. Alternatively, the at least one deletion may be a substitution (e.g., mutation) of a nucleotide with another type of nucleotide.

[0235] In some embodiments, the scaffold sequence, when aligned (or compared) to the reference polynucleotide sequence of SEQ ID NO: 500, may contain one or more nucleotide deletions. The one or more nucleotide deletions may be a single deletion. The one or more nucleotide deletions may be multiple nucleotide deletions, such as at least about 2 or less deletions, at least about 3 or less deletions, at least about 4 or less deletions, at least about 5 or less deletions, at least about 6 or less deletions, at least about 7 or less deletions, at least about 8 or less deletions, at least about 9 or less deletions, at least about 10 or less deletions, or the like. or about 10 deletions or less, at least about 11 or about 11 deletions, at least about 12 or about 12 deletions, at least about 13 or about 13 deletions, at least about 14 or about 14 deletions, at least about 15 or about 15 deletions, at least about 16 or about 16 deletions, at least about 17 or about 17 deletions, at least about 18 or about 18 deletions, at least about 19 or about 19 deletions , at least about 20 deletions or less, at least about 22 deletions or less, at least about 24 deletions or less, at least about 25 deletions or less, at least about 26 deletions or less, at least about 28 deletions or less, at least about 30 deletions or less, at least about 32 deletions or less, at least about 34 deletions or less, at least about 35 deletions or less or less than about 35 deletions, at least about 36 or less than about 36 deletions, at least about 38 or less than about 38 deletions, at least about 40 or less than about 40 deletions, at least about 42 or less than about 42 deletions, at least about 44 or less than about 44 deletions, at least about 45 or less than about 45 deletions, at least about 46 or less than about 46 deletions, at least about 48 or less than about 48 deletions, at least about 50 or less than about 50 deletions,The deletion may be at least about 52 or less, at least about 54 or less, at least about 55 or less, at least about 56 or less, at least about 58 or less, at least about 60 or less, at least about 70 or less, or at least about 80 or less. Such multiple nucleotide deletions may be adjacent to each other (e.g., contiguous) when aligned with the polynucleotide sequence of SEQ ID NO: 500. The scaffold sequence may comprise a single contiguous deletion. The scaffold sequence may comprise multiple contiguous deletions, and one contiguous deletion may not be immediately adjacent to another contiguous deletion when aligned with the polynucleotide sequence of SEQ ID NO: 500.

[0236] In some embodiments, the scaffold sequences disclosed herein, when aligned to a reference polynucleotide sequence of SEQ ID NO:500, may contain one or more nucleotide deletions at one or more positions selected from nucleotides 1-10, nucleotides 11-20, nucleotides 21-30, nucleotides 31-40, nucleotides 41-50, nucleotides 51-60, nucleotides 61-70, nucleotides 71-80, nucleotides 81-90, nucleotides 91-100, nucleotides 101-110, nucleotides 111-120, nucleotides 121-130, nucleotides 131-140, nucleotides 141-150, and / or nucleotides 151-159 of SEQ ID NO:500.

[0237] In some embodiments, a scaffold sequence disclosed herein, when aligned to a reference polynucleotide sequence of SEQ ID NO:500, may contain one or more nucleotide deletions between nucleotides 1 and 25 of the polynucleotide sequence of SEQ ID NO:500, for example, between nucleotides 1 and 23, between nucleotides 3 and 23, between nucleotides 5 and 23, between nucleotides 7 and 23, between nucleotides 9 and 23, between nucleotides 11 and 23, between nucleotides 13 and 23, between nucleotides 15 and 23, between nucleotides 17 and 23, between nucleotides 19 and 23, and / or between nucleotides 21 and 23 of the polynucleotide sequence of SEQ ID NO:500. The scaffold sequences disclosed herein, when aligned to the reference polynucleotide sequence of SEQ ID NO: 500, may comprise one or more nucleotide deletions at nucleotides 1-23, nucleotides 1-21, nucleotides 1-19, nucleotides 1-17, nucleotides 1-15, nucleotides 1-13, nucleotides 1-11, nucleotides 1-9, nucleotides 1-7, nucleotides 1-5, and / or nucleotides 1-3 of the polynucleotide sequence of SEQ ID NO: 500. The scaffold sequences disclosed herein, when aligned to the reference polynucleotide sequence of SEQ ID NO: 500, may comprise one or more nucleotide deletions at nucleotides 1-5, nucleotides 6-10, nucleotides 11-15, nucleotides 16-20, and / or nucleotides 21-23 of the polynucleotide sequence of SEQ ID NO: 500.The scaffold sequences disclosed herein, when aligned with the reference polynucleotide sequence of SEQ ID NO:500, may contain one or more nucleotide deletions at the first nucleotide, second nucleotide, third nucleotide, fourth nucleotide, fifth nucleotide, sixth nucleotide, seventh nucleotide, eighth nucleotide, ninth nucleotide, tenth nucleotide, eleventh nucleotide, twelfth nucleotide, thirteenth nucleotide, fourteenth nucleotide, fifteenth nucleotide, sixteenth nucleotide, seventeenth nucleotide, eighteenth nucleotide, nineteenth nucleotide, twentieth nucleotide, twenty-first nucleotide, twenty-second nucleotide, twenty-third nucleotide, twenty-fourth nucleotide, and / or twenty-fifth nucleotide of the polynucleotide sequence of SEQ ID NO:500.

[0238] In some embodiments, a scaffold sequence disclosed herein, when aligned to a reference polynucleotide sequence of SEQ ID NO:500, may contain one or more nucleotide deletions between 35 and 65 of the polynucleotide sequence of SEQ ID NO:500, for example, between 35 and 61, between 37 and 61, between 39 and 61, between 41 and 61, between 43 and 61, between 45 and 61, between 47 and 61, between 49 and 61, between 51 and 61, between 53 and 61, between 55 and 61, between 57 and 61, and / or between 59 and 61 of the polynucleotide sequence of SEQ ID NO:500. The scaffold sequences disclosed herein, when aligned with the reference polynucleotide sequence of SEQ ID NO:500, may contain one or more nucleotide deletions at nucleotides 35-61, nucleotides 35-59, nucleotides 35-57, nucleotides 35-55, nucleotides 35-53, nucleotides 35-51, nucleotides 35-49, nucleotides 35-47, nucleotides 35-45, nucleotides 35-43, nucleotides 35-41, nucleotides 35-39, and / or nucleotides 35-37 of the polynucleotide sequence of SEQ ID NO:500.The scaffold sequences disclosed herein, when aligned with the reference polynucleotide sequence of SEQ ID NO:500, may comprise one or more nucleotide deletions at the 35th nucleotide, 36th nucleotide, 37th nucleotide, 38th nucleotide, 39th nucleotide, 40th nucleotide, 41st nucleotide, 42nd nucleotide, 43rd nucleotide, 44th nucleotide, 45th nucleotide, 46th nucleotide, 47th nucleotide, 48th nucleotide, 49th nucleotide, 50th nucleotide, 51st nucleotide, 52nd nucleotide, 53rd nucleotide, 54th nucleotide, 55th nucleotide, 56th nucleotide, 57th nucleotide, 58th nucleotide, 59th nucleotide, 60th nucleotide, 61st nucleotide, 62nd nucleotide, 63rd nucleotide, 64th nucleotide, and / or 65th nucleotide of the polynucleotide sequence of SEQ ID NO:500.

[0239] In some embodiments, a scaffold sequence disclosed herein, when aligned to a reference polynucleotide sequence of SEQ ID NO:500, may contain one or more nucleotide deletions between nucleotides 135 and 150 of the polynucleotide sequence of SEQ ID NO:500, for example, one or more nucleotide deletions between nucleotides 136 and 149, between nucleotides 137 and 149, between nucleotides 139 and 149, between nucleotides 141 and 149, between nucleotides 143 and 149, between nucleotides 145 and 149, and / or between nucleotides 147 and 149 of the polynucleotide sequence of SEQ ID NO:500. The scaffold sequences disclosed herein, when aligned with the reference polynucleotide sequence of SEQ ID NO:500, may contain one or more nucleotide deletions at nucleotides 136-149, nucleotides 136-147, nucleotides 136-145, nucleotides 136-143, nucleotides 136-141, nucleotides 136-139, and / or nucleotides 136-137 of the polynucleotide sequence of SEQ ID NO:500. The scaffold sequences disclosed herein, when aligned with the reference polynucleotide sequence of SEQ ID NO:500, may contain one or more nucleotide deletions at nucleotide 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, and / or 150 of the polynucleotide sequence of SEQ ID NO:500.

[0240] In some embodiments, the scaffold sequences disclosed herein, when aligned to the reference polynucleotide sequence of SEQ ID NO: 500, may comprise one or more nucleotide deletions at nucleotides 136-151 of the polynucleotide sequence of SEQ ID NO: 500. In some instances, the scaffold sequences disclosed herein may comprise one or more nucleotide deletions at nucleotides T136, T137, C138, A139, T140, T141, T142, G143, A144, A145, T146, G147, A148, A149, G150 and / or G151 of the polynucleotide sequence of SEQ ID NO: 500. In some cases, the scaffold sequences disclosed herein may comprise one or more nucleotide deletions (e.g., deletion of at least 1, 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, at least 11, or all 12 of these) at nucleotide positions T136, T137, C138, A139, T140, T141, T142, A144, A145, T146, A148, and / or A149 of the polynucleotide sequence of SEQ ID NO: 500. In some cases, the scaffold sequences disclosed herein may comprise at least about one or no more than about one nucleotide, at least about two or no more than about two nucleotides, at least about three or no more than about three nucleotides, or all four nucleotides selected from the group consisting of G143, G147, G150, and G151 when aligned to the polynucleotide sequence of SEQ ID NO: 500.

[0241] In some embodiments, the scaffold sequences disclosed herein are not identical to the polynucleotide sequence of SEQ ID NO: 549 combined with SEQ ID NO: 550. For example, the polynucleotide of SEQ ID NO: 549 and the polynucleotide of SEQ ID NO: 550 may be linked to the 5' and 3' ends of a spacer sequence, respectively, and used as control sgRNA molecules for comparison with the activity of any of the scaffold sequences provided herein. In some embodiments, the scaffold sequences disclosed herein are not identical to the polynucleotide sequence of SEQ ID NO: 551 combined with SEQ ID NO: 552. For example, the polynucleotide of SEQ ID NO: 551 and the polynucleotide of SEQ ID NO: 552 may be linked to the 5' and 3' ends of a spacer sequence, respectively, and used as control sgRNA molecules for comparison with the activity of any of the scaffold sequences provided herein.

[0242] In some embodiments, the scaffold sequence (e.g., the contiguous polynucleotide sequence of the scaffold sequence) is at least about 60% or less, at least about 65% or less, at least about 70% or less, at least about 71% or less, at least about 72% or less, at least about 73% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 77% or less, at least about 78% or less, at least about 79% or less, at least about 80% or less, at least about 81% or less, at least about 82% or less, of the polynucleotide sequence of a member selected from Table 6B, Table 7B, and Table 8B. , at least about 83% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 87% or less, at least about 88% or less, at least about 89% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or substantially 100% sequence identity (or complementarity).

[0243] Optionally, the member can be selected from the group consisting of SEQ ID NOs: 503-152, 519, 524, 528, and 553. Optionally, the member can be selected from the group consisting of SEQ ID NOs: 555, 557, 558, 568, 569, 578, and 580. Optionally, the member can be selected from the group consisting of SEQ ID NOs: 555, 557, 568, 569, 576, 577, 578, 580, 593, 519, and 528.

[0244] In some cases, the length of the scaffold sequence is at least or not more than about 80 nucleotides in length, at least or not more than about 85 nucleotides in length, at least or not more than about 90 nucleotides in length, at least or not more than about 91 nucleotides in length, at least or not more than about 92 nucleotides in length, at least or not more than about 93 nucleotides in length, at least or not more than about 94 nucleotides in length, at least or not more than about 95 nucleotides in length, at least or not more than about 96 nucleotides in length, at least or not more than about 97 nucleotides in length, at least or not more than about 98 nucleotides in length, at least or not more than about 99 nucleotides in length, at least or not more than about 100 nucleotides in length, at least or not more than about 101 nucleotides in length, at least or not more than about 102 nucleotides in length, length of about 102 nucleotides or less, at least about or 103 nucleotides or less, at least about or 104 nucleotides or less, at least about or 105 nucleotides or less, at least about or 106 nucleotides or less, at least about or 107 nucleotides or less, at least about or 108 nucleotides or less, at least about or 109 nucleotides or less, at least about or 110 nucleotides or less, at least about or 112 nucleotides or less, at least about or 114 nucleotides or less, at least about or 115 nucleotides or less, at least about or 116 nucleotides or less, at least about or 118 nucleotides or less, at least about or 120 nucleotides or less,It may be at least about or less than 122 nucleotides in length, at least about or less than 124 nucleotides in length, at least about or less than 125 nucleotides in length, at least about or less than 126 nucleotides in length, at least about or less than 128 nucleotides in length, at least about or less than 130 nucleotides in length, at least about or less than 135 nucleotides in length, at least about or less than 140 nucleotides in length, at least about or less than 145 nucleotides in length, at least about or less than 150 nucleotides in length, at least about or less than 155 nucleotides in length, or at least about or less than 160 nucleotides in length.

[0245] In some embodiments, the scaffold sequence is at least about 60% or less, at least about 65% or less, at least about 70% or less, at least about 71% or less, at least about 72% or less, at least about 73% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 77% or less, at least about 78% or less, at least about 79% or less, at least about 80% or less, at least about 81% or less, at least about 82% or less, at least about 83% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 87% or less, at least about 88% or less, at least about 89% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, at least about 100% or less, at least about 101% or less, at least about 102% or less, at least about 103% or less, at least about 104% or less, at least about 105% or less, at least about 106% or less, at least about 107% or less, at least about 108% or less, at least about 109% or less, at least about 110% or less, at least about 111% or less, at least about 112% or less, at least about 113% or less, at least about 114% or less, at least about 115% or less, at least about 1 or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 87% or less, at least about 88% or less, at least about 89% or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or substantially 100% sequence identity (or complementarity).The length of the contiguous polynucleotide sequence of the scaffold sequence is at least about 15 nucleotides or less, at least about 16 nucleotides or less, at least about 17 nucleotides or less, at least about 18 nucleotides or less, at least about 19 nucleotides or less, at least about 20 nucleotides or less, at least about 21 nucleotides or less, at least about 22 nucleotides or less, at least about 23 nucleotides or less, at least about 24 nucleotides or less, at least about 25 nucleotides or less, at least about 26 nucleotides or less, or at least about 27 nucleotides or less. The contiguous polynucleotide sequence may be at most 10 nucleotides in length, at least about 28 nucleotides in length, at least about 29 nucleotides in length, at least about 30 nucleotides in length, at least about 31 nucleotides in length, at least about 32 nucleotides in length, at least about 33 nucleotides in length, at least about 34 nucleotides in length, at least about 35 nucleotides in length, at least about 36 nucleotides in length, at least about 37 nucleotides in length, at least about 38 nucleotides in length, at least about 39 nucleotides in length, or at least about 40 nucleotides in length. The contiguous polynucleotide sequence may be located at the N-terminus or C-terminus of the scaffold sequence.The contiguous polynucleotide sequence can be located in the N-terminal 50%, N-terminal 45%, N-terminal 40%, N-terminal 35%, N-terminal 30%, N-terminal 25%, N-terminal 20%, N-terminal 15%, or N-terminal 10% of the scaffold sequence. Alternatively, the contiguous polynucleotide sequence can be located in the C-terminal 50%, C-terminal 45%, C-terminal 40%, C-terminal 35%, C-terminal 30%, C-terminal 25%, C-terminal 20%, C-terminal 15%, or C-terminal 10% of the scaffold sequence. The contiguous polynucleotide sequence can be located between scaffold sequences.

[0246] In some embodiments, the length of the spacer sequence of the guide nucleic acid molecule can be at least or not more than about 12 nucleotides in length, at least or not more than about 13 nucleotides in length, at least or not more than about 14 nucleotides in length, at least or not more than about 15 nucleotides in length, at least or not more than about 16 nucleotides in length, at least or not more than about 17 nucleotides in length, at least or not more than about 18 nucleotides in length, at least or not more than about 19 nucleotides in length, at least or not more than about 20 nucleotides in length, at least or not more than about 21 nucleotides in length, or at least or not more than about 22 nucleotides in length.

[0247] In some embodiments, the length of the guide nucleic acid molecule is at least or not more than about 80 nucleotides in length, at least or not more than about 85 nucleotides in length, at least or not more than about 90 nucleotides in length, at least or not more than about 95 nucleotides in length, at least or not more than about 96 nucleotides in length, at least or not more than about 97 nucleotides in length, at least or not more than about 98 nucleotides in length, at least or not more than about 99 nucleotides in length, at least or not more than about 100 nucleotides in length, at least or not more than about 101 nucleotides in length, at least or not more than about 102 nucleotides in length, at least or not more than about 103 nucleotides in length, at least or not more than about 104 nucleotides in length, at least or not more than about 105 nucleotides in length, at least or not more than about 106 nucleotides in length. the length may be at or about 106 nucleotides or less, at least at or about 107 nucleotides or less, at least at or about 108 nucleotides or less, at least at or about 109 nucleotides or less, at least at or about 110 nucleotides or less, at least at or about 111 nucleotides or less, at least at or about 112 nucleotides or less, at least at or about 113 nucleotides or less, at least at or about 114 nucleotides or less, at least at or about 115 nucleotides or less, at least at or about 116 nucleotides or less, at least at or about 117 nucleotides or less, at least at or about 118 nucleotides or less, at least at or about 119 nucleotides or less, at least at or about 120 nucleotides or less,It may be at least about or less than 121 nucleotides in length, at least about or less than 122 nucleotides in length, at least about or less than 123 nucleotides in length, at least about or less than 124 nucleotides in length, at least about or less than 125 nucleotides in length, at least about or less than 130 nucleotides in length, at least about or less than 135 nucleotides in length, at least about or less than 140 nucleotides in length, at least about or less than 145 nucleotides in length, at least about or less than 150 nucleotides in length, at least about or less than 155 nucleotides in length, or at least about or less than 160 nucleotides in length.

[0248] In some examples, the contiguous polynucleotide sequence of the scaffold sequence may be at least about 20 nucleotides in length, at least about 25 nucleotides in length, or at least about 30 nucleotides in length, and such contiguous polynucleotide sequence may be (i) the N-terminal 30 base nucleotide sequence or (i) the N-terminal 30 base nucleotide sequence of a member selected from Table 6B, Table 7B, and Table 8B (e.g., one or more members selected from SEQ ID NOs: 555, 557, 568, 569, 576, 577, 578, 580, 593, 519, and 528). i) a polynucleotide sequence consisting of the nucleotide sequence of the C-terminal 30 bases, and a polynucleotide sequence comprising at least about 60% or less, at least about 65% or less, at least about 70% or less, at least about 71% or less, at least about 72% or less, at least about 73% or less, at least about 74% or less, at least about 75% or less, at least about 76% or less, at least about 77% or less, or at least at least about 78% or less, at least about 79% or less, at least about 80% or less, at least about 81% or less, at least about 82% or less, at least about 83% or less, at least about 84% or less, at least about 85% or less, at least about 86% or less, at least about 87% or less, at least about 88% or less, at least about 89% or less or less, at least about 90% or less, at least about 91% or less, at least about 92% or less, at least about 93% or less, at least about 94% or less, at least about 95% or less, at least about 96% or less, at least about 97% or less, at least about 98% or less, at least about 99% or less, or substantially 100% sequence identity (or complementarity).

[0249] Heterologous Polynucleotides In some embodiments, a target gene can be targeted by a system of the present disclosure (e.g., a system comprising a recombinant nuclease variant that retains at least some of its nuclease activity) to edit the target gene. In some cases, a complex comprising (i) a heterologous polypeptide comprising a recombinant nuclease disclosed herein and (ii) a guide nucleic acid (e.g., an sgRNA) can recognize and bind to a target sequence, e.g., at or near the target sequence of the complex within the target gene, to introduce a nick (single-strand break) or break (double-strand break) into the target gene. In some cases, the nick or break can be repaired by non-homologous end joining (NHEJ). In some cases, the nick or break can be repaired by homology-directed repair (HDR) or homologous recombination (HR) using a polynucleotide-modified template (e.g., a donor template, e.g., a donor DNA template). In some examples, a gene of interest can be inserted into a target gene, e.g., for gene replacement therapy, by providing a cell with a heterologous polynucleotide-modified template encoding the gene of interest.

[0250] In some embodiments, the systems and compositions of the present disclosure introduce a heterologous polynucleotide (e.g., a heterologous polynucleotide encoding a gene of interest, such as one or more genes selected from Table 1) into a cell without inserting it into the genome of the cell through the action of a recombinant nuclease of the present disclosure. In some cases, such a heterologous polynucleotide encoding a gene of interest can be inserted into the genome of the cell by other means, for example, by an adeno-associated viral vector (e.g., AAV2 or AAV8). Alternatively, such a heterologous polynucleotide encoding a gene of interest may be introduced into an intracellular portion of the cell without being integrated into the chromosome (e.g., as a non-chromosomally integrated plasmid).

[0251] Thus, the systems and compositions of the present disclosure may include a non-disease causing wild-type target gene or a variant thereof, as described above. Additionally or alternatively, the systems and compositions of the present disclosure may include a heterologous polynucleotide sequence that encodes (or includes) at least a non-disease causing wild-type target gene (e.g., an endogenous target gene) or a variant thereof, as disclosed herein.

[0252] composition In some embodiments, the present disclosure provides a composition comprising at least a portion of the system described herein for use in any of the methods disclosed herein, for example, a composition comprising (i) a heterologous polypeptide comprising a recombinant nuclease, or a heterologous polynucleotide encoding the heterologous polypeptide, and / or (ii) a guide nucleic acid, or a heterologous polynucleotide encoding the guide nucleic acid, as disclosed herein. The composition of the present disclosure can be used to modify cells in vitro, ex vivo, or in vivo. The composition of the present disclosure can be used to treat or improve a condition of a subject as disclosed herein.

[0253] The compositions disclosed herein may include active ingredients (e.g., heterologous polypeptides, including recombinant nucleases and guide nucleic acids, etc.) and, optionally, additional ingredients (e.g., additives). If necessary and / or desirable, the compositions of the present disclosure can be divided, formed, and / or packaged into desired single or multiple dose units, or desired single or multiple implant units.

[0254] In some embodiments, the compositions of the present disclosure may contain one or more heterologous polynucleotides encoding the active ingredients disclosed herein. When the active ingredients include different members, each member may be encoded by a different heterologous polynucleotide. Alternatively, two or more (e.g., all) active ingredients may be encoded by a single heterologous polynucleotide. In some cases, the single heterologous polynucleotide encodes (i) a heterologous polypeptide comprising a recombinant nuclease (e.g., a fusion protein of dCas and a transcription effector, e.g., dCas-KRAB, dCas-DNMT, dCas-ADA, etc.) and (ii) one or more guide nucleic acids (e.g., at least one, at least two, at least three, at least four, at least five, or more guide nucleic acids) for targeting specific regions or sequences of a target gene.

[0255] The one or more heterologous polynucleotides may further comprise one or more promoters (or one or more transcriptional regulators; as used herein, "promoter" and "transcriptional regulator" are used interchangeably). Different active ingredients encoded by the one or more heterologous polynucleotides may be under the control of the same promoter or different promoters. The promoters disclosed herein may be active in eukaryotic cells, mammalian cells, non-human mammalian cells, or human cells. The promoter may be an inducible promoter or a constitutively active promoter. Additionally or alternatively, the promoter may be a tissue-specific promoter or a cell-specific promoter. Examples of suitable eukaryotic promoters (i.e., promoters functional in eukaryotic cells) include, but are not limited to, the cytomegalovirus (CMV) immediate-early promoter, herpes simplex virus (HSV) thymidine kinase promoter, SV40 early and late promoters, promoters derived from retroviral long terminal repeats (LTRs), the human elongation factor 1 promoter (EF1), a hybrid construct containing the cytomegalovirus (CMV) enhancer fused to the chicken β-actin promoter (CAG), the mouse stem cell virus promoter (MSCV), the phosphoglycerate kinase 1 promoter (PGK), and the mouse metallothionein-I promoter. The promoter may also be a fungal promoter. The promoter may also be a plant promoter. Databases of plant promoters are known (e.g., PlantProm). The expression vector may further contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector may further contain appropriate sequences for amplifying expression. In some cases, the promoters disclosed herein may be promoters specific to any of the tissues provided herein, or promoters specific to any of the cell types provided herein.

[0256] The size of one heterologous polynucleotide (e.g., the single heterologous polynucleotide) among the one or more heterologous polynucleotides is at least about 2.5 kb or less, at least about 2.6 kb or less, at least about 2.7 kb or less, at least about 2.8 kb or less, at least about 2.9 kb or less, at least about 3.0 kb or less, at least about 3.1 kb or less. 3.1 kb or less, at least about 3.2 kb or less, at least about 3.3 kb or less, at least about 3.4 kb or less, at least about 3.5 kb or less, at least about 3.6 kb or less, at least about 3.7 kb or less, at least about 3.8 kb or less, at least about 3.9 kb or less, at least about 4.0 kb or less or less, at least about 4.1 kb or less, at least about 4.2 kb or less, at least about 4.3 kb or less, at least about 4.4 kb or less, at least about 4.5 kb or less, at least about 4.6 kb or less, at least about 4.7 kb or less, at least about 4.8 kb or less, at least about 4.9 kb or less, It may be at least about 5.0 kb or less, at least about 5.5 kb or less, at least about 6.0 kb or less, at least about 6.5 kb or less, at least about 7.0 kb or less, at least about 7.5 kb or less, at least about 8.0 kb or less, at least about 9.0 kb or less, or at least about 10 kb or less.In some cases, the size of one heterologous polynucleotide (e.g., the single heterologous polynucleotide) among the one or more heterologous polynucleotides is about 3 kb to about 5 kb, about 3 kb to about 4.8 kb, about 3 kb to about 4.6 kb, about 3 kb to about 4.4 kb, about 3 kb to about 4.2 kb, about 3 kb to about 4.0 kb, about 3 kb to about 3.5 kb, about 3.5 kb to about 5 kb, about 3.5 kb to about 4.8 kb, about 3 kb to about 5 ... It may be about 0.5kb to about 4.6kb, about 3.5kb to about 4.4kb, about 3.5kb to about 4.2kb, about 3.5kb to about 4kb, about 4kb to about 5kb, about 4kb to about 4.9kb, about 4kb to about 4.8kb, about 4kb to about 4.7kb, about 4kb to about 4.6kb, about 4kb to about 4.5kb, about 4kb to about 4.4kb, about 4kb to about 4.3kb, about 4kb to about 4.2kb, or about 4kb to about 4.1kb.

[0257] The vector (or expression cassette) may encode at least (i) a Cas protein and (ii) a guide nucleic acid molecule comprising a spacer sequence and a scaffold sequence as provided herein. The vector may include a first polynucleotide sequence encoding the Cas protein, a second polynucleotide sequence encoding the scaffold sequence, and / or a third polynucleotide sequence encoding the scaffold sequence.The combined length of the first polynucleotide sequence and the second polynucleotide sequence is at least about 1400 nucleotides or less, at least about 1420 nucleotides or less, at least about 1440 nucleotides or less, at least about 1450 nucleotides or less, at least about 1460 nucleotides or less, at least about 1480 nucleotides or less, at least about 1500 nucleotides or less, at least about 1520 nucleotides or less, at least about 1540 nucleotides or less, at least about 1550 nucleotides or less, at least about 1560 nucleotides or less may be at least about 1560 nucleotides or less, at least about or 1580 nucleotides or less, at least about or 1600 nucleotides or less, at least about or 1620 nucleotides or less, at least about or 1640 nucleotides or less, at least about or 1650 nucleotides or less, at least about or 1660 nucleotides or less, at least about or 1680 nucleotides or less, at least about or 1700 nucleotides or less, at least about or 1720 nucleotides or less, at least about or 1740 nucleotides or less, or at least about or 1750 nucleotides or less in length. In some embodiments, the total length of the first polynucleotide sequence plus the length of the second polynucleotide sequence may be less than 1746 nucleotides in length, less than 1737 nucleotides in length, or less than 1720 nucleotides in length.

[0258] In some embodiments, the length of the first polynucleotide sequence is at least or not more than about 1400 nucleotides in length, at least or not more than about 1420 nucleotides in length, at least or not more than about 1440 nucleotides in length, at least or not more than about 1450 nucleotides in length, at least or not more than about 1460 nucleotides in length, at least or not more than about 1480 nucleotides in length, at least or not more than about 1500 nucleotides in length, at least or not more than about 1520 nucleotides in length, at least or not more than about 1540 nucleotides in length, at least or not more than about 1 It may be 550 or less nucleotides in length, at least about or less than 1560 nucleotides in length, at least about or less than 1580 nucleotides in length, at least about or less than 1600 nucleotides in length, at least about or less than 1620 nucleotides in length, at least about or less than 1640 nucleotides in length, at least about or less than 1650 nucleotides in length, at least about or less than 1660 nucleotides in length, at least about or less than 1680 nucleotides in length, or at least about or less than 1700 nucleotides in length.

[0259] In some embodiments, the length of the second polynucleotide sequence is at least or not more than about 80 nucleotides in length, at least or not more than about 85 nucleotides in length, at least or not more than about 90 nucleotides in length, at least or not more than about 91 nucleotides in length, at least or not more than about 92 nucleotides in length, at least or not more than about 93 nucleotides in length, at least or not more than about 94 nucleotides in length, at least or not more than about 95 nucleotides in length, at least or not more than about 96 nucleotides in length, at least or not more than about 97 nucleotides in length, at least or not more than about 98 nucleotides in length, at least or not more than about 99 nucleotides in length, at least or not more than about 100 nucleotides in length, at least or not more than about 101 nucleotides in length, at least or not more than about 102 nucleotides in length. at least about 102 nucleotides in length, at least about 103 nucleotides in length, at least about 104 nucleotides in length, at least about 105 nucleotides in length, at least about 106 nucleotides in length, at least about 107 nucleotides in length, at least about 108 nucleotides in length, at least about 109 nucleotides in length, at least about 110 nucleotides in length, at least about 112 nucleotides in length, at least about 114 nucleotides in length, at least about 115 nucleotides in length, at least about 116 nucleotides in length, at least about 118 nucleotides in length, at least about 120 nucleotides in lengthIt may be at least about or less than 122 nucleotides in length, at least about or less than 124 nucleotides in length, at least about or less than 125 nucleotides in length, at least about or less than 126 nucleotides in length, at least about or less than 128 nucleotides in length, at least about or less than 130 nucleotides in length, at least about or less than 135 nucleotides in length, at least about or less than 140 nucleotides in length, at least about or less than 145 nucleotides in length, at least about or less than 150 nucleotides in length, at least about or less than 155 nucleotides in length, or at least about or less than 160 nucleotides in length.

[0260] In some embodiments, a vector encoding at least a Cas protein and a guide nucleic acid molecule may be (i) small / compact, and / or (ii) the combined length of the first polynucleotide sequence and the second polynucleotide sequence may be sufficiently short to provide sufficient space for additional cargo (e.g., a gene modulator operably linked to a Cas protein or a heterologous polynucleotide provided herein). Even if the vector is small / compact in size, a complex comprising a Cas protein and a guide nucleic acid molecule encoded by the vector may still be functional. In some cases, the complex encoded by the vector may be functionally active to bind to a target polynucleotide sequence and edit at least a portion of the target polynucleotide sequence (e.g., cleavage, nucleotide deletion, nucleotide addition, base editing, etc.). In some cases, the complex encoded by the vector may be functionally active to modulate the expression level of a target gene in a cell. Therefore, (A1) the expression level of the target gene regulated by the complex (and, in addition, or alternatively, its activity level) may be comparable to or higher than the expression level regulated by (A2) a control complex comprising the Cas protein and a control guide nucleic acid molecule.

[0261] In some cases, the expression level of the target gene regulated by (A1) said complex may be similar to (A2), such that (A1) does not differ from (A2) by more than 50%, more than 45%, more than 40%, more than 35%, more than 30%, more than 25%, more than 20%, more than 15%, more than 10%, more than 8%, more than 6%, more than 5%, more than 4%, more than 3%, more than 2%, or more than 1%.

[0262] In some cases, the expression level of a target gene can be activated by a recombinant nuclease variant and / or guide nucleic acid molecule disclosed herein, and the expression level of the target gene regulated by the complex (A1) may be higher than that of (A2), and the expression level of (A1) may be at least about 1% or at most about 1%, at least about 2% or at most about 2%, at least about 5% or at most about 5%, at least about 10% or at most about 10%, at least about 15% or at most about 15%, at least about 20% or at most about 20% higher than that of (A2). at most about 20%, at least about 30% or at most about 30%, at least about 40% or at most about 40%, at least about 50% or at most about 50%, at least about 60% or at most about 60%, at least about 70% or at most about 70%, at least about 80% or at most about 80%, at least about 90% or at most about 90%, at least about 100% or at most about 100%, at least about 150% or at most about 150%, at least about 200% or at most about 200%, at least about 250% or at most about 250%, The expression level of (A1) may be at least about 300% or at most about 300%, at least about 350% or at most about 350%, at least about 400% or at most about 400%, at least about 450% or at most about 450%, or at least about 500% or at most about 500%, or the expression level of (A1) may be at least about 0.1 times or at most about 0.1 times, at least about 0.2 times or at most about 0.2 times, at least about 0.3 times or at most about 0.3 times, at least about 0.4 times or at most about 0.4 times, at least about 0.5 times or at most about 0.5 times, at least about 0.6 times or at most about 0.7 times, at least about 0.8 times or at most about 0.9 times, at least about 0.9 ... may be at least about 0.5 times or at most about 0.5 times higher, at least about 1 times or at most about 1 times, at least about 1.5 times or at most about 1.5 times, at least about 2 times or at most about 2 times, at least about 3 times or at most about 3 times, at least about 4 times or at most about 4 times, at least about 5 times or at most about 5 times, at least about 6 times or at most about 6 times, at least about 7 times or at most about 7 times, at least about 8 times or at most about 8 times, at least about 9 times or at most about 9 times, or at least about 10 times or at most about 10 times higher.

[0263] In some cases, the expression level of a target gene can be reduced (e.g., suppressed) by a recombinant nuclease variant and / or guide nucleic acid molecule disclosed herein, and the expression level of the target gene regulated by the complex (A1) may be lower than that of (A2), and the expression level of (A1) may be at least about 1% or at most about 1%, at least about 2% or at most about 2%, at least about 5% or at most about 5%, at least about 10% or at most about 10%, at least about 15% or at most about 15%, at least about 20% or at most about 20 ... % or at most about 20%, at least about 30% or at most about 30%, at least about 40% or at most about 40%, at least about 50% or at most about 50%, at least about 60% or at most about 60%, at least about 70% or at most about 70%, at least about 80% or at most about 80%, at least about 90% or at most about 90%, at least about 100% or at most about 100%, at least about 150% or at most about 150%, at least about 200% or at most about 200%, at least about 250% or at most about 2 or the expression level of (A1) may be at least about 0.1 times or at most about 0.1 times, at least about 0.2 times or at most about 0.2 times, at least about 0.3 times or at most about 0.3 times, at least about 0.4 times or at most about 0.4 times, or at least about 0.5 times or at most about 0.5 ... It may be at least about 0.5 times lower or at most about 0.5 times lower, at least about 1 time lower or at most about 1 time lower, at least about 1.5 times lower or at most about 1.5 times lower, at least about 2 times lower or at most about 2 times lower, at least about 3 times lower or at most about 3 times lower, at least about 4 times lower or at most about 4 times lower, at least about 5 times lower or at most about 5 times lower, at least about 6 times lower or at most about 6 times lower, at least about 7 times lower or at most about 7 times lower, at least about 8 times lower or at most about 8 times lower, at least about 9 times lower or at most about 9 times lower, or at least about 10 times lower or at most about 10 times lower.

[0264] In some cases, the control guide nucleic acid molecule may be longer than the guide nucleic acid molecule encoded by the vector disclosed herein. The control scaffold sequence of the control guide nucleic acid molecule may be longer than the scaffold sequence of the guide nucleic acid molecule encoded by the vector disclosed herein, with the difference being at least or less than about 1 nucleotide, at least or less than about 2 nucleotides, at least or less than about 5 nucleotides, at least or less than about 10 nucleotides, at least or less than about 15 nucleotides, at least or less than about 20 nucleotides, at least or less than about 25 nucleotides, at least or less than about 30 nucleotides, at least or less than about 30 nucleotides. The length of the control guide nucleic acid molecule may be about or less than 35 nucleotides, at least about or less than 40 nucleotides, at least about or less than 45 nucleotides, at least about or less than 50 nucleotides, at least about or less than 55 nucleotides, at least about or less than 60 nucleotides, at least about or less than 65 nucleotides, at least about or less than 70 nucleotides, at least about or less than 75 nucleotides, or at least about or less than 80 nucleotides. For example, the control guide nucleic acid molecule may comprise the polypeptide sequence of SEQ ID NO:10.

[0265] The method provided herein for delivering one or more heterologous polynucleotides to cells can include viral or non-viral delivery methods.Therefore, the one or more heterologous polynucleotides provided herein can be one or more viral vectors (for example, one or more AAV vectors).Alternatively, the one or more heterologous polynucleotides provided herein can be non-viral vectors that are complexed with non-viral delivery moieties, such as cationic lipids and / or lipid particles (for example, lipid nanoparticles (LNP)), or non-viral vectors that are enclosed in non-viral delivery moieties.

[0266] Non-viral nucleic acid delivery methods include lipofection, nucleofection, microinjection, particle bombardment, virosomes, liposomes, immunoliposomes, polycation:nucleic acid complexes, lipid:nucleic acid complexes, naked DNA, artificial virions, and drug-enhanced DNA uptake. Cationic lipids and neutral lipids suitable for receptor-recognizing lipofection can also be used to efficiently introduce polynucleotides. Delivery can be to cells (e.g., in vitro or ex vivo administration) or to target tissues (e.g., in vivo administration).

[0267] RNA or DNA virus-based systems can be used to target specific cells in vivo and deliver the viral payload to the cell nucleus. Viral vectors can be administered directly (in vivo), or cells can be treated with the viral vector in vitro and the resulting modified cells can be administered (ex vivo). Viral systems include retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex virus vectors for gene transfer. Gene transfer methods using retroviral, lentiviral, or adeno-associated viruses can result in integration into the host genome, resulting in long-term expression of the inserted transgene. High transduction efficiency can be achieved in many different cell types and target tissues.

[0268] The tropism of retroviruses can be modified by incorporating foreign envelope proteins, thereby expanding the target cell population. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and produce high viral titers. The choice of retroviral gene transfer system depends on the target tissue type. Retroviral vectors may contain cis-acting long terminal repeats and can package foreign sequences up to 6-10 kb in length. This cis-acting minimal LTR may be sufficient for vector replication and packaging and can be used to integrate therapeutic genes into target cells to permanently express the transgene. Retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof.

[0269] Adenovirus-based systems can also be used. Adenovirus-based systems can induce transient expression of transgenes. Adenovirus-based vectors can achieve high transduction efficiency in cells, and cell division may not be required. Also, high titers and high expression levels can be obtained by using adenovirus-based vectors. Adeno-associated virus ("AAV") vectors can be used to transduce target nucleic acids into cells, for example, to produce nucleic acids and peptides in vitro, or to perform gene therapy treatments in vivo or ex vivo.

[0270] Packaging cells can be used to generate viral particles capable of infecting host cells. Examples of such cells include 293 cells (e.g., for packaging adenovirus) and Psi2 or PA317 cells (e.g., for packaging retrovirus). Viral vectors can be generated by engineering cell lines capable of packaging nucleic acid vectors into viral particles. The vector may contain the minimal viral sequences required for packaging and subsequent integration into the host. The vector may contain additional viral sequences substituted with an expression cassette encoding the polynucleotide to be expressed. Missing viral functions can be supplied in trans by the packaging cell line. For example, an AAV vector may contain ITR sequences from the AAV genome, which are required for packaging and integration into the host genome. Viral DNA can be packaged in cell lines that lack ITR sequences but may contain helper plasmids encoding other AAV genes (i.e., rep and cap). The cell lines can also be infected with adenovirus as a helper. Helper viruses can promote AAV vector replication and AAV gene expression from helper plasmids. Adenovirus contamination can be suppressed, for example, by heat treatment, to which adenovirus is more sensitive than AAV.

[0271] One or more vectors described herein can be transiently or non-transiently transfected into host cells. Cell transfection can occur naturally in a subject. Cells can be harvested from a subject or can be used to transfect cells derived from a subject. Cells (e.g., cell lines) can also be derived from cells harvested from a subject. In some embodiments, cells transfected with one or more vectors described herein are used to establish novel cell lines comprising one or more sequences from the vectors. In some embodiments, cells are modified via the activity of a heterologous polypeptide comprising a recombinant nuclease disclosed herein by transiently transfecting the disclosed composition into cells (e.g., by transient transfection of one or more vectors, or transient transfection of RNA, etc.), and the modified cells are used to establish novel cell lines comprising cells comprising the modification but without other exogenous sequences.

[0272] Any suitable vector compatible with the host cell can be used in the methods of the present disclosure. Examples of vectors for eukaryotic host cells include pXT1, pSG5 (Stratagene), and pXT2 (Stratagene). TM ), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia TM ), but are not limited to these.

[0273] In some embodiments, the additional components of the compositions disclosed herein may include additives. Examples of additives include, but are not limited to, solvents, dispersion media, diluents, or other liquid media; dispersants or suspending agents; surfactants; isotonicity agents; thickening agents or emulsifiers; preservatives, lipidoids, liposomes, lipid nanoparticles, polymers, lipoflex, core-shell nanoparticles, peptides, proteins, hyaluronidase, nanoparticle mimics, inert diluents, buffers, lubricants, oils, and combinations thereof. In some examples, the compositions disclosed herein may include one or more additives in an amount that can improve the stability of (i) a heterologous polypeptide or a heterologous gene encoding the same, and / or (ii) cells or modified cells.

[0274] In some embodiments, the present disclosure provides kits comprising such compositions and instructions for (i) contacting cells with the composition (e.g., in vitro, ex vivo, or in vivo) or (ii) administering cells comprising any one of the compositions disclosed herein to a subject, who may have or be suspected of having a condition such as a genetic disease.

[0275] In some embodiments, the compositions disclosed herein can be administered to a subject by oral administration, intraperitoneal administration, intravenous administration, intraarterial administration, transdermal administration, intramuscular administration, liposome-mediated administration, local delivery by catheter or stent, subcutaneous administration, intraadipose administration, or intrathecal administration.

[0276] The compositions (e.g., pharmaceutical compositions) disclosed herein may be suitable for administration to humans. Furthermore, such compositions may be suitable for administration to other animals, such as, for example, non-human animals, e.g., non-human mammals. Modifications of pharmaceutical compositions suitable for administration to humans to make them suitable for administration to various animals are well known, and those skilled in veterinary pharmacology can design and / or implement such modifications, if necessary, with no more than routine experimentation. Administration of the pharmaceutical compositions of the present disclosure is contemplated to include, but is not limited to, humans and / or other primates; mammals, including commercially available mammals such as cows, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, including commercially available birds such as poultry, chickens, ducks, geese, and / or turkeys.

[0277] cell In some embodiments, cells provided herein may be referred to as "target cells." In some embodiments, the systems, compositions, and methods provided herein can be used to modify target cells (e.g., to modify the expression profile of a target gene in a target cell, e.g., to modify the expression profile of one or more genes listed in Table 1). Target cells can include various types of cells. A target cell can be an in vitro cell. A target cell can be an in vivo cell. A target cell can be an ex vivo cell. A target cell can be an isolated cell. A target cell can be a cell within an organism. A target cell can be an organism. A target cell can be a cell in cell culture. A target cell can be a cell population. A target cell can be a mammalian cell or derived from a mammalian cell. A target cell can be a rodent cell or derived from a rodent cell. A target cell can be a human cell or derived from a human cell. A target cell can be a prokaryotic cell or derived from a prokaryotic cell. The target cell may be a bacterial cell or may be derived from a bacterial cell. The target cell may be an archaeal cell or may be derived from an archaeal cell. The target cell may be a eukaryotic cell or may be derived from a eukaryotic cell. The target cell may be a pluripotent stem cell. The target cell may be a plant cell or may be derived from a plant cell. The target cell may be an animal cell or may be derived from an animal cell. The target cell may be an invertebrate cell or may be derived from an invertebrate cell. The target cell may be a vertebrate cell or may be derived from a vertebrate cell. The target cell may be a microbial cell or may be derived from a microbial cell. The target cell may be a fungal cell or may be derived from a fungal cell. The target cell may be obtained from a specific organ or tissue.

[0278] The target cell may be a stem cell or progenitor cell. The target cell may be a stem cell (e.g., adult stem cell, embryonic stem cell, induced pluripotent stem (iPS) cell) or a progenitor cell (e.g., cardiac progenitor cell, neural progenitor cell, etc.). The target cell may be a mammalian stem cell or progenitor cell, such as a rodent stem cell, a rodent progenitor cell, a human stem cell, a human progenitor cell, etc. A clonal cell may be a progeny cell of a cell. The target cell may comprise a target nucleic acid. The target cell may be a living organism. The target cell may be a genetically modified cell. The target cell may be a host cell.

[0279] The target cell can be a primary cell. For example, the primary cell culture can be passaged 0, 1, 2, 4, 5, 10, 15 or more times. The cell can be a single-cell organism. The cell can be grown in culture.

[0280] The target cells may be diseased cells. Diseased cells may have altered metabolism, gene expression, and / or morphological characteristics. Diseased cells may be cancer cells, diabetic cells, or apoptotic cells. Diseased cells may be cells obtained from a subject suffering from a disease. Exemplary diseases include blood disorders, cancer, metabolic disorders, eye disorders, organ disorders, musculoskeletal disorders, heart diseases, etc.

[0281] When the target cells are primary cells, the primary cells may be obtained from an individual by any method. For example, leukocytes may be obtained by apheresis, leukapheresis, density gradient separation, etc. Cells obtained from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestinal tract, stomach, etc. may be obtained by biopsy.

[0282] Non-limiting examples of cells that can be target cells include lymphoid cells such as B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, helper T cells), natural killer cells, and cytokine-induced killer (CIK) cells; myeloid cells such as granulocytes (basophilic granulocytes, eosinophilic granulocytes, neutrophilic granulocytes / hypersegmented neutrophils), monocytes / macrophages, erythrocytes (reticulocytes), mast cells, platelets / megakaryocytes, and dendritic cells; thyroid cells (thyroid epithelial cells, parafollicular cells), parathyroid cells (chief parathyroid cells, eosinophilic cells), Endocrine system cells such as adrenal cells (chromaffin cells) and pineal cells; nervous system cells such as glial cells (astrocytes, microglia), magnocellular neurosecretory cells, astrocytes, Boettcher cells, and pituitary cells (gonadotropes, corticotropes, thyrotropes, growth hormones, and prolactin-producing cells); respiratory system cells such as alveolar epithelial cells (type I alveolar epithelial cells, type II alveolar epithelial cells), Clara cells, goblet cells, and dust cells; circulatory system cells such as cardiac myocytes and pericytes; stomach (gastric chief cells, Digestive system cells such as parietal cells, goblet cells, Paneth cells, G cells, D cells, ECL cells, I cells, K cells, and S cells; enteroendocrine cells such as enterochromaffin cells, APUD cells, and liver cells (hepatocytes, Kupffer cells); cartilage / bone / muscle; bone cells such as osteoblasts, osteocytes, osteoclasts, and teeth (cementoblasts, ameloblasts); cartilage cells such as chondrocytes and chondrocytes; skin cells such as hair follicles, keratinocytes, and melanocytes (nevus cells); muscle cells such as myocytes; podocytes, juxtaglomerular cells, intraglomerular mesangial cells / extraglomerular mesangial cells urinary system cells such as uterine lining cells, renal proximal tubule brush border cells, macula densa cells; reproductive system cells such as sperm, Sertoli cells, Leydig cells, and eggs; and other cells, such as adipocytes, fibroblasts, tendon cells, epidermal keratinocytes (differentiated epidermal cells), epidermal basal cells (stem cells), fingernail and toenail keratinocytes, nail bed basal cells (stem cells), medullary hair stem cells, cortical hair stem cells, epidermal hair stem cells, epidermal root sheath cells, root sheath cells of Huxley's layer, root sheath cells of Henle's layer, outer root sheath cells, hair matrix cells (stem cells), wet stratified barrier epithelial cells; cornea, tongue, oral cavity, esophagus, anal canal,Surface epithelial cells of the stratified squamous epithelium of the distal urethra and vagina; basal cells (stem cells) of the epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina; ureteral epithelial cells (lining the bladder and ureters), exocrine epithelial cells, salivary gland mucous cells (secreting polysaccharides), salivary gland serous cells (secreting glycoprotein enzymes), von Ebner's gland cells of the tongue (cleansing the taste buds), mammary gland cells (secreting milk), lacrimal gland cells (secreting tears), earwax gland cells of the ear (secreting earwax), eccrine sweat gland dark cells (secreting glycoproteins), eccrine sweat gland clear cells (secreting small molecules), apocrine sweat gland cells (secreting fragrance) , sex hormone sensitive), Moll's gland cells in the eyelids (specialized sweat glands), sebaceous gland cells (secreting lipid-rich sebum), Bowman's gland cells in the nasal cavity (cleansing the olfactory epithelium), Brunner's gland cells in the duodenum (enzymes and alkaline mucus), seminal vesicle cells (secreting seminal plasma components including fructose for sperm swimming), prostate cells (secreting seminal plasma components), bulbourethral gland cells (secreting mucus), Bartholin's gland cells (secreting vaginal fluid), Littré gland cells (secreting mucus), endometrial cells (secreting sugars), isolated goblet cells in the respiratory and digestive tract (secreting mucus), stomach lining mucous cells (secreting mucus), gastric gland zymogen cells ( Pepsinogen secretion), gastric oxyntic cells (secreting hydrochloric acid), pancreatic acinar cells (secreting bicarbonate and digestive enzymes), Paneth cells of the small intestine (secreting lysozyme), type II alveolar epithelial cells of the lung (secreting surfactant), Clara cells of the lung, hormone-secreting cells, anterior pituitary cells, growth hormone-secreting cells, lactotrophs, thyrotrophs, gonadotrophs, corticotrophs, intermediate pituitary cells, magnocellular neurosecretory cells, cells of the intestinal and respiratory tracts, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, chief parathyroid cells, acidophils Sex cells, adrenal gland cells, chromaffin cells, Leydig cells of the testes, theca cells of the ovarian follicles, lutein cells of ruptured follicles, granulosa lutein cells, theca lutein cells, juxtaglomerular cells (secreting renin), macula densa cells of the kidney, metabolic and storage cells, barrier function cells (lungs, intestines, exocrine glands and urogenital tract), kidney cells, type I alveolar epithelial cells (lining the alveolar lumen), pancreatic duct cells (cardiac atrial cells), unstriated duct cells (sweat glands, salivary glands, mammary glands, etc.), duct cells (seminal vesicles and prostate), epithelial cells lining the closed body cavities, ciliated cells with the function of extruding foreign bodies, extracellular matrix secreting cells, contractile cells, skeletal muscle cells,Stem cells, cardiomyocytes, blood and immune system cells, erythrocytes, megakaryocytes (platelet precursor cells), monocytes, connective tissue macrophages (various types), epidermal Langerhans cells, osteoclasts (of bone), dendritic cells (of lymphoid tissue), microglia (of the central nervous system), neutrophilic granulocytes, eosinophilic granulocytes, basophilic granulocytes, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, reticulocytes, stem cells and committed progenitor cells of the blood and immune system (various types) These include, but are not limited to, pluripotent stem cells, totipotent stem cells, induced pluripotent stem cells, adult stem cells, sensory transducer cells, autonomic nervous system neuronal cells, support cells of sensory organs and peripheral neurons, central nervous system neurons and glial cells, lens cells, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, oogonia / oocytes, spermatids, spermatocytes, spermatogonia (stem cells of spermatocytes), sperm, trophoblasts, follicle cells, Sertoli cells (of the testes), thymic epithelial cells, interstitial cells, and interstitial kidney cells.

[0283] After the cells (or target cells) have been engineered to contain (or express) any one of the systems or compositions disclosed herein, or after treatment in vitro or ex vivo with any one of the methods disclosed herein, they can be administered to a subject, e.g., for treatment of a condition in the subject. For example, a recombinant cell product of the present disclosure can be administered to a subject to treat a condition in the subject's body tissue. In some cases, the cells may be present inside the subject's body, and the cells can be contacted with (e.g., recombined with) a system / composition of the present disclosure by administering the system or composition thereof to the subject. [Example]

[0284] Example 1: Recombinant nucleases

[0285] The Cas protein encoded by the polypeptide sequence of SEQ ID NO: 1 is 529 amino acid residues in length. For example, when a gene encoding a Cas protein is delivered into a vector, such as a viral vector (e.g., an AAV vector), reducing the size of such a Cas protein can increase the cargo space within the vector (e.g., within a viral vector with cargo size or length limitations). By increasing the cargo space within the vector, it is possible to deliver (e.g., encode) at least one additional component (e.g., one or more heterologous gene effectors, one or more guide nucleic acid molecules, one or more cDNAs for therapeutic gene delivery, etc.) to achieve a desired result (e.g., a therapeutic effect). Additionally or alternatively, when the recombinant Cas protein disclosed herein is delivered encapsulated within a delivery vehicle (e.g., a lipid nanoparticle, a viral capsid, etc.), reducing the size of the Cas protein can increase the cargo space, for example, to accommodate at least one additional component, thereby achieving a desired result. Without wishing to be bound by any theory, the use of a recombinant nuclease disclosed herein in conjunction with at least one additional component can enhance the activity of the at least one additional component (e.g., binding, cleaving, editing and / or regulating a target gene) compared to the use of a control nuclease that is different from the recombinant nuclease disclosed herein.

[0286] In some embodiments, and throughout the examples of this disclosure, one or more recombinant nucleases of this disclosure can be evaluated (e.g., in vitro) to assess the activity of the one or more recombinant nucleases in binding, cleaving, and / or editing a target polynucleotide sequence, thereby modulating the expression level and / or activity level of a polypeptide (e.g., protein) encoded by or operably linked to the target polynucleotide sequence. In some examples, the efficiency of gene knockout in a target polynucleotide can be assessed by testing in cells (e.g., in vitro) a heterologous polynucleotide comprising a guide nucleic acid (e.g., sgRNA) for the target polynucleotide and a heterologous polypeptide comprising a recombinant nuclease of this disclosure. In some examples, a heterologous polypeptide comprising a nuclease-deficient variant of a recombinant nuclease of the present disclosure linked (e.g., fused) to a gene effector (e.g., a gene activator or gene repressor) heterologous to the recombinant nuclease of the present disclosure, and a heterologous polynucleotide comprising a guide nucleic acid (e.g., an sgRNA) for a target polynucleotide, can be tested in cells (e.g., in vitro) to assess its ability to modulate the expression and / or activity level of a gene linked to (or contained in) the target polynucleotide.

[0287] In some embodiments, throughout the examples of this disclosure, a library containing multiple recombinant nuclease candidates can be generated by amplifying the entire length of the plasmid using opposing primers in the deleted region.

[0288] Example 2: Recombinant nucleases based on structural comparison

[0289] A. Method

[0290] In some embodiments, the size (e.g., number of amino acid residues) of a CasMini or inactive CasMini (dCasMini) (e.g., a mutant variant of the polypeptide sequence of SEQ ID NO: 1) can be further reduced while retaining or enhancing its activity (e.g., overall epigenetic gene regulatory activity). For example, using another Cas protein smaller than SEQ ID NO: 1 (e.g., a naturally occurring Cas12f protein, e.g., Un2Cas12f1 (SEQ ID NO: 2) or AsCas12f (SEQ ID NO: 3)) as a reference, one can determine, for example, at least one amino acid residue and / or at least one tertiary structure of the Cas protein of SEQ ID NO: 1 that can be modified (e.g., deleted) with minimal or no loss of activity.

[0291] In some embodiments, the native Un1Cas12f1 nuclease encoded by SEQ ID NO:1 can be recombined with at least one deletion to generate the recombinant nucleases disclosed herein, for example, for one or more of the reasons described in Example 1.

[0292] In some cases, at least one deletion in the amino acid sequence of the recombinant nuclease may be present in one or more regions of the native Un1Cas12f1 nuclease that do not structurally align with AsCas12f (SEQ ID NO: 3). Un1Cas12f1 may contain an additional domain consisting of amino acid residues 1-71 compared to AsCas12f. For example, at least a portion of this domain and / or one or more amino acid residues near this domain may be removed without significantly reducing or minimizing the activity of the recombinant nuclease (e.g., interaction with a guide nucleic acid molecule (e.g., crRNA)). As shown in Figure 1, Un1Cas12f1 contains domain 110 (e.g., including at least a portion of this additional domain consisting of amino acid residues 1-71), which may not be conserved in AsCas12f. Meanwhile, Un1Cas12f1 contains domain 120, which may be conserved in AsCas12f.

[0293] In some cases, at least one deletion in the amino acid sequence of the recombinant nuclease may be present in one or more regions of the native Un1Cas12f1 nuclease that do not structurally align with Un2Cas12f1 (SEQ ID NO: 2). Un1Cas12f1 may contain an additional domain consisting of amino acid residues 41-71 compared to Un2Cas12f1. For example, at least a portion of this domain and / or one or more amino acid residues near this domain may be removed without significantly reducing or minimizing the activity of the recombinant nuclease (e.g., its interaction with a guide nucleic acid molecule (e.g., crRNA)). As shown in Figure 2, Un1Cas12f1 contains domain 210 (e.g., including at least a portion of this additional domain consisting of amino acid residues 41-71), which may not be conserved in Un2Cas12f1. Meanwhile, Un1Cas12f1 contains domain 220, which may be conserved in Un2Cas12f1.

[0294] B. Exemplary Library Design

[0295] Figure 3A shows a schematic representation of various regions of Un1Cas12f1 encoded by SEQ ID NO:1, and provides examples of domains (310, 320, and 330) that can be at least partially deleted in generating one or more recombinant nucleases disclosed herein. For example, domain 310 can exhibit an N-terminal deletion (e.g., deletion of amino acid residues 2-76 when aligned with SEQ ID NO:1), allowing for the generation and evaluation of 25 variants by deleting three amino acid residues from the N-terminus of the nuclease. In another example, domain 320 may exhibit a partial deletion at the N-terminus (e.g., deletion of amino acid residues 41 to 71 when aligned with SEQ ID NO: 1), and by deleting two amino acid residues at a time from the center of this region, 16 variants can be generated and evaluated (e.g., del55-56, del54-57, del54-58, del53-59, del52-60, del51-61, del50-62, del49-63, del48-64, del47-65, del46-66, del45-67, del44-68, del43-69, del42-70, del41-71, etc.). In another example, domain 330 may exhibit a C-terminal deletion (e.g., deletion of the last 75 amino acid residues when aligned with SEQ ID NO: 1), and 25 variants can be generated and evaluated by deleting 3 amino acid residues from the C-terminus.

[0296] Examples of recombinant nucleases with reduced nuclease activity produced in accordance with the present disclosure include SEQ ID NOs: 4-9 provided herein.

[0297] SEQ ID NO: 4 (including an N-terminal deletion, such as deletion of amino acid residues 2 to 21 (AKNTITKTLKLRIVRPYNSA) when aligned with SEQ ID NO: 1) JPEG2025507822000008.jpg73150

[0298] SEQ ID NO: 5 (including an N-terminal deletion, such as deletion of amino acid residues 2 to 31 (AKNTITKTLKLRIVRPYNSAEVEKIVADEK) when aligned with SEQ ID NO: 1) JPEG2025507822000009.jpg65149

[0299] SEQ ID NO: 6 (including a C-terminal deletion, such as deletion of amino acid residues 510 to 529 (DYNAALNISNPKLKSTKEEP) when aligned with SEQ ID NO: 1) JPEG2025507822000010.jpg72152

[0300] SEQ ID NO: 7 (when aligned with SEQ ID NO: 1, includes a C-terminal deletion, such as deletion of amino acid residues 500 to 529 (CEKCNFKENADYNAALNISNPKLKSTKEEP)) JPEG2025507822000011.jpg66151

[0301] SEQ ID NO: 8 (when aligned with SEQ ID NO: 1, includes a partial deletion at the N-terminus, such as the deletion of amino acid residues 47 to 66 (KEACSKHLKVAAYCTTQVER)) JPEG2025507822000012.jpg72151

[0302] SEQ ID NO: 9 (when aligned with SEQ ID NO: 1, includes a partial deletion at the N-terminus, such as the deletion of amino acid residues 41 to 71 (KNKDKVKEACSKHLKVAAYCTTQVERNACLF)) JPEG2025507822000013.jpg65151

[0303] Example 3: Recombinant nucleases based on deletion landscapes

[0304] The native Un1Cas12f1 nuclease encoded by SEQ ID NO: 1 can be recombined with at least one deletion to generate a recombinant nuclease of the present disclosure, for example, for one or more of the reasons described in Example 1. The deletion of at least one amino acid sequence of a recombinant nuclease of the present disclosure, as disclosed herein, can be determined based on the deletion landscape (or cleavage landscape) of the Cas protein (or a nuclease activity-deficient variant thereof).

[0305] For example, as shown in Figure 4, mapping of the cleavage landscape of dCas9 suggests tolerance of significant deletions, such as the deletion of amino acid residues 167-316 of dCas9. Therefore, by utilizing similar deletion landscapes, it is possible to determine one or more amino acid residues (e.g., multiple contiguous amino acid residues) in native Un1Cas12f1 (or its nuclease activity-deficient variants) that can be deleted to create deletion mutants of the Un1Cas12f1 protein without significantly affecting (e.g., affecting) the activity of native Un1Cas12f1. In some cases, approximately 20 amino acid residues can be deleted (e.g., from one contiguous region or two discontinuous regions) to create mutant variants of Un1Cas12f1 that retain comparable activity (or exhibit enhanced activity) compared to wild-type Un1Cas12f1. In some cases, small deletion windows (e.g., windows of about 3 amino acid residues to about 5 amino acid residues) can be evaluated across the entire length of the native Un1Cas12f1 protein for the deletion landscape.

[0306] Figure 3B shows a schematic representation of the deletion landscape method for generating recombinant nuclease variants of Un1Cas12f1. By scanning at least a portion of Un1Cas12f1 (e.g., the entire length of Un1Cas12f1 excluding the dimerization domain and / or PAM domain) using deletion tiles 510 consisting of multiple amino acid residues (e.g., multiple consecutive amino acid residues, e.g., 5 consecutive amino acid residues), up to 95 variants can be generated and individually screened for transfection.

[0307] Example 4: Recombinant nucleases based on deletion landscapes

[0308] In addition to deleting one or more amino acid residues from the native Un1Cas12fl nuclease encoded by SEQ ID NO: 1 to generate a recombinant nuclease, recombinant nucleases of the present disclosure can be further modified by mutating one or more amino acid residues of the native Un1Cas12fl nuclease, for example, for one or more of the reasons described in Example 1. Deletion of at least one amino acid sequence of a recombinant nuclease of the present disclosure, as disclosed herein, can be determined based on site-saturation mutagenesis (SSM). For example, a single codon or set of codons in a polynucleotide encoding a native Un1Cas12fl protein (or a nuclease activity-deficient variant thereof) can be substituted with one or more substitutable amino acids at sites of enhanced activity (e.g., sites of improved epigenetic regulation, e.g., sites of enhanced activation / repression, additional PAM recognition sites, etc.), enhanced stability, enhanced expression, enhanced binding to each guide nucleic acid molecule, etc.

[0309] Example 5: Evaluation of recombinant nucleases

[0310] Nuclease-defective variants of the recombinant nucleases disclosed herein (e.g., those prepared according to Examples 1-4) can be linked (e.g., fused) to heterologous gene effectors (e.g., VP16, VP64, p65, Rta, VPR, etc.), and one or more heterologous polypeptides and / or guide RNAs encoding such fusion proteins can be transfected into cells to assess activation of target gene expression / activity levels. For example, recombinant HEK93T cells carrying synthetic reporters can be used, and fluorescence activation can be measured as a readout. If multiple recombinant nucleases are identified, screening methods (e.g., using recombinant HEK93T cells) can be performed repeatedly to (i) confirm previous screening results and / or (ii) identify top hits.

[0311] Based on the results of the first round of recombination, one or more additional recombinant nucleases can be generated by combining multiple deletions of amino acid residues (e.g., when aligned with the polypeptide sequence of SEQ ID NO: 1). Additionally or alternatively, one or more additional recombinant nucleases can be generated by evaluating one or more more precise deletions in one or more lead nucleases obtained from the first round of recombination.

[0312] A. First recombination

[0313] Multiple sequence alignments of dCasMINI (SEQ ID NO: 10) were generated against one or more naturally occurring Cas12f protein orthologs with reported bacterial nuclease activity (e.g., Un1Cas12f1, Un2Cas12f1, AsCas12f, and other orthologs listed in Table 2) to identify one or more mutations that may be beneficial for generating recombinant nuclease variants, e.g., as disclosed herein. Furthermore, homology modeling of Cas12f orthologs was performed to identify structural conservation (e.g., based in part on amino acid conservation) and to generate recombinant nuclease variants. By combining these two methods (e.g., sequence alignment and structural conservation analysis), various amino acid sequence regions and combinations of individual residue mutations were identified as promising candidates for generating recombinant nuclease variants. Based on the identified information, various chimeric protein variants were designed by sequence swapping, and various mutant variants were designed by mutating one or more residues. In some cases, variants generated by sequence swapping al...

Claims

1. A recombinant polypeptide comprising a recombinant nuclease, wherein the recombinant nuclease comprises the amino acid sequence set forth in SEQ ID NO: 132, 134, 135, 140 or 146.

2. 2. The recombinant polypeptide of claim 1, wherein the recombinant nuclease consists of the amino acid sequence set forth in SEQ ID NO: 132, 134, 135, 140 or 146.

3. 2. The recombinant polypeptide of claim 1, wherein the recombinant nuclease comprises the amino acid sequence set forth in SEQ ID NO:

132.

4. 2. The recombinant polypeptide of claim 1, wherein the recombinant nuclease comprises the amino acid sequence set forth in SEQ ID NO:

134.

5. 2. The recombinant polypeptide of claim 1, wherein the recombinant nuclease comprises the amino acid sequence set forth in SEQ ID NO:

135.

6. 2. The recombinant polypeptide of claim 1, wherein the recombinant nuclease comprises the amino acid sequence set forth in SEQ ID NO:

140.

7. 2. The recombinant polypeptide of claim 1, wherein the recombinant nuclease comprises the amino acid sequence set forth in SEQ ID NO:

146.

8. 2. The recombinant polypeptide of claim 1, wherein the recombinant nuclease consists of the amino acid sequence set forth in SEQ ID NO:

132.

9. 2. The recombinant polypeptide of claim 1, wherein the recombinant nuclease consists of the amino acid sequence set forth in SEQ ID NO:

134.

10. 2. The recombinant polypeptide of claim 1, wherein the recombinant nuclease consists of the amino acid sequence set forth in SEQ ID NO:

135.

11. 2. The recombinant polypeptide of claim 1, wherein the recombinant nuclease consists of the amino acid sequence set forth in SEQ ID NO:

140.

12. 2. The recombinant polypeptide of claim 1, wherein the recombinant nuclease consists of the amino acid sequence set forth in SEQ ID NO:

146.

13. 2. The recombinant polypeptide of claim 1, wherein the recombinant nuclease is up to 495 amino acids in length.

14. A recombinant polypeptide comprising a recombinant nuclease, wherein the recombinant nuclease comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence set forth in SEQ ID NO: 132, 134, 135, 140 or 146.

15. 15. The recombinant polypeptide of claim 14, wherein the recombinant nuclease comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence set forth in SEQ ID NO:

132.

16. 15. The recombinant polypeptide of claim 14, wherein the recombinant nuclease comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence set forth in SEQ ID NO:

134.

17. 15. The recombinant polypeptide of claim 14, wherein the recombinant nuclease comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence set forth in SEQ ID NO:

135.

18. 15. The recombinant polypeptide of claim 14, wherein the recombinant nuclease comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence set forth in SEQ ID NO:

140.

19. 15. The recombinant polypeptide of claim 14, wherein the recombinant nuclease comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence set forth in SEQ ID NO:

146.

20. 20. The recombinant polypeptide of any one of claims 14 to 19, wherein the recombinant nuclease is up to 495 amino acids in length.