Engineered nucleases, compositions and methods of use thereof

Abstract

The present disclosure provides one or more engineered nuclease enzymes, and systems, compositions, and methods thereof, wherein the one or more engineered nuclease enzymes can be used to effect binding, cleavage, and / or editing of a target polynucleotide sequence. The one or more engineered nuclease enzymes may be engineered variants of the small CRISPR / Cas protein.

Description

Cross-references

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 315,159 filed on March 1, 2022, U.S. Provisional Application No. 63 / 380,178 filed on October 19, 2022, and U.S. Provisional Application No. 63 / 385,171 filed on November 28, 2022, which are incorporated herein by reference in their entirety. Sequence Listing Incorporated by Reference

[0002] This application is filed with a sequence listing in electronic format. The sequence listing is provided as a 426 kilobyte file named 55176-726_601_SL.XML created on February 15, 2023. The information in the electronic sequence listing is incorporated by reference in its entirety. Background Art

[0003] A variety of 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 heterologous nucleases can achieve such editing or regulation of target genes alone or together with other agents. For example, clustered regularly spaced short palindromic repeats (CRISPR) / CRISPR-associated proteins (Cas) are nuclease families that participate in specific binding, cutting and / or editing of target deoxyribonucleic acid (DNA) sequences or ribonucleic acid (RNA) sequences (e.g., foreign DNA sequences or RNA sequences). The programmable nature of these nucleases makes it possible to use them as a general technology, which has completely changed the field of target gene manipulation, for example, as gene therapy to treat or improve the subject's condition (e.g., disease). Summary of the invention

[0004] A variety of nucleases, such as CRISPR / Cas proteins (e.g., Cas12f proteins used to date) can have a smaller size than Cas9 or Cas12a. However, the size of the nuclease may not be small enough to package it with at least one additional agent (e.g., guide RNA, transgene encoding therapeutic polynucleotides or proteins, etc.) in a delivery mode (e.g., a viral vector, such as an adeno-associated virus (AAV) vector). Therefore, various aspects of the present disclosure, for example, provide engineered nucleases that are smaller but effectively bind, cut and / or edit target polynucleotide sequences, compositions thereof, and methods of use thereof.

[0005] Aspects of the present disclosure provide engineered polypeptides comprising an engineered nuclease, wherein the engineered nuclease comprises an amino acid sequence at least 80% identical to the polypeptide sequence of SEQ ID NO: 1, wherein the amino acid sequence comprises at least one deletion from amino acid residues 2-100 compared to the polypeptide sequence of SEQ ID NO: 1.

[0006] Another aspect of the present disclosure provides an engineered polypeptide comprising an engineered nuclease, wherein the engineered nuclease comprises an amino acid sequence that is more than 92% identical to the polypeptide sequence of SEQ ID NO:12.

[0007] Another aspect of the present disclosure provides an engineered polypeptide comprising an engineered nuclease variant, wherein the engineered nuclease variant comprises an amino acid sequence at least 70% identical to the polypeptide sequence of SEQ ID NO: 12, wherein the amino acid sequence of SEQ ID NO: Compared with the polypeptide sequence of NO:1, the amino acid sequence comprises a modification, wherein the modification includes 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, L522I, and at least one deletion of amino acid residues 400-529 from SEQ ID NO:1.

[0008] Another aspect of the present disclosure provides an engineered polypeptide comprising an engineered nuclease variant, wherein the engineered nuclease variant is a chimeric polypeptide, comprising: a first polypeptide sequence, the first polypeptide sequence comprising at least 3 consecutive amino acid residues in common with a first Cas protein; and a second polypeptide sequence, the second polypeptide sequence comprising at least 3 consecutive amino acid residues in common with a second Cas protein, wherein the second Cas protein is different from the first Cas protein, wherein the first Cas protein comprises an amino acid sequence at least 80% identical to the polypeptide sequence of SEQ ID NO:1.

[0009] Another aspect of the present disclosure provides an engineered polypeptide comprising an engineered nuclease variant, wherein the engineered nuclease is a chimeric polypeptide comprising: a first polypeptide sequence (CP1), the first polypeptide sequence comprising at least 3 consecutive amino acid residues in common with a portion of a first Cas protein; a second polypeptide sequence (CP2), the second polypeptide sequence comprising at least 3 consecutive amino acid residues in common with a portion of a second Cas protein different from the first Cas protein; and a third polypeptide sequence (CPx), the third polypeptide sequence comprising at least 3 consecutive amino acid residues in common with: (i) an additional portion of the first Cas protein, wherein the portion of the first Cas protein and the additional portion are not directly adjacent to each other in the first Cas protein; (ii) an additional portion of the second Cas protein, wherein the portion of the second Cas protein and the additional portion are not directly adjacent to each other in the second Cas protein; or (iii) a portion of a third Cas protein different from the first Cas protein and the second Cas protein, wherein the length of the chimeric polypeptide is less than or equal to about 1,000 amino acids.

[0010] Another aspect of the present disclosure provides an engineered polypeptide comprising an engineered nuclease variant, wherein the engineered nuclease variant is a chimeric polypeptide comprising: a first polypeptide, the first polypeptide comprising at least 3 consecutive amino acid residues in common with a first Cas protein; and a second polypeptide, the second polypeptide comprising at least 3 consecutive amino acid residues in common with a second Cas protein, wherein the second Cas protein is different from the first Cas protein, wherein the length of the second polypeptide sequence is about 20% less than the length of the first polypeptide sequence.

[0011] Another aspect of the present disclosure provides an engineered polypeptide comprising an engineered nuclease variant, wherein the engineered nuclease variant: (i) comprises an amino acid sequence at least 80% identical to a polypeptide sequence of a member selected from Table 3B, Table 4B or Table 5C; (ii) is not any of SEQ ID NO: 1-SEQ ID NO: 3, SEQ ID NO: 10 and SEQ ID NO: 13-SEQ ID NO: 19; and (iii) has a length of less than or equal to about 800 amino acids.

[0012] Another aspect of the present disclosure provides an engineered polypeptide comprising an engineered nuclease variant operably coupled to a gene regulatory factor, wherein the engineered nuclease variant: (i) comprises an amino acid sequence that is at least 70% identical to the polypeptide sequence of SEQ ID NO: 1; and (ii) when operably coupled to the gene regulatory factor, induces enhanced regulation of a target gene in a cell as compared to that induced by a control engineered polypeptide comprising SEQ ID NO: 10 operably coupled to the gene regulatory factor.

[0013] Another aspect of the present disclosure provides a method of controlling a target gene in a cell, the method comprising contacting the cell with any one of the engineered polypeptides disclosed herein.

[0014] Another aspect of the present disclosure provides a method for regulating a target gene in a cell, the method comprising: contacting the cell with an engineered polypeptide comprising an engineered nuclease variant operably coupled to a gene regulatory factor, wherein the engineered nuclease variant comprises an amino acid sequence at least 70% identical to the polypeptide sequence of SEQ ID NO: 1, wherein the contacting results in enhanced regulation of the target gene in the cell as compared to that caused by a control engineered polypeptide comprising SEQ ID NO: 10 operably coupled to the gene regulatory factor.

[0015] Another aspect of the present disclosure provides a composition comprising a guide nucleic acid molecule configured to form a complex with a Cas protein, wherein the guide nucleic acid molecule comprises:

[0016] Another aspect of the present disclosure provides a composition comprising a guide nucleic acid molecule, wherein the guide nucleic acid molecule is configured to form a complex with a Cas protein, wherein the guide nucleic acid molecule comprises: a spacer sequence, wherein the spacer sequence exhibits specific binding to a target polynucleotide sequence; and a scaffold sequence, wherein the scaffold sequence is used to form a complex with the Cas protein, wherein the scaffold sequence is characterized in that: (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) a contiguous polynucleotide sequence having at least 97% sequence identity to the polynucleotide sequence of SEQ ID NO: 557; or a contiguous polynucleotide sequence having at least 88% sequence identity to the polynucleotide sequence of SEQ ID NO: 598; (iii) a contiguous polynucleotide sequence having at least 90% sequence identity to the polynucleotide sequence of SEQ ID NO: 578; a contiguous polynucleotide sequence having at least 84% sequence identity to the polynucleotide sequence of SEQ ID NO: 597; or a contiguous polynucleotide sequence having at least 81% sequence identity to the polynucleotide sequence of SEQ ID NO: 599; (iv) a contiguous polynucleotide sequence having at least 93% sequence identity to the polynucleotide sequence of SEQ ID NO:568; a contiguous polynucleotide sequence having at least 84% sequence identity to the polynucleotide sequence of SEQ ID NO:597; or a contiguous polynucleotide sequence having at least 67% sequence identity to the polynucleotide sequence of SEQ ID NO:600; or (v) a contiguous polynucleotide sequence having at least 95% sequence identity with the polynucleotide sequence of SEQ ID NO:569; a contiguous polynucleotide sequence having at least 84% sequence identity with the polynucleotide sequence of SEQ ID NO:597; or a contiguous polynucleotide sequence having at least 71% sequence identity with the polynucleotide sequence of SEQ ID NO:601.

[0017] Another aspect of the present disclosure provides a composition comprising a guide nucleic acid molecule configured to form a complex with a Cas protein, wherein the guide nucleic acid molecule comprises: a spacer sequence, the spacer sequence exhibiting specific binding to a target polynucleotide sequence operably coupled to a target gene; and a scaffold sequence, the scaffold sequence being used to form a complex with the Cas protein, wherein 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, wherein the scaffold sequence is different from SEQ ID NO: 500, wherein binding of the complex to the target polynucleotide sequence in a cell results in a regulated expression level of the target gene in the cell, wherein (A1) the regulated expression level of the target gene caused by the complex is comparable to or better than (A2) the regulated expression level caused by a control complex comprising the Cas protein and a control guide nucleic acid molecule containing a polynucleotide sequence of SEQ ID NO: 500.

[0018] Another aspect of the present disclosure provides a composition comprising a guide nucleic acid molecule, which is configured to form a complex with a Cas protein, wherein the guide nucleic acid molecule comprises: a spacer sequence, which exhibits specific binding to a target polynucleotide sequence operably coupled to a target gene; and a scaffold sequence, which is used to form a complex with the Cas protein, wherein the length of the scaffold sequence is up to about 158 ​​nucleotides, wherein binding of the complex to the target polynucleotide sequence in a cell results in a regulated expression level of the target gene in the cell, wherein (A1) the regulated expression level of the target gene caused by the complex is comparable to or better than the regulated expression level caused by a control complex (A2) comprising the Cas protein and a control guide nucleic acid molecule containing a polynucleotide sequence of SEQ ID NO: 500.

[0019] Another aspect of the present disclosure provides a composition comprising a vector encoding a Cas protein and a guide nucleic acid molecule configured to form a complex with the Cas protein, wherein the vector comprises: a first polynucleotide sequence, the first polynucleotide sequence encoding the Cas protein; and a second polynucleotide sequence, the second polynucleotide sequence encoding a scaffold sequence of the guide nucleic acid molecule for forming a complex with the Cas protein, wherein the sum of the length of the first polynucleotide sequence and the length of the second polynucleotide sequence is at most about 1700 nucleotides.

[0020] Another aspect of the present disclosure provides a method of controlling a target gene in a cell, the method comprising contacting the cell with any one of the compositions disclosed herein.

[0021] Another aspect of the present disclosure provides a method for regulating a target gene in a cell, the method comprising: contacting the cell with a complex comprising a guide nucleic acid molecule and a Cas protein, wherein the complex exhibits specific binding to a target polynucleotide sequence operably coupled to the target gene, wherein binding of the complex to the target polynucleotide sequence results in a regulated expression level of the target gene in the cell, wherein (A1) the regulated expression level of the target gene caused by the complex is comparable to or superior to (A2) the regulated level caused by a control complex comprising the Cas protein and a control guide nucleic acid molecule containing a polynucleotide sequence of SEQ ID NO: 500.

[0022] Other aspects and advantages of the present disclosure will be readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. It will be appreciated that the present disclosure is susceptible of other different embodiments and that its several details may be modified in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative rather than restrictive. Incorporated by Reference

[0023] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. If publications and patents or patent applications incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The novel features of the present invention are particularly set forth in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description and the accompanying drawings (also referred to herein as "figures") which set forth exemplary embodiments utilizing the principles of the present invention, wherein:

[0025] Figure 1 A structural alignment between Un1Cas12f1 and AsCas12f is schematically shown to identify one or more domains in Un1Cas12f1 that may not be conserved in one or more additional Cas12f homologous structures.

[0026] Figure 2 A structural alignment between Un1Cas12f1 and Un2Cas12f1 is schematically shown to identify one or more domains in Un1Cas12f1 that may not be conserved in one or more additional Cas12f homologous structures.

[0027] Figure 3A Schematic representation of selection of various domains of Un1Cas12f1 to be at least partially deleted to generate one or more engineered nucleases. Figure 3B Schematic illustration of the deletion landscape approach to generate engineered nuclease variants of Un1Cas12f1.

[0028] Figure 4 Example deletion landscapes of Cas proteins (e.g., dCas9) are schematically shown to identify one or more domains in the Cas protein that can be deleted with minimal or essentially no effect on the activity of the Cas protein (e.g., the ability to induce transcriptional expression when a modified variant of dCas9 is operably coupled to a gene repressor).

[0029] Figure 5 Shown are enhanced expression of endogenous IFNγ (upper panel) and endogenous CD2 (lower panel) in cells resulting from multiple engineered nuclease variants disclosed herein. Engineered nuclease variants are engineered to exhibit reduced nuclease activity (e.g., dCas variants) and are fused to gene activation regulators.

[0030] Figure 6 The expression of the reduction of the target gene (e.g., GFP) in the cell caused by the multiple engineered nuclease variants disclosed herein is shown. The engineered nuclease variants are engineered to show reduced nuclease activity (e.g., dCas variants) and fused with a gene repression regulatory factor. Two different gene repression regulatory factors are used: gene repressor A (upper figure) and gene repressor B (lower figure).

[0031] Figure 7 Shown are enhanced expression of endogenous CD2 (upper panel), endogenous IFNγ (middle panel) and endogenous CXCR4 (lower panel) in cells resulting from multiple engineered nuclease variants disclosed herein. Engineered nuclease variants are engineered to exhibit reduced nuclease activity (e.g., dCas variants) and are fused to gene activation regulators.

[0032] Figure 8 A guide nucleic acid molecule configured to form a complex with a Cas protein is schematically shown.

[0033] Fig. 9 Schematically illustrated are regions of the scaffold region of a guide nucleic acid molecule that can be modified (e.g., mutated or deleted) to engineer the guide nucleic acid molecule.

[0034] Fig.10 The reduced expression of a target gene (e.g., GFP) in a cell resulting from a variety of engineered guide RNA variants disclosed herein is shown. Compared to a control guide RNA sequence ("SQ"), a variety of engineered guide RNA variants are modified at least in the scaffold region. Reduced gene expression is performed using the same dCas protein coupled to a gene repression regulator.

[0035] Fig.11 Shown are reduced expression of a target gene (e.g., GFP) in a cell resulting from additional engineered guide RNA variants disclosed herein. The additional engineered guide RNA variants are modified at least in the scaffold region compared to a control guide RNA sequence ("SQ"). Reduced gene expression is performed using the same dCas protein coupled to a gene repression regulator.

[0036] Fig.12The enhanced expression of endogenous CD2 in cells caused by additional multiple engineered guide RNA variants disclosed herein is shown. Compared with the control guide RNA sequence ("SQ"), the additional multiple engineered guide RNA variants are modified at least in the scaffold region. Enhanced gene expression is performed using the same dCas protein coupled to a gene activation regulator.

[0037] Fig.13 Examples of engineered guide nucleic acid molecules disclosed herein are schematically shown.

[0038] Fig.14 Shown is a size comparison of various DNA vectors, each encoding a Cas protein and a corresponding single guide nucleic acid molecule.

[0039] Fig.15 It is shown that the truncated nuclease variant t1 disclosed herein leads to increased expression of endogenous IFNγ (IFNγ) in cells. The engineered nuclease variant t1 is fused to the gene activation regulator VPR.

[0040] Fig.16 The chimeric nuclease variants disclosed herein result in reduced expression of endogenous CXCR4 in cells. The engineered nuclease variant t1 is fused to a gene repression regulator (ZNF10-KRAB-hDNMT3L). DETAILED DESCRIPTION

[0041] Although various embodiments of the present invention have been shown and described herein, it will be appreciated by those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art may conceive of many variations, changes and substitutions without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0042] Whenever the term "at least", "greater than", or "greater than or equal to" precedes the first value in a series of two or more values, the term "at least", "greater than", or "greater than or equal to" applies to each value in the series. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0043] Whenever the term "not greater than," "less than," or "less than or equal to" precedes the first value in a series of two or more values, the term "not greater than," "less than," or "less than or equal to" applies to every value in the series. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0044] The term "about" or "approximately" generally means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term can mean within the order of magnitude of a value, preferably within 5 times of a value, and more preferably within 2 times of a value. When describing specific values ​​in the application and claims, unless otherwise indicated, it should be assumed that the term "about" means within an acceptable error range for the specific value.

[0045] The use of alternatives such as "or" should be understood to mean one, both, or any combination of the alternatives. The term "and / or" should be understood to mean one or both of the alternatives.

[0046] The term "cell" generally refers to a biological cell. A cell can be the basic structural, functional and / or biological unit of a living organism. A cell can be derived from any organism having one or more cells. Some non-limiting examples include: prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotic organisms, protozoan cells, cells from plants (e.g., cells from plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugar cane, pumpkin, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, lycophytes, hornworts, liverworts, mosses), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. Agardh, etc.), algae (e.g., kelp), fungal cells (e.g., yeast cells, cells from mushrooms), animal cells, cells from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.), etc. Sometimes the cell is not derived from a natural organism (e.g., the cell can be synthetic, sometimes referred to as an artificial cell).

[0047] The term "nucleotide" as used herein generally refers to a base-sugar-phosphate combination. Nucleotide may include synthetic nucleotides. Nucleotide may include synthetic nucleotide analogs. Nucleotide may be a monomer unit of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside triphosphates, adenosine triphosphate (ATP), uridine triphosphate (UTP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphates, such as dATP, dCTP, dITP, dUTP, dGTP, dTTP or derivatives thereof. Such derivatives may include, for example, [αS] dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. The term nucleotide used herein may refer to dideoxyribonucleoside triphosphates (ddNTP) and derivatives thereof. Exemplary examples of dideoxyribonucleoside triphosphates may include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP and ddTTP. Nucleotide can be unlabeled, or detectably labeled by well-known technology.Labeling can also be carried out with quantum dots.Detectable labels can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels.The fluorescent labeling of nucleotides can include, but is not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxyl-X-rhodamine (ROX), 4-(4'dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, Cyanine, and 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides can include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP, which are available from Perkin Elmer, Foster City, Calif.; FluoroLink deoxynucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP, which are available from Amersham, Arlington, Mass. Heights, Ill.; fluorescein-15-dATP, fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP and fluorescein-15-2′-dATP, which can be obtained from Boehringer Mannheim, Indianapolis, Ind.; and chromosome marker nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, rhodamine green-5-UTP, rhodamine green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP, which are available from Molecular Probes, Eugene, Oregon. Nucleotides can also be labeled or marked by chemical modification. Chemically modified single nucleotides can be biotin-dNTP.Some non-limiting examples of biotinylated dNTPs can include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

[0048] The terms "polynucleotide," "oligonucleotide," or "nucleic acid" are used interchangeably herein and generally refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or analogs thereof, in single-stranded, double-stranded, or multi-stranded form. The polynucleotide may be exogenous or endogenous to the cell. The polynucleotide may be present in a cell-free environment. The polynucleotide may be a gene or a fragment thereof. The polynucleotide may be DNA. The polynucleotide may be RNA. The polynucleotide may have any three-dimensional structure and may perform any known or unknown function. The polynucleotide may contain one or more analogs (e.g., altered backbones, sugars, or nucleobases). If present, the nucleotide structure may be modified before or after polymer assembly. Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acids, xeno nucleic acids, morpholinos, locked nucleic acids, diol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to a sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine. Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, gene sites (locus) defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides (including cell-free DNA (cfDNA) and cell-free RNA (cfRNA)), nucleic acid probes and primers. The nucleotide sequence may be interrupted by non-nucleotide components.

[0049] The term "gene" generally refers to nucleic acids (e.g., DNA, such as genomic DNA and cDNA) involved in encoding RNA transcripts and their corresponding nucleotide sequences. The term used herein for genomic DNA includes intervening non-coding regions and regulatory regions, and may include 5' and 3' ends. In some uses, the term encompasses transcribed sequences including 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons and introns. In some genes, the transcribed region will include an "open reading frame" encoding a polypeptide. In some uses of the term, a "gene" only includes coding sequences (e.g., "open reading frames" or "coding regions") necessary for encoding polypeptides. In some cases, a gene does not encode a polypeptide, such as a ribosomal RNA gene (rRNA) and a transfer RNA (tRNA) gene. In some cases, the term "gene" includes not only transcribed sequences, but also non-transcribed regions, including upstream and downstream regulatory regions, enhancers and promoters. A gene may refer to an "endogenous gene" or a natural gene located in its natural position in the genome of an organism. A gene may refer to an "exogenous gene" or a non-natural gene. Non-natural genes can refer to genes that are not normally present in the host organism but are introduced into the host organism by gene transfer. Non-natural genes can also refer to genes that are not located in their natural position in the genome of an organism. Non-natural genes can also refer to naturally occurring nucleic acids or polypeptide sequences (e.g., non-natural sequences) that comprise mutations, insertions, and / or deletions.

[0050] The term "deletion" generally refers to the removal (or loss) of one or more (or a specified number of) amino acids (e.g., continuous or non-continuous amino acids) from a polypeptide sequence, or the removal (or loss) of one or more (or a specified number of) nucleic acid bases (e.g., continuous or non-continuous nucleic acid bases) from a polynucleotide sequence (e.g., a sequence encoding a polypeptide sequence). The term "internal deletion" generally refers to a deletion that does not include the N-terminus or C-terminus of a polypeptide or the 5' end or 3' end of a polynucleotide. Deletions (e.g., internal deletions) can be identified by comparison with a reference sequence, for example, by specifying the start and end positions of the deletion relative to the reference sequence. Deletions (e.g., internal deletions) are different and distinct from substitutions. For example, compared to a reference polypeptide sequence, the deletion of at least one amino acid is not followed by the insertion of at least one different amino acid at the same position as the at least one amino acid, so that the size (e.g., the number of amino acid residues) of the modified (or engineered) polypeptide sequence containing the deletion of at least one amino acid is smaller than the size of the reference polypeptide sequence by at least one amino acid deleted.

[0051] The term "sequence identity" generally refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Typically, the techniques used to determine sequence identity include determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence encoded thereby, and comparing these sequences with a second nucleotide or amino acid sequence. Two or more sequences (polynucleotides or amino acids) can be compared by determining their "percent identity". The percentage identity of two sequences (whether nucleic acid sequences or amino acid sequences) is the number of exact matches between the two aligned sequences divided by the length of the longer sequence, then multiplied by 100. Percent identity can also be determined, for example, by comparing sequence information using an advanced BLAST computer program (including version 2.2.9) available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87: 2264-2268 (1990), and as discussed in Altschul, et al., J. Mol. Biol., 215: 403-410 (1990); Karlin And Altschul, Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993); and Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997). The program can be used to determine the percent identity over the entire length of the compared proteins. Default parameters are provided to optimize searches using short query sequences, such as using the blastp program. The program also allows the use of SEG filters to mask fragments of the query sequence, as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). The desired degree of sequence identity ranges from about 50% to 100% and integer values ​​therebetween. In general, the disclosure encompasses sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to any of the sequences provided herein.

[0052] The term "expression" generally refers to one or more processes of polynucleotide transcription (e.g., transcription into mRNA or other RNA transcripts) from a DNA template and / or the process of transcribed mRNA being subsequently translated into peptides, polypeptides or proteins. Transcripts and encoded polypeptides can be collectively referred to as "gene products". If the polynucleotide is derived from genomic DNA, expression can include the splicing of mRNA in eukaryotic cells. "Upregulation" about expression generally refers to the expression level of polynucleotides (e.g., RNA, e.g., mRNA) and / or polypeptide sequences relative to their expression in the wild-type state, while "downregulation" generally refers to the expression level of polynucleotides (e.g., RNA, e.g., mRNA) and / or polypeptide sequences relative to their expression in the wild-type state. The expression of transfected genes can occur transiently or stably in cells. During "transient expression", transfected genes are not transferred to daughter cells during cell division. Since its expression is limited to transfected cells, the expression of the gene will disappear over time. On the contrary, when a gene is co-transfected with another gene that gives a selective advantage to transfected cells, stable expression of transfected genes can occur. Such a selective advantage might be resistance to a certain toxin presented by the cell.

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

[0054] The terms "peptide", "polypeptide" or "protein" are used interchangeably herein and generally refer to a polymer of at least two amino acid residues connected by a peptide bond. The term does not indicate a specific length of the polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant technology, chemical or enzymatic synthesis, or is naturally occurring. These terms apply to naturally occurring amino acid polymers and amino acid polymers comprising at least one modified amino acid. In some cases, the polymer may be interrupted by non-amino acids. These terms include amino acid chains of any length, including full-length proteins, and proteins with or without secondary and / or tertiary structures (e.g., domains). These terms also include amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other operation (e.g., conjugation with a labeling component). The term "amino acid" used herein generally refers to natural and non-natural amino acids, including but not limited to modified amino acids and amino acid analogs. Modified amino acids can include natural amino acids and non-natural amino acids that have been chemically modified to include non-naturally occurring groups or chemical moieties on amino acids. Amino acid analogs may refer to amino acid derivatives. The term "amino acid" includes both D-amino acids and L-amino acids.

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

[0056] The terms "engineered", "chimeric" or "recombinant" used herein with respect to polypeptide molecules (e.g., proteins) generally refer to polypeptide molecules having heterologous amino acid sequences or altered amino acid sequences due to the application of genetic engineering techniques to nucleic acids encoding polypeptide molecules and cells or organisms expressing the polypeptide molecules. The terms "engineered" or "recombinant" used herein with respect to polynucleotide molecules (e.g., DNA or RNA molecules) generally refer to polynucleotide molecules having heterologous nucleic acid sequences or altered nucleic acid sequences due to the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning techniques; transfection, transformation and other gene transfer techniques; homologous recombination; site-directed mutagenesis; and gene fusion. In some cases, engineered or recombinant polynucleotides (e.g., genomic DNA sequences) can be modified or altered in part by gene editing.

[0057] For example, the engineered nucleases disclosed herein (e.g., engineered Cas proteins) are not naturally occurring nucleases (e.g., are not naturally occurring Cas proteins). The terms "engineered nuclease" and "engineered nuclease variants" are used interchangeably herein.

[0058] The terms "engineered" and "modified" are used interchangeably herein. The terms "engineered" and "modified" are used interchangeably herein. The terms "engineered cells" or "modified cells" are used interchangeably herein. The terms "engineered properties" and "modified properties" are used interchangeably herein.

[0059] The terms "enhanced expression", "increased expression" or "up-regulated expression" generally refer to the production level of a portion of interest (e.g., a polynucleotide or polypeptide) in a host strain (e.g., a host cell) that is higher than the normal expression level of the portion of interest. The normal expression level can be substantially zero (or empty) or higher than zero. The portion of interest can include an endogenous gene or polypeptide construct of the host strain. The portion of interest can include a heterologous gene or polypeptide construct introduced into or entering the host strain. For example, a heterologous gene encoding a polypeptide of interest can be knocked-in (KI) into the genome of the host strain to enhance the expression of the polypeptide of interest in the host strain.

[0060] The terms "enhanced activity", "increased activity" or "up-regulated activity" generally refer to that the activity of a portion of interest (e.g., a polynucleotide or polypeptide) is modified to a level above the normal activity level of the portion of interest in a host strain (e.g., a host cell). The normal activity level can be substantially zero (or empty) or above zero. The portion of interest can include a polypeptide construct of the host strain. The portion of interest can include a heterologous polypeptide construct introduced into or into the host strain. For example, a heterologous gene encoding a polypeptide of interest can be knocked-in (KI) into the genome of a host strain to enhance the activity of the polypeptide of interest in the host strain.

[0061] The term "reduced expression", "lowered expression" or "down-regulated expression" generally refers to the production level of a portion of interest (e.g., a polynucleotide or polypeptide) in a host strain (e.g., a host cell) that is lower than the normal expression level of the portion of interest. The normal expression level is above zero. The portion of interest may include an endogenous gene or polypeptide construct of the host strain. In some cases, the portion of interest may be knocked out or knocked down in the host strain. In some instances, the reduced expression of the portion of interest may include complete inhibition of such expression in the host strain.

[0062] The term "reduced activity", "lowered activity" or "downregulated activity" generally refers to that the activity of a portion of interest (e.g., a polynucleotide or polypeptide) is modified to a level below the normal activity level of the portion of interest in a host strain (e.g., a host cell). The normal activity level is above zero. The portion of interest may include an endogenous gene or polypeptide construct of the host strain. In some cases, the portion of interest may be knocked out or knocked down in the host strain. In some instances, the reduced activity of the portion of interest may include complete inhibition of such activity in the host strain.

[0063] The terms "subject", "individual" or "patient" are used interchangeably herein and generally refer to vertebrates, preferably mammals, such as humans. Mammals include, but are not limited to, rodents, apes, humans, farm animals, sports animals, and pets. It also includes tissues, cells, and progeny of biological entities obtained in vivo or cultured in vitro.

[0064] The term "treatment" generally refers to a method of obtaining a beneficial or desired result, including but not limited to a therapeutic benefit and / or a prophylactic benefit. For example, treatment can include administering a system or cell population disclosed herein. A therapeutic benefit refers to any treatment-related improvement or effect on one or more diseases, conditions, or symptoms being treated. For a prophylactic benefit, a composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even though the disease, condition, or symptom may not yet be manifested.

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

[0066] Aspects of the present disclosure can provide engineered nucleases that are smaller but effectively bind, cut and / or edit target polynucleotide sequences, compositions thereof and methods of use thereof. Such engineered nucleases (e.g., engineered CRISPR / Cas nucleases) can, for example, achieve manipulation of expression or activity of target genes (e.g., target endogenous genes) in cells, for example, to treat or improve a subject's condition (e.g., disease). Gene expression can support a variety of physiological and pathological effects in cells and tissues, contributing to many diseases and conditions, and therefore compositions and methods utilizing engineered nucleases disclosed herein can regulate the expression of specific genes in a desired manner to have therapeutic benefits. Engineered nucleases, compositions and methods thereof

[0067] In some aspects, the present disclosure provides an engineered nuclease comprising an amino acid sequence that is at least 50% identical to the polypeptide sequence of SEQ ID NO: 1. The amino acid sequence of the engineered nuclease may comprise at least one deletion compared to the polypeptide sequence of SEQ ID NO: 1 (or SEQ ID NO: 10) (e.g., when aligned therewith). The at least one deletion may be selectively removed according to the present disclosure. As disclosed herein, SEQ ID NO: 1 encodes the polypeptide sequence of Un1Cas12f1 (or Cas14a1). As disclosed herein, SEQ ID NO: 10 encodes an engineered variant of Un1Cas12f1 with reduced nuclease activity. Therefore, the amino acid sequence of the engineered nuclease as disclosed herein may be a mutant sequence (or mutant variant) of Un1Cas12f1 (or its inactivated variant).

[0068] Without wishing to be bound by theory, at least one deletion of the amino acid sequence of the engineered nuclease disclosed herein may be present in one or more regions of the natural Un1Cas12f1 nuclease, and the one or more regions are not aligned with the control CRISPR / Cas protein in structure. The control CRISPR / Cas protein may be 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 may be divided into types I, III, and IV, and Class 2 CRISPR systems may be divided into types II, V, and VI. In some cases, the control CRISPR / Cas protein may be a V-type Cas protein, such as a VA-type Cas protein, a VB-type Cas protein, a VC-type Cas protein, a VD-type Cas protein, a VE-type Cas protein, a VF-type Cas protein, a VG-type Cas protein, a VH-type Cas protein, a VI-type Cas protein, a VJ-type Cas protein, a VK-type Cas protein, or a VU-type Cas protein. In some cases, the control CRISPR / Cas protein may be a VJ-type protein, such as a wild-type CasФ (Cas 12J) protein. In some cases, the control CRISPR / Cas protein can be Un2Cas12f1 (SEQ ID NO:2) or AsCas12f (SEQ ID NO:3).

[0069] Without wishing to be bound by theory, at least one deletion of the amino acid sequence of the engineered nuclease disclosed herein can be determined by performing a deletion landscape study (e.g., iterative 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 theory, multiple different variants of the engineered nucleases disclosed herein may exhibit different activities (e.g., different binding affinities to a control sgRNA, different binding affinities to a control target gene, different target gene cleavage levels, different target gene activation levels, different target gene repression levels, etc.). In some embodiments, a first variant among the multiple different variants may include at least one deletion at amino acid residues 1-100 (e.g., when aligned with the polypeptide sequence of SEQ ID NO: 1), and a second variant among the multiple different variants may include at least one deletion at amino acid residues 101-529 (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 first variant may achieve enhanced target gene activation and / or expression compared to the second variant, or vice versa). Alternatively, the first variant and the second variant may exhibit comparable activities. In some embodiments, a first variant among the plurality of different variants may include a single deletion at amino acid residues 1-100 (e.g., when aligned with the polypeptide sequence of SEQ ID NO: 1), and a second variant among the plurality of different variants may include multiple deletions at 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 achieve enhanced 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)

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

[0073] SEQ ID NO:2(Un2Cas12f1)

[0074] SEQ ID NO:3 (AsCas12f)

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

[0076] In some embodiments, the amino acid sequence of the engineered nuclease disclosed herein can be at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 62%, at least or up to about 64%, at least or up to about 65%, at least or up to about 66%, at least or up to about 68%, at least or up to about 70%, at least or up to about 72%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 77%, at least or up to about 78%, at least or up to about 79%, at least or up to about 80%, at least or up to about 81%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, at least or up to about 100%, at least or up to about 101%, at least or up to about 102%, at least or up to about 103%, at least or up to about 104%, at least or up to about 105%, at least or up to about 106%, at least or up to about 107%, at least or up to about 108%, at least or up to about 109%, at least or up to about To about 78%, at least or up to about 80%, at least or up to about 82%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 88%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99% identical. For example, the amino acid sequence of the engineered nuclease can be about 80% to about 100% identical to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one Cas protein selected from Table 2.

[0077] In some embodiments, the amino acid sequence of the engineered nuclease disclosed herein is not identical to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one Cas protein selected from Table 2.

[0078] In some embodiments, at least one deletion of the amino acid sequence of the engineered nuclease disclosed herein may include an N-terminal deletion, a C-terminal deletion, and / or an internal deletion. For example, the at least one deletion may not include an N-terminal deletion, but an internal deletion and / or a C-terminal deletion.

[0079] In some embodiments, at least one deletion in the amino acid sequence of the engineered nuclease disclosed herein can be 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, or amino acid residues 500-529, and / or amino acid residues 430-529, compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one Cas protein selected from Table 2 (when applicable).

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

[0081] In some embodiments, at least one deletion in the amino acid sequence of the engineered nuclease compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one Cas protein selected from Table 2 (when applicable) can be from 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, amino acid residues 2-85, amino acid residues 2-84, amino acid residues 2-82, amino acid residues 2-80, amino acid residues 2-78, amino acid residues 2-76, amino acid residues 2-75, amino acid residues 2-74, amino acid residues 2-72, amino acid residues 2-70, amino acid residues 2-68, amino acid residues 2-66, amino acid residues 2-65, amino acid residues 2-64, amino acid residues 2-62, amino acid residues 2-60, amino acid residues 2-58, or amino acid residues 2-59. -56, amino acid residues 2-55, amino acid residues 2-54, amino acid residues 2-52, amino acid residues 2-50, amino acid residues 2-48, amino acid residues 2-46, amino acid residues 2-45, amino acid residues 2-44, amino acid residues 2-42, amino acid residues 2-40, amino acid residues 2-38, amino acid residues 2-36, amino acid residues 2-35, amino acid residues 2-34, amino acid residues 2-32, amino acid residues 2-30, amino acid residues 2-28, amino acid residues 2-26, amino acid residues 2-25, amino acid residues 2-24, amino acid residues 2-22, amino acid residues 2-20, amino acid residues 2-18, amino acid residues 2-16, amino acid residues 2-15, amino acid residues 2-14, amino acid residues 2-12, amino acid residues 2-10, amino acid residues 2-8, amino acid residues 2-6, amino acid residues 2-5 or amino acid residues 2-4.

[0082] In some embodiments, at least one deletion in the amino acid sequence of the engineered nuclease can be from amino acid residues 2-100, amino acid residues 4-100, amino acid residues 5-100, amino acid residues 6-100, amino acid residues 8-100, amino acid residues 10-100, amino acid residues 12-100, amino acid residues 14-100, amino acid residues 15-100, amino acid residues 16-100, amino acid residues 18-100, amino acid residues 19-200, amino acid residues 21-200, amino acid residues 22-200, amino acid residues 23-200, amino acid residues 24-200, amino acid residues 25-200, amino acid residues 26-200, amino acid residues 27-200, amino acid residues 28-200, amino acid residues 29-300, amino acid residues 30-300, amino acid residues 31-300, amino acid residues 32-300, amino acid residues 33-300, amino acid residues 34-300, amino acid residues 35-300, amino acid residues 36-300, amino acid residues 37-300, amino acid residues 38-300 0, amino acid residues 20-100, amino acid residues 22-100, amino acid residues 24-100, amino acid residues 25-100, amino acid residues 26-100, amino acid residues 28-100, amino acid residues 30-100, amino acid residues 32-100, amino acid residues 34-100, amino acid residues 35-100, amino acid residues 36-100, amino acid residues 38-100, amino acid residues 40-100, amino acid residues 42-100, amino acid residues 44-100, amino acid residues 45 -100, amino acid residues 46-100, amino acid residues 48-100, amino acid residues 50-100, amino acid residues 52-100, amino acid residues 54-100, amino acid residues 55-100, amino acid residues 56-100, amino acid residues 58-100, amino acid residues 60-100, amino acid residues 62-100, amino acid residues 64-100, amino acid residues 65-100, amino acid residues 66-100, amino acid residues 68-100, amino acid residues 70-100, amino acid residues 100, amino acid residues 82-100, amino acid residues 84-100, amino acid residues 85-100, amino acid residues 86-100, amino acid residues 88-100, amino acid residues 90-100, amino acid residues 92-100, amino acid residues 94-100, amino acid residues 95-100, amino acid residues 96-100, or amino acid residues 98-100.

[0083] In some embodiments, at least one deletion in the amino acid sequence of the engineered nuclease compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any Cas protein selected from Table 2 (when applicable) can be from amino acid residues 30-100, amino acid residues 30-98, amino acid residues 30-96, amino acid residues 30-95, amino acid residues 30-94, amino acid residues 30-92, amino acid residues 30-90, amino acid residues 30-88, amino acid residues 30-86, amino acid residues 30-85, amino acid residues 30-84, amino acid residues 30-82, amino acid residues 30-80, amino acid residues 30-78, amino acid residues 30-76, amino acid residues 30-75, amino acid residues 30-74, or amino acid residues 30-86. 0-72, amino acid residues 30-70, amino acid residues 30-68, amino acid residues 30-66, amino acid residues 30-65, amino acid residues 30-64, amino acid residues 30-62, amino acid residues 30-60, amino acid residues 30-58, amino acid residues 30-56, amino acid residues 30-55, amino acid residues 30-54, amino acid residues 30-52, amino acid residues 30-50, amino acid residues 30-48, amino acid residues 30-46, amino acid residues 30-45, amino acid residues 30-44, amino acid residues 30-42, amino acid residues 30-40, amino acid residues 30-38, amino acid residues 30-36, amino acid residues 30-34, or amino acid residues 30-32.

[0084] In some embodiments, at least one deletion of the amino acid sequence of the engineered nuclease can include one or more deletions (e.g., deletions of substantially all amino acid residues) from amino acid residues 55-56, amino acid residues 54-57, amino acid residues 54-58, amino acid residues 53-59, amino acid residues 52-60, amino acid residues 51-61, amino acid residues 50-62, amino acid residues 49-63, amino acid residues 48-64, amino acid residues 47-65, amino acid residues 46-66, amino acid residues 45-67, amino acid residues 44-68, amino acid residues 43-69, amino acid residues 42-70, amino acid residues 41-71, amino acid residues 40-72, amino acid residues 39-73, amino acid residues 38-74, amino acid residues 37-73, amino acid residues 36-74, or amino acid residues 35-75 compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any Cas protein selected from Table 2 (when applicable).

[0085] In some embodiments, at least one deletion in the amino acid sequence of the engineered nuclease compared to the polypeptide sequence of SEQ ID NO: 1 can be from one or more members (e.g., a single member, at least 2 members, at least 3 members, at least 4 members, at least 5 members or more) selected from the group consisting of amino acid residues 2-10, amino acid residues 11-20, amino acid residues 21-30, amino acid residues 31-40, amino acid residues 41-50, amino acid residues 51-60, amino acid residues 61-70, and amino acid residues 71-80. In some embodiments, at least one deletion in the amino acid sequence of the engineered nuclease compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any Cas protein selected from Table 2 (when applicable) can be from one or more members (e.g., a single member, at least 2 members, at least 3 members, at least 4 members, at least 5 members, or more) 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.

[0086] In some embodiments, at least one deletion in the amino acid sequence of the engineered nuclease disclosed herein compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any Cas protein selected from Table 2 (when applicable) can be 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, or amino acid residues 50-60.

[0087] In some embodiments, at least one deletion in the amino acid sequence of the engineered nuclease disclosed herein compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any Cas protein selected from Table 2 (when applicable) can be 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.

[0088] In some embodiments, at least one deletion in the amino acid sequence of the engineered nuclease disclosed herein compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any Cas protein selected from Table 2 (when applicable) can be 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.

[0089] In some embodiments, at least one deletion in the amino acid sequence of the engineered nuclease disclosed herein can be from amino acid residues 430-529 compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one Cas protein selected from Table 2 (when applicable).

[0090] In some embodiments, at least one deletion in the amino acid sequence of the engineered nuclease can be from amino acid residues 430-528, amino acid residues 430-526, amino acid residues 430-525, amino acid residues 430-524, amino acid residues 430-522, amino acid residues 430-520, amino acid residues 430-518, amino acid residues 430-516, amino acid residues 430-515, amino acid residues 430-514, amino acid residues 430-512 ... amino acid residues 430-510, amino acid residues 430-508, amino acid residues 430-506, amino acid residues 430-505, amino acid residues 430-504, amino acid residues 430-502, amino acid residues 430-500, amino acid residues 430-498, amino acid residues 430-496, amino acid residues 430-495, amino acid residues 430-494, amino acid 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 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 430-466, amino acid residues 430-465, amino acid residues 430-464, amino acid residues 430-462, amino acid residues 430-460, amino acid residues 430-458, amino acid residues 430-442, amino acid residues 430-440, amino acid residues 430-438, amino acid residues 430-436, amino acid residues 430-435, amino acid residues 430-434, or amino acid residues 430-432.

[0091] In some embodiments, at least one deletion in the amino acid sequence of the engineered nuclease can be from amino acid residues 430-529, amino acid residues 432-529, amino acid residues 434-529, amino acid residues 435-529, amino acid residues 436-529, amino acid residues 438-529, amino acid residues 440-529, amino acid residues 442-529, amino acid residues 444-529, amino acid residues 445-529, amino acid residues 446-529, amino acid residues 447-548, amino acid residues 449-550, amino acid residues 451-552, amino acid residues 452-553, amino acid residues 453-554, amino acid residues 454-555 amino acid residues 448-529, amino acid residues 450-529, amino acid residues 452-529, amino acid residues 454-529, amino acid residues 455-529, amino acid residues 456-529, amino acid residues 458-529, amino acid residues 460-529, amino acid residues 462-529, amino acid residues 464-529, amino acid residues 465-529, amino acid residues 466-529, amino acid residues 468-529, amino acid residues 470-529, amino acid residues 472-529, amino acid residues 474-529, amino acid residues 475 5-529, amino acid residues 476-529, amino acid residues 478-529, amino acid residues 480-529, amino acid residues 482-529, amino acid residues 484-529, amino acid residues 485-529, amino acid residues 486-529, amino acid residues 488-529, amino acid residues 490-529, amino acid residues 492-529, amino acid residues 494-529, amino acid residues 495-529, amino acid residues 496-529, amino acid residues 498-529, amino acid residues 500-529, amino acid residues 502-529 29, amino acid residues 504-529, amino acid residues 505-529, amino acid residues 506-529, amino acid residues 508-529, amino acid residues 510-529, amino acid residues 512-529, amino acid residues 514-529, amino acid residues 515-529, amino acid residues 516-529, amino acid residues 518-529, amino acid residues 520-529, amino acid residues 522-529, amino acid residues 524-529, amino acid residues 525-529, amino acid residues 526-529, or amino acid residues 528-529.

[0092] In some embodiments, at least one deletion in the amino acid sequence of the engineered nuclease compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any Cas protein selected from Table 2 (when applicable) can be from one or more members (e.g., a single member, at least 2 members, at least 3 members, at least 4 members, at least 5 members or more) 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. In some embodiments, at least one deletion in the amino acid sequence of the engineered nuclease compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any Cas protein selected from Table 2 (when applicable) can be from one or more members (e.g., a single member, at least 2 members, at least 3 members, at least 4 members, at least 5 members, or more) selected from the group consisting of amino acid residues 450-459, amino acid residues 460-465, amino acid residues 466-469, amino acid residues 470-475, amino acid residues 476-479, amino acid residues 480-485, amino acid residues 486-489, amino acid residues 490-495, amino acid residues 496-499, amino acid residues 500-505, amino acid residues 506-509, amino acid residues 510-515, amino acid residues 516-519, amino acid residues 520-525, and amino acid residues 526-529.

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

[0094] In some embodiments, the deletion of multiple amino acid residues to generate the amino acid sequence of the engineered nuclease may include the deletion of multiple non-contiguous amino acid residues, for example, compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one Cas protein selected from Table 2 (when applicable). In some cases, when compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the polypeptide sequence of any one Cas protein selected from Table 2 (when applicable), the deletion of multiple non-contiguous amino acid residues in the amino acid sequence of the engineered nuclease may include the deletion of a first amino acid residue (e.g., a first single amino acid residue or a first plurality of amino acid residues, such as a first group of contiguous amino acid residues) and a second amino acid residue (e.g., a second single amino acid residue or a second plurality of amino acid residues, such as a second group of contiguous amino acid residues) that are not contiguous to each other. For example, the position of the first amino acid residue and the second amino acid residue (e.g., when compared to SEQ ID NO:1) can be separated by at least or up to about 1, at least or up to about 2, at least or up to about 3, at least or up to about 4, at least or up to about 5, at least or up to about 10, at least or up to about 15, at least or up to about 20, at least or up to about 30, at least or up to about 40, at least or up to about 50, at least or up to about 60, at least or up to about 70, at least or up to about 80, at least or up to about 90, at least or up to about 100, at least or up to about 110, at least or up to about 120, at least or up to about 130, at least or up to about 140, at least or up to about 151, at least or up to about 161, at least or up to about 171, at least or up to about 180, at least or up to about 190, at least or up to about 201 Up to about 90, at least or up to about 100, at least or up to about 120, at least or up to about 150, at least or up to about 200, at least or up to about 250, at least or up to about 300, at least or up to about 350, at least or up to about 400, at least or up to about 450, at least or up to about 500, at least or up to about 510, at least or up to about 520, or at least or up to about 525 amino acid residues.

[0095] In some embodiments, the deletion of multiple amino acid residues to generate the amino acid sequence of the engineered nuclease can include 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 Cas protein selected from Table 2 (when applicable).In some cases, the deletion of multiple consecutive amino acid residues can include at least or up to about 2 consecutive amino acid residues, at least or up to about 3 consecutive amino acid residues, at least or up to about 4 consecutive amino acid residues, at least or up to about 5 consecutive amino acid residues, at least or up to about 6 consecutive amino acid residues, at least or up to about 7 consecutive amino acid residues, at least or up to about 8 consecutive amino acid residues, at least or up to about 9 consecutive amino acid residues, at least or up to about 10 consecutive amino acid residues, at least or up to about 11 consecutive amino acid residues, at least or up to about 12 consecutive amino acid residues, at least or up to about 13 consecutive amino acid residues, at least or up to about 14 consecutive amino acid residues, at least or up to about 15 consecutive amino acid residues, at least or up to about 16 consecutive amino acid residues, at least or up to about 17 consecutive amino acid residues, at least or up to about 18 consecutive amino acid residues, at least or up to about 19 consecutive amino acid residues, at least or up to about 20 consecutive amino acid residues, at least or up to about 21 consecutive amino acid residues, at least or up to about 22 consecutive amino acid residues, at least or up to about 23 consecutive amino acid residues, at least or up to about 24 consecutive amino acid residues, at least or up to about 25 consecutive amino acid residues, at least or up to about 26 consecutive amino acid residues, at least or up to about 27 consecutive amino acid residues, at least or up to about 28 consecutive amino acid residues, at least or up to about 29 consecutive amino acid residues, at least or up to about 30 consecutive amino acid residues, at least or up to about 31 consecutive amino acid residues, at least or up to about 32 consecutive amino acid residues, at least or up to about 33 consecutive amino acid residues, at least or up to about 34 consecutive amino acid residues, at least or up to about 35 at least or up to about 14 consecutive amino acid residues, at least or up to about 15 consecutive amino acid residues, at least or up to about 16 consecutive amino acid residues, at least or up to about 17 consecutive amino acid residues, at least or up to about 18 consecutive amino acid residues, at least or up to about 19 consecutive amino acid residues, at least or up to about 20 consecutive amino acid residues, at least or up to about 21 consecutive amino acid residues, at least or up to about 22 consecutive amino acid residues, at least or up to about 23 consecutive amino acid residues, at least or up to about 24 consecutive amino acid residues, at least or up to about 25 consecutive amino acid residues, at least or up to about 26 consecutive amino acid residues, at least or up to about 27 consecutive amino acid residues, at least or up to about 28 consecutive amino acid residues, at least or up to about 29 consecutive amino acid residues, at least or up to about 30 consecutive amino acid residues, at least or up to about 31 consecutive amino acid residues, at least or up to about 32 consecutive amino acid residues, at least or up to about 34 consecutive amino acid residues, at least or up to about 35 consecutive amino acid residues, at least or up to about 36 consecutive amino acid residues, at least or up to about 37 consecutive amino acid residues, at least or up to about 38 consecutive amino acid residues, at least or up to about 39 consecutive amino acid residues, at least or up to about 40 consecutive amino acid residues , at least or up to about 45 consecutive amino acid residues, at least or up to about 50 consecutive amino acid residues, at least or up to about 55 consecutive amino acid residues, at least or up to about 60 consecutive amino acid residues, at least or up to about 65 consecutive amino acid residues, at least or up to about 70 consecutive amino acid residues, at least or up to about 75 consecutive amino acid residues, at least or up to about 80 consecutive amino acid residues, at least or up to about 85 consecutive amino acid residues, at least or up to about 80 consecutive amino acid residues 90, at least or up to about 95 consecutive amino acid residues, or at least or up to about 100 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 Cas protein selected from Table 2 (when applicable).

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

[0097] In some embodiments, the amino acid sequence of the engineered nuclease disclosed herein may include 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 Cas protein selected from Table 2 (when applicable). One or more heterologous amino acid residues may be at the position of at least one deletion of the engineered nuclease. Alternatively or in addition, one or more heterologous amino acid residues may not be at the position of at least one deletion of the engineered nuclease. For example, one or more heterologous amino acid residues may be upstream and / or downstream of at least one deletion of the engineered nuclease.

[0098] In some embodiments, the one or more heterologous amino acid residues of the engineered nuclease may comprise a single amino acid residue. In some embodiments, the one or more heterologous amino acid residues of the engineered nuclease may comprise a plurality of amino acid residues, e.g.at least or up to about 2 amino acid residues, at least or up to about 3 amino acid residues, at least or up to about 4 amino acid residues, at least or up to about 5 amino acid residues, at least or up to about 6 amino acid residues, at least or up to about 7 amino acid residues, at least or up to about 8 amino acid residues, at least or up to about 9 amino acid residues, at least or up to about 10 amino acid residues, at least or up to about 11 amino acid residues, at least or up to about 12 amino acid residues, at least or up to about 13 amino acid residues, at least or up to about 14 amino acid residues, at least or up to about 15 amino acid residues, at least or up to about 16 amino acid residues, at least or up to about 17 amino acid residues, at least or up to about 18 amino acid residues, at least or up to about 19 amino acid residues, at least or up to about 20 amino acid residues, at least or up to about 22 amino acid residues, at least or up to about 24 amino acid residues, at least or up to about 25 amino acid residues, at least or up to about 26 amino acid residues, at least or up to about 28 amino acid residues, at least or up to about 30 amino acid residues, at least or up to about 32 amino acid residues, at least or up to about 34 amino acid residues, at least or up to about 35 amino acid residues, at least or up to about 36 amino acid residues, at least or up to about 38 amino acid residues, at least or up to about 40 amino acid residues, at least or up to about 42 amino acid residues, at least or up to about 44 amino acid residues, at least or up to about 45 amino acid residues. 46 amino acid residues, at least or up to about 48 amino acid residues, at least or up to about 50 amino acid residues, at least or up to about 52 amino acid residues, at least or up to about 54 amino acid residues, at least or up to about 55 amino acid residues, at least or up to about 56 amino acid residues, at least or up to about 58 amino acid residues, at least or up to about 60 amino acid residues, at least or up to about 62 amino acid residues, at least or up to about 64 amino acid residues, at least or up to about 65 amino acid residues, at least or up to about 66 amino acid residues, at least or up to about 68 amino acid residues, at least or up to about 70 amino acid residues, at least or up to about 72 amino acid residues, at least or up to about 73 amino acid residues, at least or up to about 74 amino acid residues, 4 amino acid residues, at least or up to about 75 amino acid residues, at least or up to about 76 amino acid residues, at least or up to about 78 amino acid residues, at least or up to about 80 amino acid residues, at least or up to about 82 amino acid residues, at least or up to about 84 amino acid residues, at least or up to about 85 amino acid residues, at least or up to about 86 amino acid residues, at least or up to about 88 amino acid residues, at least or up to about 90 amino acid residues, at least or up to about 92 amino acid residues, at least or up to about 94 amino acid residues, at least or up to about 95 amino acid residues, at least or up to about 96 amino acid residues, at least or up to about 98 amino acid residues, or at least or up to about 100 amino acid residues.

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

[0100] In some embodiments, one or more heterologous amino acid residues of the engineered nuclease may include a heterologous polypeptide sequence (e.g., 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 (when applicable). The heterologous polypeptide sequence may exhibit a net positive charge (e.g., a net +1 charge, a net +2 charge, a net +3 charge, a net +4 charge, a net +5 charge, etc., for example, measured in a buffer at about pH 7.4). The heterologous polypeptide sequence may exhibit a net negative charge (e.g., a net -1 charge, a net -2 charge, a net -3 charge, a net -4 charge, a net -5 charge, etc., for example, measured in a buffer at about pH 7.4). The heterologous polypeptide sequence may exhibit a neutral charge, for example, measured in a buffer at about pH 7.4. In some cases, the heterologous polypeptide sequence can comprise an amino acid sequence that is 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% identical to the polypeptide sequence of SEQ ID NO: 11.

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

[0102] In some embodiments, the amino acid sequence of the engineered nuclease disclosed herein can comprise a deletion of one or more amino acid residues from (i) amino acid residues 2-100 compared to the polypeptide sequence of SEQ ID NO: 1 and / or (ii) amino acid residues 430-529 compared to the polypeptide sequence of SEQ ID NO: 1, and the amino acid sequence can further comprise a deletion of one or more additional amino acid residues (e.g., 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) from amino acid residues 101-429 as disclosed herein compared to the polypeptide sequence of SEQ ID NO: 1.

[0103] In some embodiments, the amino acid sequence of the engineered nuclease disclosed herein may comprise a deletion of one or more amino acid residues from (i) amino acid residues 2-100 compared to the polypeptide sequence of any Cas protein selected from Table 2 and / or (ii) the last 100 C-terminal amino acid residues compared to the polypeptide sequence of any Cas protein selected from Table 2, and the amino acid sequence may further comprise a deletion of one or more additional amino acid residues (e.g., 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) from the amino acid residues therebetween compared to the polypeptide sequence of any Cas protein selected from Table 2.

[0104] In some embodiments, the engineered polypeptide disclosed herein may include one or more heterologous amino acid residues (e.g., in addition to mutations) compared to the native Un1Cas12f1 nuclease provided in SEQ ID NO: 1. The presence of one or more heterologous amino acid residues may enhance, for example, the activity, stability, expression, binding to a corresponding guide nucleic acid molecule, etc. of the engineered polypeptide.

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

[0106] SEQ ID NO: 12 (Exemplary engineered nuclease polypeptide)

[0107] In some embodiments, the engineered polypeptides disclosed herein can comprise one or more of SEQ ID NOs: 20-27, 28-111, 29-30, 30-31, 31-32, 32-33, 33-34, 35-36, 37-38, 39-40, 40-41, 42-43, 44-45, 46-47, 48-49, 50-51, 52-53, 53-54, 54-55, 56-57, 58-59, 59-60, 60-71, 61-62, 62-63, 64-65, 66-67, 68-70, 69-71, 72-73, 74-75, 76-77, 78-79, In some embodiments, the invention relates to an engineered nuclease variant of the present invention that is at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or about 100% identical to the polypeptide sequence of the engineered nuclease variant of the present invention.

[0108] In some embodiments, the engineered polypeptide disclosed herein may be different from any of the following: SEQ ID NO: 1 and any Cas protein from Table 2 (SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 13-SEQ ID NO: 19).

[0109] In some embodiments, the engineered polypeptides 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 include any deletion of one or more amino acid residues relative to the polypeptide sequence of SEQ ID NO: 1 or SEQ ID NO: 10 as disclosed herein (e.g., at least one deletion of amino acid residues 400-529 from SEQ ID NO: 1 or SEQ ID NO: 10), and 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. Compared to the polypeptide sequence of SEQ ID NO: 1 or SEQ ID NO: 10, the one or more members may comprise a member 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, In some cases, the at least one amino acid modification may comprise at least one set of modifications selected from Table 5A. In some cases, the at least one amino acid modification may comprise at least one combination of modifications selected from Table 5B. For example, the at least one combination of modifications selected from Table 5B may not be cA2.55 or cA2.84.

[0110] In some embodiments, the engineered polypeptides 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, the engineered 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, the engineered nuclease variants of the engineered polypeptide disclosed herein can be chimeric polypeptides comprising different polynucleotide sequence domains derived from different Cas proteins. The chimeric polypeptide may include a first polypeptide sequence and a second polypeptide sequence, the first polypeptide sequence comprising at least 3 continuous amino acid residues common to the first Cas protein, and the second polypeptide sequence comprising at least 3 continuous amino acid residues common to the second Cas protein, wherein the second Cas protein is different from the first Cas protein. The first Cas protein and the second Cas protein may be different naturally occurring Cas proteins. The first Cas protein and the second Cas protein may have a size (or amino acid sequence length) that differs from each other by no more than 500, no more than 400, no more than 350, no more than 300, no more than 250, no more than 200, no more than 150, no more than 140, no more than 130, no more than 120, no more than 110, no more than 100, no more than 90, no more than 80, no more than 70, no more than 60, no more than 50, no more than 40, no more than 30 or no more than 20 amino acid residues.

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

[0114] In some cases, the first Cas protein and the second Cas protein can be two different members selected from SEQ ID NO: 1 and any Cas protein from Table 2 (SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 13-SEQ ID NO: 19). The first Cas protein or the second Cas protein can comprise an amino acid sequence that is at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, at least or up to about 92%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or substantially about 100% identical to the polypeptide sequence of a member selected from SEQ ID NO: 1 and any Cas protein from Table 2 (SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 13-SEQ ID NO: 19). For example, such a member can be SEQ ID NO: 1 or SEQ ID NO: 2.

[0115] In some cases, the first polypeptide of the chimeric polypeptide of the engineered nuclease variant can comprise at least or up to about 4 consecutive amino acid residues, at least or up to about 5 consecutive amino acid residues, at least or up to about 6 consecutive amino acid residues, at least or up to about 7 consecutive amino acid residues, at least or up to about 8 consecutive amino acid residues, at least or up to about 9 consecutive amino acid residues, at least or up to about 10 consecutive amino acid residues, at least or up to about 11 consecutive amino acid residues, at least or up to about 12 consecutive amino acid residues, at least or up to about 13 consecutive amino acid residues, at least or up to about 14 consecutive amino acid residues, at least or up to about 15 consecutive amino acid residues, at least or up to about 18 consecutive amino acid residues, at least or up to about 20 consecutive amino acid residues, at least or up to about 30 consecutive amino acid residues, at least or up to about 40 consecutive amino acid residues, or at least or up to about 50 consecutive amino acid residues in common with the first Cas protein.

[0116] In some cases, the second polypeptide of the chimeric polypeptide of the engineered nuclease variant can comprise at least or up to about 4 consecutive amino acid residues, at least or up to about 5 consecutive amino acid residues, at least or up to about 6 consecutive amino acid residues, at least or up to about 7 consecutive amino acid residues, at least or up to about 8 consecutive amino acid residues, at least or up to about 9 consecutive amino acid residues, at least or up to about 10 consecutive amino acid residues, at least or up to about 11 consecutive amino acid residues, at least or up to about 12 consecutive amino acid residues, at least or up to about 13 consecutive amino acid residues, at least or up to about 14 consecutive amino acid residues, at least or up to about 15 consecutive amino acid residues, at least or up to about 18 consecutive amino acid residues, at least or up to about 20 consecutive amino acid residues, at least or up to about 30 consecutive amino acid residues, at least or up to about 40 consecutive amino acid residues, or at least or up to about 50 consecutive amino acid residues in common with a second Cas protein.

[0117] In some cases, in the second polypeptide of the chimeric polypeptide of the engineered nuclease variant, the length of the first polypeptide can be greater than the length of the second polypeptide by at least or up to about 1 amino acid, at least or up to about 2 amino acids, at least or up to about 5 amino acids, at least or up to about 10 amino acids, at least or up to about 15 amino acids, at least or up to about 20 amino acids, at least or up to about 25 amino acids, at least or up to about 30 amino acids, at least or up to about 35 amino acids, at least or up to about 40 amino acids, at least or up to about 45 amino acids, at least or up to about 50 amino acids, at least or up to about 55 amino acids, at least or up to about 60 amino acids. acids, at least or up to about 70 amino acids, at least or up to about 80 amino acids, at least or up to about 90 amino acids, at least or up to about 100 amino acids, at least or up to about 110 amino acids, at least or up to about 120 amino acids, at least or up to about 130 amino acids, at least or up to about 140 amino acids, at least or up to about 150 amino acids, at least or up to about 200 amino acids, at least or up to about 250 amino acids, at least or up to about 300 amino acids, at least or up to about 350 amino acids, at least or up to about 400 amino acids, at least or up to about 450 amino acids, or at least or up to about 500 amino acids.

[0118] In some cases, the first polypeptide may be derived from 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%, N-terminal 10%, or N-terminal 5% of the first Cas protein. Alternatively or in addition, the first polypeptide may be derived from 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%, C-terminal 10%, or C-terminal 5% of the first Cas protein. In some cases, the second polypeptide may be derived from 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%, N-terminal 10%, or N-terminal 5% of the second Cas protein. Alternatively or additionally, the second polypeptide may be derived from 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%, C-terminal 10%, or C-terminal 5% of a second Cas protein.

[0119] In some cases, the first polypeptide can 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. Alternatively or additionally, 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 C-terminus of the first Cas protein.

[0120] In some cases, the second polypeptide can 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 second Cas protein. Alternatively or additionally, 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 from the C-terminus of the second Cas protein.

[0121] In some cases, the engineered nuclease variants of the engineered polypeptides disclosed herein may include a third polypeptide sequence, the third polypeptide sequence including at least 3 continuous amino acid residues (or more, as disclosed herein) in common with the first Cas protein. In this case, the first polypeptide sequence and the third polypeptide sequence may be continuous or discontinuous with each other in the chimeric polypeptide. Alternatively or in addition, the third polypeptide sequence may include at least 3 continuous amino acid residues (or more, as disclosed herein) in common with the second Cas protein. The first polypeptide sequence and the third polypeptide sequence may be discontinuous with each other in the chimeric polypeptide. Alternatively, the first polypeptide sequence and the third polypeptide sequence may be continuous with each other in the chimeric polypeptide. The second polypeptide sequence and the third polypeptide sequence may be discontinuous with each other in the chimeric polypeptide. Alternatively, the second polypeptide sequence and the third polypeptide sequence may be continuous with each other in the chimeric polypeptide. Alternatively, the third polypeptide sequence may be continuous with each other in the chimeric polypeptide. Alternatively, the third polypeptide sequence may include at least 3 continuous amino acid residues in common with the third Cas protein, and the third Cas protein 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 may be selected from SEQ ID NO: 1 and three different members of any Cas protein selected from Table 2.

[0122] In some cases, the third polypeptide of the chimeric polypeptide of the engineered nuclease variant can comprise at least or up to about 4 consecutive amino acid residues, at least or up to about 5 consecutive amino acid residues, at least or up to about 6 consecutive amino acid residues, at least or up to about 7 consecutive amino acid residues, at least or up to about 8 consecutive amino acid residues, at least or up to about 9 consecutive amino acid residues, at least or up to about 10 consecutive amino acid residues, at least or up to about 11 consecutive amino acid residues, at least or up to about 12 consecutive amino acid residues, at least or up to about 13 consecutive amino acid residues, at least or up to about 14 consecutive amino acid residues, at least or up to about 15 consecutive amino acid residues, at least or up to about 18 consecutive amino acid residues, at least or up to about 20 consecutive amino acid residues, at least or up to about 30 consecutive amino acid residues, at least or up to about 40 consecutive amino acid residues, or at least or up to about 50 consecutive amino acid residues in common with the third Cas protein.

[0123] In some cases, the third polypeptide can 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 third Cas protein. Alternatively or additionally, 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 from the C-terminus of the third Cas protein.

[0124] In some cases, in the chimeric polypeptide of the engineered 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 any of the following structures: 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), wherein "-" can be an amino acid linker (e.g., comprising one or more amino acid sequences) or a direct covalent bond. The amino acid linker disclosed herein can comprise a single amino acid, at least or up to about 2 amino acids, at least or up to about 3 amino acids, at least or up to about 4 amino acids, at least or up to about 5 amino acids, at least or up to about 8 amino acids, at least or up to about 10 amino acids, at least or up to about 12 amino acids, at least or up to about 15 amino acids, at least or up to about 16 amino acids, or at least or up to about 20 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 length of the amino acid sequence of the engineered nuclease disclosed herein (e.g., the chimeric polypeptide disclosed herein) can be at most 528 amino acids, at most 527 amino acids, at most 526 amino acids, at most 525 amino acids, at most 524 amino acids, at most 523 amino acids, at most 522 amino acids, at most 521 amino acids, at most 520 amino acids, at most 519 amino acids, at most 518 amino acids, at most 517 amino acids, at most 516 amino acids, at most 515 amino acids, at most 514 amino acids. up to 513 amino acids, up to 512 amino acids, up to 511 amino acids, up to 510 amino acids, up to 509 amino acids, up to 508 amino acids, up to 507 amino acids, up to 506 amino acids, up to 505 amino acids, up to 504 amino acids, up to 503 amino acids, up to 502 amino acids, up to 501 amino acids, up to about 500 amino acids, up to about 495 amino acids, up to about 490 amino acids, up to about 485 amino acids, up to about 480 amino acids, up to about 475 amino acids, 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 3 In some embodiments, the present invention relates to a polypeptide of the present invention to a polypeptide of the present invention. In some embodiments, the polypeptide of the present invention comprises at most about 85 amino acids, at most about 380 amino acids, at most about 375 amino acids, at most about 370 amino acids, at most about 365 amino acids, at most about 360 amino acids, at most about 355 amino acids, at most about 350 amino acids, at most about 345 amino acids, at most about 340 amino acids, at most about 335 amino acids, at most about 330 amino acids, at most about 325 amino acids, at most about 320 amino acids, at most about 315 amino acids, at most about 310 amino acids, at most about 305 amino acids, or at most about 300 amino acids.

[0126] In some embodiments, the length of the engineered nuclease comprising the amino acid sequence disclosed herein can be at most about 1000 amino acids, at most about 950 amino acids, at most about 900 amino acids, at most about 850 amino acids, at most about 800 amino acids, at most about 750 amino acids, at most about 700 amino acids, at most about 650 amino acids, at most about 640 amino acids, at most about 630 amino acids, at most about 620 amino acids, at most about 610 amino acids, at most about 600 amino acids, at most about 590 amino acids, at most about 580 amino acids, at most about 570 amino acids. , up to about 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 engineered nuclease variants disclosed herein can be derived from (e.g., by engineering) naturally occurring Cas proteins (e.g., the first Cas protein described herein, the second Cas protein, or the third Cas protein). In some cases, the length of the naturally occurring Cas protein can be at most about 800 amino acids, at most about 750 amino acids, at most about 700 amino acids, at most about 650 amino acids, at most about 600 amino acids, at most about 550 amino acids, at most about 540 amino acids, at most about 530 amino acids, at most about 510 amino acids, at most about 500 amino acids, at most about 490 amino acids, at most about 480 amino acids, at most about 470 amino acids, at most about 460 amino acids, at most about 450 amino acids, or at most about 400 amino acids. Naturally occurring Cas proteins, for example, can be members from SEQ ID NO: 1 and any Cas protein selected from Table 2.

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

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

[0130] In some embodiments, the amino acid sequence of the engineered nuclease disclosed herein may include one or more substitutions in the native amino acid sequence, wherein the position of at least some of these substitutions follows one or more specific rules, which are determined to have surprising advantages for the engineered nuclease. In some cases, specific substitution rules are selected based on their ability to produce variants of the engineered nuclease (e.g., which may be able to function in eukaryotic cells). According to these specific rules, all or part of one or more substitutions in the native amino acid sequence (1) are within the (D / E / K / N)X(R / F)(E / K)N motif of the native amino acid sequence or no more than 30 amino acids downstream of the motif, (2) at position 241 of the native amino acid sequence or no more than 30 amino acids upstream or downstream of the position, (3) at position 516 of the native amino acid sequence or no more than 30 amino acids upstream or downstream of the position, and / or (4) have a charged amino acid in the native amino acid sequence.

[0131] In some embodiments, the amino acid sequence of the engineered nuclease disclosed herein may comprise one or more substitutions at an amino acid position within a (D / E / K / N)X(R / F)(E / K)N motif or no more than a threshold length (e.g., 30 amino acid residues) upstream and / or downstream of the motif, compared to (i) the polypeptide sequence of SEQ ID NO: 1 or (ii) the amino acid sequence of an engineered nuclease disclosed herein without one or more substitutions. In some cases, at least one of the one or more substitutions may be, for example, within the motif or no more than 28 amino acids, 26 amino acids, 24 amino acids, 22 amino acids, 20 amino acids, 18 amino acids, 16 amino acids, 14 amino acids, 12 amino acids, or 10 amino acids of the motif. In some cases, at least one of the one or more substitutions may be a substitution to an R, A, S, or G amino acid residue. In some cases, one or more substitutions may include substitutions 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, one or more substitutions may include substitutions at one or more positions selected from the group consisting of N504, E507, N516, N519, E527, and E528 (e.g., N504R, E507R, N516R, N519R, E527R, and / or E528R). In some cases, the one or more substitutions may include substitutions at one or more positions selected from the group consisting of K11, K73, D143, T147, E151, K154, E241, D318, K330, K457, E425, E462, N504, E507, N516, N519, E527, and E528 (e.g., K11R, K73R, D143R, T147R, E151R, K154R, E241R, D318R, K330R, E425N, K457R, E462R, N504R, E507R, N516R, N519R, E527R, and / or E528R).

[0132] In some embodiments, the amino acid sequence of an engineered nuclease disclosed herein comprising one or more substitutions upstream and / or downstream of a (D / E / K / N)X(R / F)(E / K)N motif can exhibit a cationic charge (e.g., a positive charge) that is at least or up to about 1 cationic charge, at least or up to about 2 cationic charges, at least or up to about 3 cationic charges, at least or up to about 4 cationic charges, at least or up to about 5 cationic charges, at least or up to about 6 cationic charges, at least or up to about 7 cationic charges, at least or up to about 8 cationic charges, at least or up to about 9 cationic charges, at least or up to about 10 cationic charges, at least or up to about 11 cationic charges, at least or up to about 12 cationic charges, at least or up to about 13 cationic charges, at least or up to about 14 cationic charges, at least or up to about 15 cationic charges, at least or up to about 16 cationic charges, at least or up to about 17 cationic charges, or at least or up to about 18 cationic charges greater than a control amino acid sequence of an engineered nuclease lacking the one or more substitutions.

[0133] Without wishing to be bound by theory, the amino acid sequences of engineered nucleases disclosed herein comprising one or more substitutions upstream and / or downstream of the (D / E / K / N)X(R / F)(E / K)N motif can exhibit enhanced (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 a Cas / guide nucleic acid complex (e.g., a target gene in a cell, such as a target endogenous gene) compared to (A) a control amino acid sequence of an engineered nuclease lacking the one or more substitutions and / or (B) a target polynucleotide sequence represented by SEQ ID Compared with the CRISPR / Cas protein encoded by NO:1, at least or up to about 1%, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, at least or up to about 100% higher. %, at least or up to about 110%, at least or up to about 120%, at least or up to about 125%, at least or up to about 150%, at least or up to about 200%, at least or up to about 250%, at least or up to about 300%, at least or up to about 350%, at least or up to about 400%, at least or up to about 450%, or at least or up to about 500%, as determined by surface plasmon resonance (SPR) or isothermal titration calorimetry (IRC).

[0134] Without wishing to be bound by theory, the amino acid sequences of engineered nucleases disclosed herein comprising one or more substitutions upstream and / or downstream of the (D / E / K / N)X(R / F)(E / K)N motif can exhibit enhanced (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 a Cas / guide nucleic acid complex (e.g., a target gene in a cell, such as a target endogenous gene) compared to (A) a control amino acid sequence of an engineered nuclease lacking the one or more substitutions and / or (B) a target polynucleotide sequence represented by SEQ ID NO:1, at least or up to about 0.1 times, at least or up to about 0.2 times, at least or up to about 0.5 times, at least or up to about 1 times, at least or up to about 2 times, at least or up to about 3 times, at least or up to about 4 times, at least or up to about 5 times, at least or up to about 6 times, at least or up to about 7 times, at least or up to about 8 times, at least or up to about 9 times, at least or up to about 10 times, at least or up to about 15 times, at least or up to about 20 times, at least or up to about 25 times, at least or up to about 30 times, at least or up to about 35 times, or at least or up to about 40 times higher than the CRISPR / Cas protein encoded by NO:1, as determined by surface plasmon resonance (SPR) or isothermal titration calorimetry (IRC) assay.

[0135] In some embodiments, the disclosure provides a system comprising an engineered nuclease disclosed herein. In some embodiments, the system may include an engineered nuclease in a recombinantly expressed (or produced) form. In some embodiments, the system may include one or more polynucleotides encoding at least an engineered nuclease. In some embodiments, the system may include a cell (or cell population) comprising at least (e.g., engineered to include, e.g., transfected or transduced to express) the engineered nuclease. Methods of using engineered nucleases

[0136] In some embodiments, the engineered nuclease disclosed herein can be used to achieve the binding, cutting and / or editing of the target polynucleotide sequence, for example, to regulate the expression and / or activity level of the target polynucleotide sequence of the polypeptide (e.g., protein) encoded by the target polynucleotide sequence or operably coupled to the target polynucleotide sequence. In some cases, a heterologous polypeptide comprising an engineered nuclease can be introduced into a cell (e.g., a mammalian cell) to achieve the binding, cutting and / or editing of the target polynucleotide sequence of the cell (e.g., an endogenous gene or a heterologous gene of the cell). In some embodiments, the engineered nuclease disclosed herein or a protein comprising the engineered nuclease can be referred to as an actuator moiety.

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

[0138] In some embodiments, the engineered nuclease disclosed herein can be a nuclease-deficient type. In some embodiments, the engineered nuclease can be a nuclease-invalid DNA binding protein that does not induce transcriptional activation or repression of a target DNA sequence unless it is present as a complex with one or more heterologous gene effectors disclosed herein. In some embodiments, the engineered nuclease can be a nuclease-invalid DNA binding protein that can induce transcriptional activation or repression of a target DNA sequence (e.g., it can be altered or enhanced by the presence of a heterologous gene effector provided herein). The terms "gene effector" and "gene modulator" are used interchangeably herein. The terms "gene effector polypeptide" and "gene regulatory polypeptide" are used interchangeably herein.

[0139] In some embodiments, the engineered nuclease disclosed herein can be an RNA nuclease, such as an engineered (e.g., programmable or targetable) RNA nuclease. In some embodiments, the engineered nuclease disclosed herein can be a nuclease-ineffective RNA binding protein that does not induce transcriptional activation or repression of a target RNA sequence unless it is present as a complex with one or more heterologous gene effectors disclosed herein. In some embodiments, the engineered nuclease disclosed herein can be a nuclease-ineffective RNA binding protein that can induce transcriptional activation or repression of a target RNA sequence (e.g., it can be altered or enhanced by the presence of heterologous gene effectors provided herein).

[0140] In some embodiments, the engineered nuclease can be a nucleic acid-guided targeting system. In some embodiments, the engineered nuclease can be a DNA-guided targeting system. In some embodiments, the engineered nuclease can be an RNA-guided targeting system. The nucleic acid-guided targeting system can include and utilize, for example, a guide nucleic acid sequence that promotes the CRISPR-Cas system (e.g., its nuclease-deficient form, such as dCas9 or dCas14) to specifically bind to a target gene (e.g., a target endogenous gene) or a target gene regulatory sequence. For example, the target gene can be any one of the genes listed in Table 1, and the target gene regulatory sequence can be operably coupled to any one of the genes listed in Table 1. Binding specificity can be determined by using a guide nucleic acid (e.g., a single guide RNA (sgRNA) or a portion thereof). In some embodiments, the use of different sgRNAs allows the compositions and methods of the present disclosure to be used together with different target genes (e.g., target endogenous genes) or target gene regulatory sequences (e.g., targeting them).

[0141] In some embodiments, the engineered nuclease can form a complex with a guide nucleic acid (e.g., a guide RNA or a portion thereof). In some embodiments, the engineered nuclease can form a complex with a single guide nucleic acid (e.g., a single guide RNA (sgRNA)). In some embodiments, the engineered nuclease can be an RNA binding protein (RBP), which is optionally compounded with a guide nucleic acid (e.g., a guide RNA (e.g., sgRNA)), and the guide nucleic acid can form a complex with the Cas protein. In some embodiments, the engineered nuclease can be a nuclease-invalid DNA binding protein, which can induce transcriptional activation or repression of a target DNA sequence. In some embodiments, the engineered nuclease can be an RNA binding protein derived from an RNA-invalid nuclease.

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

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

[0144] In some embodiments, the guide nucleic acid used in the compositions and methods of the present disclosure is about 8 to about 40 nucleotides, about 10 to about 40 nucleotides, about 11 to about 40 nucleotides, about 12 to about 40 nucleotides, about 13 to about 40 nucleotides, about 14 to about 40 nucleotides, about 15 to about 40 nucleotides, about 16 to about 40 nucleotides, about 17 to about 40 nucleotides, about 18 to about 40 nucleotides, about 19 to about 40 nucleotides, about 20 to about 40 nucleotides, about 22 to about 40 nucleotides, about 24 to about 40 nucleotides, about 26 to about 40 nucleotides, about 28 to about 40 nucleotides, about 30 to about 40 nucleotides, about 8 to about 30 nucleotides, about 10 to about 30 nucleotides, about 11 to about 30 nucleotides, about 12 to about 30 nucleotides, about 13 to about 30 nucleotides, about 14 to about 30 nucleotides, about 15 to about 30 nucleotides, about 16 to about 30 nucleotides, about 17 to about 30 nucleotides, about 18 to about 30 nucleotides, about 19 to about 30 nucleotides, about 20 to about 30 nucleotides, about 22 to about 30 nucleotides, about 24 to about 30 nucleotides, about 26 to about 30 nucleotides, about 28 to about 30 nucleotides, about 8 to about 25 nucleotides, about 10 to about 25 nucleotides, about 11 to about 30 nucleotides, about 25 nucleotides, about 12 to about 25 nucleotides, about 13 to about 25 nucleotides, about 14 to about 25 nucleotides, about 15 to about 25 nucleotides, about 16 to about 25 nucleotides, about 17 to about 25 nucleotides, about 18 to about 25 nucleotides, about 19 to about 25 nucleotides, about 20 to about 25 nucleotides, about 22 to about 25 nucleotides, about 24 to about 25 nucleotides, about 8 to about 20 nucleotides, about 10 to about 20 nucleotides, about 11 to about 20 nucleotides, about 12 to about 20 nucleotides, about 13 to about 20 nucleotides, about 14 to about 20 nucleotides, about 15 to about 20 nucleotides, about 1 In some embodiments, the guide nucleic acid can be a guide RNA or a portion thereof.

[0145] The engineered nuclease disclosed herein can be modified to enhance the regulation of gene expression by the compositions and methods disclosed herein (e.g., as part of the complex disclosed herein). The engineered nuclease can be modified to increase or decrease nucleic acid binding affinity, nucleic acid binding specificity, enzymatic activity and / or binding and inducing ligands with other factors (e.g., heterodimerization or oligomerization domains). The engineered nuclease can also be modified to change any other activity or property of the protein, such as stability. For example, one or more nuclease domains of the engineered nuclease can be modified, deleted or inactivated, or at least a portion of the engineered nuclease can be truncated to remove domains that are not important for the desired function of the protein or complex. The engineered nuclease can be modified to regulate (e.g., enhance or reduce) the activity of the engineered nuclease in regulating gene expression through the complex of the present disclosure comprising a heterologous gene effector.

[0146] For example, an engineered nuclease can be coupled (e.g., fused, covalently coupled or non-covalently coupled) to a heterologous gene effector (e.g., an epigenetic modification domain, a transcriptional activation domain, and / or a transcriptional repression domain). An engineered nuclease can be coupled (e.g., fused, covalently coupled or non-covalently coupled) to an oligomerization or dimerization domain (e.g., a heterodimerization domain) disclosed herein. An engineered nuclease can be coupled (e.g., fused, covalently coupled or non-covalently coupled) to a heterologous polypeptide providing improved or reduced stability. An engineered nuclease can be coupled (e.g., fused, covalently coupled or non-covalently coupled) to a sequence that can promote the degradation of an engineered nuclease or a complex comprising an engineered nuclease. An engineered nuclease can be coupled (e.g., fused, covalently coupled or non-covalently coupled) to a gene editing portion (e.g., a heterologous protein or its domain or functional fragment) that edits, mutates or modifies (directly or indirectly) a target polynucleotide sequence.

[0147] The engineered nucleic acid enzyme can be coupled (e.g., fused, covalently coupled or non-covalently coupled) to any suitable number of partners, such as at least one, at least two, at least three, at least four, or at least five, at least six, at least seven, or at least eight partners. In some embodiments, the engineered nucleic acid enzyme of the present disclosure is coupled (e.g., fused, covalently coupled or non-covalently coupled) to at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, or at most ten partners. In some embodiments, the engineered nucleic acid enzyme of the present disclosure is coupled (e.g., fused, covalently coupled or non-covalently coupled) to 1-5, 1-4, 1-3, 1-2, 2-5, 2-4, 2-3, 3-5, 3-4, or 4-5 partners. In some embodiments, the engineered nucleic acid enzyme of the present disclosure is coupled (e.g., fused, covalently coupled or non-covalently coupled) to one partner. In some embodiments, the engineered nuclease of the present invention is coupled (e.g., fused, covalently coupled, or non-covalently coupled) to two partners. In some embodiments, the engineered nuclease of the present invention is coupled (e.g., fused, covalently coupled, or non-covalently coupled) to three partners. In some embodiments, the engineered nuclease of the present invention is coupled (e.g., fused, covalently coupled, or non-covalently coupled) to four partners. In some embodiments, the engineered nuclease of the present invention is coupled (e.g., fused, covalently coupled, or non-covalently coupled) to five partners. In some embodiments, the engineered nuclease of the present invention is coupled (e.g., fused, covalently coupled, or non-covalently coupled) to six partners.

[0148] The engineered nuclease disclosed herein can be a fusion protein, for example, a fusion comprising an engineered nuclease and one or more of the partners disclosed herein. The fusion domain or heterologous polypeptide can be located at the N-terminus, C-terminus or internal of the engineered nuclease.

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

[0150] In some embodiments, the transcription effector can be a histone epigenetic modifier (or histone modifier). In some cases, histone epigenetic modifier can be methylated (for example, histone methylation modifier, such as amino acid methyltransferase, such as KRAB) to regulate histone. In some cases, histone epigenetic modifier can be acetylated to regulate histone. In some cases, histone epigenetic modifier can be phosphorylated to regulate histone. In some cases, histone epigenetic modifier can be ADP-ribosylation regulated histone. In some cases, histone epigenetic modifier can be glycosylated to regulate histone. In some cases, histone epigenetic modifier can be SUMOylation regulated histone. In some cases, histone epigenetic modifier can be ubiquitinated to regulate histone. In some cases, histone epigenetic modifier can be remodeled histone structure (for example, by ATP hydrolysis dependent process) to regulate histone.

[0151] In some embodiments, the transcription effector can be a gene epigenetic modifier (or gene modifier). In some cases, the gene modifier can regulate the gene by methylation (e.g., a gene methylation modifier such as DNA methyltransferase or DNMT). In some cases, the gene modifier can regulate the gene by acetylation.

[0152] In some embodiments, the transcriptional effector is from a family of related histone acetyltransferases. Non-limiting examples of histone acetyltransferases include the GNAT subfamily, the MYST subfamily, the p300 / CBP subfamily, the HAT1 subfamily, GCN5, PCAF, Tip60, MOZ, MORF, MOF, HBO1, p300, CBP, HAT1, ATF-2, SRC1, and TAFII250.

[0153] In some embodiments, the transcription effector may include an epigenetic modifier. In some embodiments, the transcription effector includes a histone epigenetic modifier (e.g., a histone lysine methyltransferase, a histone lysine demethylase, or a DNA methylase). Non-limiting examples of epigenetic modifiers may include EZH subfamily, non-SET subfamily, other SET subfamily, PRDM subfamily, SET1 subfamily, SET2 subfamily, SUV39 subfamily, SYMD subfamily, ASH1L, EHMT1, EHMT2, EZH1, EZH2, MLL, MLL2, MLL3, MLL4, MLL5, NSD1, NSD2, NSD3, PRDM1, PRDM10, PRDM11, PRDM12, PRDM13, PRDM14, PRDM 15, PRDM16, PRDM2, PRDM4, PRDM5, PRDM6, PRDM7, PRDM8, PRDM9, SET1, SET1L, SET2L, SETD2, SETD3, SETD4, SETD5, SETD6, SETD7, SETD8, SETDB1, SETDB2, SETMAR, SUV39H1, SUV39H2, SUV420H1, SUV420H2, SYMD1, SYMD2, SYMD3, SYMD4 and SYMD5.

[0154] Examples of proteins (or fragments thereof) that can be used as fusion partners to increase transcription include, but are not limited to, transcriptional activators such as VP16, VP64, VP48, VP160, p65 subdomains (e.g., from NFkB), and activation domains of EDLL and / or TAL activation domains (e.g., for activity in plants), SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, JHDM2a / b, UTX, JMJD3, GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZMYST3, MORFMYST4, SRC1, ACTR, PI 60, CLOCK, 10-11 translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, etc. Another example of such a gene activation regulator 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, but are not limited to, transcriptional repressors, such as Kruppel-associated box (KRAB or SKD); KOX1 repression domain; Mad mSIN3 interaction domain (SID); ERF repressor domain (ERD), SRDX repression domain (e.g., for repression in plants), etc.; histone lysine methyltransferases, such as Pr-SET7 / 8, SUV4-20H1, RIZ1, etc.; histone lysine demethylases, such as JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARJD 1A / RBP2, JARID1B / PLU-1, JARID1C / SMCX, JARIDID / SMCY, etc.; histone lysine deacetylase, such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, etc.; DNA methylase, such as Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), etc.; and peripheral recruitment elements, such as lamin A, lamin B, etc.

[0155] In many aspects, the engineered nuclease provided herein can achieve the editing or mutation of the target polynucleotide, as described herein. In some embodiments, editing or mutating the target polynucleotide sequence involves changing one or more nucleotides in the target polynucleotide to one or more different nucleotides. In some embodiments, editing or mutating the target polynucleotide involves changing guanine (G) to different nucleotides. In some cases, guanine (G) can be changed to adenine (A). In some cases, guanine (G) can be changed to thymine (T). In some cases, guanine (G) can be changed to cytosine (C). In some cases, guanine (G) can be changed to uracil (U). In some cases, guanine (G) can be changed to inosine (I). In some embodiments, editing or mutating the target polynucleotide involves changing cytosine (C) to different nucleotides. In some cases, cytosine (C) can be changed to guanine (G). In some cases, cytosine (C) can be changed to adenine (A). In some cases, cytosine (C) can be changed to thymine (T). In some cases, cytosine (C) can be changed to uracil (U). In some cases, cytosine (C) can be changed to inosine (I). In some embodiments, editing or mutating a target polynucleotide involves changing thymine (T) to a different nucleotide. In some cases, thymine (T) can be changed to cytosine (C). In some cases, thymine (T) can be changed to guanine (G). In some cases, thymine (T) can be changed to adenine (A). In some cases, thymine (T) can be changed to uracil (U). In some cases, thymine (T) can be changed to inosine (I). In some embodiments, editing or mutating a target polynucleotide involves changing adenine (A) to a different nucleotide. In some cases, adenine (A) can be changed to guanine (G). In some cases, adenine (A) can be changed to cytosine (C). In some cases, adenine (A) can be changed to thymine (T). In some cases, adenine (A) can be changed to uracil (U). In some cases, adenine (A) can be changed to inosine (I). In some embodiments, editing or mutating a target polynucleotide involves changing uracil (U) to a different nucleotide. In some cases, uracil (U) can be changed to guanine (G). In some cases, uracil (U) can be changed to cytosine (C). In some cases, uracil (U) can be changed to thymine (T). In some cases, uracil (U) can be changed to adenine (A). In some cases, uracil (U) can be changed to inosine (I). In some embodiments, editing or mutating a target polynucleotide involves changing inosine (I) to a different nucleotide. In some cases, inosine (I) can be changed to guanine (G). In some cases, inosine (I) can be changed to cytosine (C).In some cases, inosine (I) can be changed to thymine (T). In some cases, inosine (I) can be changed to adenine (A). In some cases, inosine (I) can be changed to uracil (U).

[0156] In some embodiments, editing or mutating a target polynucleotide involves introducing one or more point mutations into a target polynucleotide. In some embodiments, editing or mutating a target polynucleotide involves introducing one or more deletions (e.g., deletions of one or more nucleotides) into a target polynucleotide. In some embodiments, editing or mutating a target polynucleotide involves introducing one or more insertions (e.g., insertions of one or more nucleotides) into a target polynucleotide. In some embodiments, editing or mutating a target polynucleotide involves introducing one or more inversions (e.g., inversions of two or more nucleotides) into a target polynucleotide. In some embodiments, editing or mutating a target polynucleotide involves introducing one or more translocations (e.g., translocations of one or more nucleotides) into a target polynucleotide. In some embodiments, editing or mutating a target polynucleotide involves introducing one or more transpositions into a target polynucleotide.

[0157] In some cases, the engineered nuclease described herein can be coupled to a partner (e.g., a gene editing portion) that affects target polynucleotide editing or mutation, as described herein. In some cases, the engineered nuclease provided herein is covalently or non-covalently coupled to a gene editing portion (e.g., a protein, or its functional domain or functional fragment) that affects target polynucleotide editing or mutation. In some embodiments, the gene editing portion (e.g., a protein, or its functional domain or functional fragment) that affects target polynucleotide editing or mutation is a gene editing portion that changes one or more nucleotides to different nucleotides. In some embodiments, the gene editing portion (e.g., a protein, or its functional domain or functional fragment) that affects target polynucleotide editing or mutation is a gene editing portion that changes guanine (G) to different nucleotides. In some cases, the gene editing portion that affects target polynucleotide editing or mutation changes guanine (G) to cytosine (C). In some cases, the gene editing portion that affects target polynucleotide editing or mutation changes guanine (G) to thymine (T). In some cases, the gene editing portion that affects the editing or mutation of the target polynucleotide changes guanine (G) to adenine (A). In some cases, the gene editing portion that affects the editing or mutation of the target polynucleotide changes guanine (G) to uracil (U). In some cases, the gene editing portion that affects the editing or mutation of the target polynucleotide changes guanine (G) to inosine (I). In some embodiments, the gene editing portion (e.g., protein, or its functional domain or functional fragment) that affects the editing or mutation of the target polynucleotide sequence is a gene editing portion that changes cytosine (C) to a different nucleotide. In some cases, the gene editing portion that affects the editing or mutation of the target polynucleotide changes cytosine (C) to guanine (G). In some cases, the gene editing portion that affects the editing or mutation of the target polynucleotide changes cytosine (C) to thymine (T). In some cases, the gene editing portion that affects the editing or mutation of the target polynucleotide changes cytosine (C) to adenine (A). In some cases, the gene editing portion that affects the editing or mutation of the target polynucleotide changes cytosine (C) to uracil (U). In some cases, the gene editing portion that affects the editing or mutation of the target polynucleotide changes cytosine (C) to inosine (I). In some embodiments, the gene editing portion (e.g., protein, or its functional domain or functional fragment) that affects the editing or mutation of the target polynucleotide sequence is a gene editing portion that changes thymine (T) to a different nucleotide. In some cases, the gene editing portion that affects the editing or mutation of the target polynucleotide changes thymine (T) to cytosine (C). In some cases, the gene editing portion that affects the editing or mutation of the target polynucleotide changes thymine (T) to guanine (G). In some cases, the gene editing portion that affects the editing or mutation of the target polynucleotide changes thymine (T) to adenine (A).In some cases, the gene editing portion that affects the editing or mutation of the target polynucleotide changes thymine (T) to uracil (U). In some cases, the gene editing portion that affects the editing or mutation of the target polynucleotide changes thymine (T) to inosine (I). In some embodiments, the gene editing portion (e.g., protein, or its functional domain or functional fragment) that affects the editing or mutation of the target polynucleotide sequence is a gene editing portion that changes adenine (A) to a different nucleotide. In some cases, the gene editing portion that affects the editing or mutation of the target polynucleotide changes adenine (A) to cytosine (C). In some cases, the gene editing portion that affects the editing or mutation of the target polynucleotide changes adenine (A) to thymine (T). In some cases, the gene editing portion that affects the editing or mutation of the target polynucleotide changes adenine (A) to guanine (G). In some cases, the gene editing portion that affects the editing or mutation of the target polynucleotide changes adenine (A) to uracil (U). In some cases, the gene editing portion that affects the editing or mutation of the target polynucleotide changes adenine (A) to inosine (I). In some embodiments, the gene editing portion (e.g., a protein, or a functional domain or functional fragment thereof) that affects the editing or mutation of the target polynucleotide sequence is a gene editing portion that changes uracil (U) to a different nucleotide. In some cases, the gene editing portion that affects the editing or mutation of the target polynucleotide changes uracil (U) to cytosine (C). In some cases, the gene editing portion that affects the editing or mutation of the target polynucleotide changes uracil (U) to thymine (T). In some cases, the gene editing portion that affects the editing or mutation of the target polynucleotide changes uracil (U) to adenine (A). In some cases, the gene editing portion that affects the editing or mutation of the target polynucleotide changes uracil (U) to guanine (G). In some cases, the gene editing portion that affects the editing or mutation of the target polynucleotide changes uracil (U) to inosine (I). In some embodiments, the gene editing portion (e.g., a protein, or a functional domain or functional fragment thereof) that affects the editing or mutation of a target polynucleotide sequence is a gene editing portion that changes inosine (I) to a different nucleotide. In some cases, the gene editing portion that affects the editing or mutation of a target polynucleotide changes inosine (I) to cytosine (C). In some cases, the gene editing portion that affects the editing or mutation of a target polynucleotide changes inosine (I) to thymine (T). In some cases, the gene editing portion that affects the editing or mutation of a target polynucleotide changes inosine (I) to adenine (A). In some cases, the gene editing portion that affects the editing or mutation of a target polynucleotide changes inosine (I) to uracil (U). In some cases, the gene editing portion that affects the editing or mutation of a target polynucleotide changes inosine (I) to guanine (G).

[0158] In some embodiments, the engineered nucleases described herein (e.g., nuclease-deficient or nuclease-ineffective engineered nucleases described herein) can be coupled (e.g., covalently or non-covalently) to a partner (e.g., a gene editing portion) that introduces one or more point mutations into a target polynucleotide. In some embodiments, the engineered nucleases described herein (e.g., nuclease-deficient or nuclease-ineffective engineered nucleases described herein) can be coupled (e.g., covalently or non-covalently) to a partner (e.g., a gene editing portion) that introduces one or more deletions (e.g., deletions of one or more nucleotides) into a target polynucleotide. In some embodiments, the engineered nucleases described herein (e.g., nuclease-deficient or nuclease-ineffective engineered nucleases described herein) can be coupled (e.g., covalently or non-covalently) to a partner (e.g., a gene editing portion) that introduces one or more insertions (e.g., insertions of one or more nucleotides) into a target polynucleotide. In some embodiments, the engineered nucleases described herein (e.g., nuclease-deficient or nuclease-ineffective engineered nucleases described herein) can be coupled (e.g., covalently or non-covalently) to a partner (e.g., a gene editing portion) that introduces one or more inversions (e.g., an inversion of two or more nucleotides) into a target polynucleotide. In some embodiments, the engineered nucleases described herein (e.g., nuclease-deficient or nuclease-ineffective engineered nucleases described herein) can be coupled (e.g., covalently or non-covalently) to a partner (e.g., a gene editing portion) that introduces one or more translocations (e.g., a translocation of one or more nucleotides) into a target polynucleotide. In some embodiments, the engineered nucleases described herein (e.g., nuclease-deficient or nuclease-ineffective engineered nucleases described herein) can be coupled (e.g., covalently or non-covalently) to a partner (e.g., a gene editing portion) that introduces one or more transpositions 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, cytidine deaminase catalyzes the reaction of cytosine (C) to uracil (U), which has the base pairing properties of thymine. In some embodiments, for example, when the polynucleotide is double-stranded (e.g., double-stranded DNA), the uridine base can then be replaced by a thymidine base (e.g., by a cell repair mechanism) to produce a C·G to T·A transition. In some embodiments, the deaminase is an adenine deaminase. In some cases, adenine deaminase catalyzes the reaction of adenosine (A) to inosine (I). Non-limiting examples of deaminases suitable for use herein include, but are not limited to, APOBEC 1 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 engineered nucleases described herein can be used for prime editing. For example, the engineered nucleases described herein can be coupled to a reverse transcriptase (e.g., an engineered M-MLV reverse transcriptase) and a prime editing RNA (pegRNA). In this case, the engineered nuclease can include nickase activity. In some embodiments, prime editing can be used to mediate targeted insertions, deletions, or base-to-base conversions.

[0161] In some embodiments, the engineered nucleases described herein can be used for gene writing.

[0162] In some embodiments, the engineered nuclease described herein can be coupled to a partner (e.g., a protein, or its functional domain or functional fragment) that methylates the target substrate. In some cases, the partner (e.g., a protein, or its functional domain or functional fragment) that methylates the 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 engineered nuclease described herein can be coupled to a partner (e.g., a protein, or a functional domain or functional fragment thereof) having demethylase activity (e.g., a methyl group can be removed from a nucleic acid, protein, or other molecule). In some cases, the partner (e.g., a protein, or a functional domain or functional fragment thereof) having demethylase activity is a histone lysine demethylase, such as, but not limited to, KDM1, KDM2, KDM3, KDM4, KDM5, and KDM6.

[0164] In some embodiments, the engineered nucleic acid enzymes described herein can be coupled to a partner with dismutase activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with dismutase activity (e.g., a protein, or a functional domain or functional fragment thereof) is superoxide dismutase, formaldehyde dismutase, or chlorite dismutase.

[0165] In some embodiments, the engineered nuclease described herein can be coupled to a partner with alkylation activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with alkylation activity (e.g., a protein, or a functional domain or functional fragment thereof) is a prenyltransferase, a terpene cyclase, or a terpene synthase.

[0166] In some embodiments, the engineered nuclease described herein can be coupled to a partner with depurinic activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with depurinic activity (e.g., a protein, or a functional domain or functional fragment thereof) is a DNA glycosylase.

[0167] In some embodiments, the engineered nuclease described herein can be coupled to a partner with oxidation activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with oxidation activity (e.g., a protein, or a functional domain or functional fragment thereof) is a peroxidase or an oxidase.

[0168] In some embodiments, the engineered nucleases described herein can be coupled to a partner (eg, a protein, or a functional domain or functional fragment thereof) having pyrimidine dimer-forming activity.

[0169] In some embodiments, the engineered nuclease described herein can be coupled to a partner with integrase activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with integrase activity (e.g., a protein, or a functional domain or functional fragment thereof) is a retroviral integrase or HIV integrase.

[0170] In some embodiments, the engineered nuclease described herein can be coupled to a partner with transposase activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with transposase activity (e.g., a protein, or a functional domain or functional fragment thereof) is ty1, Mariner transposase, Tn3, transposase (Tnp) Tn5 or Tn7 transposon.

[0171] In some embodiments, the engineered nuclease described herein can be coupled to a partner with recombinase activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with recombinase activity (e.g., a protein, or a functional domain or functional fragment thereof) is a tyrosine recombinase, a Rad51 recombinase, a RecA recombinase, or a Dmc1 recombinase.

[0172] In some embodiments, the engineered nuclease described herein can be coupled to a partner with polymerase activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with polymerase activity (e.g., a protein, or a functional domain or functional fragment thereof) is a DNA polymerase, an RNA polymerase, a reverse transcriptase, or an RdRp replicase.

[0173] In some embodiments, the engineered nuclease described herein can be coupled to a partner with ligase activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with ligase activity (e.g., a protein, or a functional domain or functional fragment thereof) is a ubiquitin ligase, a glutamate-cysteine ​​ligase, an aminoacyl-tRNA synthetase, a succinyl-CoA 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 engineered nuclease described herein can be coupled to a partner with helicase activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with helicase activity (e.g., a protein, or a functional domain or functional 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 engineered nucleic acid enzyme described herein can be coupled to a partner with photolyase activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with photolyase activity (e.g., a protein, or a functional domain or functional fragment thereof) is a photoreactivation enzyme, a DNA photolyase, a DNA photoreactivation enzyme, a DNA cyclobutane dipyrimidine photolyase, a DNA photolyase, a deoxyribonucleic acid photolyase, a deoxyribonucleic acid dipyrimidine photolyase, a photolyase, a PRE, a PhrB photolyase, a deoxyribonucleic acid cyclobutane dipyrimidine photolyase, a phr A photolyase, a dipyrimidine photolyase (light-sensitive), or a deoxyribonucleic acid pyrimidine dimer lyase (light-sensitive).

[0176] In some embodiments, the engineered nuclease described herein can be coupled to a partner with glycosylase activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with glycosylase activity (e.g., a protein, or a functional domain or functional 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 engineered nuclease described herein can be coupled to a partner with acetyltransferase activity (e.g., protein, or its functional domain or functional fragment). In some cases, the partner with acetyltransferase activity (e.g., protein, or its functional domain or functional fragment) is CBP histone acetyltransferase, choline acetyltransferase, chloramphenicol acetyltransferase, serotonin N-acetyltransferase, NatA acetyltransferase or NatB acetyltransferase.

[0178] In some embodiments, the engineered nuclease described herein can be coupled to a partner with deacetylase activity (e.g., protein, or its functional domain or functional fragment). In some cases, the partner with deacetylase activity (e.g., protein, or its functional domain or functional fragment) is HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-6, HDAC-7 or HDAC-8.

[0179] In some embodiments, the engineered nuclease described herein can be coupled to a partner with kinase activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with kinase activity (e.g., a protein, or a functional domain or functional fragment thereof) is a Ca2+ / calmodulin-dependent protein kinase, a cyclin-dependent kinase, a nucleoside diphosphate kinase, a phosphatidylinositol phosphate kinase, a thymidine kinase, a thymidylate kinase, or a wall-associated kinase.

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

[0181] In some embodiments, the engineered nuclease described herein can be coupled to a partner (e.g., a protein, or a functional domain or functional fragment thereof) having ubiquitin ligase activity. In some cases, the partner (e.g., a protein, or a functional domain or functional fragment thereof) having ubiquitin ligase activity is 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, HERC4, HERC 5. HERC6, HUWE1, ITCH, NEDD4, NEDD4L, PPIL2, PRPF19, PIAS1, PIAS2, PIAS3, PIAS4, RANBP2, RNF4, RBX1, SMURF1, SMUR F2, STUB1, TOPORS, TRIP12, UBE3A, UBE3B, UBE3C, UBE3D, UBE4A, UBE4B, UBOX5, UBR5, VHL, WWP1, WWP2, Parkin or MKRN1.

[0182] In some embodiments, the engineered nuclease described herein can be coupled to a partner with deubiquitinating activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with deubiquitinating activity (e.g., a protein, or a functional domain or functional fragment thereof) 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 engineered nuclease described herein can be coupled to a partner with adenylylation activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with adenylylation activity (e.g., a protein, or a functional domain or functional fragment thereof) is a carboxylic acid reductase.

[0184] In some embodiments, the engineered nuclease described herein can be coupled to a partner with deadenylation activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with deadenylation activity (e.g., a protein, or a functional domain or functional fragment thereof) is a 5'-deadenylate enzyme, a CNOT6 deadenylase, a CNOT6L deadenylase, or a CCR4-NOT deadenylase.

[0185] In some embodiments, the engineered nuclease described herein can be coupled to a partner with SUMOylation activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with SUMOylation activity (e.g., a protein, or a functional domain or functional fragment thereof) is a small ubiquitin-related modifier (SUMO-1), SUMO-2 or SUMO-3.

[0186] In some embodiments, the engineered nuclease described herein can be coupled to a partner with deSUMOylation activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with deSUMOylation activity (e.g., a protein, or a functional domain or functional fragment thereof) is SENP1, SENP2, SENP3 or SENP5.

[0187] In some embodiments, the engineered nuclease described herein can be coupled to a partner with ribosylation activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with ribosylation activity (e.g., a protein, or a functional domain or functional fragment thereof) is a single (ADP-ribosyl) transferase, a poly (ADP-ribose) polymerase, or a histone ribosylase.

[0188] In some embodiments, the engineered nuclease described herein can be coupled to a partner with deribosylation activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with deribosylation activity (e.g., a protein, or a functional domain or functional fragment thereof) is a histone lysine deribosylase or an ADP-ribose deribosylase.

[0189] In some embodiments, the engineered nuclease described herein can be coupled to a partner with myristoylation activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with myristoylation activity (e.g., a protein, or a functional domain or functional fragment thereof) is N-myristoyltransferase (NMT) 1, N-myristoyltransferase (NMT) 2, or glycyl peptide N-tetradecanoyltransferase.

[0190] In some embodiments, the engineered nuclease described herein can be coupled to a partner with remodeling activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with remodeling activity (e.g., a protein, or a functional domain or functional fragment thereof) is a histone acetyltransferase (HAT), a deacetylase, or a methyltransferase.

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

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

[0193] In some embodiments, the engineered nuclease described herein can be coupled to a partner with transferase activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with transferase activity (e.g., a protein, or a functional domain or functional fragment thereof) is a coenzyme A transferase, an acyltransferase, a peptidyltransferase, an N-acetyltransferase, or a pyruvate dehydrogenase.

[0194] In some embodiments, the engineered nucleic acid enzyme described herein can be coupled to a partner with hydrolase activity (e.g., a protein, or its functional domain or functional fragment). In some cases, the partner with hydrolase activity (e.g., a protein, or its functional domain or functional fragment) is an esterase, a protease, a glycosidase or a lipase.

[0195] In some embodiments, the engineered nuclease described herein can be coupled to a partner (e.g., a protein, or its functional domain or functional fragment) with lyase activity. In some cases, the partner (e.g., a protein, or its functional domain or functional fragment) with lyase activity 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 engineered nucleic acid enzyme described herein can be coupled to a partner with isomerase activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with isomerase activity (e.g., a protein, or a functional domain or functional fragment thereof) is a ribose phosphate isomerase, a bisphosphoglycerate mutase, or a photoisomerase.

[0197] In some embodiments, the engineered nuclease described herein can be coupled to a partner with synthase activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with synthase activity (e.g., a protein, or a functional domain or functional fragment thereof) is ATP synthase, citrate synthase, tryptophan synthase, pseudouridine synthase or fatty acid synthase.

[0198] In some embodiments, the engineered nuclease described herein can be coupled to a partner with demyristoylation activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with demyristoylation activity (e.g., a protein, or a functional domain or functional fragment thereof) is a T3SS effector protein.

[0199] In some embodiments, the engineered nuclease described herein can be coupled to a partner with transposition activity (e.g., a protein, or a functional domain or functional fragment thereof). In some cases, the partner with transposition activity (e.g., a protein, or a functional domain or functional fragment thereof) is a transposase Tn5 or a sleeping beauty transposase.

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

[0201] The nucleic acid encoding at least the engineered nuclease disclosed herein, its fragment or derivative can be stably integrated into the genome of the cell. The nucleic acid encoding at least the engineered nuclease can be operably connected to a promoter, for example, a promoter of constitutive or inducible activity in the cell. The nucleic acid encoding at least the engineered nuclease can be operably connected to a promoter in an expression construct. The expression construct can include any nucleic acid construct that can direct the expression of a gene or other nucleic acid sequence of interest (for example, at least the engineered nuclease) and can transfer this nucleic acid sequence of interest to a target cell.

[0202] In some embodiments, the engineered nuclease disclosed herein can be associated with a single guide RNA (sgRNA) to activate or repress transcription of a target gene (e.g., a target endogenous gene), for example, in combination with a heterologous gene effector disclosed herein. The sgRNA can be introduced into a cell expressing an engineered nuclease or a variant thereof provided herein. In some cases, such cells can contain one or more different sgRNAs targeting the same target gene (e.g., a target endogenous gene) or a target gene regulatory sequence. In other cases, the sgRNA targets different nucleic acids in the cell (e.g., different target genes, different target gene regulatory sequences, or different sequences within the same target gene or target gene regulatory sequence).

[0203] Enzyme inactivation (e.g., nuclease deficiency) can refer to a nuclease that can bind to a nucleic acid sequence in a polynucleotide in a sequence-specific manner, but may not cut the target polynucleotide or will cut it at a significantly reduced frequency. The enzyme inactivation guide portion can include an enzyme inactivation domain (e.g., a nuclease domain). Enzyme inactivation can refer to inactivity. Enzyme inactivation can refer to substantially inactivity. Enzyme inactivation can refer to substantially inactivity. Enzyme inactivity can refer to activity no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 6%, no more than 7%, no more than 8%, no more than 9% or no more than 10% activity compared to a comparable wild-type activity (e.g., nucleic acid cleavage activity, wild-type Cas activity).

[0204] In some embodiments, the target nucleic acid of the engineered nuclease disclosed herein can be dsDNA. In such an embodiment, the dsDNA targeting specificity is determined at least in part by two parameters: the gRNA spacer targeting the original spacer sequence (protospacer) in the target dsDNA (the sequence corresponding to the gRNA spacer on the non-complementary DNA strand in the target dsDNA) and the short sequence protospacer adjacent motif (PAM) located immediately 5' (upstream) of the original spacer sequence on the non-complementary DNA strand. In some embodiments, PAM is 5'-TTTG-3', 5'-TTTA-3' or 5'-TTTR-3'. In some embodiments, PAM is 5'-TTTG-3'. In some embodiments, PAM is 5'-TTTA-3'. In some embodiments, PAM is 5'-TTTR-3'.

[0205] In some embodiments, the target nucleic acid of the engineered nuclease disclosed herein can be RNA. In such an embodiment, the RNA targeting specificity is at least partially determined by the gRNA spacer of the original spacer-like sequence in the target RNA (sequence complementary to the gRNA spacer in the target RNA), and is independent of the sequence immediately 5' (upstream) of the original spacer-like sequence. In some embodiments, the engineered nuclease can also be able to target dsDNA molecules, wherein the gRNA spacer is selected to target the original spacer sequence in the target dsDNA molecule with a PAM selected from 5'-TTTG-3', 5'-TTTA-3' and 5'-TTTR-3'. In other embodiments, the engineered nuclease cannot target dsDNA molecules, wherein the gRNA spacer is selected so that any original spacer sequence in the dsDNA molecule targeted by the gRNA spacer does not have a PAM selected from 5'-TTTG-3', 5'-TTTA-3' and 5'-TTTR-3'.

[0206] In some embodiments, a heterologous polypeptide comprising an engineered nuclease (e.g., and / or a complex comprising a heterologous polypeptide) can regulate the expression and / or activity of a target gene (e.g., a target endogenous gene). In some embodiments, a heterologous polypeptide and / or its complex can edit the sequence of a nucleic acid (e.g., a gene and / or a gene product). Nuclease activity variants of engineered nucleases can edit nucleic acid sequences by generating double-strand breaks or single-strand breaks in a target polynucleotide.

[0207] In some embodiments, a heterologous polypeptide comprising an engineered nuclease (e.g., and / or a complex comprising a heterologous polypeptide) can generate a double-strand break in a target polynucleotide (e.g., DNA). Double-strand breaks in DNA can lead to DNA break repair, thereby allowing the introduction of genetic modifications (e.g., nucleic acid editing). In some embodiments, the nuclease induces site-specific single-stranded DNA breaks or gaps, resulting in HDR.

[0208] Double-strand breaks in DNA can lead to DNA break repair, thereby allowing the introduction of genetic modifications (e.g., nucleic acid editing). DNA break repair can occur by non-homologous end joining (NHEJ) or homology-directed repair (HDR). In HDR, a donor DNA repair template or template polynucleotide can be provided, which includes homology arms flanking the target DNA site.

[0209] In some embodiments, a heterologous polypeptide comprising an engineered nuclease (e.g., and / or a complex comprising a heterologous polypeptide) does not generate double-strand breaks in a target polynucleotide (e.g., DNA). Binding of a heterologous polypeptide or a complex comprising a heterologous polypeptide (e.g., a complex comprising a nuclease-deficient variant of an engineered nuclease and a guide RNA) in the absence of nucleic acid breaks may be sufficient to modulate (e.g., enhance or inhibit) the expression of a target gene (e.g., an endogenous target gene). Target Gene

[0210] The present disclosure provides compositions, methods and systems for regulating the expression of one or more target genes.The target gene can be one or more endogenous target genes, such as (i) pathogenic alleles, such as mutant alleles, and / or (ii) non-pathogenic alleles, such as wild-type alleles.For example, one or more complexes are disclosed herein, which include a guide portion and one or more heterologous polypeptides, and the heterologous polypeptide includes an engineered polypeptide disclosed herein (such as an engineered nuclease), which can regulate (such as increase or decrease) the activity or expression level of the target gene (such as in a cell). Such complexes including a guide portion (such as a small guide RNA) and an engineered nuclease can achieve the regulation of target gene expression by cutting the target gene. Alternatively or in addition, such complexes include a gene regulator that is operably coupled to (for example, fused to) an engineered nuclease, so that the complex can achieve the regulation of target gene expression without cutting the target gene. In some cases, the gene regulator can achieve an increase in the expression of the target gene. In some cases, the gene regulator can achieve a reduction in the expression of the target gene. In some cases, a gene regulatory factor (e.g., a gene editing moiety described herein) can effect editing of a target gene, e.g., to correct an undesirable mutation in a target gene such that expression of the mutant gene is reduced and expression of the corrected gene is increased.

[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, for example, a pathogen target gene or an exogenous gene expressed as a result of a therapeutic intervention (e.g., 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 of a target gene (e.g., after introducing a complex comprising a heterologous polypeptide into a cell or cell population) or its duration. In some embodiments, the expression level is an RNA expression level, which can be measured by, for example, RNAseq, qPCR, microarrays, gene arrays, FISH, etc. In some embodiments, the expression level is a protein expression level, which can be measured by, for example, Western blot, ELISA, multiplex immunoassay, mass spectrometry, NMR, proteomics, flow cytometry, mass spectrometry flow cytometry, etc.

[0214] In some embodiments, the systems and methods disclosed herein can modulate (e.g., increase or decrease) the expression of a target gene (e.g., after introducing a complex comprising a heterologous polypeptide into a cell or cell population) or its duration by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 21-fold, at least about 22-fold, at least about 23-fold, at least about 24-fold, at least about 25-fold, at least about 26-fold, at least about 27-fold, at least about 28-fold, at least about 29-fold, at least about 30-fold, at least about 31-fold, at least about 32-fold, at least about 33-fold, at least about 34-fold, at least about 35-fold, at least about 36-fold, at least about 37-fold, at least about 38-fold, at least about 39-fold, at least about 40-fold, at least about 41-fold, at least about 42-fold, at least about 43-fold At least about 13 times, at least about 14 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times, at least about 150 times, at least about 200 times, at least about 250 times, at least about 300 times, at least about 350 times, at least about 400 times, at least about 500 times, at least about 600 times, at least about 700 times, at least about 800 times, at least about 900 times, at least about 1000 times, at least about 1500 times, at least about 2000 times, or at least about 3000 times.

[0215] In some embodiments, the systems and methods disclosed herein can modulate (e.g., increase or decrease) the expression of a target gene (e.g., after introducing a complex comprising a heterologous polypeptide into a cell or cell population) or its duration by up to about 50%, up to about 60%, up to about 70%, up to about 80%, up to about 90%, up to about 2-fold, up to about 3-fold, up to about 4-fold, up to about 5-fold, up to about 6-fold, up to about 7-fold, up to about 8-fold, up to about 9-fold, up to about 10-fold, up to about 11-fold, up to about 12-fold, up to about 13-fold, up to about 14-fold, up to about 15-fold, up to about 20-fold, or up to about 25-fold. 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 of a target gene (e.g., after introducing a complex comprising a heterologous polypeptide into a cell or cell population) or its duration by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, about 20-fold, about 21-fold, about 22-fold, about 23-fold, about 24-fold, about 25-fold, about 26-fold, about 27-fold, about 28-fold, about 29-fold, about 30-fold, about 31-fold, about 32-fold, about 33-fold, about 34-fold, about 35-fold, about 36-fold, about 37-fold, about 38-fold, about 39-fold, about 40-fold, about 41-fold, about 42-fold, about 43-fold, about 44-fold, about 45-fold, about 46-fold, about 47-fold, about 48-fold, about 49-fold, about 50-fold, about 51-fold, about 52-fold, about 53-fold, about 54-fold, about 55-fold, about 56-fold, about 57-fold, about 58-fold, about 59-fold, about 60-fold, about 61- times, about 14 times, about 15 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, about 100 times, about 150 times, about 200 times, about 250 times, about 300 times, about 350 times, about 400 times, about 500 times, about 600 times, about 700 times, about 800 times, about 900 times, about 1000 times, about 1500 times, about 2000 times, about 3000 times, about 5000 times, or about 10000 times.

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

[0218] In some embodiments, the extent of change in expression or its duration is relative to before the system of the disclosure (e.g., a complex comprising a heterologous polypeptide) is introduced into the cell or cell population. In some embodiments, the extent of change in expression or its duration is relative to a corresponding control cell or cell population not treated with the system of the disclosure. In some embodiments, the extent of change in expression or its duration is relative to a corresponding control cell or cell population treated with a substitute for the system of the disclosure.

[0219] In some embodiments, the degree of change in expression or its duration is relative to a control nuclease. The control nuclease may include a naturally occurring nuclease (e.g., (i) a polypeptide sequence of SEQ ID NO: 1 or (ii) a polypeptide sequence of any Cas protein selected from Table 2) or any modification thereof (e.g., a variant thereof having reduced nuclease activity and operably coupled to a gene regulator). For example, the control nuclease may be a dCasMINI coupled (e.g., fused) to the same gene regulator, wherein the same gene regulator is coupled to an engineered nuclease disclosed herein.

[0220] In some embodiments, the systems and methods disclosed herein can modulate (e.g., increase or decrease) the activity level of a target gene (e.g., after introducing a complex comprising a heterologous polypeptide comprising an engineered nuclease disclosed herein into a cell or cell population) or its duration. The activity level can be determined by a suitable functional assay for the target gene in question, depending on the functional characteristics of the target gene. For example, the activity level of a target gene that is a mitogen can be determined by measuring cell proliferation; the activity level of a target gene that induces apoptosis can be measured by an Annexin V assay or other suitable cell death assay; the activity level of an anti-inflammatory cytokine can be measured by an LPS-induced cytokine release assay.

[0221] In some cases, the systems and methods of the present disclosure can cause changes in the expression and / or activity levels of a target gene (e.g., a target endogenous gene) that last longer than can be achieved using alternative compositions and methods (e.g., by RNAi inhibition, e.g., using siRNA). In some embodiments, sustained regulation of gene expression is advantageous over transient regulation.

[0222] 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 for at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 18 hours, at least about 20 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 18 hours, at least about 20 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 5 days, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 18 hours, at least about 20 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 ... At least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 28 days, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 12 weeks, at least about 14 weeks, at least about 18 weeks, at least about 20 weeks, at least about 26 weeks, or at least about 5 months, at least about 6 months, at least about 9 months, at least about 12 months, or longer.

[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., a target endogenous gene) to above a certain threshold for at least or up to about 1 hour, at least or up to about 2 hours, at least or up to about 3 hours, at least or up to about 4 hours, at least or up to about 5 hours, at least or up to about 6 hours, at least or up to about 7 hours, at least or up to about 8 hours, at least or up to about 9 hours, at least or up to about 10 hours, at least or up to about 12 hours, at least or up to about 14 hours, at least or up to about 18 hours, at least or up to about 20 hours, at least or up to about 1 day, at least or up to about 2 days, at least or up to about 3 days, at least or up to about 4 days, at least or up to about 5 days, at least or up to about 6 days, at least or up to about 7 days, at least or up to about 8 days, at least or up to about 9 days, at least or up to about 10 days, at least or up to about 14 days, at least or up to about 21 days, at least or up to about 28 days, at least or up to about 5 weeks, at least or up to about 6 weeks, at least or up to about 7 weeks, at least or up to about 8 weeks, at least or up to about 9 weeks, at least or up to about 10 weeks, at least or up to about 12 weeks, at least or up to about 14 weeks, at least or up to about 18 weeks, at least or up to about 20 weeks, at least or up to about 26 weeks, or at least or up to about 5 months, at least or up to about 6 months, at least or up to about 9 months, or at least or up to 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., a target endogenous gene) to be above a certain threshold for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 14 hours, about 18 hours, about 20 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 14 days, about 21 days, about 28 days, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 12 weeks, about 14 weeks, about 18 weeks, about 20 weeks, about 26 weeks, about 5 months, about 6 months, about 9 months, or about 12 months.

[0225] In some embodiments, the engineered polypeptide disclosed herein (e.g., an engineered nuclease operably coupled to a gene regulator) may be able to enhance the regulation of a target gene or may achieve enhanced regulation of a target gene compared to the regulation of a target gene by a control polypeptide (e.g., a control nuclease operably coupled to the same gene regulator). The control nuclease may be a naturally occurring nuclease (e.g., (i) a polypeptide sequence of SEQ ID NO: 1 or (ii) a polypeptide sequence of any Cas protein selected from Table 2) or any modification thereof (e.g., dCasMINI disclosed herein).

[0226] In some cases, enhanced regulation of the target gene can be characterized by a change in the expression level of the target gene that is greater than the change caused by the control polypeptide. In some examples, this change can be an increased expression level of the target gene. The increased expression level of the target gene caused by the engineered polypeptides disclosed herein can be at least or up to about 1%, at least or up to about 2%, at least or up to about 3%, at least or up to about 4%, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, at least or up to about 100%, at least or up to about 110%, at least or up to about 120%, at least or up to about 150%, at least or up to about 200%, at least or up to about 300%, at least or up to about 400%, or at least or up to about 500% higher than that caused by a control polypeptide. The increased expression level of the target gene caused by the engineered polypeptide disclosed herein can be at least or more than about 0.1 times, at least or more than about 0.2 times, at least or more than about 0.5 times, at least or more than about 1 times, at least or more than about 1.5 times, at least or more than about 2 times, at least or more than about 3 times, at least or more than about 4 times, at least or more than about 5 times, at least or more than about 6 times, at least or more than about 7 times, at least or more than about 8 times, at least or more than about 9 times, at least or more than about 10 times, at least or more than about 15 times, at least or more than about 20 times, at least or more than about 30 times, at least or more than about 40 times, or at least or more than about 100 times higher than the control polypeptide. In some examples, this change can be a reduced (or decreased) expression level of the target gene. The reduced expression level of a target gene caused by the engineered polypeptides disclosed herein can be at least or up to about 1%, at least or up to about 2%, at least or up to about 3%, at least or up to about 4%, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, at least or up to about 100%, at least or up to about 110%, at least or up to about 120%, at least or up to about 150%, at least or up to about 200%, at least or up to about 300%, at least or up to about 400%, or at least or up to about 500% lower than that caused by a control polypeptide.The reduced (or decreased) expression level of a target gene caused by the engineered polypeptides disclosed herein can be at least or up to about 0.1 times, at least or up to about 0.2 times, at least or up to about 0.5 times, at least or up to about 1 times, at least or up to about 1.5 times, at least or up to about 2 times, at least or up to about 3 times, at least or up to about 4 times, at least or up to about 5 times, at least or up to about 6 times, at least or up to about 7 times, at least or up to about 8 times, at least or up to about 9 times, at least or up to about 10 times, at least or up to about 15 times, at least or up to about 20 times, at least or up to about 30 times, at least or up to about 40 times, or at least or up to about 100 times lower than that caused by a control polypeptide.

[0227] In some cases, enhanced regulation of a target gene can be characterized by a prolonged change in the expression level of the target gene (e.g., increased expression or decreased expression above a threshold as disclosed herein) longer than that caused by a control polypeptide. The prolonged change in target gene expression levels caused by the engineered polypeptides disclosed herein can be at least or up to about 1%, at least or up to about 2%, at least or up to about 3%, at least or up to about 4%, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, at least or up to about 100%, at least or up to about 110%, at least or up to about 120%, at least or up to about 150%, at least or up to about 200%, at least or up to about 300%, at least or up to about 400%, or at least or up to about 500% longer than that caused by a control polypeptide. The prolonged change in target gene expression levels caused by the engineered polypeptides disclosed herein can be at least or up to about 0.1 times, at least or up to about 0.2 times, at least or up to about 0.5 times, at least or up to about 1 times, at least or up to about 1.5 times, at least or up to about 2 times, at least or up to about 3 times, at least or up to about 4 times, at least or up to about 5 times, at least or up to about 6 times, at least or up to about 7 times, at least or up to about 8 times, at least or up to about 9 times, at least or up to about 10 times, at least or up to about 15 times, at least or up to about 20 times, at least or up to about 30 times, at least or up to about 40 times, or at least or up to about 100 times longer than that caused by a control polypeptide. For example, the threshold level can be relative to (i) the expression level of the target gene before the enhanced regulation, (ii) 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%), or (iii) the maximum decrease in the expression level of the target gene for repressing 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%). In some embodiments, the target gene (e.g., an endogenous target gene) can be a pathogenic allele, such as a mutant variant of a wild-type allele. The disease can be a genetic disease, such as a genetic disorder.The non-limiting examples of genetic diseases can include Duchenne muscular dystrophy (DMD), hemophilia, cystic fibrosis, Huntington's disease, familial hypercholesterolemia (LDL receptor defect), 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 can be a gene encoding a protein. In some cases, the target gene can be a gene regulatory sequence (e.g., promoter, enhancer, repressor, silencer, insulator, cis-regulatory element, trans-regulatory element, epigenetic modification (e.g., DNA methylation) site, etc.), which can affect the expression of the gene encoding the protein of interest provided herein. For example, a target gene regulatory sequence may be physically located outside the transcription unit or open reading frame encoding the target gene product.

[0228] In some embodiments, the target gene regulatory sequence does not include a nucleotide sequence that is foreign to the subject or host cell. In some embodiments, the target gene regulatory sequence does not include an engineered or artificially generated or introduced nucleotide sequence.

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

[0230] In some embodiments, the target gene (e.g., target endogenous gene) is a gene that is overexpressed or underexpressed in cancer, such as acute leukemia, astrocytoma, biliary cancer (cholangiocarcinoma), bone cancer, breast cancer, brain stem glioma, bronchoalveolar cell lung cancer, adrenal cancer, anal region cancer, bladder cancer, endocrine system cancer, esophageal cancer, head and neck cancer, kidney cancer, parathyroid cancer, penis cancer, pleural / peritoneal cancer, salivary gland cancer, small intestine cancer, thyroid cancer, ureter cancer, urethral cancer, cervical cancer, endometrial cancer, fallopian tube cancer, renal pelvis cancer, vaginal cancer, vulvar cancer, cervical cancer, chronic leukemia, colon cancer, colorectal cancer, skin melanoma, ependymoma, epidermoid tumor, Ewing's sarcoma, gastric cancer, glioblastoma The invention relates to a type of cancer comprising: a neoplasm of the lung, a glioma of the ovarian, a prostate, a colorectal, a glioma, a hematological malignancy, a hepatocellular (liver) carcinoma, a hepatoma, a Hodgkin's disease, an intraocular melanoma, a Kaposi's sarcoma, a lung cancer, a lymphoma, a medulloblastoma, a melanoma, a meningioma, a mesothelioma, a multiple myeloma, a muscle cancer, a central nervous system (CNS) neoplasm, a neuronal cancer, a small cell lung cancer, a non-small cell lung cancer, an osteosarcoma, an ovarian cancer, a pancreatic cancer, a childhood malignancy, a pituitary adenoma, a prostate cancer, a colorectal cancer, a renal cell carcinoma, a soft tissue sarcoma, a schwannoma, a skin cancer, a spinal tumor, a squamous cell carcinoma, a stomach cancer, a synovial sarcoma, a testicular cancer, a uterine cancer, or a tumor and its metastasis, including a refractory version of any of the foregoing cancers, or a combination thereof.

[0231] As disclosed herein, non-limiting examples of target genes or genes encoding proteins of interest are included in Table 1. Guide nucleic acid molecules

[0232] In some aspects, the present disclosure provides guide nucleic acid molecules (e.g., engineered guide nucleic acid molecules), which are configured to form a complex with Cas proteins. Cas proteins can be naturally occurring proteins. Cas proteins can be engineered nuclease variants provided herein. Guide nucleic acid molecules can include spacer sequences that exhibit specific binding to a target polynucleotide sequence that is operably coupled to a target gene (e.g., in a cell). The target polynucleotide sequence can be a part of a target gene. Alternatively, the target polynucleotide sequence can be upstream of the target gene (e.g., a part of a promoter sequence of a target gene or a promoter sequence adjacent to a target gene) or downstream (e.g., a part of a termination sequence of a target gene or a termination sequence adjacent to a target gene). Guide nucleic acid molecules can include a scaffold sequence for forming a complex with Cas proteins. The spacer sequence and the scaffold sequence can be a part of a single polynucleotide sequence (e.g., a single guide nucleic acid molecule, such as sgRNA). Alternatively, the spacer sequence and the scaffold sequence can be separate molecules that hybridize to form a complex with the Cas protein.

[0233] Without wishing to be bound by theory, the guide nucleic acid molecules disclosed herein can be operably coupled 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, or any engineered nuclease variant provided throughout the present disclosure (e.g., a 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 compared to the polynucleotide sequence of SEQ ID NO: 500 (e.g., when aligned). Without wishing to be bound by theory, at least one deletion of the scaffold sequence may be determined by performing a deletion landscape study (e.g., iterative and / or comprehensive deletion) on a control scaffold sequence of SEQ ID NO: 500. The scaffold sequence may comprise at least one mutation compared to the polynucleotide sequence of SEQ ID NO: 500 (e.g., when aligned). Without wishing to be bound by theory, at least one mutation of the scaffold sequence may be determined by performing a mutation landscape study (e.g., iterative and / or comprehensive mutation) on a control scaffold sequence of SEQ ID NO: 500. At least one deletion disclosed herein may be the removal of a nucleotide. Alternatively, at least one deletion may be a replacement of a nucleotide with a different nucleotide (e.g., a mutation).

[0235] In some embodiments, when compared (or compared) to a control polynucleotide sequence of SEQ ID NO: 500, the scaffold sequence may include one or more nucleotide deletions. One or more nucleotide deletions may include a single deletion. One or more nucleotide deletions may include multiple nucleotide deletions, such as at least or up to about 2 deletions, at least or up to about 3 deletions, at least or up to about 4 deletions, at least or up to about 5 deletions, at least or up to about 6 deletions, at least or up to about 7 deletions, at least or up to about 8 deletions, at least or up to about 9 deletions, at least or up to about 10 deletions, at least or up to about 11 deletions, at least or up to about 12 deletions, at least or up to about 13 deletions, at least or up to about 14 deletions, at least or up to about 15 deletions, at least or up to about 16 deletions, at least or up to about 17 deletions, at least or up to about 18 deletions, at least or up to about 19 deletions, at least or up to about 20 deletions, at least or up to about 22 deletions, at least or up to about 24 deletions, at least or up to about 25 deletions. 54 deletions, at least or up to about 55 deletions, at least or up to about 56 deletions, at least or up to about 58 deletions, at least or up to about 60 deletions, at least or up to about 70 deletions, or at least or up to about 80 deletions. When aligned with the polynucleotide sequence of SEQ ID NO: 500, multiple nucleotide deletions can be adjacent to each other (e.g., continuous). The scaffold sequence may comprise a single contiguous deletion.The scaffold sequence may comprise multiple contiguous deletions, wherein one contiguous deletion is not directly adjacent to another contiguous deletion when aligned with the polynucleotide sequence of SEQ ID NO: 500.

[0236] In some embodiments, when aligned with a control polynucleotide sequence of SEQ ID NO:500, a scaffold sequence disclosed herein can comprise one or more nucleotide deletions in one or more members of 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 from SEQ ID NO:500.

[0237] In some embodiments, when aligned with a control polynucleotide sequence of SEQ ID NO: 500, a scaffold sequence disclosed herein can comprise one or more nucleotide deletions among nucleotides 1-25 of the polynucleotide sequence of SEQ ID NO: 500, such as nucleotides 1-23, nucleotides 3-23, nucleotides 5-23, nucleotides 7-23, nucleotides 9-23, nucleotides 11-23, nucleotides 13-23, nucleotides 15-23, nucleotides 17-23, nucleotides 19-23, and / or nucleotides 21-23 of the polynucleotide sequence of SEQ ID NO: 500. When aligned with a control polynucleotide sequence of SEQ ID NO: 500, a scaffold sequence disclosed herein can comprise one or more nucleotide deletions among 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. When aligned with a control polynucleotide sequence of SEQ ID NO: 500, the scaffold sequences disclosed herein may comprise one or more nucleotide deletions in 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. When aligned with a control polynucleotide sequence of SEQ ID NO: 500, the scaffold sequences disclosed herein may comprise one or more nucleotide deletions in nucleotides 1, nucleotide 2, nucleotide 3, nucleotide 4, nucleotide 5, nucleotide 6, nucleotide 7, nucleotide 8, nucleotide 9, nucleotide 10, nucleotide 11, nucleotide 12, nucleotide 13, nucleotide 14, nucleotide 15, nucleotide 16, nucleotide 17, nucleotide 18, nucleotide 19, nucleotide 20, nucleotide 21, nucleotide 22, nucleotide 23, nucleotide 24, and / or nucleotide 25 of the polynucleotide sequence of SEQ ID NO: 500.

[0238] In some embodiments, when aligned with a control polynucleotide sequence of SEQ ID NO: 500, a scaffold sequence disclosed herein can comprise nucleotides 35-65 of the polynucleotide sequence of SEQ ID NO: 500, such as one or more nucleotide deletions among nucleotides 35-61, nucleotides 37-61, nucleotides 39-61, nucleotides 41-61, nucleotides 43-61, nucleotides 45-61, nucleotides 47-61, nucleotides 49-61, nucleotides 51-61, nucleotides 53-61, nucleotides 55-61, nucleotides 57-61 and / or nucleotides 59-61 of the polynucleotide sequence of SEQ ID NO: 500. When aligned with a control polynucleotide sequence of SEQ ID NO:500, the scaffold sequence disclosed herein may comprise one or more nucleotide deletions among 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. When aligned with a control polynucleotide sequence of SEQ ID NO: 500, the scaffold sequence disclosed herein can comprise one or more nucleotide deletions in nucleotide 35, nucleotide 36, nucleotide 37, nucleotide 38, nucleotide 39, nucleotide 40, nucleotide 41, nucleotide 42, nucleotide 43, nucleotide 44, nucleotide 45, nucleotide 46, nucleotide 47, nucleotide 48, nucleotide 49, nucleotide 50, nucleotide 51, nucleotide 52, nucleotide 53, nucleotide 54, nucleotide 55, nucleotide 56, nucleotide 57, nucleotide 58, nucleotide 59, nucleotide 60, nucleotide 61, nucleotide 62, nucleotide 63, nucleotide 64 and / or nucleotide 65 of the polynucleotide sequence of SEQ ID NO: 500.

[0239] In some embodiments, when aligned with a control polynucleotide sequence of SEQ ID NO: 500, a scaffold sequence disclosed herein can comprise one or more nucleotide deletions among nucleotides 135-150 of the polynucleotide sequence of SEQ ID NO: 500, such as nucleotides 136-149, nucleotides 137-149, nucleotides 139-149, nucleotides 141-149, nucleotides 143-149, nucleotides 145-149, and / or nucleotides 147-149 of the polynucleotide sequence of SEQ ID NO: 500. When aligned with a control polynucleotide sequence of SEQ ID NO: 500, a scaffold sequence disclosed herein can comprise one or more nucleotide deletions among 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. When aligned with a control polynucleotide sequence of SEQ ID NO:500, the scaffold sequence disclosed herein may comprise one or more nucleotide deletions in nucleotide 135, nucleotide 136, nucleotide 137, nucleotide 138, nucleotide 139, nucleotide 140, nucleotide 141, nucleotide 142, nucleotide 143, nucleotide 144, nucleotide 145, nucleotide 146, nucleotide 147, nucleotide 148, nucleotide 149 and / or nucleotide 150 of the polynucleotide sequence of SEQ ID NO:500.

[0240] In some embodiments, the scaffold sequences disclosed herein may comprise one or more nucleotide deletions from nucleotides 136-151 of the polynucleotide sequence of SEQ ID NO: 500 when aligned with a control polynucleotide sequence of SEQ ID NO: 500. In some cases, the scaffold sequences disclosed herein may comprise one or more nucleotide deletions from 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 sequence disclosed herein may comprise one or more nucleotide deletions (e.g., 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 nucleotides 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, when aligned with the polynucleotide sequence of SEQ ID NO: 500, the scaffold sequence disclosed herein may comprise at least or up to about 1 nucleotide, at least or up to about 2 nucleotides, at least or up to about 3 nucleotides, or all 4 nucleotides selected from the group consisting of G143, G147, G150, and G151.

[0241] In some embodiments, the scaffold sequence disclosed herein is not identical to the polynucleotide sequence of the combination of SEQ ID NO: 549 and SEQ ID NO: 550. For example, the polynucleotides of SEQ ID NO: 549 and SEQ ID NO: 550 can be coupled to the 5' end and 3' end of the spacer sequence, respectively, to serve as control sgRNA molecules to compare the activity of any scaffold sequence provided herein. In some embodiments, the scaffold sequence disclosed herein is not identical to the polynucleotide sequence of the combination of SEQ ID NO: 551 and SEQ ID NO: 552. For example, the polynucleotides of SEQ ID NO: 551 and SEQ ID NO: 552 can be coupled to the 5' end and 3' end of the spacer sequence, respectively, to serve as control sgRNA molecules to compare the activity of any scaffold sequence provided herein.

[0242] In some embodiments, a scaffold sequence (e.g., a contiguous polynucleotide sequence of a scaffold sequence) can be characterized as exhibiting at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 71%, at least or up to about 72%, at least or up to about 73%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 77%, at least or up to about 78%, at least or up to about 79%, at least or up to about 80%, at least or up to about 81%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, at least or up to about 100%, at least or up to about 101%, at least or up to about 102%, at least or up to about 103%, at least or up to about 104%, at least or up to about 105%, at least or up to about 106%, at least or up to about 107%, at least or up to about 108%, at least or up to about 109%, at least or up to about 110%, at least or up to about 111%, at least or up to about 112%, at least or up to about 113%, at least or up to about 114 %, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or substantially 100% sequence identity (or complementarity).

[0243] In some cases, the member may be selected from the group consisting of SEQ ID NOs: 503-152, 519, 524, 528, and 553. In some cases, the member may be selected from the group consisting of SEQ ID NOs: 555, 557, 558, 568, 569, 578, and 580. In some cases, the member may 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 can be at least or up to about 80 nucleotides, at least or up to about 85 nucleotides, at least or up to about 90 nucleotides, at least or up to about 91 nucleotides, at least or up to about 92 nucleotides, at least or up to about 93 nucleotides, at least or up to about 94 nucleotides, at least or up to about 95 nucleotides, at least or up to about 96 nucleotides, at least or up to about 97 nucleotides, at least or up to about 98 nucleotides, at least or up to about 99 nucleotides, at least or up to about 100 nucleotides, at least or up to about 101 nucleotides, at least or up to about 102 nucleotides, at least or up to about 103 nucleotides, at least or up to about 104 nucleotides, at least or up to about 105 nucleotides, at least or up to about 106 nucleotides, at least or up to about 107 nucleotides, at least or up to about 108 nucleotides, at least or up to about 110 nucleotides, at least or up to about 111 nucleotides, at least or up to about 112 nucleotides, at least or up to about 113 nucleotides, at least or up to about 114 nucleotides, at least or up to about 115 nucleotides, at least or up to about 116 nucleotides, at least or up to about 117 nucleotides, at least or up to about 118 nucleotides, at least or up to about 119 nucleotides, at least or up to about 120 nucleotides, at least or up to about 121 nucleotides, at least or up to about 122 nucleotides, at least or up to about 123 nucleotides, at least or up to about 124 nucleotides about 108 nucleotides, at least or up to about 109 nucleotides, at least or up to about 110 nucleotides, at least or up to about 112 nucleotides, at least or up to about 114 nucleotides, at least or up to about 115 nucleotides, at least or up to about 116 nucleotides, at least or up to about 118 nucleotides, at least or up to about 120 nucleotides, at least or up to about 122 nucleotides, at least or up to about 124 nucleotides, at least or up to about 125 nucleotides, at least or up to about 126 nucleotides, at least or up to about 128 nucleotides, at least or up to about 130 nucleotides, at least or up to about 135 nucleotides, at least or up to about 140 nucleotides, at least or up to about 145 nucleotides, at least or up to about 150 nucleotides, at least or up to about 155 nucleotides, or at least or up to about 160 nucleotides.

[0245] In some embodiments, the scaffold sequence can comprise a contiguous polynucleotide sequence that exhibits at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 71%, at least or up to about 72%, at least or up to about 73%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 77%, at least or up to about 78%, at least or up to about 79%, at least or up to about 80%, at least or up to about 81%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99, or essentially 100% sequence identity (or complementarity). The length of the contiguous polynucleotide sequence of the scaffold sequence can be at least or up to about 15 nucleotides, at least or up to about 16 nucleotides, at least or up to about 17 nucleotides, at least or up to about 18 nucleotides, at least or up to about 19 nucleotides, at least or up to about 20 nucleotides, at least or up to about 21 nucleotides, at least or up to about 22 nucleotides, at least or up to about 23 nucleotides, at least or up to about 24 nucleotides, at least or up to about 25 nucleotides, at least or up to about 26 nucleotides, at least or up to about 27 nucleotides, at least or up to about 28 nucleotides, at least or up to about 29 nucleotides, at least or up to about 30 nucleotides, at least or up to about 31 nucleotides, at least or up to about 32 nucleotides, at least or up to about 33 nucleotides, at least or up to about 34 nucleotides, at least or up to about 35 nucleotides, at least or up to about 36 nucleotides, at least or up to about 37 nucleotides, at least or up to about 38 nucleotides, at least or up to about 39 nucleotides, or at least or up to about 40 nucleotides. The continuous polynucleotide sequence can be configured at the N-terminus or C-terminus of the scaffold sequence. The continuous polynucleotide sequence can be configured at 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 continuous polynucleotide sequence can be configured at 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 continuous polynucleotide sequence can be configured between the scaffold sequences.

[0246] In some embodiments, the spacer sequence of the guide nucleic acid molecule can have a length of at least or up to about 12 nucleotides, at least or up to about 13 nucleotides, at least or up to about 14 nucleotides, at least or up to about 15 nucleotides, at least or up to about 16 nucleotides, at least or up to about 17 nucleotides, at least or up to about 18 nucleotides, at least or up to about 19 nucleotides, at least or up to about 20 nucleotides, at least or up to about 21 nucleotides, or at least or up to about 22 nucleotides.

[0247] In some embodiments, the guide nucleic acid molecule can have at least or up to about 80 nucleotides, at least or up to about 85 nucleotides, at least or up to about 90 nucleotides, at least or up to about 95 nucleotides, at least or up to about 96 nucleotides, at least or up to about 97 nucleotides, at least or up to about 98 nucleotides, at least or up to about 99 nucleotides, at least or up to about 100 nucleotides, at least or up to about 101 nucleotides, at least or up to about 102 nucleotides, at least or up to about 103 nucleotides, at least or up to about 104 nucleotides, at least or up to about 105 nucleotides, at least or up to about 106 nucleotides, at least or up to about 107 nucleotides, at least or up to about 108 nucleotides, at least or up to about 109 nucleotides, at least or up to about 110 nucleotides, at least or up to about 111 nucleotides, at least or up to about 112 nucleotides, at least or up to about 113 nucleotides, at least or up to about 114 nucleotides, A length of up to about 112 nucleotides, at least or up to about 113 nucleotides, at least or up to about 114 nucleotides, at least or up to about 115 nucleotides, at least or up to about 116 nucleotides, at least or up to about 117 nucleotides, at least or up to about 118 nucleotides, at least or up to about 119 nucleotides, at least or up to about 120 nucleotides, at least or up to about 121 nucleotides, at least or up to about 122 nucleotides, at least or up to about 123 nucleotides, at least or up to about 124 nucleotides, at least or up to about 125 nucleotides, at least or up to about 130 nucleotides, at least or up to about 135 nucleotides, at least or up to about 140 nucleotides, at least or up to about 145 nucleotides, at least or up to about 150 nucleotides, at least or up to about 155 nucleotides, or at least or up to about 160 nucleotides.

[0248] In some examples, the contiguous polynucleotide sequence of the scaffold sequence can be at least about 20, at least about 25, or at least about 30 nucleotides in length, and such contiguous polynucleotide sequence can exhibit at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 71%, at least or up to about 72%, at least or up to about 73%, at least or up to about 74%, at least or up to about 75%, at least or up to about 76%, at least or up to about 77%, at least or up to about 78%, at least or up to about 79%, at least or up to about 80%, at least or up to about 81%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, at least or up to about 100%, at least or up to about 101%, at least or up to about 102%, at least or up to about 103%, at least or up to about 104%, at least or up to about 105%, at least or up to about 106%, at least or up to about 107%, at least or up to about 108%, at least or up to about 109%, at least or up to about 110%, at least or up to about 111%, at least Up to about 80%, at least or up to about 81%, at least or up to about 82%, at least or up to about 83%, at least or up to about 84%, at least or up to about 85%, at least or up to about 86%, at least or up to about 87%, at least or up to about 88%, at least or up to about 89%, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99, or essentially 100% sequence identity (or complementarity). Heterologous polynucleotide

[0249] In some embodiments, the system of the present disclosure (e.g., a variant of an engineered nuclease that retains at least a portion of its nuclease activity) can target a target gene to edit the target gene. In some cases, a complex comprising (i) a heterologous polypeptide containing an engineered nuclease disclosed herein and (ii) a guide nucleic acid (e.g., sgRNA) can recognize, bind to, and produce a nick (one strand) or break (two strands) in the target gene, such as at or near the target sequence of the complex within 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-mediated repair (HDR) or by homologous recombination (HR) with a polynucleotide modified template (e.g., a donor template, such as a donor DNA template). In some instances, a heterologous polynucleotide modified template encoding a gene of interest can be provided to a cell so that the gene of interest can be inserted into the target gene, such as for gene replacement therapy.

[0250] In some embodiments, the systems and compositions of the present disclosure introduce heterologous polynucleotides (e.g., encoding a gene of interest, such as one or more genes selected from Table 1) into a cell without entering the genome of the cell through the action of an engineered nuclease of the present disclosure. In some cases, such heterologous polynucleotides encoding a gene of interest may enter the genome of the cell by other means, such as through an adeno-associated viral vector (e.g., AAV2 or AAV8). Alternatively, such heterologous polynucleotides encoding a gene of interest may be introduced into the intracellular portion of the cell and remain non-chromosomal (e.g., as a non-chromosomal plasmid).

[0251] Therefore, the system and composition can include a non-pathogenic wild type or variant of the target gene as described above.Alternatively or additionally, the system and composition can include a heterologous polynucleotide sequence encoding (or comprising) at least a non-pathogenic wild type or variant of a target gene (e.g., an endogenous target gene) disclosed herein. Composition

[0252] In some aspects, the disclosure provides compositions comprising at least a portion of a system as described above, such as (i) a heterologous polypeptide comprising an engineered 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, for use in any of the methods disclosed herein. The subject compositions can be used to modify cells in vitro, ex vivo, or in vivo. The subject compositions can be used to treat or enhance a subject's condition, as disclosed herein.

[0253] The compositions disclosed herein can include active ingredients (e.g., heterologous polypeptides including engineered nucleases, guide nucleic acids, etc.) and optional additional ingredients (e.g., excipients). If necessary and / or desired, the compositions can be divided, molded and / or packaged into desired single or multiple dose units or single or multiple implant units.

[0254] In some embodiments, the composition may include one or more heterologous polynucleotides encoding active ingredients disclosed herein. When different members are present in the active ingredient, each member may be encoded by different heterologous polynucleotides. Alternatively, two or more (e.g., all) components may be encoded by a single heterologous polynucleotide. In some cases, a single heterologous polynucleotide may encode (i) a heterologous polypeptide comprising an engineered nuclease (e.g., dCas-transcription effector fusion protein, such as dCas-KRAB, dCas-DNMT, dCas-ADA) and (ii) one or more guide nucleic acids (e.g., at least 1, at least 2, at least 3, at least 4, at least 5 or more guide nucleic acids) for targeting a specific region or sequence of a target gene.

[0255] One or more heterologous polynucleotides may also include one or more promoters (or one or more transcription control elements, as used interchangeably herein). Different active ingredients encoded by one or more heterologous polynucleotides may be under the control of the same promoter or different promoters. The promoter disclosed herein may be active in eukaryotic cells, mammalian cells, non-human mammalian cells, or human cells. The promoter may be an inducible or constitutively active promoter. Alternatively or in addition, the promoter may be tissue-specific or cell-specific. Non-limiting examples of suitable eukaryotic promoters (i.e., promoters functional in eukaryotic cells) may include long terminal repeats (LTRs) from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, retroviruses, human elongation factor-1 promoter (EF1), hybrid constructs comprising cytomegalovirus (CMV) enhancers fused to chicken beta actin promoter (CAG), mouse stem cell virus promoter (MSCV), phosphoglycerate kinase-1 locus promoter (PGK), and mouse metallothionein-I. The promoter may be a fungal promoter. The promoter can be a plant promoter. A database of plant promoters can be found (e.g., PlantProm). The expression vector can also include a ribosome binding site and a transcription terminator for translation initiation. The expression vector can also include an appropriate sequence for amplifying expression. In some cases, the promoter disclosed herein can be any tissue-specific promoter provided herein, or any cell type-specific promoter provided herein.

[0256] The size of the heterologous polynucleotide in the one or more heterologous polynucleotides (e.g., a single heterologous polynucleotide) can be at least or up to about 2.5 kilobases, at least or up to about 2.6 kilobases, at least or up to about 2.7 kilobases, at least or up to about 2.8 kilobases, at least or up to about 2.9 kilobases, at least or up to about 3.0 kilobases, at least or up to about 3.1 kilobases, at least or up to about 3.2 kilobases, at least or up to about 3.3 kilobases, at least or up to about 3.4 kilobases, at least or up to about 3.5 kilobases, at least or up to about 3.6 kilobases, at least or up to about 3.7 kilobases, at least or up to about 3.8 kilobases, at least or up to about 3.9 kilobases, at least or up to about 4. .0 kilobases, at least or up to about 4.1 kilobases, at least or up to about 4.2 kilobases, at least or up to about 4.3 kilobases, at least or up to about 4.4 kilobases, at least or up to about 4.5 kilobases, at least or up to about 4.6 kilobases, at least or up to about 4.7 kilobases, at least or up to about 4.8 kilobases, at least or up to about 4.9 kilobases, at least or up to about 5.0 kilobases, at least or up to about 5.5 kilobases, at least or up to about 6.0 kilobases, at least or up to about 6.5 kilobases, at least or up to about 7.0 kilobases, at least or up to about 7.5 kilobases, at least or up to about 8.0 kilobases, at least or up to about 9.0 kilobases, or at least or up to about 10 kilobases. In some cases, the size of the heterologous polynucleotide in one or more heterologous polynucleotides (e.g., a single heterologous polynucleotide) can be about 3 kilobases to about 5 kilobases, about 3 kilobases to about 4.8 kilobases, about 3 kilobases to about 4.6 kilobases, about 3 kilobases to about 4.4 kilobases, about 3 kilobases to about 4.2 kilobases, about 3 kilobases to about 4.0 kilobases, about 3 kilobases to about 3.5 kilobases, about 3.5 kilobases to about 5 kilobases, about 3.5 kilobases to about 4.8 kilobases, about 3.5 kilobases to about 4.6 kilobases. , about 3.5 kilobases to about 4.4 kilobases, about 3.5 kilobases to about 4.2 kilobases, about 3.5 kilobases to about 4 kilobases, about 4 kilobases to about 5 kilobases, about 4 kilobases to about 4.9 kilobases, about 4 kilobases to about 4.8 kilobases, about 4 kilobases to about 4.7 kilobases, about 4 kilobases to about 4.6 kilobases, about 4 kilobases to about 4.5 kilobases, about 4 kilobases to about 4.4 kilobases, about 4 kilobases to about 4.3 kilobases, about 4 kilobases to about 4.2 kilobases, or about 4 kilobases to about 4.1 kilobases.

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

[0258] In some embodiments, the length of the first polynucleotide sequence can be at least or up to about 1400 nucleotides, at least or up to about 1420 nucleotides, at least or up to about 1440 nucleotides, at least or up to about 1450 nucleotides, at least or up to about 1460 nucleotides, at least or up to about 1480 nucleotides, at least or up to about 1500 nucleotides, at least or up to about 1520 nucleotides, at least or up to about 1540 nucleotides, at least or up to about 1550 nucleotides, at least or up to about 1560 nucleotides, at least or up to about 1580 nucleotides, at least or up to about 1600 nucleotides, at least or up to about 1620 nucleotides, at least or up to about 1640 nucleotides, at least or up to about 1650 nucleotides, at least or up to about 1660 nucleotides, at least or up to about 1680 nucleotides, or at least or up to about 1700 nucleotides.

[0259] In some embodiments, the length of the second polynucleotide sequence can be at least or up to about 80 nucleotides, at least or up to about 85 nucleotides, at least or up to about 90 nucleotides, at least or up to about 91 nucleotides, at least or up to about 92 nucleotides, at least or up to about 93 nucleotides, at least or up to about 94 nucleotides, at least or up to about 95 nucleotides, at least or up to about 96 nucleotides, at least or up to about 97 nucleotides, at least or up to about 98 nucleotides, at least or up to about 99 nucleotides, at least or up to about 100 nucleotides, at least or up to about 101 nucleotides, at least or up to about 102 nucleotides, at least or up to about 103 nucleotides, at least or up to about 104 nucleotides, at least or up to about 105 nucleotides, at least or up to about 106 nucleotides, at least or up to about 107 nucleotides, at least or up to about 108 nucleotides, at least or up to about 109 nucleotides, at least or up to about 110 nucleotides, at least or up to about 112 nucleotides, at least or up to about 114 nucleotides, at least or up to about 115 nucleotides, at least or up to about 116 nucleotides, at least or up to about 118 nucleotides, at least or up to about 120 nucleotides, at least or up to about 122 nucleotides, at least or up to about 124 nucleotides, at least or up to about 125 nucleotides, at least or up to about 126 nucleotides, at least or up to about 128 nucleotides, at least or up to about 130 nucleotides, at least or up to about 135 nucleotides, at least or up to about 140 nucleotides, at least or up to about 145 nucleotides, at least or up to about 150 nucleotides, at least or up to about 155 nucleotides, or at least or up to about 160 nucleotides.

[0260] In some embodiments, the sum of the length of the combined first polynucleotide sequence and the length of the second polynucleotide sequence can be small enough so that the vector encoding at least the Cas protein and the guide nucleic acid molecule can be (i) small / compact and / or (ii) have enough space for additional cargo (e.g., a gene regulatory factor operably coupled to the Cas protein, or a heterologous polynucleotide provided herein). Even if the vector has a small / compact size, the complex encoded by the vector containing the Cas protein and the guide nucleic acid molecule can also be functional. In some cases, the complex encoded by the vector can have functional activity to bind to the target polynucleotide sequence and edit (e.g., cut, delete nucleotides, add nucleotides, edit bases, etc.) at least a portion of the target polynucleotide sequence. In some cases, the complex encoded by the vector can have functional activity to achieve the regulated expression level of the target gene in the cell. Therefore, the regulated expression level of the target gene caused by the (A1) complex (alternatively or additionally, its activity level) can be comparable to or better than the control complex (A2) containing the Cas protein and the control guide nucleic acid molecule.

[0261] In some cases, the regulated expression level of the target gene caused by the (A1) complex can be comparable to (A2), such that (A1) differs from (A2) by no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 8%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2% or no more than 1%.

[0262] In some cases, the expression level of the target gene can be activated by the engineered nuclease variants and / or guide nucleic acid molecules disclosed herein, and the regulated expression level of the target gene caused by the (A1) complex can be better than that of (A2), such that (A1) is greater than (A2) by at least or up to about 1%, at least or up to about 2%, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, at least or up to about 100%, at least or up to about 150%, at least or up to about 20 ... %, at least or up to about 250%, at least or up to about 300%, at least or up to about 350%, at least or up to about 400%, at least or up to about 450%, or at least or up to about 500%, or such that (A1) is at least or up to about 0.1 times, at least or up to about 0.2 times, at least or up to about 0.3 times, at least or up to about 0.4 times, at least or up to about 0.5 times, at least or up to about 1 times, at least or up to about 1.5 times, at least or up to about 2 times, at least or up to about 3 times, at least or up to about 4 times, at least or up to about 5 times, at least or up to about 6 times, at least or up to about 7 times, at least or up to about 8 times, at least or up to about 9 times, or at least or up to about 10 times greater than (A2).

[0263] In some cases, the expression level of the target gene can be reduced (e.g., suppressed) by the engineered nuclease variants and / or guide nucleic acid molecules disclosed herein, and the regulated expression level of the target gene caused by the (A1) complex can be superior to that of (A2), such that (A1) is less than (A2) by at least or up to about 1%, at least or up to about 2%, at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, at least or up to about 100%, at least or up to about 150 ...150%, at least or up to about 160%, at least or up to about 170%, at least or up to about 180%, at least or up to about 190%, at least or up to about 200%, at least or up to about 210%, at least or up to about 220%, at least or up to about 230%, at least or up to about 240%, at least or up to about 250%, at least or up to about 260%, at least or up to about 270%, at least or up to about 280%, at least or up to about 290%, at least or up to about 300%, at to about 200%, at least or up to about 250%, at least or up to about 300%, at least or up to about 350%, at least or up to about 400%, at least or up to about 450%, or at least or up to about 500%, or such that (A1) is at least or up to about 0.1 times, at least or up to about 0.2 times, at least or up to about 0.3 times, at least or up to about 0.4 times, at least or up to about 0.5 times, at least or up to about 1 times, at least or up to about 1.5 times, at least or up to about 2 times, at least or up to about 3 times, at least or up to about 4 times, at least or up to about 5 times, at least or up to about 6 times, at least or up to about 7 times, at least or up to about 8 times, at least or up to about 9 times, or at least or up to about 10 times smaller than (A2).

[0264] In some cases, the control guide nucleic acid molecule can 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 can be longer than the scaffold sequence of the guide nucleic acid molecule encoded by the vector by at least or up to about 1 nucleotide, at least or up to about 2 nucleotides, at least or up to about 5 nucleotides, at least or up to about 10 nucleotides, at least or up to about 15 nucleotides, at least or up to about 20 nucleotides, at least or up to about 25 nucleotides, at least or up to about 30 nucleotides, at least or up to about 35 nucleotides, at least or up to about 40 nucleotides, at least or up to about 45 nucleotides, at least or up to about 50 nucleotides, at least or up to about 55 nucleotides, at least or up to about 60 nucleotides, at least or up to about 65 nucleotides, at least or up to about 70 nucleotides, at least or up to about 75 nucleotides, or at least or up to about 80 nucleotides. For example, the control guide nucleic acid molecule can include the polypeptide sequence of SEQ ID NO:10.

[0265] The method for delivering one or more heterologous polynucleotides provided herein to a cell can be related to a viral delivery method or a non-viral delivery method. Therefore, one or more heterologous polynucleotides can be one or more viral vectors (e.g., one or more AAV vectors). Alternatively, one or more heterologous polynucleotides can be a non-viral vector that is compounded with a non-viral delivery portion (e.g., a cationic lipid and / or a lipid particle (e.g., a lipid nanoparticle (LNP))) or is wrapped by a non-viral delivery portion.

[0266] Non-viral delivery methods for nucleic acids can include lipofection, nuclear transfection, microinjection, gene guns, viral particles, liposomes, immunoliposomes, polycations or lipids: nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced DNA uptake. Cationic and neutral lipids suitable for effective receptor recognition lipofection of polynucleotides can be used. Can be delivered to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration).

[0267] RNA or DNA virus-based systems can be used to target specific cells in vivo and deliver viral payloads to the nucleus of cells. Viral vectors can be directly applied (in vivo), or viral vectors can be used for in vitro treatment of cells, and modified cells can be optionally applied (ex vivo). Virus-based systems can include retroviruses, slow viruses, adenoviruses, adeno-associated viruses, and herpes simplex virus vectors for gene transfer. Integration in the host genome can be performed using retroviruses, slow viruses, and adeno-associated viruses gene transfer methods, which can result in long-term expression of the transgenic inserted. High transduction efficiency can be observed in many different cell types and target tissues.

[0268] The tropism of retrovirus can be changed by introducing foreign envelope proteins, thereby expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and produce high viral titers. The selection of retroviral gene transfer systems may depend on the target tissue. Retroviral vectors may include cis-acting long terminal repeats with the ability to package up to 6-10kb foreign sequences. The smallest cis-acting LTR may be sufficient to replicate and package vectors, which can be used to integrate therapeutic genes into target cells to provide permanent transgenic expression. Retroviral vectors may include retroviral vectors 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 be used. Adenovirus-based systems can result in transient expression of transgenes. Adenovirus-based vectors can have high transduction efficiencies in cells and may not require cell division. High titers and expression levels can be obtained using adenovirus-based vectors. Adeno-associated virus ("AAV") vectors can be used to transduce cells with target nucleic acids, for example, to produce nucleic acids and peptides in vitro, and for in vivo and ex vivo gene therapy procedures.

[0270] Packaging cells can be used to form viral particles capable of infecting host cells. Such cells can include 293 cells (e.g., for packaging adenovirus) and Psi2 cells or PA317 cells (e.g., for packaging retrovirus). Viral vectors can be produced by producing cell lines that package nucleic acid vectors into viral particles. The vector can contain the minimum viral sequence required for packaging and subsequent integration into the host. The vector can contain other viral sequences replaced by the expression cassette of the polynucleotide to be expressed. The missing viral function can be provided in trans by the packaging cell line. For example, the AAV vector can contain ITR sequences from the AAV genome, which are necessary for packaging and integration into the host genome. Viral DNA can be packaged in a cell line, which can contain helper plasmids encoding other AAV genes (i.e., rep and cap), but lacks ITR sequences. The cell line can also be used as an auxiliary adenovirus infection. Helper viruses can promote the replication of AAV vectors and the expression of AAV genes in helper plasmids. Adenovirus contamination can be reduced by, for example, heat treatment, and adenovirus is more sensitive to heat treatment than AAV.

[0271] Host cells can be transiently or non-transiently transfected with one or more vectors described herein. Cells can be transfected in a manner that occurs naturally in a subject. Cells can be taken out or derived from a subject and transfected. Cells can be derived from cells taken out from a subject, such as cell lines. In some embodiments, cells transfected with one or more vectors described herein are used to establish new cell lines comprising one or more vector-derived sequences. In some embodiments, cells transiently transfected with a composition disclosed herein (e.g., transiently transfected by one or more vectors or transfected with RNA) and modified by the activity of a heterologous polypeptide comprising an engineered nuclease disclosed herein are used to establish new cell lines comprising cells containing modifications but lacking any other exogenous sequences.

[0272] Any suitable vector compatible with the host cell can be used with the methods of the present disclosure. Non-limiting examples of vectors for eukaryotic host cells include pXT1, pSG5 (Stratagene TM ), pSVK3, pBPV, pMSG and pSVLSV40 (Pharmacia TM ).

[0273] In some embodiments, the additional ingredients of the compositions disclosed herein may include excipients. Non-limiting examples of excipients may include solvents, dispersion media, diluents or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, lipids (lipidoids), liposomes, lipid nanoparticles, polymers, lipid complexes (lipoplexes), core-shell nanoparticles, peptides, proteins, hyaluronidase, nanoparticle mimics, inert diluents, buffers, lubricants, oils and combinations thereof. In some instances, the compositions disclosed herein may include one or more excipients, each of which is in a certain amount to jointly increase the stability of (i) heterologous polypeptides or heterologous genes encoding them and / or (ii) cells or modified cells.

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

[0275] In some embodiments, any of the compositions disclosed herein can be administered to a subject orally, intraperitoneally, intravenously, intraarterially, transdermally, intramuscularly, liposomally, locally delivered via a catheter or stent, subcutaneously, intraadiposally, or intrathecally.

[0276] The compositions disclosed herein (e.g., pharmaceutical compositions) may be suitable for administration to humans. In addition, such compositions may be suitable for administration to any other animal, e.g., non-human animals, e.g., non-human mammals. It is well known that pharmaceutical compositions suitable for administration to humans are modified so that the compositions are suitable for administration to various animals, and ordinary skilled veterinary pharmacologists can design and / or perform such modifications only by ordinary (if any) experiments. Subjects contemplated for administration of the pharmaceutical compositions include, but are not limited to, humans and / or other primates; mammals, including commercially relevant mammals, such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or poultry, including commercially relevant poultry, such as poultry, chickens, ducks, geese, and / or turkeys. cell

[0277] In some embodiments, the cells provided herein can 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., modify the expression profile of a target gene (e.g., one or more genes in Table 1) of a target cell). The target cell can include a variety of cell types. The target cell can be in vitro. The target cell can be in vivo. The target cell can be ex vivo. The target cell can be a separated cell. The target cell can be a cell in an organism. The target cell can be an organism. The target cell can be a cell in a cell culture. The target cell can be one of a collection of cells. The target cell can be a mammalian cell or derived from a mammalian cell. The target cell can be a rodent cell or derived from a rodent cell. The target cell can be a human cell or derived from a human cell. The target cell can be a prokaryotic cell or derived from a prokaryotic cell. The target cell can be a bacterial cell or can be derived from a bacterial cell. The target cell can be an archaeal cell or derived from an archaeal cell. The target cell can be a eukaryotic cell or derived from a eukaryotic cell. The target cell can be a pluripotent stem cell. The target cell can be a plant cell or derived from a plant cell. The target cell can be an animal cell or derived from an animal cell. The target cell can be an invertebrate cell or derived from an invertebrate cell. The target cell can be a vertebrate cell or derived from a vertebrate cell. The target cell can be a microbial cell or derived from a microbial cell. The target cell can be a fungal cell or derived from a fungal cell. The target cell can be from a specific organ or tissue.

[0278] The target cell can be a stem cell or a progenitor cell. The target cell can include stem cells (e.g., adult stem cells, embryonic stem cells, induced pluripotent stem cells (iPS) cells) and progenitor cells (e.g., cardiac progenitor cells, neural progenitor cells, etc.). The target cell can include mammalian stem cells and progenitor cells, including rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, etc. Cloned cells can include the offspring of cells. The target cell can contain a target nucleic acid. The target cell can be present in a living organism. The target cell can be a genetically modified cell. The target cell can be a host cell.

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

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

[0281] If the target cells are primary cells, they can be harvested from the individual by any method. For example, leukocytes can be harvested by apheresis, leukocytapheresis, density gradient separation, etc. Cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. can be harvested by biopsy.

[0282] Non-limiting examples of cells that can serve as target cells include, but are not limited to, lymphocytes, such as B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, T helper cells), natural killer cells, cytokine-induced killer (CIK) cells; myeloid cells, such as granulocytes (basophils, eosinophils, neutrophils / hypersegmented neutrophils); neutrophil), monocytes / macrophages, red blood cells (reticulocytes), mast cells, thrombin / megakaryocytes, dendritic cells; cells from the endocrine system, including thyroid (thyroid epithelial cells, parafollicular cells), parathyroid (parathyroid chief cells, eosinophils), adrenal glands (chromaffin cells), pineal gland (pinealocytes) cells; nervous system cells, including glial cells (astrocytes, microglia), magnocellular neurosecretory cells, stellate cells, Boettcher cells, pituitary (gonadotropes, corticotropes, thyrotropes, somatotropes, lactotrophs); respiratory system cells, including alveolar epithelial cells (Pneumocytes) (type I alveolar epithelial cells, type II alveolar epithelial cells), Clara cells, goblet cells, dust cells (Dust cells) cell); circulatory system cells, including myocardiocytes and pericytes; digestive system cells, including stomach (chief cells and parietal cells), goblet cells, Paneth cells, G cells, D cells, ECL cells, I cells, K cells and S cells; enteroendocrine cells, including enterochromaffin cells and APUD cells, liver (hepatocytes and Kupffer cells), cartilage / bone / muscle; bone cells, including osteoblasts, osteocytes, osteoclasts, teeth (cementoblasts and ameloblasts); cartilage tissue cells, including chondroblasts and chondrocytes; skin cells, including hair cells (Trichocytes), keratinocytes and melanocytes (nevus cells); muscle cells, including myocytes; urinary system cells, including podocytes, juxtaglomerular cells, glomerular mesangial cells / extraglomerular mesangial cells, and proximal tubule brush border cells (Kidney proximal tubule brush border cells). cells, macula densa cells; reproductive system cells, including sperm, Sertoli cells, interstitial cells, and oocytes; and other cells,Including 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 shaft cells, cortical hair shaft cells, cuticular hair shaft cells, epidermal hair root sheath cells, hair root sheath cells of Huxley's layer, hair root sheath cells of Henle's layer, external hair root sheath cells, hair matrix cells (stem cells), wet stratified barrier epithelial cells (Wet stratified barrier epithelial cells epithelial cells), surface epithelial cells of the stratified squamous epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, basal cells (stem cells) of the epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, urothelial cells (lining the bladder and urethra), exocrine secretory epithelial cells, salivary mucous cells (polysaccharide-rich secretions), salivary serous cells (glycoprotein-rich secretions), Von Ebner gland cells in the tongue (rinsing taste buds), mammary gland cells (milk secretion), lacrimal gland cells (tear secretion), cerumen gland cells in the ear (secreting cerumen), eccrine sweat gland dark cells (secreting glycoproteins), eccrine sweat gland clear cells (secreting small molecules). Apocrine sweat gland cells (smellous secretion, sensitive to sex hormones), Moll gland cells in the eyelids (specialized sweat glands), sebaceous gland cells (lipid-rich sebum secretion), Bowman gland cells in the nose (rinsing the olfactory epithelium), Brunner gland cells in the duodenum (enzymes and alkaline mucus), seminal vesicle cells (secreting semen components,Including fructose for motile sperm), prostate cells (secreting semen components), bulbourethral gland cells (mucus secretion), Bartholin gland cells (vaginal lubricant secretion), Littre gland cells (mucus secretion), endometrial cells (carbohydrate secretion), goblet cells separated from the respiratory and digestive tracts (mucus secretion), gastric wall mucous cells (mucus secretion), gastric gland zymogen cells (pepsinogen secretion), gastric gland acid secretory cells (hydrochloric acid secretion), pancreatic acinar cells (bicarbonate and digestive enzyme secretion), small intestinal Paneth cells (lysozyme secretion), lung type II alveolar cells (surfactant secretion), lung Clara cells, hormone-secreting cells, anterior pituitary cells, somatotropin cells, lactotropin cells, thyroid stimulating cells, gonadotropin cells, corticotropin cells, middle pituitary cells, large cell neurosecretory cells, intestinal and respiratory cells, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid chief cells, eosinophils, adrenal cells, chromaffin cells, testicular Ley dig cells, ovarian follicle theca cells, ruptured ovarian follicle luteal cells, granulosa luteal cells, theca luteal cells, juxtaglomerular cells (renin secretion), macula densa cells of the kidney, metabolic and storage cells, barrier function cells (lungs, intestines, exocrine glands and urogenital tract), kidney, type I alveolar cells (lining of lung air spaces), pancreatic duct cells (alveolar heart cells), non-striated duct cells (sweat glands, salivary glands, breast glands, etc.), duct cells (spermatic vesicles, prostate, etc.), closed internal body cavity lining epithelial cells, ciliated cells with propulsion function, extracellular matrix secreting cells, contractile cells; skeletal muscle cells, stem cells, cardiomyocytes, blood and immune system cells, red blood cells (erythrocytes), megakaryocytes (platelet precursors), monocytes, connective tissue macrophages (various types), epidermal Langerhans cells, osteoclasts (in bones), dendritic cells (in lymphoid tissues) , microglia (in the central nervous system), neutrophils, eosinophils, basophils, 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 (all types) of the blood and immune systems, pluripotent stem cells, totipotent stem cells, induced pluripotent stem cells, adult stem cells, sensory sensory cells, autonomic neuronal cells, sensory organ and peripheral neuronal supporting cells, central nervous system neurons and glial cells, lens cells, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, oogonia / oocytes, sperm cells, spermatocytes, spermatogonia (stem cells of spermatocytes), sperm, nurse cells, follicle cells, Sertoli cells (in the testes), thymic epithelial cells, interstitial cells and interstitial kidney cells. ,

[0283] Cells (or target cells) can be engineered to contain (or express) any system or composition disclosed herein, or can be processed in vitro or ex vivo by any method disclosed herein and then administered to a subject, e.g., to treat a condition in the subject. For example, any subject modified cell product can be administered to a subject to treat a condition in the subject's body tissue. In some cases, cells can reside in a subject, and any of its systems or compositions can be administered to a subject to contact the cell through the system / composition (e.g., to engineer the cell with the system / composition). Example

[0284] Example 1: Engineered Nucleases

[0285] The Cas protein encoded by the polypeptide sequence of SEQ ID NO: 1 has a size of 529 amino acid residues. When the gene encoding the Cas protein is delivered in a vector (e.g., a viral vector, such as an AAV vector), reducing the size of such Cas proteins can provide more cargo space in the vector (e.g., a viral vector with cargo size or length restrictions). The increased cargo space in the vector can be used 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 the desired result (e.g., therapeutic effect). Alternatively or in addition, when a recombinant version of the Cas protein disclosed herein is delivered in a delivery vehicle (e.g., lipid nanoparticles, viral capsids, etc.), reducing the size of the Cas protein can provide more cargo space to accommodate, for example, at least one additional component to achieve the desired result. Without wishing to be bound by theory, the use of the engineered nuclease disclosed herein and at least one additional component can enhance its activity (e.g., its targeted gene binding, cutting, editing and / or regulation) compared to different control nucleases.

[0286] In some embodiments, throughout the examples of the present disclosure, one or more engineered nucleases of the present disclosure can be evaluated (e.g., in vitro) to evaluate the activity of each of the one or more engineered nucleases in binding, cutting and / or editing a target polynucleotide sequence, for example, regulating the expression and / or activity level of a target polynucleotide sequence of a polypeptide (e.g., protein) encoded by or operably coupled to a target polynucleotide sequence. In some examples, a heterologous polypeptide comprising an engineered nuclease and a heterologous polynucleotide comprising a guide nucleic acid (e.g., sgRNA) for a target polynucleotide can be tested in a cell (e.g., in vitro) to evaluate the gene knockout efficiency at the target polynucleotide. In some examples, a heterologous polypeptide comprising a nuclease-deficient variant of an engineered nuclease coupled (e.g., fused) to a gene effector (e.g., a gene activator, a gene repressor) that is heterologous to the engineered nuclease and a heterologous polynucleotide comprising a guide nucleic acid (e.g., sgRNA) to a target polynucleotide can be tested in cells (e.g., in vitro) to assess the ability to regulate the expression and / or activity level of a gene coupled to (or comprising) the target polynucleotide.

[0287] In some embodiments, throughout the disclosed examples, a library of multiple engineered nuclease candidates can be generated using whole plasmid amplification using primers in opposite orientations spanning the deleted region. Example 2: Engineering nucleases based on structural comparison

[0289] A. Methods

[0290] In some embodiments, the size (e.g., the number of amino acid residues) of a CasMini or an inactivated CasMini (dCasMini) (e.g., a mutant variant of the polypeptide sequence of SEQ ID NO: 1) can be further reduced while maintaining or enhancing its activity (e.g., overall epigenetic gene regulatory activity). For example, other Cas proteins that are smaller than the Cas protein of SEQ ID NO: 1 (e.g., naturally occurring Cas12f proteins, such as Un2Cas12f1 (SEQ ID NO: 2) or AsCas12f (SEQ ID NO: 3)) can be used as reference points to determine 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), such that its activity is minimally impaired or unaffected.

[0291] In some embodiments, the native Un1Cas12f1 nuclease encoded by SEQ ID NO: 1 can be engineered by creating at least one deletion to produce an engineered nuclease of the present disclosure, for example, due to one or more of the reasons described in Example 1.

[0292] In some cases, at least one deletion of the amino acid sequence of the engineered nuclease may be present in one or more regions of the native Un1Cas12f1 nuclease that are not structurally aligned with AsCas12f (SEQ ID NO: 3). When compared to AsCas12f, Un1Cas12f1 comprises an additional amino acid residue 1-71 domain, and at least a portion of the domain and / or one or more amino acid residues near the domain may be removed, for example, so that the engineered nuclease activity (e.g., the interaction of the engineered nuclease with a guide nucleic acid molecule (e.g., crRNA)) is minimally reduced or substantially not reduced. Figure 1 As shown, Un1Cas12f1 comprises a domain 110 that may not be conserved in AsCas12f (e.g., comprising at least a portion of an additional amino acid residue 1-71 domain). On the other hand, Un1Cas12f1 comprises a domain 120 that may be conserved in AsCas12f.

[0293] In some cases, at least one deletion of the amino acid sequence of the engineered nuclease can be present in one or more regions of the native Un1Cas12f1 nuclease that are not structurally aligned with Un2Cas12f1 (SEQ ID NO: 2). When compared to Un2Cas12f1, Un1Cas12f1 comprises an additional amino acid residue 41-71 domain, and at least a portion of the domain and / or one or more amino acid residues near the domain can be removed, for example, so that the activity of the engineered nuclease (e.g., the interaction of the engineered nuclease with a guide nucleic acid molecule (e.g., crRNA)) is minimal or substantially not reduced. Figure 2 As shown, Un1Cas12f1 comprises a domain 210 that may not be conserved in Un2Cas12f1 (e.g., comprising at least a portion of an additional domain of amino acid residues 41-71). On the other hand, Un1Cas12f1 comprises a domain 220 that may be conserved in Un2Cas12f1.

[0294] B. Exemplary Library Design

[0295] Figure 3ASchematically illustrates different regions of Un1Cas12f1 as encoded by SEQ ID NO: 1 and exemplary domains (310, 320, and 330) that can be at least partially deleted to generate one or more engineered nucleases disclosed herein. For example, domain 310 can involve an N-terminal deletion (e.g., amino acid residues 2-76 when aligned with SEQ ID NO: 1), which can be evaluated by removing 3 amino acids at a time from the N-terminus of the protein to generate 25 variants. In another example, domain 320 may involve a partial deletion of the N-terminus (e.g., amino acid residues 41-71 when aligned with SEQ ID NO: 1), which may be evaluated by removing an additional 2 amino acids from the middle of this region at each increment to generate 16 variants (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 a different example, domain 330 may involve a C-terminal deletion (e.g., the last 75 amino acid residues when aligned with SEQ ID NO: 1), which may be evaluated by removing 3 amino acids from the C-terminus at a time to generate 25 variants.

[0296] Examples of engineered nucleases with reduced nuclease activity produced according to the present disclosure can include SEQ ID NOs: 4-9 as provided herein.

[0297] SEQ ID NO:4 (comprising an N-terminal deletion, e.g., amino acid residues 2-21 when aligned with SEQ ID NO:1 (AKNTITKTLKLRIVRPYNSA))

[0298] SEQ ID NO:5 (comprising an N-terminal deletion, e.g., amino acid residues 2-31 (AKNTITKTLKLRIVRPYNSAEVEKIVADEK) when aligned with SEQ ID NO:1)

[0299] SEQ ID NO:6 (comprising a C-terminal deletion, e.g., amino acid residues 510-529 (DYNAALNISNPKLKSTKEEP) when aligned with SEQ ID NO:1)

[0300] SEQ ID NO:7 (comprising a C-terminal deletion, e.g., amino acid residues 500-529 (CEKCNFKENADYNAALNISNPKLKSTKEEP) when aligned with SEQ ID NO:1)

[0301] SEQ ID NO:8 (comprising a partial N-terminal deletion, e.g., amino acid residues 47-66 (KEACSKHLKVAAYCTTQVER) when aligned with SEQ ID NO:1)

[0302] SEQ ID NO:9 (comprising a partial N-terminal deletion, e.g., amino acid residues 41-71 (KNKDKVKEACSKHLKVAAYCTTQVERNACLF) when aligned with SEQ ID NO:1)

[0303] Example 3: Engineering nucleases based on deletion landscape

[0304] The native Un1Cas12f1 nuclease encoded by SEQ ID NO: 1 can be engineered by generating at least one deletion to produce the engineered nuclease of the present disclosure, for example due to one or more of the reasons described in Example 1. As disclosed herein, at least one deletion of the amino acid sequence of the engineered nuclease can be determined based on the deletion (or truncation) landscape of the Cas protein (or its nuclease-deficient variant).

[0305] For example, Figure 4 As shown, the mapping of the truncation landscape of dCas9 indicates tolerance to significant deletions (e.g., deletions of amino acid residues 167-316 of dCas9). Therefore, a similar deletion landscape can be used to determine one or more amino acid residues (e.g., multiple continuous amino acid residues) of the natural Un1Cas12f1 (or its nuclease-deficient variant) to produce a deletion mutant of the Un1Cas12f1 protein without significantly affecting the activity (e.g., not affecting the activity). In some cases, the deletion of about 20 amino acid residues (e.g., from one continuous region, or from two non-continuous regions) can be determined to produce a mutant variant of Un1Cas12f1 that still exhibits comparable (or enhanced) activity compared to wild-type Un1Cas12f1. In some cases, a small deletion window (e.g., a window of about 3 to about 5 amino acids) of the entire natural Un1Cas12f1 protein can be evaluated for deletion landscape.

[0306] Figure 3BSchematically illustrated is a deletion landscape method for generating engineered nuclease variants of Un1Cas12f1. A deletion block 510 of multiple amino acid residues (e.g., multiple consecutive amino acid residues, such as 5 consecutive amino acid residues) throughout at least a portion of Un1Cas12f1 (e.g., the entire Un1Cas12f1, excluding the dimerization domain and / or the PAM domain) can be scanned to generate, for example, up to 95 variants for individual transfection screening.

[0307] Example 4: Engineering nucleases based on deletion landscape

[0308] In addition to deleting one or more amino acid residues from the native Un1Cas12f1 nuclease encoded by SEQ ID NO: 1 to produce an engineered nuclease, one or more amino acid residues can be mutated compared to the native Un1Cas12f1 nuclease to further modify the engineered nuclease of the present disclosure, for example, due to one or more of the reasons described in Example 1. As disclosed herein, at least one deletion of the amino acid sequence of the engineered nuclease can be determined based on site-saturation mutagenesis (SSM). For example, a single codon or a group of codons of a polynucleotide encoding a native Un1Cas12f1 protein (or a nuclease-deficient variant thereof) can be replaced by one or more possible amino acids at a position that enhances activity (e.g., for improved epigenetic regulation, such as enhanced activation / repression, different PAM recognition, etc.), stability, expression, binding to a corresponding guide nucleic acid molecule, etc.

[0309] Example 5: Evaluation of engineered nucleases

[0310] Nuclease-deficient variants of engineered nucleases disclosed herein (e.g., produced according to Examples 1-4) can be coupled (e.g., fused) to heterologous gene effectors (e.g., VP16, VP64, p65, Rta, VPR, etc.), and one or more heterologous polypeptides encoding such fusion proteins and / or guide RNAs can be transfected into cells to assess the activation of the expression / activity level of the target gene. For example, engineered HEK93T cells with synthetic reporters can be used, in which fluorescence activation can be measured as a readout. When multiple engineered nucleases are identified, the screening method can be repeated (e.g., by using engineered HEK93T cells) to (i) confirm the previous screening results and / or (ii) identify the highest hits.

[0311] Based on the results of the first round of engineering, the deletion of multiple amino acid residues can be combined (e.g., when aligned with the polypeptide sequence of SEQ ID NO: 1) to generate one or more additional engineered nucleases. Alternatively or in addition, one or more additional engineered nucleases can be generated by evaluating one or more particle deletions around one or more leads in the first round of engineering.

[0312] A. First round of engineering

[0313] Multiple sequence alignment of dCasMINI (SEQ ID NO: 10) with one or more naturally occurring Cas12f protein orthologs (e.g., Un1Cas12f1, Un2Cas12f1, AsCas12f and other orthologs provided in Table 2) with reported nuclease activity in bacteria, for example, identifying one or more potentially beneficial mutations in generating engineered nuclease variants disclosed herein. Homology modeling was also performed on Cas12f orthologs to identify structural conservation (e.g., not entirely based on amino acid-based conservation), thereby generating engineered nuclease variants. Combining these two methods (e.g., sequence alignment and structural conservation analysis), the extension of amino acid sequences and the combination of single residue mutations were determined to be promising candidates for generating the engineered nuclease variants. Based on the identified information, chimeric protein variants were designed by sequence exchange, and mutant variants were designed by one or more residue mutations. In some cases, sequence exchange variants also result in an overall reduced protein size.

[0314] In the first round of engineering, engineered nuclease variants (e.g., truncated variants, chimeric protein variants and / or mutant variants) are generated, tested and screened to identify lead hits. For example, a variety of chimeric engineered nuclease variants with reduced nuclease activity (as provided in Table 3B) are designed and tested in combination (e.g., fusion) with gene regulators (e.g., gene activators or gene repressors) to test their efficacy in gene regulation compared to the control dCasMINI (SEQ ID NO: 10).

[0315] Gene activation is assessed by fusing each engineered nuclease variant with a gene activator. Each engineered nuclease variant with reduced nuclease activity is cloned into a dCas plasmid in frame with a gene activator (e.g., VPR) for transcriptional activation. HEK293T cells are transfected in a 96-well plate format with the same sgRNA plasmid and individual dCas variant plasmids in triplicate or quadruplicate. After transfection for a few days (e.g., 2 days, 3 days, or 4 days), CD2 protein levels are quantified by cell surface antibody staining of living cells followed by flow cytometry, while ELISA is used to measure secreted IFNγ (IFNγ) protein levels in cell culture supernatants. The gene activation levels of each engineered nuclease variant are compared with the activity of the dCasMINI fused with the same gene activator as a control.

[0316] Gene repression is assessed by fusing each engineered nuclease variant with a gene repressor. Each engineered nuclease variant with reduced nuclease activity is cloned into a dCas plasmid in frame with a gene repressor alone for transcriptional inhibition. In a 96-well plate format, HEK293T GFP reporter cells (eg, ESR221) are transfected with the same sgRNA plasmid and individual dCas variant plasmids in triplicate or quadruplicate. After a few days (eg, 5 to 7 days) after transfection, the inhibition of GFP expression is measured by flow cytometry. The gene repression level of each engineered nuclease variant is compared with the activity of the dCasMINI fused to the same gene activator as a control.

[0317] like Figure 5 As shown, among the chimeric engineered nuclease variants cA1 to cA9, the chimeric engineered nuclease variant cA2 resulted in the greatest activation of IFNγ expression (e.g., greater than that of the dCasMINI control) as well as CD2 activation (as summarized in Table 3A). Additional engineered nuclease variants were designed with one or more mutations relative to Un1Cas12f1, while the overall size of the engineered nuclease variant was not significantly changed compared to dCasMINI. See the amino acid sequences of such engineered nuclease variants in Table 4B. Figure 5 As shown, when fused to a gene activator, a variety of engineered nucleases (e.g., mD2, mD4) exhibited comparable or higher efficacy than the control dCasMINI in activating a target gene (e.g., IFNγ or CD2) (as summarized in Table 4A). Figure 6As shown, some of the various engineered nucleases (e.g., mD2, mD4) exhibited comparable or higher efficacy in repressing target genes (e.g., eGFP) compared to the control dCasMINI (as summarized in Table 4A). Another engineered nuclease variant (t1) was also identified that contained a deletion in the C-terminal region compared to SEQ ID NO: 1, but did not sacrifice gene regulatory activity. Fig.15 As shown, when fused to a gene activator, the engineered nuclease variant t1 exhibited comparable efficacy to the control dCasMINI in activating a target gene (e.g., IFNγ).

[0318] A chimeric engineered nuclease variant cA2 was designed that has a sequence deletion at the zinc binding motif. Without wishing to be bound by theory, such deletions at specific zinc binding motifs result in smaller nuclease variants that maintain or even improve gene regulatory activity (e.g., epigenetic modification activity) when operably linked (e.g., fused) to one or more gene regulatory factors.

[0319] Table 5A summarizes each set of modifications relative to the amino acid sequence of Un1Cas12f1 (SEQ ID NO: 1) embedded in each engineered nuclease variant that resulted in enhanced gene regulatory activity compared to dCasMINI (SEQ ID NO: 10).

[0320] Without wishing to be bound by theory, one or more additional modifications may be made to any of the engineered nuclease variants from Table 3A, Table 4A, Table 5A, and Table 5B to enhance a desired activity (e.g., complex formation with a guide nucleic acid molecule, target sequence nuclease activity, increased expression of a target gene, decreased expression of a target gene, etc.).

[0321] B. Second round of engineering

[0322] In the second round of engineering, the engineered nuclease variant "cA2" (SEQ ID NO: 12) was selected as a scaffold, and a new library of engineered nuclease variants was generated by grafting one or more combinations of mutations and / or truncations identified and listed in Table 5A onto the cA2 scaffold. Table 5B shows a list of engineered nuclease variants in the new library, indicating the combination of modifications that have been grafted onto each engineered nuclease variant. Table 5C shows the corresponding amino acid sequence of each engineered nuclease variant listed in Table 5B. Figure 7As shown, when tested for their ability to activate target genes (e.g., CD2, IFNγ, and CXCR4) in combination with gene activators (e.g., VPR), multiple engineered nuclease variants exhibited comparable or enhanced gene activation to the control dCasMINI, with some variants outperforming the starting scaffold cA2 (e.g., cA2.6, cA2.39, cA2.69, cA2.29, cA2.10, cA2.4, cA2.21, cA2.13, cA2.3, cA2.16, cA2.23, cA2.8 , cA2.31, cA2.30, cA2.11, cA2.5, cA2.41, cA2.49, cA2.26, cA2.14, cA2.20, cA2.1, cA2.24, cA2.58, cA2.61, cA2.38, cA2.88, cA2.2, cA2.51, cA2.34, cA2.25, cA2.85, cA2.54, cA2.15, cA2.75, cA2.32, cA2.90, cA2.89 or cA2.46). In contrast, some of the newly engineered nuclease variants did not show enhanced activity compared to the control dCasMINI (e.g., cA2.55, cA2.84). Fig.16 As shown, when tested for their ability to inhibit target genes (e.g., CXCR4) in combination with gene repression regulators (e.g., ZNF10-KRAB-hDNMT3L), multiple engineered nuclease variants exhibited comparable or enhanced gene repression to the control dCasMINI, with some of the engineered nuclease variants performing comparable to the starting scaffold cA2 (e.g., cA2.69, cA.2.29, cA2.4, cA2.2, cA2.34, or cA2.7). In contrast, some of the new engineered nucleases did not exhibit enhanced gene repression activity compared to the control dCasMINI (e.g., cA2.26, cA2.63).

[0323] In summary, two sequence deletion regions were identified as compact Cas variants herein. Additional point mutations were also identified that contribute to improved gene regulatory activity when used in combination with gene regulatory factors and guide nucleic acid molecules. Combined with these sequence modifications, cA2 protein (SEQ ID NO: 12) and its variants (with additional point mutations and sequence deletions, see Table 5C) outperformed the control dCasMINI in both transcriptional activation and repression activity at multiple endogenous loci.

[0324] Without wishing to be bound by theory, any amino acid deletion, structural deletion, or amino acid modification (e.g., mutation / substitution) identified herein for enhancing the gene regulatory efficacy of an engineered nuclease variant can be "transplanted" back into any other naturally or non-naturally occurring Cas protein (e.g., a naturally occurring Cas protein selected from Table 2 or an inactivated nuclease variant thereof) to produce one or more additional engineered nuclease variants that can produce higher gene regulatory efficacy than a control Cas protein (e.g., a dCasMINI disclosed herein).

[0325] Example 6: Evaluation of engineered nucleases

[0326] The nuclease activity of engineered nuclease variants of engineered nucleases disclosed herein (e.g., produced according to Examples 1-4) that retain at least a portion of the nuclease activity (e.g., compared to SEQ ID NO: 1) can be tested in cells. Polypeptides comprising engineered nuclease variants and / or guide RNAs can be transfected into cells to assess the ability of complexes comprising engineered nucleases and guide RNAs to produce breaks in target polynucleotide sequences (e.g., double-strand breaks in or near target polynucleotide sequences comprising appropriate PAMs). Such nuclease activity can be observed by in vitro enzymatic assays (e.g., with purified enzymes or cell lysates) or in vivo (e.g., in E. coli or eukaryotic cells).

[0327] Example 7: Guided Nucleic Acid Scaffold Engineering

[0328] The Cas protein may comprise a naturally occurring Cas protein (e.g., (i) a polypeptide sequence of SEQ ID NO: 1 or (ii) a polypeptide sequence of any Cas protein selected from Table 2) or any modification thereof (e.g., an engineered polypeptide comprising an engineered nuclease variant disclosed herein). The Cas protein may form a complex (e.g., a ribonucleoprotein (RNP) complex) with a guide nucleic acid molecule (e.g., sgRNA), which may bind to a target polynucleotide sequence to regulate a target gene. The guide nucleic acid molecule may comprise (i) a scaffold sequence to at least form a complex with the Cas protein; and (ii) a spacer sequence that exhibits at least partial sequence complementarity with a target polynucleotide sequence. Without wishing to be bound by theory, the generation of a smaller and more compact guide nucleic acid scaffold sequence (e.g., compared to that of dCasMINI) may enhance the overall biological activity of the complex (e.g., enhanced target gene regulation, reduced off-target effects, etc.).

[0329] The control guide nucleic acid used throughout this example is denoted as "SQ" (see Figure 8 Schematic diagram in and its scaffold polynucleotide sequence in Table 6B). The control guide nucleic acid SQ is 179 nucleotides (nt) long, including a 20 nt spacer sequence.

[0330] Without wishing to be bound by theory, one or more of the following regions from the guide nucleic acid sequence (e.g., SQ) may be modified (e.g., mutated or deleted) while maintaining the biological activity of the resulting RNP complex or improving such biological activity. Fig. 9 As shown, such regions may include loops (e.g., stem loops) within the scaffold sequence that are structurally disoriented and / or solvent exposed. For an SQ guide nucleic acid sequence, non-limiting examples of such regions may include region 1 (e.g., 1-23 nt), region 2 (e.g., 35-61 nt), and region 3 (e.g., 138-143 nt), each nt position relative to the polynucleotide sequence of the SQ.

[0331] A. Truncated Guide Nucleic Acid Molecules

[0332] Guide RNA scaffold variants were generated to contain combinations of stepwise deletions in the three regions discussed above. For region 1, stepwise base pair trimming was performed. For region 2, in addition to removing misdirected stem-loops, we also installed stable hairpin structures commonly found in RNA (e.g., cUUCGg). For region 3 and its adjacent sequences, one or more deletions (e.g., a deletion of 136-149 nt) were made, and additional stepwise truncations of other sequences were performed to further reduce the scaffold size near region 3.

[0333] Gene repression is evaluated by testing gRNA scaffold variants and engineered nuclease variants fused to gene repressors. Each gRNA scaffold variant is cloned individually into a sgRNA plasmid with the same spacer sequence targeting the gene of interest. In a 96-well plate format, HEK293T GFP reporter cells ESR221 (in each well) are transfected with the same dCas plasmid (e.g., encoding a gene repression regulator, such as dCas fused to a gene repressor (e.g., KRAB) (e.g., dCasMINI disclosed herein)) and individual sgRNA variant plasmids in triplicate or quadruplicate. Five to seven days after transfection, the inhibition of GFP expression is measured by flow cytometry. The GFP inhibition level of each gRNA scaffold variant is compared with the activity of the SQ control gRNA.

[0334] Gene activation is assessed by testing gRNA scaffold variants and engineered nuclease variants fused to gene activators. Each gRNA scaffold variant is cloned individually into an sgRNA plasmid with the same spacer sequence targeting a gene of interest. In a 96-well plate format, HEK293T cells are transfected in triplicate or quadruplicate with the same dCas plasmid (e.g., encoding a gene activation regulator, such as a dCas fused to a gene activator (e.g., VPR) (e.g., dCasMINI disclosed herein) and individual sgRNA variant plasmids. Two to four days after transfection, target gene activation is measured (e.g., by cell surface antibody staining of live cells followed by quantification of CD2 protein levels by flow cytometry, while secreted IFNγ protein levels are measured using ELISA t...

Claims

1. An engineered polypeptide comprising an engineered nuclease, wherein: The engineered nuclease comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1, wherein the amino acid sequence comprises at least one deletion from amino acid residues 2-100 compared to the polypeptide sequence of SEQ ID NO:

1.

2. The engineered polypeptide according to claim 1, wherein Compared to the amino acid sequence of SEQ ID NO: 1, the at least one deletion is from amino acid residues 31-40.

3. The engineered polypeptide according to claim 1, wherein Compared to the amino acid sequence of SEQ ID NO: 1, the at least one deletion is from amino acid residues 41-60.

4. The engineered polypeptide according to claim 1, wherein Compared to the amino acid sequence of SEQ ID NO: 1, the at least one deletion is from amino acid residues 61-80.

5. The engineered polypeptide according to claim 1, wherein Compared to the amino acid sequence of SEQ ID NO: 1, the at least one deletion comprises at least 10 amino acid residues.

6. The engineered polypeptide according to claim 1, wherein Compared to the amino acid sequence of SEQ ID NO: 1, the at least one deletion comprises at least 30 amino acid residues.

7. The engineered polypeptide according to claim 1, wherein The amino acid sequence of the engineered nuclease has at least 85% sequence identity to the amino acid sequence of SEQ ID NO:

1.

8. The engineered polypeptide according to claim 1, wherein The engineered nuclease is up to about 528 amino acids in length.

9. The engineered polypeptide according to claim 1, wherein Compared to the amino acid sequence of SEQ ID NO: 1, the engineered nuclease comprises an amino acid substitution at D326 or D510, thereby reducing the nuclease activity of the engineered nuclease.

10. The engineered polypeptide according to claim 1, wherein Compared to the amino acid sequence of SEQ ID NO: 1, the engineered nuclease comprises one or more amino acid substitutions selected from the group consisting of D143R, T147R, K330R and E528R.

11. The engineered polypeptide according to claim 1, wherein The engineered nuclease comprises an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO:

11.

12. The engineered polypeptide according to claim 1, wherein The engineered nuclease comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:

11.

13. A method for regulating the expression and / or activity of a target gene in a cell, the method comprising: contacting the cell with an engineered polypeptide capable of forming a complex with a guide nucleic acid molecule that exhibits specific binding to the target gene, wherein the engineered polypeptide comprises an engineered nuclease, the engineered nuclease comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1, wherein the amino acid sequence comprises at least one deletion from amino acid residues 2-100 compared to the amino acid sequence of SEQ ID NO: 1, Wherein, after the contact, the binding of the complex to the target gene leads to the regulation of the expression and / or activity of the target gene.

14. The method according to claim 13, wherein: The binding of the complex to the target gene results in enhanced regulation of the expression and / or activity of the target gene in the cell, compared to a control cell contacted by a control complex, wherein the control complex comprises (i) a control engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 10 and (ii) the guide nucleic acid molecule.

15. The method according to claim 13, wherein: Compared to the amino acid sequence of SEQ ID NO: 1, the at least one deletion is from amino acid residues 31-40.

16. The method according to claim 13, wherein: Compared to the amino acid sequence of SEQ ID NO: 1, the at least one deletion is from amino acid residues 41-60.

17. The method according to claim 13, wherein: Compared to the amino acid sequence of SEQ ID NO: 1, the at least one deletion is from amino acid residues 61-80.

18. The method according to claim 13, wherein: Compared to the amino acid sequence of SEQ ID NO: 1, the at least one deletion comprises at least 10 amino acid residues.

19. The method according to claim 13, wherein: Compared to the amino acid sequence of SEQ ID NO: 1, the at least one deletion comprises at least 30 amino acid residues.

20. The method according to claim 13, wherein: The amino acid sequence of the engineered nuclease is at least 85% identical to the amino acid sequence of SEQ ID NO:

1.

21. The method according to claim 13, wherein: The engineered nuclease is up to about 528 amino acids in length.

22. The method according to claim 13, wherein: Compared to the amino acid sequence of SEQ ID NO: 1, the engineered nuclease comprises an amino acid substitution at D326 or D510, thereby reducing the nuclease activity of the engineered nuclease.

23. The method according to claim 13, wherein: Compared to the amino acid sequence of SEQ ID NO: 1, the engineered nuclease comprises one or more amino acid substitutions selected from the group consisting of D143R, T147R, K330R and E528R.

24. The method according to claim 13, wherein: The amino acid sequence is at least 60% identical to the amino acid sequence of SEQ ID NO:

11.

25. The method of claim 13, wherein: The amino acid sequence is at least 80% identical to the amino acid sequence of SEQ ID NO:

11.

26. An engineered polypeptide comprising an engineered nuclease, wherein: The engineered nuclease comprises an amino acid sequence that is greater than 92% identical to the amino acid sequence of SEQ ID NO:

12.

27. The engineered polypeptide according to claim 26, wherein The amino acid sequence of the engineered nuclease is at least about 93% identical to the amino acid sequence of SEQ ID NO:

12.

28. The engineered polypeptide according to claim 26, wherein The amino acid sequence of the engineered nuclease is at least about 95% identical to the amino acid sequence of SEQ ID NO:

12.

29. The engineered polypeptide according to claim 26, wherein The amino acid sequence is up to about 528 amino acids in length.

30. The engineered polypeptide according to claim 26, wherein Compared with the amino acid sequence of SEQ ID NO: 1, the amino acid sequence comprises an amino acid substitution at D326 or D510, thereby reducing the nuclease activity of the engineered nuclease.

31. The engineered polypeptide according to claim 26, wherein Compared to the amino acid sequence of SEQ ID NO: 1, the amino acid sequence comprises one or more amino acid substitutions selected from the group consisting of D143R, T147R, K330R and E528R.

32. The engineered polypeptide according to claim 26, wherein The amino acid sequence is at least 60% identical to the amino acid sequence of SEQ ID NO:

11.

33. The engineered polypeptide according to claim 26, wherein The amino acid sequence is at least 80% identical to the amino acid sequence of SEQ ID NO:

11.

34. A method for regulating the expression and / or activity of a target gene in a cell, the method comprising: contacting the cell with an engineered polypeptide capable of forming a complex with a guide nucleic acid molecule that exhibits specific binding to the target gene, wherein the engineered polypeptide comprises an engineered nuclease, wherein the engineered nuclease comprises an amino acid sequence that is greater than 92% identical to the amino acid sequence of SEQ ID NO: 12, Wherein, after the contact, the binding of the complex to the target gene leads to the regulation of the expression and / or activity of the target gene.

35. The method of claim 34, wherein: The binding of the complex to the target gene results in enhanced regulation of the expression and / or activity of the target gene in the cell, compared to a control cell contacted by a control complex, wherein the control complex comprises (i) a control engineered polypeptide comprising the amino acid sequence of SEQ ID NO: 10 and (ii) the guide nucleic acid molecule.

36. The method of claim 34, wherein: The amino acid sequence of the engineered nuclease is at least about 93% identical to the amino acid sequence of SEQ ID NO:

12.

37. The method of claim 34, wherein: The amino acid sequence of the engineered nuclease is at least about 95% identical to the amino acid sequence of SEQ ID NO:

12.

38. The method of claim 34, wherein: The amino acid sequence is up to about 528 amino acids in length.

39. The method of claim 34, wherein: Compared to the amino acid sequence of SEQ ID NO: 1, the engineered nuclease comprises an amino acid substitution at D326 or D510, thereby reducing the nuclease activity of the engineered nuclease.

40. The method of claim 34, wherein: Compared to the amino acid sequence of SEQ ID NO: 1, the engineered nuclease comprises one or more amino acid substitutions selected from the group consisting of D143R, T147R, K330R and E528R.

41. The method of claim 34, wherein: The amino acid sequence is at least 60% identical to the amino acid sequence of SEQ ID NO:

11.

42. The method of claim 34, wherein: The amino acid sequence is at least 80% identical to the amino acid sequence of SEQ ID NO:

11.

43. A composition comprising a guide nucleic acid molecule configured to form a complex with a Cas protein, wherein: The guide nucleic acid molecule comprises: a spacer sequence that exhibits specific binding to a target gene; and A scaffold sequence, wherein the scaffold sequence is used to form the complex with the Cas protein, wherein the scaffold sequence comprises a continuous polynucleotide sequence having at least 74% sequence identity with the polynucleotide sequence of SEQ ID NO:

597.

44. The composition according to claim 43, wherein The contiguous polynucleotide sequence has at least 75% sequence identity with the polynucleotide sequence of SEQ ID NO:

597.

45. The composition according to claim 43, wherein The contiguous polynucleotide sequence has at least 80% sequence identity with the polynucleotide sequence of SEQ ID NO:

597.

46. ​​The composition according to claim 43, wherein The contiguous polynucleotide sequence has at least 90% sequence identity with the polynucleotide sequence of SEQ ID NO:

597.

47. The composition according to claim 43, wherein The scaffold sequence is up to about 110 nucleotides in length.

48. The composition according to claim 43, wherein The scaffold sequence is at most about 105 nucleotides in length.

49. The composition of claim 43, comprising the Cas protein.

50. The composition according to claim 49, wherein The Cas protein is up to about 528 amino acids in length.

51. The composition according to claim 49, wherein The Cas protein is up to about 520 amino acids in length.

52. A method for regulating the expression and / or activity of a target gene in a cell, the method comprising: The cell is contacted with a composition comprising a guide nucleic acid molecule, wherein the guide nucleic acid molecule is configured to form a complex with a Cas protein, wherein the guide nucleic acid molecule comprises: A spacer sequence, wherein the spacer molecule exhibits specific binding to the target gene; and A scaffold sequence, wherein the scaffold sequence is used to form the complex with the Cas protein, wherein the scaffold sequence comprises a continuous polynucleotide sequence having at least 74% sequence identity with the polynucleotide sequence of SEQ ID NO:

597.

53. The method of claim 52, wherein: The binding of the complex to the target gene results in regulation of the target gene in the cell, which is comparable to the regulation in a control cell contacted with a control, wherein the control comprises (i) a control guide nucleic acid molecule comprising a polynucleotide sequence of SEQ ID NO: 500 and (ii) the Cas protein.

54. The method of claim 52, wherein: The contiguous polynucleotide sequence has at least 75% sequence identity with the polynucleotide sequence of SEQ ID NO:

597.

55. The method of claim 52, wherein: The contiguous polynucleotide sequence has at least 80% sequence identity with the polynucleotide sequence of SEQ ID NO:

597.

56. The method of claim 52, wherein: The contiguous polynucleotide sequence has at least 90% sequence identity with the polynucleotide sequence of SEQ ID NO:

597.

57. The method of claim 52, wherein: The scaffold sequence is up to about 110 nucleotides in length.

58. The method of claim 52, wherein: The scaffold sequence is at most about 105 nucleotides in length.

59. The method of claim 52, wherein: The composition comprises the Cas protein.

60. The method of claim 59, wherein: The Cas protein is up to about 528 amino acids in length.

61. The method of claim 59, wherein: The Cas protein is up to about 520 amino acids in length.

Images