Engineered CAS12 polypeptides, compositions, and methods of use thereof

Abstract

Provided herein are one or more engineered polypeptides and systems, compositions, and methods thereof, wherein the one or more engineered polypeptides exhibits broadened targeting range. The engineered polypeptide can be used to effect binding, cleaving, and / or editing a target polynucleotide sequence. The one or more engineered polypeptides can be engineered variants of a small CRISPR / Cas protein (e.g., a non-naturally occurring Cas polypeptide).

Description

ENGINEERED CAS12 POLYPEPTIDES, COMPOSITIONS, AND METHODS OF USE THEREOFTHEREOFREFERENCE TO PRIORITY APPLICATION

[0001] This application claims priority to U.S. provisional application number: 63 / 580938, filed September 6, 2023. The content of the aforementioned application is expressly incorporated herein by reference in its entirety.REFERENCE TO SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled EPICR_031WOSequenceListing.xml created August 23, 2024, which is 2,720,665 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.BACKGROUND

[0003] Various nucleases (e.g., endonucleases) can be utilized to edit a target sequence in a cell, or regulate expression or activity of the target gene in the cell. For example, a heterologous nuclease can be introduced (e.g., delivered, expressed, etc.) to the cell, and the heterologous nuclease, either alone or along with an additional agent, can effect such editing or regulation of the target gene. For example, clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein (Cas) is a family of nucleases that are involved in specifically binding, cleaving, and / or editing a target deoxyribonucleic acid (DNA) sequence or ribonucleic acid (RNA) sequence (e.g., foreign DNA sequence or RNA sequence). The programmable nature of these nucleases has facilitated their use as a versatile technology' that is revolutionizing the field of target gene manipulation, e.g., as gene therapy to treat or ameliorate a condition (e.g., a disease) of a subject.SUMMARY

[0004] Disclosed herein are non-naturally occurring Cas polypeptides comprising: a substitution at one or more amino acid positions selected from the group consisting of: (a) S135, S136, V137, E138, H139, Y140, L141, S142, D143, V144, C145, Y146, T147, R148, A149, A150, E151, L152, F153, K154, N155, A156, A157, I158, A159, S160, G161, L162, R163, S164, K165, I166, K167, S168, N169, F170, R171, P191, I192, P193, L194, V195, K196, Q197, K198, G199, G200, Q201, Y202, T203,G204, F205, F240, E241, Q242, V243, Q244, K245, S246, P247, K248, P249, and I250, relative to the amino acid numbering position of SEQ ID NO: 1 or 2 or 2027; or (b) S101, S102, V103, E104, H105, Y106, L107, S108, D109, V110, C111, Y112, T113, R114, A115, A116, E117, L118, F119, K120, N121, A122, A123, I124, A125, S126, G127, L128, R129, S130, K131, I132, K133, S134, N135, F136, R137, P157, I158, P159, L160, V161, K162, Q163, K164, G165, G166, Q167, Y168, T169, G170, F171, F206, E207, Q208, V209, Q210, K211, S212, P213, K214, P215, or I216, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the substitution is at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63 of the amino acid positions in (a) or (b). In some embodiments, the substitution at any one of the amino acid positions of (a) or (b) is a substitution to an amino acid residue selected from the group consisting of: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, the non-naturally occurring polypeptide exhibits an increased binding to a protospacer- adjacent motif (PAM) site other than a canonical PAM site, relative to the Cas polypeptide of SEQ ID NOS: 1-3. In some embodiments, the canonical PAM site is 5’-TTTR-3’, where R is A or G. In some embodiments, the non-naturally occurring Cas polypeptide comprises or consists of an amino acid sequence listed in Tables 3-7. In some embodiments, the non-naturally occurring Cas polypeptide comprises or consists of an amino acid sequence listed in Table 11. In some embodiments, the non-naturally occurring Cas polypeptide comprises or consists of an amino acid sequence listed in Table 12.

[0005] Disclosed herein are non-naturally occurring Cas polypeptides comprising: a substitution at one or more amino acid positions selected from the group consisting of: (a) Q115, E119, M178, K179, S180, G181, P183, T184, T185, K186, S187, I364, L365, L366, K367, K368, N369, R370, H371, K372, R373, A374, G375, and H376, relative to the amino acid numbering position of SEQ ID NO: 1 or 2 or 2027; or (b) Q81, E85, M144, K145, S146, G147, P149, T150, T151, K152, S153, I330, L331, L332, K333, K334, N335, R336, H337, K338, R339, A340, G341, or H342, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the substitution is at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 of the amino acid positions in (a) or (b). In some embodiments, the substitution at any one of the amino acid positions of (a) or (b) is a substitution to an amino acid residue selected from the group consisting of: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, the non- naturally occurring Cas polypeptide comprises or consists of an amino acid sequence listed in Tables 3-7. In some embodiments, the non-naturally occurring Cas polypeptide comprises or consists of an amino acidsequence listed in Table 11. In some embodiments, the non-naturally occurring Cas polypeptide comprises or consists of an amino acid sequence listed in Table 12.

[0006] Also provided herein are non-naturally occurring Cas polypeptides comprising: a substitution at amino acid position C297 relative to the amino acid numbering position of SEQ ID NO: 2 or 2027, or a substitution at amino acid position C263 relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the substitution at amino acid position C297 relative to the amino acid numbering position of SEQ ID NO: 2 or 2027 is C297G. In some embodiments, the substitution at amino acid position C263 relative to the amino acid numbering position of SEQ ID NO: 3 or 2026 is C263G. In some embodiments, the Cas polypeptide comprises an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to any one of SEQ ID NOS: 1-3, 2026, or 2027. In some embodiments, the non-naturally occurring polypeptide has nuclease activity. In some embodiments, the non-naturally occurring polypeptide has nickase activity. In some embodiments, the non-naturally occurring polypeptide is partially or fully nuclease deficient.

[0007] Also provided is a fusion protein comprising the Cas polypeptide of any one of the embodiments described herein; and a heterologous gene effector, optionally wherein the heterologous gene effector comprises an epigenetic modification domain, a transcriptional activation domain, and / or a transcriptional repressor domain.

[0008] Also provided is a nucleic acid-guided targeting system comprising: the non-naturally occurring Cas polypeptide of any one of the embodiments described herein or the fusion protein of any one of the embodiments described herein; and a guide nucleic acid configured to form a complex with the non- naturally occurring Cas polypeptide and that exhibits specific binding to a target gene or a regulatory region thereof in a target cell.

[0009] Additional embodiments include a composition comprising a first nucleic acid encoding the non-naturally occurring Cas polypeptide of any one of the embodiments described herein or the fusion protein of any one of the embodiments described herein; and a second nucleic acid encoding a guide nucleic acid configured to form a complex with the non-naturally occurring Cas polypeptide and that exhibits specific binding to a target gene or a regulatory region thereof in a target cell, optionally wherein the first nucleic acid and the second nucleic acid are comprised in a single nucleic acid molecule, optionally wherein the single nucleic acid molecule is a nucleic acid vector.

[0010] Additional embodiments include a composition comprising the non-naturally occurring Cas polypeptide of any one of the embodiments described herein or the fusion protein of any one of the embodiments described herein; and a guide nucleic acid configured to form a complex with the non-naturally occurring Cas polypeptide and that exhibits specific binding to a target gene or a regulatory region thereof in a target cell.

[0011] Also provided are methods of modulating expression or activity of a target gene, comprising contacting a cell with: the non-naturally occurring Cas polypeptide of any one of the embodiments described herein or the fusion protein of any one of the embodiments described herein, or a nucleic acid encoding the non-naturally occurring Cas polypeptide of any one of the embodiments described herein or the fusion protein of any one of the embodiments described herein; and a guide nucleic acid exhibiting specific binding to the target gene or a regulatory region thereof, thereby modulating expression or activity of the target gene.

[0012] Disclosed herein are methods of modulating expression or activity of a target gene, comprising contacting a cell with the non-naturally occurring Cas polypeptide disclosed herein, or a nucleic acid encoding the non-naturally occurring Cas polypeptide disclosed herein, and a guide nucleic acid exhibiting specific binding to the target gene or a regulatory region thereof, thereby modulating expression or activity of the target gene.

[0013] Additional aspects of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0015] FIG. 1 schematically illustrates occurrence of selected protospacer-adjacent motifs (PAMs) near TSS (either within 2 kb flanking region, or within optimal region (-400bp to +300bp relative to TSS) in the human genome. TCTG showed more frequent occurrences than the canonical PAM.

[0016] FIG.2 shows a cell-based reporter system for directed evolution towards TCTG PAM.

[0017] FIG.3 illustrates residues 101-137 predicted interaction with the PAM.

[0018] FIG.4 illustrates residues 157-171, adjacent to the double helix structure of 101-137, and the PAM.

[0019] FIG. 5 shows residues 206-216, which are predicted to lie directly beneath the predicted PAM interaction region.

[0020] FIG. 6 shows cysteine in 297 in the engineered polypeptide variant as predicted by ESMFold shows that it is in a free and partially buried position.

[0021] FIG.7 shows the structure of Un1Cas12f1 (PDB:7L49).

[0022] FIG. 8 shows a magnified view of residues involved in hydrophobic interactions between Rec1 domains.

[0023] FIG. 9 shows a magnified view of residues involved in electrostatic interactions between Rec2 domains.

[0024] FIG.10 shows gene modulation by a deactivated variant of the engineered polypeptide (SEQ ID NO.3).

[0025] FIG.11 shows gene modulation by a deactivated variant of the engineered polypeptide (SEQ ID NO.2).

[0026] FIG. 12 shows a schematic of the engineered polypeptide variant directed evolution workflow.

[0027] FIG. 13 shows a heat map of activation levels, expressed as log2 fold changes (log2FC), for engineered polypeptide variants across the deactivated variant of the engineered polypeptide (SEQ ID NO: 2) PAM recognition region in sorted GFP+ cells as compared to unsorted cells.

[0028] FIGs.14A-14B shows validation data for top mutations identified in the site-saturated mutagenesis (SSM) screen.

[0029] FIG. 15 is a collection of graphs showing the effect of stacked mutation variants on TTTA and TCTG PAM recognition.

[0030] FIG.16 is a collection of graphs showing a suppression assay to validate combinatorial mutation engineered polypeptide variants. DETAILED DESCRIPTION

[0031] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed. Terms

[0032] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or“greater than or equal to” applies to each of the numerical values in that series of numerical values. 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.

[0033] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. 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.

[0034] The term “about” or “approximately” generally means within an acceptable error range for the 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, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. 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 with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2- fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0035] The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. The term “and / or” should be understood to mean either one, or both of the alternatives.

[0036] 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 originate from any organism having one or more cells. Some non-limiting examples include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soybean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, hornworts, liverworts, mosses), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, and the like), seaweeds (e.g., kelp), a fungal cell (e.g., a yeast cell, a cell from a mushroom), an animal cell, a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), or a cell from a mammal (e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.). Sometimes a cell is not originating from a natural organism (e.g., a cell can be a synthetically made, sometimes termed an artificial cell).

[0037] The term “nucleotide,” as used herein, generally refers to a base-sugar-phosphate combination. A nucleotide can comprise a synthetic nucleotide. A nucleotide can comprise a synthetic nucleotide analog. Nucleotides can be monomeric units of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide can include ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives can include, for example, [ĮS]dATP, 7-deaza-dGTP and 7- deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them. The term nucleotide as used herein can refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of dideoxyribonucleoside triphosphates can include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. A nucleotide may be unlabeled or detectably labeled by well-known techniques. Labeling can also be carried out with quantum dots. Detectable labels can include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels. Fluorescent labels of nucleotides may include but are not limited fluorescein, 5-carboxyfluorescein (FAM), 2ƍ7ƍ-dimethoxy-4ƍ5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,Nƍ,Nƍ-tetramethyl-6-carboxyrhodamine (TAMRA), 6- carboxy-X-rhodamine (ROX), 4-(4ƍdimethylaminophenylazo) benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, Cyanine and 5-(2ƍ-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 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 available from Amersham, Arlington Heights, Ill.; Fluorescein-15-dATP, Fluorescein-12-dUTP, Tetramethyl-rodamine-6-dUTP, IR770-9-dATP, Fluorescein-12-ddUTP, Fluorescein-12-UTP, and Fluorescein-15-2ƍ-dATP available from Boehringer Mannheim, Indianapolis, Ind.; and Chromosome Labeled 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 available from Molecular Probes, Eugene, Oreg. Nucleotides can also be labeled or marked by chemical modification. A chemically-modified single nucleotide 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).

[0038] The term “polynucleotide,” “oligonucleotide,” or “nucleic acid,” as used interchangeably herein, generally refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multi-stranded form. A polynucleotide can be exogenous or endogenous to a cell. A polynucleotide can exist in a cell-free environment. A polynucleotide can be a gene or fragment thereof. A polynucleotide can be DNA. A polynucleotide can be RNA. A polynucleotide can have any three dimensional structure, and can perform any function, known or unknown. A polynucleotide can comprise one or more analogs (e.g., altered backbone, sugar, or nucleobase). If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, or wyosine. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from 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, or primers. The sequence of nucleotides can be interrupted by non-nucleotide components.

[0039] The term “gene” generally refers to a nucleic acid (e.g., DNA such as genomic DNA and cDNA) and its corresponding nucleotide sequence that is involved in encoding an RNA transcript. The term as used herein with reference to genomic DNA includes intervening, non-coding regions as well as regulatory regions and can include 5ƍ and 3ƍ ends. In some uses, the term encompasses the transcribed sequences, including 5ƍ and 3ƍ untranslated regions (5ƍ-UTR and 3ƍ-UTR), exons and introns. In some genes, the transcribed region will contain “open reading frames” that encode polypeptides. In some uses of the term, a “gene” comprises only the coding sequences (e.g., an “open reading frame” or “coding region”) necessary for encoding a polypeptide. In some cases, genes do not encode a polypeptide, for example, ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term “gene” includes not only the transcribed sequences, but in addition, also includes non-transcribed regions including upstream and downstream regulatory regions, enhancers and promoters. A gene can refer to an “endogenous gene” or a native gene in its natural location in the genome of an organism. A gene can referto an “exogenous gene” or a non-native gene. A non-native gene can refer to a gene not normally found in the host organism, but which is introduced into the host organism by gene transfer. A non-native gene can also refer to a gene not in its natural location in the genome of an organism. A non-native gene can also refer to a naturally occurring nucleic acid or polypeptide sequence that comprises mutations, insertions and / or deletions (e.g., non-native sequence).

[0040] The term “substitution” has its customary and ordinary meaning as understood by one of ordinary skill in the art in view of the present disclosure, and generally refers to the removal (or loss) of one or more (or a specified number of) amino acids (e.g., contiguous or non-contiguous amino acids) from a polypeptide sequence followed by an insertion of at least one different amino acid at the same position as the at least one amino acid as compared to a reference polypeptide sequence, such that the size (e.g., a number of the amino acid residue(s)) of a modified (or engineered) polypeptide sequence comprising the substitution of the at least one amino acid is the same as the reference polypeptide sequence; or the removal (or loss) of one or more (or a specified number of) nucleic acid bases (e.g., contiguous or non-contiguous nucleic acid bases) from a polynucleotide sequence followed by an insertion of at least one different nucleic acid base at the same position as the at least one nucleic acid base as compared to a reference polynucleotide sequence, such that the size (e.g., a number of the nucleic acid base(s)) of a modified (or engineered) polynucleotide sequence comprising the substitution of the at least one nucleic acid base is the same as the reference polynucleotide sequence. Substitutions can be conservative, non-conservative, or a combination thereof. The term “conservative amino acid substitution” generally refers to a substitution of one amino acid for another amino acid of similar biochemical properties (e.g., charge, size, and / or hydrophobicity). The term “non-conservative amino acid substitution” generally refers to a substitution of one amino acid for another amino acid with different biochemical properties (e.g., charge, size, and / or hydrophobicity). A conservative amino acid change can be, for example, a substitution that has minimal effect on the secondary or tertiary structure of a polypeptide. In some embodiments, conservative substitutions are shown in Table A under the heading of “preferred substitutions.” More substantial changes are provided in Table A under the heading of “exemplary substitutions,” and as further described below in reference to amino acid side chain classes. In some embodiments, a non-conservative substitution involves a substitution of an amino acid residue in a reference sequence (“Original Residue” in Table A) with a different amino acid residue that is not listed in Table A as one of the “preferred substitutions” or as one of the “exemplary substitutions.” TABLE A Original Exemplary Substitutions Preferred Substitutions Residue Ala (A) Val; Leu; Ile ValOriginal Exemplary Substitutions Preferred Substitutions Residue Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

[0041] The term “deletion” generally refers to the removal (or loss) of one or more (or a specified number of) amino acids (e.g., contiguous or non-contiguous amino acids) from a polypeptide sequence, or the removal (or loss) one or more (or a specified number of) nucleic acid bases (e.g., contiguous or non-contiguous nucleic acid bases) from a polynucleotide sequence (e.g., that encodes the polypeptide sequence. The term “internal deletion” generally refers to a deletion that does not include the N- or C-terminus of a polypeptide or the 5ƍ or 3ƍ end of a polynucleotide. A deletion (e.g., an internal deletion) can be identified by comparing to a reference sequence, e.g., by specifying the start and endpositions of the deletion relative to the reference sequence. A deletion (e.g., an internal deletion) is different and distinct from a substitution. For example, deletion of at least one amino acid is not followed by an insertion of at least one different amino acid at the same position as the at least one amino acid as compared to a reference polypeptide sequence, such that the size (e.g., a number of the amino acid residue(s)) of a modified (or engineered) polypeptide sequence comprising the deletion of the at least one amino acid is smaller than the reference polypeptide sequence by the size of the at least one amino acid that has been deleted.

[0042] The term “sequence identity” generally refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid) can be compared by determining their “percent identity.” The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the longer sequence and multiplied by 100. Percent identity may also be determined, for example, by comparing sequence information using the 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 may be used to determine percent identity over the entire length of the proteins being compared. Default parameters are provided to optimize searches with short query sequences in, for example, with the blastp program. The program also allows use of an SEG filter to mask-off segments of the query sequences as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17:149-163 (1993). Ranges of desired degrees of sequence identity are approximately 50% to 100% and integer values therebetween. In general, this disclosure encompasses sequences with 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 with any sequence provided herein.

[0043] The term “expression” generally refers to one or more processes by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides can be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell. “Up-regulated,” with reference to expression, generally refers to an increased expression level of apolynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence relative to its expression level in a wild-type state while “down-regulated” generally refers to a decreased expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence relative to its expression in a wild- type state. Expression of a transfected gene can occur transiently or stably in a cell. During “transient expression” the transfected gene is not transferred to the daughter cell during cell division. Since its expression is restricted to the transfected cell, expression of the gene is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selection advantage to the transfected cell. Such a selection advantage may be a resistance towards a certain toxin that is presented to the cell.

[0044] The term “expression profile” generally refers to quantitative (e.g., abundance) and qualitative expression of one or more genes in a sample (e.g., a cell). The one or more genes can be expressed and ascertained in the form of a nucleic acid molecule (e.g., an mRNA or other RNA transcript). Alternatively or in addition to, the one or more genes can be expressed and ascertained in the form of a polypeptide (e.g., a protein measured via Western blot). An expression profile of a gene may be defined as a shape of an expression level of the gene over a time period (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 up to about 5 days, at least up to about 6 days, at least up to about 7 days, at least up to about 8 days, at least up to about 9 days, at least up to about 10 days, at least up to about 11 days, at least up to about 12 days, at least up to about 13 days, or at least up to about 14 days, etc.). Alternatively, an expression profile of a gene may be defined as an expression level of the gene at a time point of interest (e.g., the expression level of the gene measured 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 up to about 5 days, at least up to about 6 days, at least up to about 7 days, at least up to about 8 days, at least up to about 9 days, at least up to about 10 days, at least up to about 11 days, at least up to about 12 days, at least up to about 13 days, or at least up to about 14 days after treating a cell to induce such expression level.)

[0045] The term “peptide,” “polypeptide,” or “protein,” as used interchangeably herein, generally refers to a polymer of at least two amino acid residues joined by peptide bond(s). This term does not connote a specific length of polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. In some cases, the polymer can be interrupted by non-amino acids. The terms include amino acid chains of any length, including full length proteins, and proteins with or without secondary and / or tertiary structure (e.g., domains). The terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation such as conjugation with a labeling component. The terms “amino acid” and “amino acids,” as used herein, generally refer to natural and non-natural amino acids, including, but not limited to, modified amino acids and amino acid analogues. Modified amino acids can include natural amino acids and non-natural amino acids, which have been chemically modified to include a group or a chemical moiety not naturally present on the amino acid. Amino acid analogues can refer to amino acid derivatives. The term “amino acid” includes both D-amino acids and L-amino acids.

[0046] The term “derivative,” “variant,” or “fragment,” as used herein with reference to a polypeptide, generally refers to a polypeptide related to a wild type polypeptide, for example either 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 can comprise one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof compared to a wild type polypeptide.

[0047] The term “engineered,” “chimeric,” or “recombinant,” as used herein with respect to a polypeptide molecule (e.g., a protein), generally refers to a polypeptide molecule having a heterologous amino acid sequence or an altered amino acid sequence as a result of the application of genetic engineering techniques to nucleic acids which encode the polypeptide molecule, as well as cells or organisms which express the polypeptide molecule. The term “engineered” or “recombinant,” as used herein with respect to a polynucleotide molecule (e.g., a DNA or RNA molecule), generally refers to a polynucleotide molecule having a heterologous nucleic acid sequence or an altered nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation and other gene transfer technologies; homologous recombination; site-directed mutagenesis; or gene fusion. In some cases, an engineered or recombinant polynucleotide (e.g., a genomic DNA sequence) can be modified or altered by a gene editing moiety.

[0048] For example, an engineered polypeptide (e.g., an engineered Cas protein) as disclosed herein is not a naturally occurring nuclease (e.g., not a naturally occurring Cas protein). The terms “engineered polypeptide” and “engineered polypeptide variant” may be used interchangeable herein.

[0049] The terms “engineered” and “modified” are used interchangeably herein. The terms “engineering” and “modifying” are used interchangeably herein. The terms “engineered cell” or “modified cell” are used interchangeably herein. The terms “engineered characteristic” and “modified characteristic” are used interchangeably herein.

[0050] The term “enhanced expression,” “increased expression,” or “upregulated expression” generally refers to production of a moiety of interest (e.g., a polynucleotide or a polypeptide) to a level that is above a normal level of expression of the moiety of interest in a host strain (e.g., a host cell). The normal level of expression can be substantially zero (or null) or higher than zero. The moiety of interest can comprise an endogenous gene or polypeptide construct of the host strain. The moiety of interest can comprise a heterologous gene or polypeptide construct that is introduced to or into the host strain. For example, a heterologous gene encoding a polypeptide of interest can be knocked-in (KI) to a genome of the host strain for enhanced expression of the polypeptide of interest in the host strain.

[0051] The term “enhanced activity,” “increased activity,” or “upregulated activity” generally refers to activity of a moiety of interest (e.g., a polynucleotide or a polypeptide) that is modified to a level that is above a normal level of activity of the moiety of interest in a host strain (e.g., a host cell). The normal level of activity can be substantially zero (or null) or higher than zero. The moiety of interest can comprise a polypeptide construct of the host strain. The moiety of interest can comprise a heterologous polypeptide construct that is introduced to or into the host strain. For example, a heterologous gene encoding a polypeptide of interest can be knocked-in (KI) to a genome of the host strain for enhanced activity of the polypeptide of interest in the host strain.

[0052] The term “reduced expression,” “decreased expression,” or “downregulated expression” generally refers to a production of a moiety of interest (e.g., a polynucleotide or a polypeptide) to a level that is below a normal level of expression of the moiety of interest in a host strain (e.g., a host cell). The normal level of expression is higher than zero. The moiety of interest can comprise an endogenous gene or polypeptide construct of the host strain. In some cases, the moiety of interest can be knocked-out or knocked-down in the host strain. In some examples, reduced expression of the moiety of interest can include a complete inhibition of such expression in the host strain.

[0053] The term “reduced activity,” “decreased activity,” or “downregulated activity” generally refers to activity of a moiety of interest (e.g., a polynucleotide or a polypeptide) that is modified to a level that is below a normal level of activity of the moiety of interest in a host strain (e.g., a host cell). The normal level of activity is higher than zero. The moiety of interest can comprise an endogenous geneor polypeptide construct of the host strain. In some cases, the moiety of interest can be knocked-out or knocked-down in the host strain. In some examples, reduced activity of the moiety of interest can include a complete inhibition of such activity in the host strain.

[0054] The term “subject,” “individual,” or “patient,” as used interchangeably herein, generally refers to a vertebrate, preferably a mammal such as a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, or pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[0055] The term “treatment” or “treating” generally refers to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and / or a prophylactic benefit. For example, a treatment can comprise administering a system or cell population disclosed herein. By therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. For 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 of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested.

[0056] The term “effective amount” or “therapeutically effective amount” generally refers to the quantity of a composition, for example a composition comprising heterologous polypeptides, heterologous polynucleotides, and / or modified cells (e.g., modified stem cells), that is sufficient to result in a desired activity upon administration to a subject in need thereof. Within the context of the present disclosure, the term “therapeutically effective” generally refers to that quantity of a composition that is sufficient to delay the manifestation, arrest the progression, relieve or alleviate at least one symptom of a disorder treated by the methods of the present disclosure. Overview

[0057] Various aspects of the present disclosure provide engineered polypeptides that are smaller, yet effective, in binding, cleaving, and / or editing a target polynucleotide sequence, compositions thereof, and methods of use thereof. Such engineered polypeptides (e.g., engineered CRISPR / Cas nuclease), for example, effect manipulation of expression or activity of a target gene (e.g., a target endogenous gene) in a cell, e.g., to treat, mitigate,inhibit, or ameliorate a condition (e.g., a disease) of a subject. For example, the engineered polypeptide described herein can be used for targeted gene modification, targeted insertion, perturbation of gene transcripts, nucleic acid editing. The compositions, methods, and systems described herein can include CRISPR associated transposable elements (e.g., an engineered polypeptide and a transposase (or subunits thereof) that catalyze RNA-guided DNA transposition). In some aspects, the engineered polypeptides described herein recognize a PAM sequencethat is different than a canonical PAM sequence (e.g., 5’-TTTG-3’ or 5’-TTTA-3’), thereby increasing the likelihood of finding optimal guide RNAs. In some embodiments, the engineered polypeptides described herein have an enhanced CRISPR activation (CRISPRa) and / or CRISPR interference (CRISPRi) activity compared to a non-engineered polypeptide. Gene expression can underpin various physiological and pathological effects in cells and tissues, contributing to many diseases and conditions, and thus compositions and methods utilizing the engineered polypeptides of the present disclosure can modulate expression of specific genes in a desirable way to have therapeutic benefit. In any embodiment described herein, reference to an engineered polypeptide can refer to a non-naturally occurring Cas polypeptide such as a modified derivative of a Cas polypeptide.

[0058] Provided herein are engineered polypeptides (e.g., non-naturally occurring Cas polypeptides) that exhibit increased binding to a PAM site other than a canonical PAM site, relative to the Cas polypeptide of SEQ ID NOS: 1-3, 2026, or 2027. Engineered polypeptides, compositions, and methods thereof

[0059] In some aspects, the present disclosure provides an engineered polypeptide (e.g., a non- naturally occurring Cas polypeptide) comprising an amino acid sequence that is at least 50% identical (e.g., 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%, or at least or up to about 99%) to the polypeptide sequence of SEQ ID NO: 1. The amino acid sequence of the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) can comprise at least one mutation (e.g., at least one amino acid addition, deletion, or substitution) as compared to (e.g., when aligned to) the polypeptide sequence of SEQ ID NO: 1 or (ii) one or more orthologs or homologs of SEQ ID NO: 1 (e.g., a Cas protein selected from TABLE 2).

[0060] The engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) described herein can comprise at least one mutation (e.g., at least one amino acid addition, deletion, or substitution) in a region that effects (e.g., increases) the inter-molecular and / or intra-molecular interactions of the Cas protein. For example, the engineered polypeptide disclosed herein can have at least one mutation (e.g., at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more mutations) in or near a region that binds to the PAM and / or in or near the dimerization interface (e.g., within Rec1, Rec2, RuvC1, and / or RuvC2).

[0061] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) as disclosed herein comprises an amino acid substitution at one or more amino acid positionsrelative to the amino acid position numbering of SEQ ID NO: 1, or the corresponding amino acid position of a homolog or ortholog of SEQ ID NO: 1 (e.g., SEQ ID NOs: 4-12). In some cases, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises at least one amino acid substitution in or near a region that binds to or interfaces with a PAM. In some cases, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at one or more of amino acid positions 135-171, 191-205, or 240-250 of SEQ ID NO: 1, or the corresponding amino acid positions of a homolog or ortholog of SEQ ID NO: 1. In some cases, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises at least one amino acid substitution in or near the dimerization interface (e.g., within Rec1, Rec2, RuvC2, and / or RuvC2). In some cases, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at one or more of amino acid positions 115, 119178-181, 183-187, or 364-376 of SEQ ID NO: 1, or the corresponding amino acid positions of a homolog or ortholog of SEQ ID NO: 1.

[0062] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) described herein has at least one amino acid mutation (e.g., as compared to the polypeptide sequence of SEQ ID NOs: 1-3, 2026, 2027, or one or more orthologs or homologs of SEQ ID NO: 1), which can alter PAM preference. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at one or more of amino acid positions S135, S136, V137, E138, L141, L141, L141, S142, D143, V144, C145, Y146, T147, R148, A149, A150, E151, L152, F153, K154, N155, A156, A157, I158, A159, S160, G161, L162, R163, S164, K165, I166, K167, S168, N169, F170, R171, P191, I192, P193, L194, V195, K196, Q197, K198, G199, G200, Q201, Y202, T203, G204, F205, F240, E241, Q242, V243, Q244, K245, S246, P247, K248, P249, or I250 of SEQ ID NO: 1 (or the polypeptide sequence of any one of a homolog or ortholog of SEQ ID NO: 1 as shown in TABLE 2). In some embodiments, the substitution a is a non-conservative substitution.

[0063] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at one or more of amino acid positions S135, S136, V137, E138, L141, L141, L141, S142, R143, V144, C145, Y146, R147, R148, A149, A150, E151, L152, F153, K154, N155, A156, A157, I158, A159, S160, G161, L162, R163, S164, K165, I166, K167, S168, N169, F170, R171, P191, I192, P193, L194, V195, K196, Q197, K198, G199, G200, Q201, Y202, T203, G204, F205, F240, E241, Q242, V243, Q244, K245, S246, P247, K248, P249, I250 of SEQ ID NO: 2 or the corresponding variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at one or more of amino acid positions S135, S136, V137, E138, L141, L141, L141, S142, R143, V144, C145, Y146, R147, R148, A149, A150, E151, L152, F153, K154, N155, A156, A157, I158, A159, S160, G161, L162, R163, S164, K165, I166, K167, S168, N169, F170, R171, P191, I192, P193,L194, V195, K196, Q197, K198, G199, G200, Q201, Y202, T203, G204, F205, F240, E241, Q242, V243, Q244, K245, S246, P247, K248, P249, or I250 relative to the amino acid numbering position of SEQ ID NO: 2027. In some embodiments, the substitution is a non-conservative substitution.

[0064] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at one or more of amino acid positions S101, S102, V103, E104, L107, L107, L107, S108, R109, V110, C111, Y112, R113, R114, A115, A116, E117, L118, F119, K120, N121, A122, A123, I124, A125, S126, G127, L128, R129, S130, K131, I132, K133, S134, N135, F136, R137, P157, I158, P159, L160, V161, K162, Q163, K164, G165, G166, Q167, Y168, T169, G170, F171, F206, E207, Q208, V209, Q210, K211, S212, P213, K214, P215, or I216 of SEQ ID NO: 3 or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104), or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at one or more of amino acid positions S101, S102, V103, E104, L107, L107, L107, S108, R109, V110, C111, Y112, R113, R114, A115, A116, E117, L118, F119, K120, N121, A122, A123, I124, A125, S126, G127, L128, R129, S130, K131, I132, K133, S134, N135, F136, R137, P157, I158, P159, L160, V161, K162, Q163, K164, G165, G166, Q167, Y168, T169, G170, F171, F206, E207, Q208, V209, Q210, K211, S212, P213, K214, P215, or I216 relative to the amino acid numbering position of SEQ ID NO:2026. In some embodiments, the substitution is a non-conservative substitution.

[0065] In any embodiment described herein, any suitable amino acid substitution can be present in the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide), relative to the corresponding reference sequence (e.g., SEQ ID NOs: 1-3, 2026, or 2027). In some embodiments, the substitution at any one of the noted amino acid positions is a substitution to an amino acid residue selected from the group consisting of: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. For example, a substitution of a tyrosine residue at the amino acid position in the reference sequence can include replacing the tyrosine with an amino acid residue other than tyrosine (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, or valine). In some embodiments, a substitution involves replacing an amino acid residue in a reference sequence with a different amino acid residue that is present in the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) at the amino acid position. In some embodiments, the substitution at any one of the amino acid positions of the noted positions is a substitution to an amino acid residue selected from the group consisting of: arginine, glutamine, histidine, lysine, and tyrosine. In some embodiments, the substitution at any of the noted amino acid positions relative to the reference sequence is a non-conservative substitution.

[0066] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position S135 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:2026, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: S135A, S135R, S135N, S135D, S135C, S135Q, S135E, S135G, S135H, S135I, S135L, S135K, S135M, S135F, S135P, S135T, S135W, S135Y, and S135V, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the S135 position of SEQ ID NO: 1 corresponds to amino acid position S101 of SEQ ID NO: 3. In some embodiments, the S135 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position S101 of SEQ ID NO:2026. In some cases, the amino acid substitution is selected from the group consisting of S101A, S101R, S101N, S101D, S101C, S101Q, S101E, S101G, S101H, S101I, S101L, S101K, S101M, S101F, S101P, S101T, S101W, S101Y, and S101V, relative to the amino acid position numbering of SEQ ID NO: 3, SEQ ID NO:2026, the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0067] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position S135. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position S101.

[0068] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position S136 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: S136A, S136R, S136N, S136D, S136C, S136Q, S136E, S136G, S136H, S136I, S136L, S136K, S136M, S136F, S136P, S136T, S136W, S136Y, and S136V, relative to the aminoacid position numbering of SEQ ID NOs: 1, 2, or 13-96 or 2027. In some cases, the S136 position of SEQ ID NO: 1 corresponds to amino acid position S102 of SEQ ID NO: 3. In some embodiments, the S136 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position S102 of SEQ ID NO:2026. In some cases, the amino acid substitution is selected from the group consisting of S102A, S102R, S102N, S102D, S102C, S102Q, S102E, S102G, S102H, S102I, S102L, S102K, S102M, S102F, S102P, S102T, S102W, S102Y, and S102V, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0069] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position S136. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position S102.

[0070] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position V137 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: V137A, V137R, V137N, V137D, V137C, V137Q, V137E, V137G, V137H, V137I, V137L, V137K, V137M, V137F, V137P, V137T, V137W, V137Y, and V137S, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, or 13-96. In some cases, the V137 position of SEQ ID NO: 1 corresponds to amino acid position V103 of SEQ ID NO: 3. In some embodiments, the V137 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position V103 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of V103A, V103R, V103N, V103D, V103C, V103Q, V103E, V103G, V103H, V103I, V103L, V103K, V103M, V103F, V103P, V103T, V103W, V103Y, and V103S, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0071] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position V137. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position V103.

[0072] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position E138 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: E138A, E138R, E138N, E138D, E138C, E138Q, E138V, E138G, E138H, E138I, E138L, E138K, E138M, E138F, E138P, E138T, E138W, E138Y, and E138S, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the E138 position of SEQ ID NO: 1 corresponds to amino acid position E104 of SEQ ID NO: 3. In some embodiments, the E138 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position E104 of SEQ ID NO:2026. In some cases, the amino acid substitution is selected from the group consisting of E104A, E104R, E104N, E104D, E104C, E104Q, E104V, E104G, E104H, E104I, E104L, E104K, E104M, E104F, E104P, E104T, E104W, E104Y, and E104S, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0073] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position E138. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at leastabout 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position E104.

[0074] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position H139 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: H139A, H139R, H139N, H139D, H139C, H139Q, H139V, H139G, H139E, H139I, H139L, H139K, H139M, H139F, H139P, H139T, H139W, H139Y, and H139S, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the H139 position of SEQ ID NO: 1 corresponds to amino acid position H105 of SEQ ID NO: 3. In some cases, the H139 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position H105 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of H105A, H105R, H105N, H105D, H105C, H105Q, H105V, H105G, H105E, H105I, H105L, H105K, H105M, H105F, H105P, H105T, H105W, H105Y, and H105S, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the amino acid substitution is or includes H105Y, relative to the amino acid position numbering of SEQ ID NO: 3 or 2026. In some embodiments, the substitution is a non- conservative substitution.

[0075] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, or 13-96, and an amino acid substitution at position H139. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NO: 2027, and an amino acid substitution at position H139. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3 or 97-194, and an amino acid substitutionat position H105. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NO:2026, and an amino acid substitution at position H105. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 1, 2, 3, 4, 5, or 6 amino acid substitutions thereto, including an amino acid substitution at position H105. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NO:2026, and an amino acid substitution of H105Y. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 1, 2, 3, 4, 5, 6 or more amino acid substitutions thereto, including an amino acid substitution of H105Y.

[0076] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position Y140 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: Y140A, Y140R, Y140N, Y140D, Y140C, Y140Q, Y140V, Y140G, Y140E, Y140I, Y140L, Y140K, Y140M, Y140F, Y140P, Y140T, Y140W, Y140H, and Y140S, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the Y140 position of SEQ ID NO: 1 corresponds to amino acid position Y106 of SEQ ID NO: 3. In some embodiments, the Y140 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position Y106 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of Y106A, Y106R, Y106N, Y106D, Y106C, Y106Q, Y106V, Y106G, Y106E, Y106I, Y106L, Y106K, Y106M, Y106F, Y106P, Y106T, Y106W, Y106H, and Y106S, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0077] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, atleast about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position Y140. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position Y106.

[0078] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position L141 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: L141A, L141R, L141N, L141D, L141C, L141Q, L141V, L141G, L141E, L141I, L141Y, L141K, L141M, L141F, L141P, L141T, L141W, L141H, and L141S, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the L141 position of SEQ ID NO: 1 corresponds to amino acid position L107 of SEQ ID NO: 3. In some embodiments, the L141 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position L107 of SEQ ID NO:2026. In some cases, the amino acid substitution is selected from the group consisting of L107A, L107R, L107N, L107D, L107C, L107Q, L107V, L107G, L107E, L107I, L107Y, L107K, L107M, L107F, L107P, L107T, L107W, L107H, and L107S, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the amino acid substitution is or includes L107Y, relative to the amino acid position numbering of SEQ ID NO: 3 or 2026. In some embodiments, the substitution is a non-conservative substitution.

[0079] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, or 13-96, and an amino acid substitution at position L141. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NO:2027, and an amino acid substitution at position L141. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3 or 97-194, and an amino acid substitution at position L107. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NO: 2026, and an amino acid substitution at position L107. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 1, 2, 3, 4, 5, or 6 amino acid substitutions thereto, including an amino acid substitution at position L107. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NO:2026, and an amino acid substitution of L107Y. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 1, 2, 3, 4, 5, or 6 amino acid substitutions thereto, including an amino acid substitution of L107Y.

[0080] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position S142 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: S142A, S142R, S142N, S142D, S142C, S142Q, S142V, S142G, S142E, S142I, S142Y, S142K, S142M, S142F, S142P, S142T, S142W, S142H, and S142L, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, or 13-96. In some cases, the S142 position of SEQ ID NO: 1 corresponds to amino acid position S108 of SEQ ID NO: 3. In some embodiments, the S142 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position S108 of SEQ ID NO:2026. In some cases, the amino acid substitution is selected from the group consisting of S108A, S108R, S108N, S108D, S108C, S108Q, S108V, S108G, S108E, S108I, S108Y, S108K, S108M, S108F, S108P, S108T, S108W, S108H, and S108L, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the correspondingvariant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0081] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position S142. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position S108.

[0082] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position D143 (or R143 of SEQ ID NO: 2) relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: D143A, D143R, D143N, D143S, D143C, D143Q, D143V, D143G, D143E, D143I, D143Y, D143K, D143M, D143F, D143P, D143T, D143W, D143H, and D143L, relative to the amino acid position numbering of SEQ ID NO 1. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: R143A, R143S, R143N, R143D, R143C, R143Q, R143V, R143G, R143E, R143I, R143Y, R143K, R143M, R143F, R143P, R143T, R143W, R143H, and R143L, relative to the amino acid position numbering of 2, or 13-96. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: R143A, R143S, R143N, R143D, R143C, R143Q, R143V, R143G, R143E, R143I, R143Y, R143K, R143M, R143F, R143P, R143T, R143W, R143H, and R143L, relative to the amino acid position numbering of SEQ ID NO: 2027. In some cases, the D143 position of SEQ ID NO: 1 corresponds to amino acid position R109 of SEQ ID NO: 3. In some embodiments, the R143 position of SEQ ID NO: 2 or 2027 corresponds to amino acid position R109 of SEQ ID NO: 3. In some embodiments, the R143 position of SEQ ID NO: 2 or 2027 corresponds to amino acid position R109 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of R109A, R109S, R109N, R109D, R109C, R109Q, R109V, R109G, R109E, R109I, R109Y, R109K, R109M, R109F, R109P, R109T,R109W, R109H, and R109L, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0083] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NO: 1, and an amino acid substitution at position D143. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 2, 2027, or 13-96, and an amino acid substitution at position R143. In some embodiments, the engineered polypeptide (e.g., non- naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position R109.

[0084] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position V144 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: V144A, V144R, V144N, V144D, V144C, V144Q, V144S, V144G, V144E, V144I, V144Y, V144K, V144M, V144F, V144P, V144T, V144W, V144H, and V144L, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the V144 position of SEQ ID NO: 1 corresponds to amino acid position V110 of SEQ ID NO: 3. In some embodiments, the V144 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position V110 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of V110A, V110R, V110N, V110D, V110C, V110Q, V110S, V110G, V110E, V110I, V110Y, V110K, V110M, V110F, V110P, V110T, V110W, V110H, and V110L, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0085] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position V144. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position V110.

[0086] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position C145 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: C145A, C145R, C145N, C145D, C145V, C145Q, C145S, C145G, C145E, C145I, C145Y, C145K, C145M, C145F, C145P, C145T, C145W, C145H, and C145L, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, or 13-96. In some cases, the C145 position of SEQ ID NO: 1 corresponds to amino acid position C111 of SEQ ID NO: 3. In some cases, the C145 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position C111 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of C111A, C111R, C111N, C111D, C111V, C111Q, C111S, C111G, C111E, C111I, C111Y, C111K, C111M, C111F, C111P, C111T, C111W, C111H, and C111L, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0087] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position C145. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at leastabout 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position C111.

[0088] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position Y146 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: Y146A, Y146R, Y146N, Y146D, Y146V, Y146Q, Y146S, Y146G, Y146E, Y146I, Y146C, Y146K, Y146M, Y146F, Y146P, Y146T, Y146W, Y146H, and Y146L, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, or 13-96. In some cases, the Y146 position of SEQ ID NO: 1 corresponds to amino acid position Y112 of SEQ ID NO: 3. In some cases, the amino acid substitution is selected from the group consisting of Y112A, Y112R, Y112N, Y112D, Y112V, Y112Q, Y112S, Y112G, Y112E, Y112I, Y112C, Y112K, Y112M, Y112F, Y112P, Y112T, Y112W, Y112H, and Y112L, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0089] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position Y146. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position Y112.

[0090] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position T147 (or R147 of SEQ ID NO: 2) relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: T147A, T147R, T147N, T147S, T147C,T147Q, T147V, T147G, T147E, T147I, T147Y, T147K, T147M, T147F, T147P, T147D, T147W, T147H, and T147L, relative to the amino acid position numbering of SEQ ID NO 1. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: R147A, R147S, R147N, R147D, R147C, R147Q, R147V, R147G, R147E, R147I, R147Y, R147K, R147M, R147F, R147P, R147T, R147W, R147H, and R147L, relative to the amino acid position numbering of 2, or 13-96. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: R147A, R147S, R147N, R147D, R147C, R147Q, R147V, R147G, R147E, R147I, R147Y, R147K, R147M, R147F, R147P, R147T, R147W, R147H, and R147L, relative to the amino acid position numbering of SEQ ID NO: 2027. In some cases, the T147 position of SEQ ID NO: 1 corresponds to amino acid position R113 of SEQ ID NO: 3. In some embodiments, the R147 position of SEQ ID NO: 2 or 2027 corresponds to amino acid position R113 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of R113A, R113S, R113N, R113D, R113C, R113Q, R113V, R113G, R113E, R113I, R113Y, R113K, R113M, R113F, R113P, R113T, R113W, R113H, and R113L, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0091] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NO: 1, and an amino acid substitution at position T147. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 2, 2027, or 13-96, and an amino acid substitution at position R147. In some embodiments, the engineered polypeptide (e.g., non- naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position R113.

[0092] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position R148 relative to the amino acidposition numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: R148A, R148Y, R148N, R148D, R148V, R148Q, R148S, R148G, R148E, R148I, R148C, R148K, R148M, R148F, R148P, R148T, R148W, R148H, and R148L, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, or 13-96. In some cases, the R148 position of SEQ ID NO: 1 corresponds to amino acid position R114 of SEQ ID NO: 3. In some embodiments, the R148 position of SEQ ID NO: 2027 corresponds to amino acid position R114 of SEQ ID NO: 3 or 2026. In some cases, the amino acid substitution is selected from the group consisting of R114A, R114Y, R114N, R114D, R114V, R114Q, R114S, R114G, R114E, R114I, R114C, R114K, R114M, R114F, R114P, R114T, R114W, R114H, and R114L, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0093] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position R148. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position R114.

[0094] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position A149 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: A149R, A149Y, A149N, A149D, A149V, A149Q, A149S, A149G, A149E, A149I, A149C, A149K, A149M, A149F, A149P, A149T, A149W, A149H, and A149L, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, or 13-96. In some cases, the A149 position of SEQ ID NO: 1 corresponds to amino acid position A115 of SEQ ID NO: 3 or 2026. In some cases, the aminoacid substitution is selected from the group consisting of A115R, A115Y, A115N, A115D, A115V, A115Q, A115S, A115G, A115E, A115I, A115C, A115K, A115M, A115F, A115P, A115T, A115W, A115H, and A115L, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0095] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position A149. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position A115.

[0096] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position A150 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: A150R, A150Y, A150N, A150D, A150V, A150Q, A150S, A150G, A150E, A150I, A150C, A150K, A150M, A150F, A150P, A150T, A150W, A150H, and A150L, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, or 13-96. In some cases, the A150 position of SEQ ID NO: 1 corresponds to amino acid position A116 of SEQ ID NO: 3. In some cases, the A150 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position A116 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of A116R, A116Y, A116N, A116D, A116V, A116Q, A116S, A116G, A116E, A116I, A116C, A116K, A116M, A116F, A116P, A116T, A116W, A116H, and A116L, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0097] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, atleast about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position A150. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position A116.

[0098] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position E151 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: E151R, E151Y, E151N, E151D, E151V, E151Q, E151S, E151G, E151A, E151I, E151C, E151K, E151M, E151F, E151P, E151T, E151W, E151H, and E151L, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the E151 position of SEQ ID NO: 1 corresponds to amino acid position E117 of SEQ ID NO: 3. In some cases, the E151 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position E117 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of E117R, E117Y, E117N, E117D, E117V, E117Q, E117S, E117G, E117A, E117I, E117C, E117K, E117M, E117F, E117P, E117T, E117W, E117H, and E117L, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0099] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position E151. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position E117.

[0100] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position L152 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: L152R, L152Y, L152N, L152D, L152V, L152Q, L152S, L152G, L152A, L152I, L152C, L152K, L152M, L152F, L152P, L152T, L152W, L152H, and L152E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, or 13-96. In some cases, the L152 position of SEQ ID NO: 1 corresponds to amino acid position L118 of SEQ ID NO: 3. In some embodiments, the L152 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position L118 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of L118R, L118Y, L118N, L118D, L118V, L118Q, L118S, L118G, L118A, L118I, L118C, L118K, L118M, L118F, L118P, L118T, L118W, L118H, and L118E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0101] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position L152. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position L118.

[0102] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position F153 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: F153R, F153Y, F153N, F153D, F153V, F153Q, F153S, F153G, F153A, F153I, F153C, F153K, F153M, F153L, F153P, F153T, F153W, F153H, and F153E, relative to the aminoacid position numbering of SEQ ID NOs: 1, 2, or 13-96. In some cases, the F153 position of SEQ ID NO: 1 corresponds to amino acid position F119 of SEQ ID NO: 3. In some embodiments, the F153 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position F119 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of F119R, F119Y, F119N, F119D, F119V, F119Q, F119S, F119G, F119A, F119I, F119C, F119K, F119M, F119L, F119P, F119T, F119W, F119H, and F119E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0103] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position F153. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position F119.

[0104] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position K154 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: K154R, K154Y, K154N, K154D, K154V, K154Q, K154S, K154G, K154A, K154I, K154C, K154F, K154M, K154L, K154P, K154T, K154W, K154H, and K154E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the K154 position of SEQ ID NO: 1 corresponds to amino acid position K120 of SEQ ID NO: 3. In some cases, the amino acid substitution is selected from the group consisting of K120R, K120Y, K120N, K120D, K120V, K120Q, K120S, K120G, K120A, K120I, K120C, K120F, K120M, K120L, K120P, K120T, K120W, K120H, and K120E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the amino acid substitution is or includes K120H, relative to the amino acid positionnumbering of SEQ ID NO: 3 or 2026. In some embodiments, the substitution is a non-conservative substitution.

[0105] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, or 13-96, and an amino acid substitution at position K154. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NO: 2027, and an amino acid substitution at position K154. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3 or 97-194, and an amino acid substitution at position K120. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 1, 2, 3, 4, 5, or 6 amino acid substitutions thereto, including an amino acid substitution at position K120. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NO: 2026, and an amino acid substitution at position K120. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NO:2026, and an amino acid substitution of K120H. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 1, 2, 3, 4, 5, or 6 amino acid substitutions thereto, including an amino acid substitution of K120H.

[0106] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position N155 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid positioncorresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: N155R, N155Y, N155K, N155D, N155V, N155Q, N155S, N155G, N155A, N155I, N155C, N155F, N155M, N155L, N155P, N155T, N155W, N155H, and N155E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the N155 position of SEQ ID NO: 1 corresponds to amino acid position N121 of SEQ ID NO: 3. In some cases, the amino acid substitution is selected from the group consisting of N121R, N121Y, N121K, N121D, N121V, N121Q, N121S, N121G, N121A, N121I, N121C, N121F, N121M, N121L, N121P, N121T, N121W, N121H, and N121E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0107] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position N155. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position N121.

[0108] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position A156 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: A156R, A156Y, A156K, A156D, A156V, A156Q, A156S, A156G, A156N, A156I, A156C, A156F, A156M, A156L, A156P, A156T, A156W, A156H, and A156E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, or 13-96. In some cases, the A156 position of SEQ ID NO: 1 corresponds to amino acid position A122 of SEQ ID NO: 3. In some cases, the A156 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position A122 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of A122R, A122Y, A122K, A122D, A122V,A122Q, A122S, A122G, A122N, A122I, A122C, A122F, A122M, A122L, A122P, A122T, A122W, A122H, and A122E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0109] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position A156. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position A122.

[0110] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position A157 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: A157R, A157Y, A157K, A157D, A157V, A157Q, A157S, A157G, A157N, A157I, A157C, A157F, A157M, A157L, A157P, A157T, A157W, A157H, and A157E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, or 13-96. In some cases, the A157 position of SEQ ID NO: 1 corresponds to amino acid position A123 of SEQ ID NO: 3. In some embodiments, the A157 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position A123 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of A123R, A123Y, A123K, A123D, A123V, A123Q, A123S, A123G, A123N, A123I, A123C, A123F, A123M, A123L, A123P, A123T, A123W, A123H, and A123E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0111] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acidsequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position A157. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position A123.

[0112] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position I158 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: I158R, I158Y, I158K, I158D, I158V, I158Q, I158S, I158G, I158N, I158A, I158C, I158F, I158M, I158L, I158P, I158T, I158W, I158H, and I158E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the I158 position of SEQ ID NO: 1 corresponds to amino acid position I124 of SEQ ID NO: 3. In some embodiments, the I158 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position I124 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of I124R, I124Y, I124K, I124D, I124V, I124Q, I124S, I124G, I124N, I124A, I124C, I124F, I124M, I124L, I124P, I124T, I124W, I124H, and I124E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0113] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position I158. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position I124.

[0114] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position A159 relative to the amino acidposition numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: A159R, A159Y, A159K, A159D, A159V, A159Q, A159S, A159G, A159N, A159I, A159C, A159F, A159M, A159L, A159P, A159T, A159W, A159H, and A159E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the A159 position of SEQ ID NO: 1 corresponds to amino acid position A125 of SEQ ID NO: 3. In some cases, the A159 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position A125 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of A125R, A125Y, A125K, A125D, A125V, A125Q, A125S, A125G, A125N, A125I, A125C, A125F, A125M, A125L, A125P, A125T, A125W, A125H, and A125E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0115] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position A159. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position A125.

[0116] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position S160 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: S160R, S160Y, S160K, S160D, S160V, S160Q, S160A, S160G, S160N, S160I, S160C, S160F, S160M, S160L, S160P, S160T, S160W, S160H, and S160E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, or 13-96. In some cases, the S160 position of SEQ ID NO: 1 corresponds to amino acid position S126 of SEQ ID NO: 3. In some cases, the S160 position of SEQ IDNO: 1 or 2027 corresponds to amino acid position S126 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of S126R, S126Y, S126K, S126D, S126V, S126Q, S126A, S126G, S126N, S126I, S126C, S126F, S126M, S126L, S126P, S126T, S126W, S126H, and S126E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the amino acid substitution is or includes S126Q, relative to the amino acid position numbering of SEQ ID NO: 3 or 2026. In some embodiments, the substitution is a non-conservative substitution.

[0117] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, or 13-96, and an amino acid substitution at position S160. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NO: 2027, and an amino acid substitution at position S160. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3 or 97-194, and an amino acid substitution at position S126. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 1, 2, 3, 4, 5, or 6 amino acid substitutions thereto, including an amino acid substitution at position S126. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NO:2026, and an amino acid substitution at position S126. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NO:2026, and an amino acid substitution at position S126Q. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises theamino acid sequence of SEQ ID NO:2026 having 1, 2, 3, 4, 5, or 6 amino acid substitutions thereto, including an amino acid substitution at position S126Q.

[0118] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position G161 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: G161R, G161Y, G161K, G161D, G161V, G161Q, G161A, G161S, G161N, G161I, G161C, G161F, G161M, G161L, G161P, G161T, G161W, G161H, and G161E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the G161 position of SEQ ID NO: 1 corresponds to amino acid position G127 of SEQ ID NO: 3. In some cases, the G161 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position G127 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of G127R, G127Y, G127K, G127D, G127V, G127Q, G127A, G127S, G127N, G127I, G127C, G127F, G127M, G127L, G127P, G127T, G127W, G127H, and G127E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0119] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position G161. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position G127.

[0120] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position L162 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selectedfrom the group consisting of: L162R, L162Y, L162K, L162D, L162V, L162Q, L162A, L162S, L162N, L162I, L162C, L162F, L162M, L162G, L162P, L162T, L162W, L162H, and L162E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the L162 position of SEQ ID NO: 1 corresponds to amino acid position L128 of SEQ ID NO: 3. In some embodiments, the L162 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position L128 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of L128R, L128Y, L128K, L128D, L128V, L128Q, L128A, L128S, L128N, L128I, L128C, L128F, L128M, L128G, L128P, L128T, L128W, L128H, and L128E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0121] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position L162. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position L128.

[0122] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position R163 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: R163L, R163Y, R163K, R163D, R163V, R163Q, R163A, R163S, R163N, R163I, R163C, R163F, R163M, R163G, R163P, R163T, R163W, R163H, and R163E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the R163 position of SEQ ID NO: 1 corresponds to amino acid position R129 of SEQ ID NO: 3. In some embodiments, the R163 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position R129 of SEQ ID NO:2026. In some cases, the amino acid substitution is selected from the group consisting of R129L, R129Y, R129K, R129D, R129V, R129Q, R129A, R129S, R129N, R129I, R129C, R129F, R129M, R129G, R129P, R129T, R129W, R129H, and R129E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or thecorresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0123] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, or 13-96, and an amino acid substitution at position R163. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3 or 97-194, and an amino acid substitution at position R129.

[0124] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position S164 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: S164L, S164Y, S164K, S164D, S164V, S164Q, S164A, S164R, S164N, S164I, S164C, S164F, S164M, S164G, S164P, S164T, S164W, S164H, and S164E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the S164 position of SEQ ID NO: 1 corresponds to amino acid position S130 of SEQ ID NO: 3. In some embodiments, the S164 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position S130 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of S130L, S130Y, S130K, S130D, S130V, S130Q, S130A, S130R, S130N, S130I, S130C, S130F, S130M, S130G, S130P, S130T, S130W, S130H, and S130E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0125] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position S164. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises orconsists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position S130.

[0126] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position K165 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: K165L, K165Y, K165S, K165D, K165V, K165Q, K165A, K165R, K165N, K165I, K165C, K165F, K165M, K165G, K165P, K165T, K165W, K165H, and K165E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some embodiments, the K165 position of SEQ ID NO: 1 corresponds to amino acid position K131 of SEQ ID NO: 3. In some cases, the K165 position of SEQ ID NO: 1 corresponds to amino acid position K131 of SEQ ID NO: 3. In some cases, the amino acid substitution is selected from the group consisting of K131L, K131Y, K131S, K131D, K131V, K131Q, K131A, K131R, K131N, K131I, K131C, K131F, K131M, K131G, K131P, K131T, K131W, K131H, and K131E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0127] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position K165. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position K131.

[0128] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position I166 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position correspondingthereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: I166L, I166Y, I166S, I166D, I166V, I166Q, I166A, I166R, I166N, I166K, I166C, I166F, I166M, I166G, I166P, I166T, I166W, I166H, and I166E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the I166 position of SEQ ID NO: 1 corresponds to amino acid position I132 of SEQ ID NO: 3. In some cases, the I166 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position I132 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of I132L, I132Y, I132S, I132D, I132V, I132Q, I132A, I132R, I132N, I132K, I132C, I132F, I132M, I132G, I132P, I132T, I132W, I132H, and I132E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0129] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position I166. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position I132.

[0130] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position K167 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: K167L, K167Y, K167S, K167D, K167V, K167Q, K167A, K167R, K167N, K167I, K167C, K167F, K167M, K167G, K167P, K167T, K167W, K167H, and K167E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, or 13-96. In some cases, the K167 position of SEQ ID NO: 1 corresponds to amino acid position K133 *** of SEQ ID NO: 3. In some embodiments, the K167 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position K133 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of K133L, K133Y, K133S, K133D,K133V, K133Q, K133A, K133R, K133N, K133I, K133C, K133F, K133M, K133G, K133P, K133T, K133W, K133H, and K133E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0131] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position K167. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position K133.

[0132] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position S168 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: S168L, S168Y, S168K, S168D, S168V, S168Q, S168A, S168R, S168N, S168I, S168C, S168F, S168M, S168G, S168P, S168T, S168W, S168H, and S168E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the S168 position of SEQ ID NO: 1 corresponds to amino acid position S134 of SEQ ID NO: 3. In some embodiments, the S168 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position S134 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of S134L, S134Y, S134K, S134D, S134V, S134Q, S134A, S134R, S134N, S134I, S134C, S134F, S134M, S134G, S134P, S134T, S134W, S134H, and S134E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0133] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acidsequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position S168. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position S134.

[0134] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position N169 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: N169L, N169Y, N169K, N169D, N169V, N169Q, N169A, N169R, N169S, N169I, N169C, N169F, N169M, N169G, N169P, N169T, N169W, N169H, and N169E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the N169 position of SEQ ID NO: 1 corresponds to amino acid position N135 of SEQ ID NO: 3. In some cases, the N169 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position N135 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of N135L, N135Y, N135K, N135D, N135V, N135Q, N135A, N135R, N135S, N135I, N135C, N135F, N135M, N135G, N135P, N135T, N135W, N135H, and N135E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0135] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position N169. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position N135.

[0136] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position F170 relative to the amino acidposition numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: F170L, F170Y, F170K, F170D, F170V, F170Q, F170A, F170R, F170S, F170I, F170C, F170N, F170M, F170G, F170P, F170T, F170W, F170H, and F170E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the F170 position of SEQ ID NO: 1 corresponds to amino acid position F136 of SEQ ID NO: 3. In some cases, the F170 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position F136 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of F136L, F136Y, F136K, F136D, F136V, F136Q, F136A, F136R, F136S, F136I, F136C, F136N, F136M, F136G, F136P, F136T, F136W, F136H, and F136E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0137] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position F170. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position F136.

[0138] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position R171 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: R171L, R171Y, R171K, R171D, R171V, R171Q, R171A, R171F, R171S, R171I, R171C, R171N, R171M, R171G, R171P, R171T, R171W, R171H, and R171E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the R171 position of SEQ ID NO: 1 corresponds to amino acid position R137 of SEQ ID NO: 3. In some cases, the R171 position ofSEQ ID NO: 1 or 2027 corresponds to amino acid position R137 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of R137L, R137Y, R137K, R137D, R137V, R137Q, R137A, R137F, R137S, R137I, R137C, R137N, R137M, R137G, R137P, R137T, R137W, R137H, and R137E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0139] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position R171. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position R137.

[0140] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position P191 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: P191L, P191Y, P191K, P191D, P191V, P191Q, P191A, P191F, P191S, P191I, P191C, P191N, P191M, P191G, P191R, P191T, P191W, P191H, and P191E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the P191 position of SEQ ID NO: 1 corresponds to amino acid position P157 of SEQ ID NO: 3. In some cases, the P191 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position P157 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of P157L, P157Y, P157K, P157D, P157V, P157Q, P157A, P157F, P157S, P157I, P157C, P157N, P157M, P157G, P157R, P157T, P157W, P157H, and P157E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0141] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position P191. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position P157.

[0142] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position I192 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: I192L, I192Y, I192K, I192D, I192V, I192Q, I192A, I192F, I192S, I192P, I192C, I192N, I192M, I192G, I192R, I192T, I192W, I192H, and I192E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the I192 position of SEQ ID NO: 1 corresponds to amino acid position I158 of SEQ ID NO: 3. In some cases, the I192 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position I158 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of I158L, I158Y, I158K, I158D, I158V, I158Q, I158A, I158F, I158S, I158P, I158C, I158N, I158M, I158G, I158R, I158T, I158W, I158H, and I158E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0143] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position I192. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position I158.

[0144] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position P193 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: P193L, P193Y, P193K, P193D, P193V, P193Q, P193A, P193F, P193S, P193I, P193C, P193N, P193M, P193G, P193R, P193T, P193W, P193H, and P193E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the P193 position of SEQ ID NO: 1 corresponds to amino acid position P159 of SEQ ID NO: 3. In some cases, the P193 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position P159 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of P159L, P159Y, P159K, P159D, P159V, P159Q, P159A, P159F, P159S, P159I, P159C, P159N, P159M, P159G, P159R, P159T, P159W, P159H, and P159E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0145] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position P193. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position P159.

[0146] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position L194 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: L194P, L194Y, L194K, L194D, L194V, L194Q, L194A, L194F, L194S, L194I, L194C, L194N, L194M, L194G, L194R, L194T, L194W, L194H, and L194E, relative to the aminoacid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the L194 position of SEQ ID NO: 1 corresponds to amino acid position L160 of SEQ ID NO: 3. In some cases, the L194 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position L160 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of L160P, L160Y, L160K, L160D, L160V, L160Q, L160A, L160F, L160S, L160I, L160C, L160N, L160M, L160G, L160R, L160T, L160W, L160H, and L160E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0147] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position L194. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position L160.

[0148] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position V195 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: V195P, V195Y, V195K, V195D, V195L, V195Q, V195A, V195F, V195S, V195I, V195C, V195N, V195M, V195G, V195R, V195T, V195W, V195H, and V195E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the V195 position of SEQ ID NO: 1 corresponds to amino acid position V161 of SEQ ID NO: 3. In some cases, the V195 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position V161 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of V161P, V161Y, V161K, V161D, V161L, V161Q, V161A, V161F, V161S, V161I, V161C, V161N, V161M, V161G, V161R, V161T, V161W, V161H, and V161E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0149] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position V195. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position V161.

[0150] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position K196 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: K196P, K196Y, K196V, K196D, K196L, K196Q, K196A, K196F, K196S, K196I, K196C, K196N, K196M, K196G, K196R, K196T, K196W, K196H, and K196E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the K196 position of SEQ ID NO: 1 corresponds to amino acid position K162 of SEQ ID NO: 3. In some cases, the K196 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position K162 of SEQ ID NO: 3. In some cases, the amino acid substitution is selected from the group consisting of K162P, K162Y, K162V, K162D, K162L, K162Q, K162A, K162F, K162S, K162I, K162C, K162N, K162M, K162G, K162R, K162T, K162W, K162H, and K162E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0151] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position K196. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at leastabout 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position K162.

[0152] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position Q197 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: Q197P, Q197Y, Q197V, Q197D, Q197L, Q197K, Q197A, Q197F, Q197S, Q197I, Q197C, Q197N, Q197M, Q197G, Q197R, Q197T, Q197W, Q197H, and Q197E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the Q197 position of SEQ ID NO: 1 corresponds to amino acid position Q163 of SEQ ID NO: 3. In some cases, the Q197 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position Q163 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of Q163P, Q163Y, Q163V, Q163D, Q163L, Q163K, Q163A, Q163F, Q163S, Q163I, Q163C, Q163N, Q163M, Q163G, Q163R, Q163T, Q163W, Q163H, and Q163E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the amino acid substitution is or includes Q163K or Q163R, relative to the amino acid position numbering of SEQ ID NO: 3 or 2026. In some embodiments, the amino acid substitution is or includes Q163K relative to the amino acid position numbering of SEQ ID NO: 3 or 2026. In some embodiments, the amino acid substitution is or includes Q163R, relative to the amino acid position numbering of SEQ ID NO: 3 or 2026. In some embodiments, the substitution is a non-conservative substitution.

[0153] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, or 13-96, and an amino acid substitution at position Q197. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NO: 2027, and an amino acid substitution at position Q197. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having atleast 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3 or 97-194, and an amino acid substitution at position Q163. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 1, 2, 3, 4, 5, or 6 amino acid substitutions thereto, including an amino acid substitution at position Q163. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NO: 2026, and an amino acid substitution at position Q163. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NO: 2026, and an amino acid substitution of Q163K. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 1, 2, 3, 4, 5, or 6 amino acid substitutions thereto, including an amino acid substitution of Q163K. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NO: 2026, and an amino acid substitution of Q163R. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 1, 2, 3, 4, 5, or 6 amino acid substitutions thereto, including an amino acid substitution of Q163R.

[0154] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position K198 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: K198P, K198Y, K198V, K198D, K198L, K198Q, K198A, K198F, K198S, K198I, K198C, K198N, K198M, K198G, K198R, K198T, K198W, K198H, and K198E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the K198 position ofSEQ ID NO: 1 corresponds to amino acid position K164 of SEQ ID NO: 3. In some cases, the K198 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position K164 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of K164P, K164Y, K164V, K164D, K164L, K164Q, K164A, K164F, K164S, K164I, K164C, K164N, K164M, K164G, K164R, K164T, K164W, K164H, and K164E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0155] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position K198. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position K164.

[0156] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position G199 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: G199P, G199Y, G199V, G199D, G199L, G199Q, G199A, G199F, G199S, G199I, G199C, G199N, G199M, G199K, G199R, G199T, G199W, G199H, and G199E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the G199 position of SEQ ID NO: 1 corresponds to amino acid position G165 of SEQ ID NO: 3. In some cases, the G199 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position G165 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of G165P, G165Y, G165V, G165D, G165L, G165Q, G165A, G165F, G165S, G165I, G165C, G165N, G165M, G165K, G165R, G165T, G165W, G165H, and G165E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0157] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position G199. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position G165.

[0158] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position G200 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: G200P, G200Y, G200V, G200D, G200L, G200Q, G200A, G200F, G200S, G200I, G200C, G200N, G200M, G200K, G200R, G200T, G200W, G200H, and G200E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the G200 position of SEQ ID NO: 1 corresponds to amino acid position G166 of SEQ ID NO: 3. In some cases, the G200 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position G166 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of G166P, G166Y, G166V, G166D, G166L, G166Q, G166A, G166F, G166S, G166I, G166C, G166N, G166M, G166K, G166R, G166T, G166W, G166H, and G166E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0159] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position G200. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at leastabout 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position G166.

[0160] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position Q201 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: Q201P, Q201Y, Q201V, Q201D, Q201L, Q201G, Q201A, Q201F, Q201S, Q201I, Q201C, Q201N, Q201M, Q201K, Q201R, Q201T, Q201W, Q201H, and Q201E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the Q201 position of SEQ ID NO: 1 corresponds to amino acid position Q167 of SEQ ID NO: 3. In some cases, the Q201 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position Q167 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of Q167P, Q167Y, Q167V, Q167D, Q167L, Q167g, Q167A, Q167F, Q167S, Q167I, Q167C, Q167N, Q167M, Q167K, Q167R, Q167T, Q167W, Q167H, and Q167E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0161] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position Q201. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position Q167.

[0162] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position Y202 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selectedfrom the group consisting of: Y202P, Y202Q, Y202V, Y202D, Y202L, Y202G, Y202A, Y202F, Y202S, Y202I, Y202C, Y202N, Y202M, Y202K, Y202R, Y202T, Y202W, Y202H, and Y202E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the Y202 position of SEQ ID NO: 1 corresponds to amino acid position Y168 of SEQ ID NO: 3. In some cases, the Y202 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position Y168 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of Y168P, Y168Q, Y168V, Y168D, Y168L, Y168G, Y168A, Y168F, Y168S, Y168I, Y168C, Y168N, Y168M, Y168K, Y168R, Y168T, Y168W, Y168H, and Y168E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the amino acid substitution is or includes any one of Y168H, Y168K or Y168R, relative to the amino acid position numbering of SEQ ID NO: 3 or 2026. In some embodiments, the amino acid substitution is or includes Y168H, relative to the amino acid position numbering of SEQ ID NO: 3 or 2026. In some embodiments, the amino acid substitution is or includes Y168K, relative to the amino acid position numbering of SEQ ID NO: 3 or 2026. In some embodiments, the amino acid substitution is or includes Y168R, relative to the amino acid position numbering of SEQ ID NO: 3 or 2026. In some embodiments, the amino acid substitution is or includes Y168H, relative to the amino acid position numbering of SEQ ID NO: 3 or 2026. In some embodiments, the substitution is a non-conservative substitution.

[0163] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, or 13-96, and an amino acid substitution at position Y202. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NO: 2027, and an amino acid substitution at position Y202. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3 or 97-194, and an amino acid substitution at position Y168. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NO: 2026, and an amino acid substitution at position Y168. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 1, 2, 3, 4, 5, or 6 amino acid substitutions thereto, including an amino acid substitution at position Y168. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NO: 2026, and an amino acid substitution of Y168H. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 1, 2, 3, 4, 5, or 6 amino acid substitutions thereto, including an amino acid substitution of Y168H. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NO: 2026, and an amino acid substitution of Y168R. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 1, 2, 3, 4, 5, or 6 amino acid substitutions thereto, including an amino acid substitution of Y168R. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NO: 2026, and an amino acid substitution of Y168K. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 1, 2, 3, 4, 5, or 6 amino acid substitutions thereto, including an amino acid substitution of Y168K.

[0164] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position T203 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: T203P, T203Q, T203V, T203D, T203L, T203G, T203A, T203F, T203S,T203I, T203C, T203N, T203M, T203K, T203R, T203Y, T203W, T203H, and T203E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the T203 position of SEQ ID NO: 1 corresponds to amino acid position T169 of SEQ ID NO: 3. In some cases, the T203 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position T169 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of T169P, T169Q, T169V, T169D, T169L, T169G, T169A, T169F, T169S, T169I, T169C, T169N, T169M, T169K, T169R, T169Y, T169W, T169H, and T169E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0165] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position T203. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position T169.

[0166] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position G204 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: G204P, G204Q, G204V, G204D, G204L, G204T, G204A, G204F, G204S, G204I, G204C, G204N, G204M, G204K, G204R, G204Y, G204W, G204H, and G204E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the G204 position of SEQ ID NO: 1 corresponds to amino acid position G170 of SEQ ID NO: 3. In some cases, the G204 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position G170 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of G170P, G170Q, G170V, G170D, G170L, G170T, G170A, G170F, G170S, G170I, G170C, G170N, G170M, G170K, G170R, G170Y, G170W, G170H, and G170E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or thecorresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0167] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position G204. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position G170.

[0168] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position F205 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: F205P, F205Q, F205V, F205D, F205L, F205T, F205A, F205G, F205S, F205I, F205C, F205N, F205M, F205K, F205R, F205Y, F205W, F205H, and F205E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the F205 position of SEQ ID NO: 1 corresponds to amino acid position F171 of SEQ ID NO: 3. In some cases, the F205 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position F171 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of F171P, F171Q, F171V, F171D, F171L, F171T, F171A, F171G, F171S, F171I, F171C, F171N, F171M, F171K, F171R, F171Y, F171W, F171H, and F171E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0169] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position F205. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises orconsists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position F171.

[0170] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position F240 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: F240P, F240Q, F240V, F240D, F240L, F240T, F240A, F240G, F240S, F240I, F240C, F240N, F240M, F240K, F240R, F240Y, F240W, F240H, and F240E, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the F240 position of SEQ ID NO: 1 corresponds to amino acid position F206 of SEQ ID NO: 3. In some cases, the F240 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position F206 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of F206P, F206Q, F206V, F206D, F206L, F206T, F206A, F206G, F206S, F206I, F206C, F206N, F206M, F206K, F206R, F206Y, F206W, F206H, and F206E, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0171] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position F240. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position F206.

[0172] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position E241 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position correspondingthereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: E241P, E241Q, E241V, E241D, E241L, E241T, E241A, E241G, E241S, E241I, E241C, E241N, E241M, E241K, E241R, E241Y, E241W, E241H, and E241F, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the E241 position of SEQ ID NO: 1 corresponds to amino acid position E207 of SEQ ID NO: 3. In some cases, the E241 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position E207 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of E207P, E207Q, E207V, E207D, E207L, E207T, E207A, E207G, E207S, E207I, E207C, E207N, E207M, E207K, E207R, E207Y, E207W, E207H, and E207F, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0173] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position E241. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position E207.

[0174] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position Q242 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: Q242P, Q242E, Q242V, Q242D, Q242L, Q242T, Q242A, Q242G, Q242S, Q242I, Q242C, Q242N, Q242M, Q242K, Q242R, Q242Y, Q242W, Q242H, and Q242F, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the Q242 position of SEQ ID NO: 1 corresponds to amino acid position Q208 of SEQ ID NO: 3. In some cases, the Q242 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position Q208 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of Q208P, Q208E, Q208V, Q208D,Q208L, Q208T, Q208A, Q208G, Q208S, Q208I, Q208C, Q208N, Q208M, Q208K, Q208R, Q208Y, Q208W, Q208H, and Q208F, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0175] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position Q242. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position Q208.

[0176] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position V243 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: V243P, V243E, V243Q, V243D, V243L, V243T, V243A, V243G, V243S, V243I, V243C, V243N, V243M, V243K, V243R, V243Y, V243W, V243H, and V243F, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the V243 position of SEQ ID NO: 1 corresponds to amino acid position V209 of SEQ ID NO: 3. In some cases, the V243 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position V209 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of V209P, V209E, V209Q, V209D, V209L, V209T, V209A, V209G, V209S, V209I, V209C, V209N, V209M, V209K, V209R, V209Y, V209W, V209H, and V209F, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0177] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acidsequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position V243. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position V209.

[0178] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position Q244 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: Q244P, Q244E, Q244V, Q244D, Q244L, Q244T, Q244A, Q244G, Q244S, Q244I, Q244C, Q244N, Q244M, Q244K, Q244R, Q244Y, Q244W, Q244H, and Q244F, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the Q244 position of SEQ ID NO: 1 corresponds to amino acid position Q210 of SEQ ID NO: 3. In some cases, the Q244 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position Q210 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of Q210P, Q210E, Q210V, Q210D, Q210L, Q210T, Q210A, Q210G, Q210S, Q210I, Q210C, Q210N, Q210M, Q210K, Q210R, Q210Y, Q210W, Q210H, and Q210F, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0179] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position Q244. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position Q210.

[0180] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position K245 relative to the amino acidposition numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: K245P, K245E, K245V, K245D, K245L, K245T, K245A, K245G, K245S, K245I, K245C, K245N, K245M, K245Q, K245R, K245Y, K245W, K245H, and K245F, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the K245 position of SEQ ID NO: 1 corresponds to amino acid position K211 of SEQ ID NO: 3. In some cases, the K245 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position K211 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of K211P, K211E, K211V, K211D, K211L, K211T, K211A, K211G, K211S, K211I, K211C, K211N, K211M, K211Q, K211R, K211Y, K211W, K211H, and K211F, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0181] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position K245. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position K211.

[0182] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position S246 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: S246P, S246E, S246V, S246D, S246L, S246T, S246A, S246G, S246K, S246I, S246C, S246N, S246M, S246Q, S246R, S246Y, S246W, S246H, and S246F, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the S246 position of SEQ ID NO: 1 corresponds to amino acid position S212 of SEQ ID NO: 3. In some cases, the S246 position ofSEQ ID NO: 1 or 2027 corresponds to amino acid position S212 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of S212P, S212E, S212V, S212D, S212L, S212T, S212A, S212G, S212K, S212I, S212C, S212N, S212M, S212Q, S212R, S212Y, S212W, S212H, and S212F, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0183] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position S246. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position S212.

[0184] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position P247 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: P247S, P247E, P247V, P247D, P247L, P247T, P247A, P247G, P247K, P247I, P247C, P247N, P247M, P247Q, P247R, P247Y, P247W, P247H, and P247F, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the P247 position of SEQ ID NO: 1 corresponds to amino acid position P213 of SEQ ID NO: 3. In some cases, the P247 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position P213 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of P213S, P213E, P213V, P213D, P213L, P213T, P213A, P213G, P213K, P213I, P213C, P213N, P213M, P213Q, P213R, P213Y, P213W, P213H, and P213F, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0185] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position P247. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position P213.

[0186] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position K248 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: K248S, K248E, K248V, K248D, K248L, K248T, K248A, K248G, K248P, K248I, K248C, K248N, K248M, K248Q, K248R, K248Y, K248W, K248H, and K248F, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the K248 position of SEQ ID NO: 1 corresponds to amino acid position K214 of SEQ ID NO: 3. In some cases, the K248 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position K214 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of K214S, K214E, K214V, K214D, K214L, K214T, K214A, K214G, K214P, K214I, K214C, K214N, K214M, K214Q, K214R, K214Y, K214W, K214H, and K214F, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105- 194). In some embodiments, the substitution is a non-conservative substitution.

[0187] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position K248. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position K214.

[0188] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position P249 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: P249S, P249E, P249V, P249D, P249L, P249T, P249A, P249G, P249K, P249I, P249C, P249N, P249M, P249Q, P249R, P249Y, P249W, P249H, and P249F, relative to the amino acid position numbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the P249 position of SEQ ID NO: 1 corresponds to amino acid position P215 of SEQ ID NO: 3. In some cases, the P249 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position P215 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of P215S, P215E, P215V, P215D, P215L, P215T, P215A, P215G, P215K, P215I, P215C, P215N, P215M, P215Q, P215R, P215Y, P215W, P215H, and P215F, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0189] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position P249. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position P215.

[0190] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution at amino acid position I250 relative to the amino acid position numbering of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 2027, an amino acid position corresponding thereto of a homolog or ortholog of SEQ ID NO: 1, or an amino acid position corresponding thereto of a variant from TABLE 3 (e.g., SEQ ID NOs: 13-96). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises an amino acid substitution selected from the group consisting of: I250S, I250E, I250V, I250D, I250L, I250T, I250A, I250G, I250K, I250P, I250C, I250N, I250M, I250Q, I250R, I250Y, I250W, I250H, and I250F, relative to the amino acid positionnumbering of SEQ ID NOs: 1, 2, 2027, or 13-96. In some cases, the I250 position of SEQ ID NO: 1 corresponds to amino acid position I216 of SEQ ID NO: 3. In some cases, the I250 position of SEQ ID NO: 1 or 2027 corresponds to amino acid position I216 of SEQ ID NO: 2026. In some cases, the amino acid substitution is selected from the group consisting of I216S, I216E, I216V, I216D, I216L, I216T, I216A, I216G, I216K, I216P, I216C, I216N, I216M, I216Q, I216R, I216Y, I216W, I216H, and I216F, relative to the amino acid position numbering of SEQ ID NO: 3, 2026, or the corresponding variant from TABLE 4 (e.g., SEQ ID NOs: 97-104) or TABLE 5 (e.g., SEQ ID NOs: 105-194). In some embodiments, the substitution is a non-conservative substitution.

[0191] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 2027, or 13-96, and an amino acid substitution at position I250. In some embodiments, the engineered polypeptide comprises or consists of an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to the amino acid sequence of SEQ ID NOs: 3, 2026, or 97-194, and an amino acid substitution at position I216.

[0192] The engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) can include any suitable number of substitutions relative to the amino acid numbering position of the reference sequence (e.g., SEQ ID NO: 1, 2, 3, 13-104, 2026, or 2027). In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes 1, 2, 3, 4, 5, 6 or more amino acid substitutions relative to the amino acid numbering position of SEQ ID NO: 1, 2, 13-96, or 2027. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes 1, 2, 3, 4, 5, 6 or more amino acid substitutions relative to the amino acid numbering position of SEQ ID NO: 3, 97-104, or 2026.

[0193] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes at least two amino acid substitutions relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes a substitution at any two of the following amino acid positions: S101, S102, V103, E104, L107, L107, L107, S108, R109, V110, C111, Y112, R113, R114, A115, A116, E117, L118, F119, K120, N121, A122, A123, I124, A125, S126, G127, L128, R129, S130, K131, I132, K133, S134, N135, F136, R137, P157, I158, P159, L160, V161, K162, Q163, K164, G165, G166, Q167, Y168, T169, G170, F171, F206, E207, Q208, V209, Q210, K211, S212, P213, K214, P215, or I216. In someembodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes a substitution at one of the following combinations of amino acid positions: L107 / Y168, K120 / Y168, S126 / Y168, Q163 / Y168, or H105 / Y168, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes one of the following combinations of substitutions: L107Y / Y168H, L107Y / Y168K, L107Y / Y168R, K120H / Y168H, K120H / Y168K, K120H / Y168R, S126Q / Y168H, S126Q / Y168K, S126Q / Y168R, H105Y / Y168H, H105Y / Y168K, H105Y / Y168R, Q163K / Y168H, Q163K / Y168K, Q163K / Y168R, Q163R / Y168H, Q163R / Y168K, or Q163R / Y168R, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, each substitution is a non-conservative substitution.

[0194] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes a combination of substitutions at L107 / Y168, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, 85-97%, etc.), and includes the combination of substitutions L107Y / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, 85-97%, etc.), and includes the combination of substitutions L107Y / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, 85-97%, etc.), and includes the combination of substitutions L107Y / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 2, 3, 4, 5, or 6 amino acid substitutions thereto, including a combination of two amino acid substitutions selected from: L107Y / Y168H, L107Y / Y168K, and L107Y / Y168R.

[0195] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes a combination of substitutions at K120 / Y168, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions K120H / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, 85-97%, etc.), and includes the combination of substitutions K120H / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions K120H / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, 85-97%, etc.), and includes the combination of substitutions K120H / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions K120H / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, 85- 97%, etc.), and includes the combination of substitutions K120H / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 2, 3, 4, 5, or 6 amino acid substitutions thereto, including a combination of two amino acid substitutions selected from: K120H / Y168H, K120H / Y168K, and K120H / Y168R.

[0196] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes a combination of substitutions at S126 / Y168, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions S126Q / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, 85-97%, etc.), and includes the combination of substitutions S126Q / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions S126Q / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, 85-97%, etc.), and includes the combination of substitutions S126Q / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions S126Q / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, 85- 97%, etc.), and includes the combination of substitutions S126Q / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 2, 3, 4, 5, or 6 amino acid substitutions thereto, including a combination of two amino acid substitutions selected from: S126Q / Y168H, S126Q / Y168K, and S126Q / Y168R.

[0197] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes a combination of substitutions at H105 / Y168, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions H105Y / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide(e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, 85-97%, etc.), and includes the combination of substitutions H105Y / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions H105Y / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, 85-97%, etc.), and includes the combination of substitutions H105Y / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions H105Y / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, 85- 97%, etc.), and includes the combination of substitutions H105Y / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 2, 3, 4, 5, or 6 amino acid substitutions thereto, including a combination of two amino acid substitutions selected from: H105Y / Y168H, H105Y / Y168K, and H105Y / Y168R.

[0198] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes a combination of substitutions at Q163 / Y168, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions Q163K / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, 85-97%, etc.), and includes the combination of substitutions Q163K / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. Insome embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions Q163K / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions Q163K / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions Q163K / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85- 97%, etc.), and includes the combination of substitutions Q163K / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions Q163R / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions Q163R / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions Q163R / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions Q163R / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions Q163R / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Caspolypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions Q163R / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 2, 3, 4, 5, or 6 amino acid substitutions thereto, including a combination of two amino acid substitutions selected from: Q163K / Y168H, Q163K / Y168K, Q163K / Y168R, Q163R / Y168H, Q163R / Y168K, and Q163R / Y168R.

[0199] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes at least three amino acid substitutions relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes a substitution at any three of the following amino acid positions: S101, S102, V103, E104, L107, L107, L107, S108, R109, V110, C111, Y112, R113, R114, A115, A116, E117, L118, F119, K120, N121, A122, A123, I124, A125, S126, G127, L128, R129, S130, K131, I132, K133, S134, N135, F136, R137, P157, I158, P159, L160, V161, K162, Q163, K164, G165, G166, Q167, Y168, T169, G170, F171, F206, E207, Q208, V209, Q210, K211, S212, P213, K214, P215, or I216. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes a substitution at one of the following combinations of amino acid positions: L107 / K120 / Y168, L107 / S126 / Y168, L107 / Q163 / Y168, K120 / S126 / Y168, K120 / Q163 / Y168, H105 / Q163 / Y168, or S126 / Q163 / Y168, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes one of the following combinations of substitutions: L107Y / K120H / Y168H, L107Y / K120H / Y168K, L107Y / K120H / Y168R, L107Y / S126Q / Y168H, L107Y / S126Q / Y168K, L107Y / S126Q / Y168R, L107Y / Q163K / Y168H, L107Y / Q163K / Y168K, L107Y / Q163K / Y168R, L107Y / Q163R / Y168H, L107Y / Q163R / Y168K, L107Y / Q163R / Y168R, K120H / S126Q / Y168H, K120H / S126Q / Y168K, K120H / S126Q / Y168R, K120H / Q163K / Y168H, K120H / Q163K / Y168K, K120H / Q163K / Y168R, K120H / Q163R / Y168H, K120H / Q163R / Y168K, K120H / Q163R / Y168R, H105Y / Q163K / Y168H, H105Y / Q163K / Y168K, H105Y / Q163K / Y168R, H105Y / Q163R / Y168H, H105Y / Q163R / Y168K, H105Y / Q163R / Y168R, S126Q / Q163K / Y168H, S126Q / Q163K / Y168K, S126Q / Q163K / Y168R, S126Q / Q163R / Y168H, S126Q / Q163R / Y168K, or S126Q / Q163R / Y168R, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, each substitution is a non- conservative substitution.

[0200] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes a combination of substitutions at L107 / K120 / Y168, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / K120H / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions L107Y / K120H / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / K120H / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions L107Y / K120H / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / K120H / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions L107Y / K120H / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / S126Q / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions L107Y / S126Q / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, theengineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 3, 4, 5, or 6 amino acid substitutions thereto, including a combination of three amino acid substitutions selected from: L107Y / K120H / Y168H, L107Y / K120H / Y168K, or L107Y / K120H / Y168R.

[0201] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes a combination of substitutions at L107 / S126 / Y168, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / S126Q / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions L107Y / S126Q / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / S126Q / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions L107Y / S126Q / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 3, 4, 5, or 6 amino acid substitutions thereto, including a combination of three amino acid substitutions selected from: L107Y / S126Q / Y168H, or L107Y / S126Q / Y168K.

[0202] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / Q163K / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions L107Y / Q163K / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / Q163K / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions L107Y / Q163K / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / Q163K / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions L107Y / Q163K / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / Q163R / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions L107Y / Q163R / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / Q163R / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85- 97%, etc.), and includes the combination of substitutions L107Y / Q163R / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / Q163R / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, theengineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions L107Y / Q163R / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 3, 4, 5, or 6 amino acid substitutions thereto, including a combination of three amino acid substitutions selected from: L107Y / S126Q / Y168R, L107Y / Q163K / Y168H, L107Y / Q163K / Y168K, L107Y / Q163K / Y168R, L107Y / Q163R / Y168H, L107Y / Q163R / Y168K, or L107Y / Q163R / Y168R.

[0203] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes a combination of substitutions at K120 / S126 / Y168, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions K120H / S126Q / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions K120H / S126Q / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions K120H / S126Q / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions K120H / S126Q / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions K120H / S126Q / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values(e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions K120H / S126Q / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 3, 4, 5, or 6 amino acid substitutions thereto, including a combination of three amino acid substitutions selected from: K120H / S126Q / Y168H, K120H / S126Q / Y168K, or K120H / S126Q / Y168R.

[0204] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes a combination of substitutions at K120 / Q163 / Y168, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions K120H / Q163K / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions K120H / Q163K / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions K120H / Q163K / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions K120H / Q163K / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions K120H / Q163K / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions K120H / Q163K / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions K120H / Q163R / Y168H relative to the amino acid numbering position of SEQID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions K120H / Q163R / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions K120H / Q163R / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions K120H / Q163R / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions K120H / Q163R / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85- 97%, etc.), and includes the combination of substitutions K120H / Q163R / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 3, 4, 5, or 6 amino acid substitutions thereto, including a combination of three amino acid substitutions selected from: K120H / Q163K / Y168H, K120H / Q163K / Y168K, K120H / Q163K / Y168R, K120H / Q163R / Y168H, K120H / Q163R / Y168K, or K120H / Q163R / Y168R.

[0205] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes a combination of substitutions at H105 / Q163 / Y168, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions H105Y / Q163K / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a rangedefined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions H105Y / Q163K / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions H105Y / Q163K / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions H105Y / Q163K / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions H105Y / Q163K / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions H105Y / Q163K / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions H105Y / Q163R / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions H105Y / Q163R / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions H105Y / Q163R / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions H105Y / Q163R / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide(e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions H105Y / Q163R / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, 85- 97%, etc.), and includes the combination of substitutions H105Y / Q163R / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 3, 4, 5, or 6 amino acid substitutions thereto, including a combination of three amino acid substitutions selected from: H105Y / Q163K / Y168H, H105Y / Q163K / Y168K, H105Y / Q163K / Y168R, H105Y / Q163R / Y168H, H105Y / Q163R / Y168K, or H105Y / Q163R / Y168R.

[0206] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes a combination of substitutions at S126 / Q163 / Y168, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions S126Q / Q163K / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions S126Q / Q163K / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions S126Q / Q163K / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions S126Q / Q163K / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions S126Q / Q163K / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96,97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions S126Q / Q163K / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions S126Q / Q163R / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions S126Q / Q163R / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions S126Q / Q163R / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions S126Q / Q163R / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions S126Q / Q163R / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85- 97%, etc.), and includes the combination of substitutions S126Q / Q163R / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 3, 4, 5, or 6 amino acid substitutions thereto, including a combination of three amino acid substitutions selected from: S126Q / Q163K / Y168K, S126Q / Q163K / Y168R, S126Q / Q163R / Y168H, or S126Q / Q163R / Y168K, S126Q / Q163R / Y168R.

[0207] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes at least four amino acid substitutions relative to the amino acid numbering positionof SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes a substitution at any two of the following amino acid positions: S101, S102, V103, E104, L107, L107, L107, S108, R109, V110, C111, Y112, R113, R114, A115, A116, E117, L118, F119, K120, N121, A122, A123, I124, A125, S126, G127, L128, R129, S130, K131, I132, K133, S134, N135, F136, R137, P157, I158, P159, L160, V161, K162, Q163, K164, G165, G166, Q167, Y168, T169, G170, F171, F206, E207, Q208, V209, Q210, K211, S212, P213, K214, P215, orI216. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes a substitution at one of the following combinations of amino acid positions: L107 / K120 / S126 / Y168, L107 / K120 / Q163 / Y168, L107 / S126 / Q163 / Y168, or K120 / S126 / Q163 / Y168, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes one of the following combinations of substitutions: L107Y / K120H / S126Q / Y168H, L107Y / K120H / S126Q / Y168K, L107Y / K120H / S126Q / Y168R, L107Y / K120H / Q163K / Y168H, L107Y / K120H / Q163K / Y168K, L107Y / K120H / Q163K / Y168R, L107Y / K120H / Q163R / Y168H, L107Y / K120H / Q163R / Y168K, L107Y / K120H / Q163R / Y168R, L107Y / S126Q / Q163K / Y168H, L107Y / S126Q / Q163K / Y168K, L107Y / S126Q / Q163K / Y168R, L107Y / S126Q / Q163R / Y168H, L107Y / S126Q / Q163R / Y168K, L107Y / S126Q / Q163R / Y168R, K120H / S126Q / Q163K / Y168H, K120H / S126Q / Q163K / Y168K, K120H / S126Q / Q163K / Y168R, K120H / S126Q / Q163R / Y168H, K120H / S126Q / Q163R / Y168K, or K120H / S126Q / Q163R / Y168R, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, each substitution is a non-conservative substitution.

[0208] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes a combination of substitutions at L107 / K120 / S126 / Y168 relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / K120H / S126Q / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85- 97%, etc.), and includes the combination of substitutions L107Y / K120H / S126Q / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / K120H / S126Q / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes anamino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85- 97%, etc.), and includes the combination of substitutions L107Y / K120H / S126Q / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / K120H / S126Q / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85- 97%, etc.), and includes the combination of substitutions L107Y / K120H / S126Q / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 4, 5, or 6 amino acid substitutions thereto, including a combination of four amino acid substitutions selected from: L107Y / K120H / S126Q / Y168H, L107Y / K120H / S126Q / Y168K, or L107Y / K120H / S126Q / Y168R.

[0209] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes a combination of substitutions at L107 / K120 / Q163 / Y168 relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / K120H / Q163K / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85- 97%, etc.), and includes the combination of substitutions L107Y / K120H / Q163K / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / K120H / Q163K / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85-97%, etc.), and includes the combination of substitutions L107Y / K120H / Q163K / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / K120H / Q163K / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85- 97%, etc.), and includes the combination of substitutions L107Y / K120H / Q163K / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / K120H / Q163R / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85- 97%, etc.), and includes the combination of substitutions L107Y / K120H / Q163R / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / K120H / Q163R / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85- 97%, etc.), and includes the combination of substitutions L107Y / K120H / Q163R / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / K120H / Q163R / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85- 97%, etc.), and includes the combination of substitutions L107Y / K120H / Q163R / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineeredpolypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 4, 5, or 6 amino acid substitutions thereto, including a combination of four amino acid substitutions selected from: L107Y / K120H / Q163K / Y168H, L107Y / K120H / Q163K / Y168K, L107Y / K120H / Q163K / Y168R, L107Y / K120H / Q163R / Y168H, L107Y / K120H / Q163R / Y168K, or L107Y / K120H / Q163R / Y168R.

[0210] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes a combination of substitutions at L107 / S126 / Q163 / Y168 relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / S126Q / Q163K / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85- 97%, etc.), and includes the combination of substitutions L107Y / S126Q / Q163K / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / S126Q / Q163K / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85- 97%, etc.), and includes the combination of substitutions L107Y / S126Q / Q163K / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / S126Q / Q163K / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85- 97%, etc.), and includes the combination of substitutions L107Y / S126Q / Q163K / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / S126Q / Q163R / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. Insome embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85- 97%, etc.), and includes the combination of substitutions L107Y / S126Q / Q163R / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / S126Q / Q163R / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85- 97%, etc.), and includes the combination of substitutions L107Y / S126Q / Q163R / Y168K relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions L107Y / S126Q / Q163R / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQ ID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85- 97%, etc.), and includes the combination of substitutions L107Y / S126Q / Q163R / Y168R relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) comprises the amino acid sequence of SEQ ID NO:2026 having 4, 5, or 6 amino acid substitutions thereto, including a combination of four amino acid substitutions selected from: L107Y / S126Q / Q163K / Y168H, L107Y / S126Q / Q163K / Y168K, L107Y / S126Q / Q163K / Y168R, L107Y / S126Q / Q163R / Y168H, L107Y / S126Q / Q163R / Y168K, or L107Y / S126Q / Q163R / Y168R.

[0211] In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes a combination of substitutions at K120 / S126 / Q163 / Y168 relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions K120H / S126Q / Q163K / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes an amino acid sequence that is, is at least, or is about 85, 90, 95, 96, 97, 98, 99%, or 100% identical to SEQID NO: 3 or 2026, optionally, where the amino acid sequence is identical to SEQ ID NO: 3 or 2026 by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 95-97%, or 85- 97%, etc.), and includes the combination of substitutions K120H / S126Q / Q163K / Y168H relative to the amino acid numbering position of SEQ ID NO: 3 or 2026. In some embodiments, the engineered polypeptide (e.g., non-naturally occurring Cas polypeptide) includes the combination of substitutions K120H / S126Q / Q163K / Y168K relative to the amino acid numbering position of S...

Claims

WHAT IS CLAIMED IS:

1. A non-naturally occurring Cas polypeptide comprising: a substitution at one or more amino acid position selected from the group consisting of: (a) S135, S136, V137, E138, H139, Y140, L141, S142, D143, V144, C145, Y146, T147, R148, A149, A150, E151, L152, F153, K154, N155, A156, A157, I158, A159, S160, G161, L162, R163, S164, K165, I166, K167, S168, N169, F170, R171, P191, I192, P193, L194, V195, K196, Q197, K198, G199, G200, Q201, Y202, T203, G204, F205, F240, E241, Q242, V243, Q244, K245, S246, P247, K248, P249, and I250, relative to the amino acid numbering position of SEQ ID NO: 1 or 2 or 2027; or (b) S101, S102, V103, E104, H105, Y106, L107, S108, D109, V110, C111, Y112, T113, R114, A115, A116, E117, L118, F119, K120, N121, A122, A123, I124, A125, S126, G127, L128, R129, S130, K131, I132, K133, S134, N135, F136, R137, P157, I158, P159, L160, V161, K162, Q163, K164, G165, G166, Q167, Y168, T169, G170, F171, F206, E207, Q208, V209, Q210, K211, S212, P213, K214, P215, and I216, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026.

2. The non-naturally occurring Cas polypeptide of claim 1, comprising the substitution at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63 of the amino acid positions in (a) or (b).

3. The non-naturally occurring Cas polypeptide of claim 1 or 2, wherein the substitution at any one of the amino acid positions of (a) or (b) is a substitution to an amino acid residue selected from the group consisting of: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, optionally wherein the substitution at any one of the one or more amino acid position is a non-conservative substitution.

4. The non-naturally occurring Cas polypeptide of claim 3, wherein the substitution at any one of the amino acid positions of (a) or (b) is a substitution to an amino acid residue selected from the group consisting of: arginine, glutamine, histidine, lysine, and tyrosine.

5. The non-naturally occurring Cas polypeptide of any one of the preceding claims, wherein the non-naturally occurring polypeptide exhibits increased binding to a protospacer-adjacent motif (PAM) site other than a canonical PAM site, relative to the Cas polypeptide of SEQ ID NOS: 1-3, 2026, or 2027.

6. The non-naturally occurring Cas polypeptide of claim 5, wherein the canonical PAM site is 5’-TTTR-3’, where R is A or G.

7. The non-naturally occurring Cas polypeptide of any one of the preceding claims, wherein the non-naturally occurring Cas polypeptide comprises or consists of an amino acid sequence listed in Tables 3-7 or 12.

8. The non-naturally occurring Cas polypeptide of any one of the preceding claims, wherein the substitution at one or more amino acid position is selected from the group consisting of: H105, L107, K120, S126, Q163, Y168, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026.

9. The non-naturally occurring Cas polypeptide of claim 8, comprising the substitution at one amino acid position selected from H105, L107, K120, S126, Q163, or Y168, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026.

10. The non-naturally occurring Cas polypeptide of claim 9, comprising any one of the following substitutions: Y168H, Y168K, Y168R, K120H, S126Q, Q163K, Q163R, L107Y, or H105Y.

11. The non-naturally occurring Cas polypeptide of any one of the preceding claims, comprising the substitution at two, three, four, five or more of the one or more amino acid position.

12. The non-naturally occurring Cas polypeptide of claim 11, comprising the substitution at two of the one or more amino acid position.

13. The non-naturally occurring Cas polypeptide of claim 12, comprising the substitution at one of the following combinations of two of the one or more amino acid position: L107 / Y168, K120 / Y168, S126 / Y168, Q163 / Y168, or H105 / Y168, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026.

14. The non-naturally occurring Cas polypeptide of claim 12 or 13, comprising one of the following combinations of substitutions: L107Y / Y168H, L107Y / Y168K, L107Y / Y168R, K120H / Y168H, K120H / Y168K, K120H / Y168R, S126Q / Y168H, S126Q / Y168K, S126Q / Y168R, H105Y / Y168H, H105Y / Y168K, H105Y / Y168R, Q163K / Y168H, Q163K / Y168K, Q163K / Y168R, Q163R / Y168H, Q163R / Y168K, or Q163R / Y168R, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026.

15. The non-naturally occurring Cas polypeptide of claim 11, comprising the substitution at three of the one or more amino acid position.

16. The non-naturally occurring Cas polypeptide of claim 15, comprising the substitution at one of the following combinations of three of the one or more amino acid position: L107 / K120 / Y168, L107 / S126 / Y168, L107 / Q163 / Y168, K120 / S126 / Y168, K120 / Q163 / Y168, H105 / Q163 / Y168, or S126 / Q163 / Y168, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026.

17. The non-naturally occurring Cas polypeptide of claim 15 or 16, comprising one of the following combinations of substitutions: L107Y / K120H / Y168H, L107Y / K120H / Y168K, L107Y / K120H / Y168R, L107Y / S126Q / Y168H, L107Y / S126Q / Y168K, L107Y / S126Q / Y168R, L107Y / Q163K / Y168H, L107Y / Q163K / Y168K, L107Y / Q163K / Y168R, L107Y / Q163R / Y168H, L107Y / Q163R / Y168K, L107Y / Q163R / Y168R, K120H / S126Q / Y168H, K120H / S126Q / Y168K, K120H / S126Q / Y168R, K120H / Q163K / Y168H, K120H / Q163K / Y168K, K120H / Q163K / Y168R, K120H / Q163R / Y168H, K120H / Q163R / Y168K, K120H / Q163R / Y168R, H105Y / Q163K / Y168H, H105Y / Q163K / Y168K, H105Y / Q163K / Y168R, H105Y / Q163R / Y168H, H105Y / Q163R / Y168K, H105Y / Q163R / Y168R, S126Q / Q163K / Y168H, S126Q / Q163K / Y168K, S126Q / Q163K / Y168R, S126Q / Q163R / Y168H, S126Q / Q163R / Y168K, or S126Q / Q163R / Y168R, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026.

18. The non-naturally occurring Cas polypeptide of claim 11, comprising the substitution at four of the one or more amino acid position.

19. The non-naturally occurring Cas polypeptide of claim 18, comprising the substitution at one of the following combinations of four of the one or more amino acid position: L107 / K120 / S126 / Y168, L107 / K120 / Q163 / Y168, L107 / S126 / Q163 / Y168, or K120 / S126 / Q163 / Y168, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026.

20. The non-naturally occurring Cas polypeptide of claim 18 or 19, wherein comprising one of the following combinations of substitutions: L107Y / K120H / S126Q / Y168H, L107Y / K120H / S126Q / Y168K, L107Y / K120H / S126Q / Y168R, L107Y / K120H / Q163K / Y168H, L107Y / K120H / Q163K / Y168K, L107Y / K120H / Q163K / Y168R, L107Y / K120H / Q163R / Y168H, L107Y / K120H / Q163R / Y168K, L107Y / K120H / Q163R / Y168R, L107Y / S126Q / Q163K / Y168H, L107Y / S126Q / Q163K / Y168K, L107Y / S126Q / Q163K / Y168R, L107Y / S126Q / Q163R / Y168H, L107Y / S126Q / Q163R / Y168K, L107Y / S126Q / Q163R / Y168R, K120H / S126Q / Q163K / Y168H, K120H / S126Q / Q163K / Y168K, K120H / S126Q / Q163K / Y168R, K120H / S126Q / Q163R / Y168H, K120H / S126Q / Q163R / Y168K, or K120H / S126Q / Q163R / Y168R, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026.

21. The non-naturally occurring Cas polypeptide of claim 11, comprising the substitution at five of the one or more amino acid position.

22. The non-naturally occurring Cas polypeptide of claim 21, comprising the substitution at one of the following combinations of five of the one or more amino acid position: L107 / K120 / S126 / Q163 / Y168 relative to the amino acid numbering position of SEQ ID NO: 3 or 2026.

23. The non-naturally occurring Cas polypeptide of claim 21 or 22, wherein comprising one of the following combinations of substitutions: L107Y / K120H / S126Q / Q163K / Y168H, L107Y / K120H / S126Q / Q163K / Y168K, L107Y / K120H / S126Q / Q163K / Y168R, L107Y / K120H / S126Q / Q163R / Y168H, L107Y / K120H / S126Q / Q163R / Y168K, or L107Y / K120H / S126Q / Q163R / Y168R, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026.

24. A non-naturally occurring Cas polypeptide comprising: a substitution at one or more amino acid position selected from the group consisting of: (a) Q115, E119, M178, K179, S180, G181, P183, T184, T185, K186, S187, I364, L365, L366, K367, K368, N369, R370, H371, K372, R373, A374, G375, and H376, relative to the amino acid numbering position of SEQ ID NO: 1 or 2 or 2027; or (b) Q81, E85, M144, K145, S146, G147, P149, T150, T151, K152, S153, I330, L331, L332, K333, K334, N335, R336, H337, K338, R339, A340, G341, and H342, relative to the amino acid numbering position of SEQ ID NO: 3 or 2026.

25. The non-naturally occurring Cas polypeptide of claim 24, comprising the substitution at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 of the amino acid positions in (a) or (b).

26. The non-naturally occurring Cas polypeptide of claim 24 or 25, wherein the substitution at any one of the amino acid positions of (a) or (b) is a substitution to an amino acid residue selected from the group consisting of: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

27. The non-naturally occurring Cas polypeptide of any one of claims 24-26, wherein the non- naturally occurring Cas polypeptide comprises or consists of an amino acid sequence listed in Tables 3-7.

28. A non-naturally occurring Cas polypeptide comprising: (a) a substitution at amino acid position C297 relative to the amino acid numbering position of SEQ ID NO: 2 or 2027, or a substitution at amino acid position C263 relative to the amino acid numbering position of SEQ ID NO: 3 or 2026.

29. The non-naturally occurring Cas polypeptide of claim 11, wherein the substitution at amino acid position C297 relative to the amino acid numbering position of SEQ ID NO: 2 or 2027 is C297G or C297L.

30. The non-naturally occurring Cas polypeptide of claim 11, wherein the substitution at amino acid position C263 relative to the amino acid numbering position of SEQ ID NO: 3 is C263G.

31. The non-naturally occurring Cas polypeptide of any one of the preceding claims, wherein the Cas polypeptide comprises an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to any one of SEQ ID NOS: 1-3, 2026, or 2027, optionally wherein the Cas polypeptide comprises an amino acid sequence having 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 96%, at least about 97%, at least about 98%, at least about 99%, or greater sequence identity to SEQ ID NO:3 or 2026.

32. The non-naturally occurring Cas polypeptide of any one of the preceding claims, wherein the non-naturally occurring polypeptide has nuclease activity.

33. The non-naturally occurring polypeptide of any one of claims 1-31, wherein the non- naturally occurring polypeptide has nickase activity.

34. The non-naturally occurring polypeptide of any one of claims 1-31, wherein the non- naturally occurring polypeptide is partially or fully nuclease deficient.

35. A non-naturally occurring Cas polypeptide comprising an amino acid sequence at least 85% identical to any one of the sequences in Table 12, optionally comprising the amino acid sequence of any one of the sequences in Table 12.

36. The non-naturally occurring Cas polypeptide of any one of the preceding claims that is coupled to a heterologous gene effector.

37. A fusion protein comprising the Cas polypeptide of any one of claims 1-35; and a heterologous gene effector, optionally wherein the heterologous gene effector comprises an epigenetic modification domain, a transcriptional activation domain, and / or a transcriptional repressor domain.

38. The fusion protein of claim 37, wherein the heterologous gene effector comprises a transcriptional activation domain, optionally wherein the transcriptional activation domain comprises VPR or XV1.

48.

39. The fusion protein of claim 37, wherein the heterologous gene effector comprises a transcriptional repressor domain, optionally wherein the transcriptional repressor domain comprises KRAB.

40. A nucleic acid-guided targeting system comprising: the non-naturally occurring Cas polypeptide of any one of claims 1-36 or the fusion protein of any one of claims 37-39; and a guide nucleic acid configured to form a complex with the non-naturally occurring Cas polypeptide and that exhibits specific binding to a target gene or a regulatory region thereof in a target cell.

41. The nucleic acid-guided targeting system of claim 40, wherein the guide nucleic acid comprises a scaffold sequence having, having about, or having at least 80% identical to any one of the polynucleotide sequences set forth in TABLE 8, TABLE 9, or TABLE 10, optionally wherein the scaffold sequence is selected from SEQ ID NOs: 1899, 1901, 1902, 1914, 1915, 1924, or 1926.

42. A composition comprising: a first nucleic acid encoding the non-naturally occurring Cas polypeptide of any one of claims 1-36 or the fusion protein of any one of claims 37-39; and a second nucleic acid encoding a guide nucleic acid configured to form a complex with the non-naturally occurring Cas polypeptide and that exhibits specific binding to a target gene or a regulatory region thereof in a target cell, optionally wherein the first nucleic acid and the second nucleic acid are comprised in a single nucleic acid molecule, optionally wherein the single nucleic acid molecule is a nucleic acid vector.

43. A composition comprising: the non-naturally occurring Cas polypeptide of any one of claims 1-36 or the fusion protein of any one of claims 37-39; and a guide nucleic acid configured to form a complex with the non-naturally occurring Cas polypeptide and that exhibits specific binding to a target gene or a regulatory region thereof in a target cell.

44. The composition of claim 42 or 43, wherein the guide nucleic acid comprises a scaffold sequence having, having about, or having at least 80% identical to any one of the polynucleotide sequences set forth in TABLE 8, TABLE 9, or TABLE 10, optionally wherein the scaffold sequence is selected from SEQ ID NOs: 1899, 1901, 1902, 1914, 1915, 1924, or 1926.

45. A method of modulating expression or activity of a target gene, the method comprising: contacting a cell with: the non-naturally occurring Cas polypeptide of any one of claims 1-36 or the fusion protein of any one of claims 37-39, or a nucleic acid encoding the non-naturally occurring Cas polypeptide of any one of claims 1-36 or the fusion protein of any one of claims 37-39; and a guide nucleic acid exhibiting specific binding to the target gene or a regulatory region thereof, thereby modulating expression or activity of the target gene.

46. The method of claim 45, wherein the guide nucleic acid forms a complex with the Cas polypeptide, and wherein specific binding of the guide nucleic acid to the target gene or a regulatory region thereof comprises binding of the complex to a target sequence in the target gene or a regulatory region thereof in the cell.

47. The method of claim 45, wherein the target sequence is adjacent a protospacer-adjacent motif (PAM) sequence that is different than a canonical PAM sequence, wherein the canonical PAM sequence is TTTR, wherein R is A or G, optionally wherein the PAM sequence that is different than a canonical PAM sequence is TCTG.

48. The method of any one of claims 45-47, wherein the guide nucleic acid comprises a scaffold sequence having, having about, or having at least 80% identical to any one of the polynucleotide sequences set forth in TABLE 8, TABLE 9, or TABLE 10, optionally wherein the scaffold sequence is selected from SEQ ID NOs: 1899, 1901, 1902, 1914, 1915, 1924, or 1926.

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