Editing systems and components and uses thereof

Abstract

Provided are guided nucleic acid binding agents, systems comprising guided nucleic acid binding agents, and methods of use thereof. Such guided nucleic acid binding agents and systems are useful for gene editing and epigenetic modulation of gene expression.

Description

[0001] EDITING SYSTEMS AND COMPONENTS AND USES THEREOF Sequence Listing The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled EDIT0001SEQ.xml created on September 13, 2024, which is 2,646 KB in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. Field The present embodiments provide guided nucleic acid binding agents, such as Cas proteins; guides; editing systems comprising guided nucleic acid binding agents and guides; and methods of use thereof. Background Cas proteins along with their associated Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) guide ribonucleic acids (gRNAs) appear to be a pervasive (~45% of bacteria, ~84% of archaea) component of prokaryotic immune systems. These CRISPR-gRNA cleavage systems serve to protect such microorganisms against non-self nucleic acids, such as infectious viruses and plasmids. CRISPR-associated (Cas) proteins are highly diverse, containing a wide variety of nucleic acid-binding domains. Cas nucleic acid-binding domains can be harnessed for a variety of applications, including, but not limited to, gene editing and epigenetic modulation of gene expression. Summary Certain embodiments provided herein are directed to guided nucleic acid binding agents (Cas proteins, dead Cas proteins, and Cas fusion proteins); guides (single guides and dual guides); and editing systems comprising such guided nucleic acid binding agents and guides. Certain embodiments are directed to methods of using such editing systems. In certain embodiments, provided herein are polypeptides. In certain embodiments, polypeptides are guided nucleic binding agents. In certain embodiments, polypeptides provided herein are Cas proteins. In certain such embodiments, a Cas protein is a Cas enzyme and is capable of inducing cleavage of both strands of double-stranded DNA target (a double-strand break). In certain embodiment, a Cas protein is a Cas enzyme and is capable of inducing cleavage of one strand of a double-stranded DNA target (a nick). In certain embodiments, a Cas protein is a dead Cas protein, which does not induce cleavage of double-stranded DNA. In certain embodiments, polypeptides comprise Cas proteins. Certain Cas proteins may be fused with one or more other proteins or protein domains, also known as ‘heterologous domains’, to create Cas fusion proteins. In certain embodiments, provided herein are nucleic acids that encode the polypeptides described above. In certain embodiments, such nucleic acids are DNA (e.g., template DNA) or RNA (e.g., mRNA). In certain embodiments, provided herein are guides. In certain embodiments, a guide consists of a single oligonucleotide (a single guide). In certain embodiments, a guide consists of two oligonucleotides (a first oligonucleotide and a second oligonucleotide) that are partially complementary to one another, allowing them to hybridize together (a dual guide). Guides comprise a target-recognition region and a protein- recognition region. For single guides, the target-recognition region and protein-recognition region are regions of the single oligonucleotide. For dual guides consisting of a first oligonucleotide and a second oligonucleotide, the first oligonucleotide comprises the target-recognition region and a first portion of the protein-recognition region, and the second oligonucleotide comprises a second portion of the protein- recognition region. In certain such embodiments, the first oligonucleotide can be called a “crRNA” and the second oligonucleotide can be called a “tracrRNA”. The target-recognition region of a guide is a region of an oligonucleotide (either of the single guide or of the crRNA in the case of a dual guide) that has a nucleobase sequence that is complementary to an equal length portion of a target sequence of a target nucleic acid. In certain embodiments, the target nucleic acid is a target DNA. In certain embodiments, editing systems comprise a guided nucleic acid binding agent and a guide. In certain such embodiments, the guided nucleic acid binding agent is a Cas protein. In certain embodiments, the guide is a single guide. In certain embodiments, an editing system comprises a Cas protein and a single guide. In certain embodiments, provided herein are compositions comprising a template DNA encoding both a guided nucleic acid binding agent and a guide. In certain embodiments, provided herein are compositions comprising a template DNA encoding both a Cas protein and a guide. In certain embodiments, provided herein are compositions comprising a guide and an mRNA encoding a guided nucleic acid binding agent. In certain embodiments, provided herein are compositions comprising a guide and an mRNA encoding a Cas protein. In certain embodiments, provided herein are compositions comprising a single guide and an mRNA encoding a guided nucleic acid binding agent. In certain embodiments, provided herein are compositions comprising a dual guide and an mRNA encoding a guided nucleic acid binding agent. In certain embodiments, provided herein are compositions comprising a single guide and an mRNA encoding a Cas protein. In certain embodiments, provided herein are compositions comprising a dual guide and an mRNA encoding a Cas protein. In such embodiments, the Cas protein is selected from a Cas enzyme, a dead Cas protein, and a Cas fusion protein. In certain embodiments, such compositions are administered to a subject to induce gene editing. Brief Description of the Drawings Figure 1 shows a gel showing cleavage activity of ION-CAS proteins in HEK293T nuclear extracts using a general target-recognition region for the guide. Figure 2 shows a gel showing cleavage activity of ION-CAS proteins in HEK293T nuclear extract using Cas-specific target-recognition regions and Cas-specific protein-recognition regions in the guide RNA Figure 3 shows the consensus PAM sequences for IONCAS008, IONCAS009, and IONCAS0016. Figure 4a-4c show gels showing cleavage activity of ION-CAS proteins in HEK293T nuclear extracts using a general target-recognition region for the guide. Figure 5 provides a schematic for a Cas editing system. Figure 6 provides a schematic for labeling the secondary structure of a guide for SpCas9. Detailed Description It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the embodiments, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and / or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, treatises, and GenBank and NCBI reference sequence records are hereby expressly incorporated by reference for the portions of the document discussed herein, as well as in their entirety. It is understood that throughout the specification, the first letter in a peptide sequence is the first amino acid of the peptide at the N-terminus and the last letter in a peptide sequence is the last amino acid of the peptide at the C-terminus unless indicated otherwise. Similarly, the first nucleoside in a nucleotide sequence represents the 5’-end of the nucleotide, and the last letter in the nucleotide sequence represents the 3’ end, unless indicated otherwise. Unless specific definitions are provided, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Where permitted, all patents, applications, published applications and other publications and other data referred to throughout in the disclosure are incorporated by reference herein in their entirety. Unless otherwise indicated, the following terms have the following meanings: As used herein, “2’-deoxynucleoside” means a nucleoside comprising a 2’-H(H) deoxyfuranosyl sugar moiety. In certain embodiments, a 2’-deoxynucleoside is a 2’-β-D-deoxynucleoside and comprises a 2’-β-D-deoxyribosyl sugar moiety, which has the β-D ribosyl configuration as found in naturally occurring deoxyribonucleic acids (DNA). In certain embodiments, a 2’-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (uracil). As used herein, “2’-F” means a 2’-F group in place of the 2’-OH group of a furanosyl sugar moiety. A “2’-fluoro sugar moiety” or “2’-F sugar moiety” means a sugar moiety with a 2’-F group in place of the 2’- OH group of a furanosyl sugar moiety. Unless otherwise indicated, a 2’-F sugar moiety is in the β-D-ribosyl configuration. As used herein, “2’-F nucleoside” means a nucleoside comprising a 2’-F sugar moiety. As used herein, “2’-MOE” means a 2’-OCH2CH2OCH3group in place of the 2’-OH group of a furanosyl sugar moiety. A “2’-MOE sugar moiety” means a sugar moiety with a 2’-OCH2CH2OCH3group in place of the 2’-OH group of a furanosyl sugar moiety. Unless otherwise indicated, a 2’-MOE sugar moiety is in the β-D-ribosyl configuration. “MOE” means O-methoxyethyl. As used herein, “2’-MOE nucleoside” means a nucleoside comprising a 2’-MOE sugar moiety. As used herein, “2’-NMA” means a 2’–O-CH2-C(=O)-NH-CH3group in place of the 2’-OH group of a ribosyl sugar moiety. A “2’-NMA sugar moiety” is a sugar moiety with a 2’–O-CH2-C(=O)-NH-CH3group in place of the 2’-OH group of a ribosyl sugar moiety. Unless otherwise indicated, a 2’-NMA sugar moiety is in the β-D configuration. “NMA” means O-(N-methyl)acetamide. As used herein, “2’-NMA nucleoside” means a nucleoside comprising a 2’-NMA sugar moiety. As used herein, “2’-OMe” means a 2’-OCH3group in place of the 2’-OH group of a furanosyl sugar moiety. A“2’-O-methyl sugar moiety” or “2’-OMe sugar moiety” means a sugar moiety with a 2’-OCH3group in place of the 2’-OH group of a furanosyl sugar moiety. Unless otherwise indicated, a 2’-OMe sugar moiety is in the β-D-ribosyl configuration. As used herein, “2’-OMe nucleoside” means a nucleoside comprising a 2’-OMe sugar moiety. As used herein, “2’-substituted nucleoside” means a nucleoside comprising a 2’-substituted sugar moiety. As used herein, “2’-substituted” in reference to a sugar moiety means a sugar moiety comprising at least one 2'-substituent group other than H or OH. As used herein, “5-methyl cytosine” means a cytosine modified with a methyl group attached to the 5 position. A 5-methyl cytosine is a modified nucleobase. As used herein, “active site” means a region of a protein where a substrate molecule binds to undergo enzymatic catalysis of a chemical reaction. An active site comprises one or more catalytic residues. As used herein, “catalytic residue” means an amino acid that is involved in the enzymatic catalysis of a chemical reaction at its active site. A catalytic residue may be directly involved in the catalytic mechanism (e.g., as a nucleophile), may exert an effect on another residue or water molecule which is directly involved in the catalytic mechanism which aids catalysis (e.g., by electrostatic or acid-base action), may stabilize a transition-state intermediate, and / or may exert an effect on a substrate or cofactor which aids catalysis (e.g., by polarizing a bond which is to be broken), including steric and electrostatic effects. As used herein, “Cas enzyme” means a guided nucleic acid binding agent that is capable of cleaving one or both strands of double-stranded DNA. In certain embodiments, the target nucleic acid is a double- stranded “target DNA”. A “Cas enzyme” may be an enzyme capable of cleaving both strands of a double- stranded DNA or may be an enzyme capable of cleaving only one strand of a double-stranded DNA (a “Cas nickase”), (e.g., containing a mutation in one but not both of the catalytic residues). As used herein, a “Cas protein” means a “Cas enzyme” or a “dead Cas protein”. As used herein, a “dead Cas protein” means a guided nucleic acid binding agent that cannot cleave either strand of a double-stranded DNA. As used herein, “cEt” or “constrained ethyl” means a bicyclic sugar moiety, wherein the first ring of the bicyclic sugar moiety is a ribosyl sugar moiety, the second ring of the bicyclic sugar is formed via a bridge connecting the 4’-carbon and the 2’-carbon, the bridge has the formula 4'-CH(CH3)-O-2', and the bridge is in the S configuration. A cEt bicyclic sugar moiety is in the β-D configuration. As used herein, “cleavable moiety” means a bond or group of atoms that is cleaved under physiological conditions, for example, inside a cell, or a subject. As used herein, “complementary” in reference to an oligonucleotide means that at least 70% of the nucleobases of the oligonucleotide and the nucleobases of another nucleic acid or one or more regions thereof are capable of hydrogen bonding with one another when the nucleobase sequence of the oligonucleotide and the other nucleic acid are aligned in opposing directions. “Complementary region” in reference to a region of an oligonucleotide means that at least 70% of the nucleobases of that region and the nucleobases of another nucleic acid or one or more regions thereof are capable of hydrogen bonding with one another when the nucleobase sequence of the oligonucleotide and the other nucleic acid are aligned in opposing directions. Complementary nucleobases mean nucleobases that are capable of forming hydrogen bonds with one another. Complementary nucleobase pairs include adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), 5-methyl cytosine (mC) and guanine (G). Complementary oligonucleotides and / or nucleic acids need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated. As used herein, “fully complementary” or “100% complementary” in reference to oligonucleotides means that oligonucleotides are complementary to another oligonucleotide or nucleic acid at each nucleoside of the oligonucleotide. As used herein, the “complementary strand” of a target DNA is the strand of a double-stranded DNA that is complementary to the target-recognition region of a guide. As used herein, the “non-complementary strand” of a target DNA is the strand of a double-stranded DNA that is complementary to the “complementary strand”. The PAM sequence is found within the “non-complementary strand”. As used herein, “domain” means a subset of linked amino acids in a polypeptide. A domain may be comprised of multiple smaller domains called subdomains, for example the RuvC domain of Streptococcus pyogenes SpyCas9 is comprised of three discontiguous subdomains: RuvC-I, RuvC-II, and RuvC-III. As used herein, “double-stranded break”, “double-strand break”, or “DSB” refers to a break in both strands of a double-stranded DNA. As used herein, “exogenous mRNA” means any mRNA that is introduced into an organism or cell and that is not synthesized by the recipient organism or cell itself. An exogenous mRNA can be isolated or purified from an organism or cell, can be transcribed in vitro, or can be produced by synthetic means. An exogenous mRNA comprises a coding region (e.g., an open reading frame (ORF)) encoding a polypeptide sequence. As used herein, “gene editing”, “genome editing”, or “genomic editing” means a process of changing the nucleobase sequence of a genome (e.g., insertions, deletions, mutations, or changes in a nucleobase, including epigenetic status (e.g., methylation states for A or G)), either directly or through innate cellular processes, e.g., following the introduction of a double-stranded break or a nick. In certain embodiments, gene editing is mediated by a complex comprising a guided nucleic acid binding agent and a guide. In certain embodiments, gene editing is mediated by a complex comprising a Cas protein, a dead Cas protein, or Cas fusion protein and a guide. As used herein, “guide” means an oligonucleotide (a “single guide”) or a complex consisting of two oligonucleotides that are partially hybridized to one another (a “dual guide”), in both cases comprising a target-recognition region and a protein-recognition region. In embodiments in which a guide is a single guide, the target-recognition region and the protein-recognition region are regions of the one oligonucleotide that constitutes the single guide. In embodiments in which a guide is a dual guide, the target-recognition region is a region of a first oligonucleotide and the protein-recognition region is a portion of the complex that includes regions of each of the two oligonucleotides. In certain embodiments, a guide directs a guided nucleic acid binding agent to a target sequence of a target DNA. As used herein, “guided nucleic acid binding agent” means a polypeptide comprising (1) a region that interacts with the protein-recognition region of a guide; and (2) a region that interacts with the PAM of a target DNA. In certain embodiments, the target-recognition region of the guide causes the guided nucleic acid binding agent to specifically interact with a target DNA. A guided nucleic acid binding agent may comprise one or more active or inactive nuclease domains. A guided nucleic acid binding agent may comprise a heterologous domain. Guided nucleic acid binding agents include Cas enzymes, dead Cas proteins, and Cas fusion proteins. As used herein, “HNH domain” or “HNH” means a cation-dependent endonuclease domain of a protein with an active site comprising a histidine (H) catalytic residue. In certain embodiments, the active site of an HNH domain comprises histidine (H) and asparagine (N) catalytic residues. In certain embodiments, an HNH domain of a Cas protein cleaves the complementary strand of a target DNA. An HNH domain may have one or more substitutions that render it catalytically inactive. As used herein, “hybridization” means the annealing of oligonucleotides and / or nucleic acids. While not limited to a particular mechanism, the most common mechanism of hybridization involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense compound and a nucleic acid target. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an oligonucleotide and a nucleic acid target. As used herein, “identity,” or “percent identity”, with regard to an amino acid sequence, means the percentage of amino acids that are identical between two amino acid sequences when the amino acid sequences are aligned for maximal similarity. Percent identity can be determined by a program such as BLASTP (Altschul, et al., J. Mol. Biol., 1990; Altschul, et al., Nucleic Acids Research, 1997) or Clustal Omega (Sievers, et al., Molecular Sys.Biol., 2011; Goujon et al., Nucleic Acids Research, 2010; McWilliam, et al., Nucleic Acids Research 2013). As used herein, the term “interact with”, with regards to two macromolecules (e.g. a polypeptide and an oligonucleotide; two polypeptides with each other; or a polypeptide and a nucleic acid), means that multiple non-covalent stabilizing interactions form between the two macromolecules, including but not limited to dipolar interactions (hydrogen bonds), cation- ^ or ^- ^ stacking interactions, electrostatic interactions (salt bridges), and hydrophobic interactions. As used herein, the term “internucleoside linkage” is the covalent linkage between adjacent nucleosides in an oligonucleotide. As used herein “modified internucleoside linkage” means any internucleoside linkage other than a phosphodiester internucleoside linkage. As used herein, “mismatch” or “non-complementary” means a nucleobase of a first oligonucleotide that is not complementary with the corresponding nucleobase of a second oligonucleotide or target nucleic acid when the first and second oligonucleotide are aligned. As used herein, “motif” means the pattern of unmodified and / or modified sugar moieties, nucleobases, and / or internucleoside linkages, in an oligonucleotide. A “modified guide” means a guide comprising a modified internucleoside linkage, a modified sugar moiety, and / or a modified nucleobase. As used herein, “natural amino acid” means Gly or theL-isomer of each of the following: Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Lys, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val. As used herein, “nick” refers to a break in one strand of a double-stranded DNA. As used herein, “non-natural amino acid” means any amino acid other than the standard twenty amino acids encoded by the human genetic code, includingD-isomers of each of the following: Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Lys, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val. A non-natural amino acid may have a modified or functionalized side chain (e.g., through the attachment of a linker) Examples of non- natural amino acids include, but are not limited to, allo-isoleucine, 2-amino-3-ethyl-pentanoic acid, aminoisobutyric acid, aminobutyric acid, azetidine, 7-azatryptophan, 6-azidolysine, β-cyclobutylalanine, β- methyl isoleucine, 4, 4-biphenylalanine, cis-hydroxyproline, cyclobutyl glycine, cyclohexyl glycine, cyclopentyl alanine, cyclopentyl glycine, 2,6-dimethyl tyrosine, 3,3-diphenyl alanine, 4-trans-hydroxy-L- proline, 1-napthaylalanine, 2-napthylalanine, N-methyl alanine, 1-methyl histidine, 3-methyl histidine, N- methyl-tryptophan, pipecolic acid,4-pyridylalanine, sarcosine, t-butyl alanine, or 3-t-butyl tyrosine. As used herein, “nucleobase” means an unmodified nucleobase or a modified nucleobase. A nucleobase is a heterocyclic moiety. As used herein an “unmodified nucleobase” is adenine (A), thymine (T), cytosine (C), uracil (U), or guanine (G). As used herein, a “modified nucleobase” is a group of atoms other than unmodified A, T, C, U, or G capable of pairing with at least one other nucleobase. A “5-methyl cytosine” is a modified nucleobase. A universal base is a modified nucleobase that can pair with any one of the five unmodified nucleobases. As used herein, “nucleobase sequence” means the order of contiguous nucleobases in a nucleic acid or oligonucleotide independent of any sugar or internucleoside linkage modification. As used herein, “the nucleobase sequence of” or “the sequence of” a reference nucleobase SEQ ID NO, refers only to the nucleobase sequence provided in such SEQ ID NO and therefore, unless otherwise indicated, includes compounds wherein each sugar moiety and each internucleoside linkage, independently, may be modified or unmodified, irrespective of the presence or absence of modifications, indicated in the referenced SEQ ID NO. As used herein, “nucleoside” means a compound or fragment of a compound comprising a nucleobase and a sugar moiety. The nucleobase and sugar moiety are each, independently, unmodified or modified. As used herein, “oligonucleotide” means a strand of linked nucleosides connected via internucleoside linkages, wherein each nucleoside and internucleoside linkage independently may be modified or unmodified. Unless otherwise indicated, oligonucleotides consist of 8-150 linked nucleosides. As used herein, “modified oligonucleotide” means an oligonucleotide, wherein at least one nucleoside or internucleoside linkage is modified. As used herein, “unmodified oligonucleotide” means an oligonucleotide that does not comprise any nucleoside modifications or internucleoside modifications. As used herein, “PAM interacting domain” or “PI domain” means a domain of a guided nucleic acid binding agent that can bind to or associate with one or more PAM sequences in the non-complementary strand of a target DNA. The PAM-interacting domain may confer specificity to one or more PAM sequences. As used herein, “PAM sequence”, “PAM”, or “protospacer adjacent motif” refers to 2-9 linked nucleosides within the non-complementary strand of a target DNA that interacts with a guided nucelic acid binding agent. The PAM sequence may be adjacent to the 5’-end of a protospacer sequence. The PAM sequence may be adjacent to the 3’-end of a protospacer sequence. As used herein, “the peptide sequence of” or “the polypeptide sequence of” or “the sequence of” a reference peptide / polypeptide SEQ ID NO refers to the linear, amide-bond-linked amino acid sequence provided in such SEQ ID NO, even in cases where the given peptide or polypeptide contains one or more modified side chains that that link to another moiety. As used herein, “pharmaceutically acceptable carrier or diluent” means any substance suitable for use in administering to a subject. Certain such carriers enable pharmaceutical compositions to be formulated as, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspension, and lozenges for the oral ingestion by a subject. In certain embodiments, a pharmaceutically acceptable carrier or diluent is sterile water, distilled water for injection, sterile saline, sterile buffer solution or sterile artificial cerebrospinal fluid. As used herein “pharmaceutically acceptable salt(s)” means physiologically and pharmaceutically acceptable salt(s) of compounds. Pharmaceutically acceptable salts retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. As used herein “pharmaceutical composition” means a mixture of substances suitable for administering to a subject. For example, a pharmaceutical composition may comprise an active agent and a sterile aqueous solution. In certain embodiments, a pharmaceutical composition shows activity in free uptake assay in certain cell lines. As used herein, “protein recognition region” means a portion of a guide that interacts with a guided nucleic acid binding agent. As used herein, the “protospacer sequence” of the target DNA refers to the reverse complement of the “target sequence” and is found in the non-complementary strand of the target DNA. In certain embodiments, the sequence of the target-recognition region of a guide has at least 90%, 95%, 98%, or 99% identity to the protospacer sequence of the target DNA. As used herein, “RuvC domain” or “RuvC” means a cation-dependent endonuclease domain of a protein with an active site comprising an aspartic acid (D) catalytic residue. In certain embodiments, the active site of a RuvC domain comprises aspartic acid (D) and glutamic acid (E) catalytic residues. In certain embodiments, a RuvC domain cleaves the non-complementary strand of a target DNA . In certain embodiments, a RuvC domain cleaves both strands of a target DNA. A RuvC domain may be formed from non-contiguous amino acids; for example, the RuvC domain of SpCas9 is split into RuvCI, RuvCII, and RuvCIII portions. A RuvC domain may have one or more substitutions that render it catalytically inactive. As used herein, a “standard length internucleoside linkage” refers to an internucleoside linkage that has a structure represented byFormula Z1, Z2, or Z3: wherein independently for each internucleoside linking group of Formula Z1, Z2, or Z3: each X1is independently selected from O and S; X2is selected from O, NR1, CH2, and S; X3is selected from O, NR1, CH2, and S; L is absent, NR1, N(R1)SO2, –N=, O, C1-C6alkylene, or C1-C6heteroalkylene; each R1is independently selected from H, C1-C6alkyl, and substituted C1-C6alkyl, or two R1on the same atom together form =O; and R2is selected from -OH, -SH, C1-C22alkyl, substituted C1-C22alkyl, C2-C22alkenyl, substituted C2-C22alkenyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, heteroaryl, substituted heteroaryl, aryl, and substituted aryl; wherein when a group is substituted, it comprises one or more substituent groups selected from halo, -OH, -N(R1)2, -O-C1-C6alkyl, C1-C22alkyl, C2-C22alkenyl, cycloalkyl, heterocyclyl, heteroaryl, and aryl. As used herein, “subject” refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees. As used herein, “sugar moiety” means an unmodified sugar moiety or a modified sugar moiety. As used herein, “unmodified sugar moiety” means a 2’-OH(H) ribosyl moiety, as found in RNA (an “unmodified RNA sugar moiety”), or a 2’-H(H) deoxyribosyl sugar moiety, as found in DNA (an “unmodified DNA sugar moiety”). Unmodified sugar moieties have one hydrogen at each of the 1’, 3’, and 4’ positions, an oxygen at the 3’ position, and two hydrogens at the 5’ position. As used herein, “modified sugar moiety” or “modified sugar” means a modified furanosyl sugar moiety or a sugar surrogate. As used herein, "sugar surrogate" means a modified sugar moiety having other than a furanosyl moiety that can link a nucleobase to an internucleoside linkage. Modified nucleosides comprising sugar surrogates can be incorporated into one or more positions within an oligonucleotide and such oligonucleotides are capable of hybridizing to complementary oligonucleotides or target nucleic acids. As used herein, “target-recognition region” refers to a 12-30 linked nucleoside region of a guide that is complementary to the “target sequence” within the complementary strand of a target DNA. A target- recognition region may be fully complementary to the target sequence within the complementary strand of the target DNA. As used herein, “target sequence” refers to the 12-30 linked nucleoside portion of a nucleic target that is complementary to the “target-recognition region” of a guide. The target sequence is within the “complementary strand” of a target DNA. As used herein, “therapeutically effective amount” means an amount of an agent or pharmaceutical composition that provides a therapeutic benefit to a subject. For example, a therapeutically effective amount improves a symptom of a disease. As used herein, “treating” means improving a subject’s disease or condition by administering an agent or compound described herein. In certain embodiments, treating a subject improves a symptom relative to the same symptom in the absence of the treatment. In certain embodiments, treatment reduces in the severity or frequency of a symptom, or delays the onset of a symptom, slows the progression of a symptom, or slows the severity or frequency of a symptom. CERTAIN EMBODIMENTS The present disclosure provides the following non-limiting numbered embodiments: Embodiment 1. A polypeptide, comprising a region of at least 900, at least 1000, at least 1100, at least 1200, or at least 1300 linked amino acids, wherein the amino sequence of the region has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal length portion of any of SEQ ID NOs: 12, 13, or 20; wherein the polypeptide comprises a RuvC domain and an HNH domain; optionally wherein the polypeptide is isolated. Embodiment 2. A polypeptide, comprising a region, wherein the amino acid sequence of the region has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NOs: 12, 13, or 20; optionally wherein the polypeptide is isolated. Embodiment 3. A polypeptide, wherein the amino acid sequence of the polypeptide has at least 85%, at least 90%, at least 95%, or 100% identity to any of SEQ ID NOs: 12, 13, or 20; optionally wherein the polypeptide is isolated. Embodiment 4. The polypeptide of embodiment 1 or 2, consisting of the region. Embodiment 5. The polypeptide of embodiment 1 or 2, additionally comprising an heterologous domain. Embodiment 6. The polypeptide of embodiment 5, wherein the heterologous domain is selected from a transcriptional activator, a transcriptional repressor, a methyltransferase, a demethylase, a deaminase, an acetyltransferase, or a deacetylase. Embodiment 7. The polypeptide of any of embodiments 1-6, wherein the RuvC domain is comprised of subdomains having an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 85, 92, and 94; or SEQ ID NOs: 97, 104, and 106; or SEQ ID NOs: 109, 116, and 118. Embodiment 8. The polypeptide of any of embodiments 1-7, wherein the HNH domain has an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NO: 93, 105, or 117. Embodiment 9. The polypeptide of any of embodiments 1-8, wherein the polypeptide comprises a REC lobe. Embodiment 10. The polypeptide of embodiment 9, wherein the REC lobe has an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 91, 103, or 115. Embodiment 11. The polypeptide of any of embodiments 1-10, wherein the polypeptide comprises a NUC lobe. Embodiment 12. The polypeptide of embodiment 11, wherein the NUC lobe has an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 96, 108, or 120. Embodiment 13. The polypeptide of any of embodiments 1-12, wherein the polypeptide comprises a REC lobe and a NUC lobe. Embodiment 14. The polypeptide of embodiment 13, wherein the REC Lobe has an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 91 and the NUC Lobe has an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 96. Embodiment 15. The polypeptide of embodiment 14, wherein the REC Lobe has an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 103 and the NUC Lobe has an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 108. Embodiment 16. The polypeptide of embodiment 14, wherein the REC Lobe has an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 115 and the NUC Lobe has an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 120. Embodiment 17. The polypeptide of any of embodiments 1-16, wherein the polypeptide has exactly two catalytically active nuclease active sites. Embodiment 18. The polypeptide of any of embodiments 1-16, wherein the polypeptide has exactly one catalytically active nuclease active site. Embodiment 19. The polypeptide of any of embodiments 1-17, wherein the polypeptide has zero catalytically active nuclease active sites. Embodiment 20. A polypeptide, comprising a region having at least 800, at least 900, at least 1000, or at least 1050 linked amino acids, wherein the amino acid sequence has at least 85%, at least 90%, at least 95%, or 100% identity to an equal length portion of any of SEQ ID NOs: 241, 242, 245247, 314, or 323; optionally wherein the polypeptide is isolated. Embodiment 21. A polypeptide, comprising a region having at least 900, at least 1000, at least 1100, or at least 1200, linked amino acids, wherein the amino acid sequence has at least 85%, at least 90%, at least 95%, or 100% identity to an equal length portion of SEQ ID NO: 260; optionally wherein the polypeptide is isolated. Embodiment 22. A polypeptide, comprising a region having at least 900, at least 1000, at least 1100, at least 1200, or at least 1300 linked amino acids, wherein the amino acid sequence has at least 85%, at least 90%, at least 95%, or 100% identity to an equal length portion of any of SEQ ID NOs: 266- 268, 271, 274-275, 279, 282-284, 288-289, 292, 297, or 299-302; optionally wherein the polypeptide is isolated. Embodiment 23. A polypeptide, comprising a region, wherein the region has an amino acid sequence that has at least 85%, at least 90%, at least 95%, or 100% identity to an equal length portion of any of SEQ ID NOs: 241, 242, 245, 247, 260, 266-268, 271, 274-275, 279, 282-284, 288-289, 292, 297, 299-302, 314, or 323; optionally wherein the polypeptide is isolated. Embodiment 24. A polypeptide, wherein amino acid sequence of the polypeptide has at least 85%, at least 90%, at least 95%, or 100% identity to any of SEQ ID NOs: 241, 242, 245, 247, 260, 266-268, 271, 274-275, 279, 282-284, 288-289, 292, 297, 299-302, 314, or 323; optionally wherein the polypeptide is isolated. Embodiment 25. A guide, wherein the guide comprises a protein-recognition element, wherein the protein-recognition element binds to the polypeptide of any of embodiments 1-24. Embodiment 26. The guide of embodiment 25, wherein the guide is a dual guide consisting of two oligonucleotides joined by a duplex region. Embodiment 27. The guide of embodiment 25, wherein the guide is a single guide comprising an oligonucleotide, and the protein-recognition element is a protein-recognition region of the oligonucleotide. Embodiment 28. The guide of embodiment 27, wherein the nucleobase sequence of the protein- recognition region of the guide has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 129, 130, 131, or 139. Embodiment 29. The guide of embodiment 27, wherein the nucleobase sequence of the protein- recognition region of the guide has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 520-521, 524, 526, 539, 545-547, 550, 553-554, 558, 561- 563, 567, 568, 571, 576, 578-581, 593, or 602. Embodiment 30. The guide any of embodiments 26-29, wherein the guide comprises a modified oligonucleotide. Embodiment 31. The guide of any of embodiments 26-30, wherein the guide comprises at least one modified sugar moiety. Embodiment 32. The guide of embodiment 31, wherein the modified sugar moiety is selected from a 2’-OMe and a 2’-F. Embodiment 33. The guide of embodiment 32, wherein the guide comprises a 2’-OMe or 2’-F sugar moiety within the first five nucleosides of the 5’ end or the last five nucleosides 3’ end of the guide. Embodiment 34. The guide of any of embodiments 26-33, wherein the guide comprises a modified internucleoside linkage. Embodiment 35. The guide of embodiment 34, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage. Embodiment 36. The guide of embodiment 34 or 35, wherein the guide comprises at least one modified internucleoside linkage within the first five nucleosides of the 5’ end or the last five nucleosides at the 3’ end of the guide. Embodiment 37. The guide of any of embodiments 27-36, wherein the guide consists of an oligonucleotide. Embodiment 38. The guide of any of embodiments 25-37, wherein the guide comprises a DNA- recognition element that is at least 90%, at least 95%, or 100% complementary to a target sequence. Embodiment 39. An editing system comprising the polypeptide of any of embodiments 1-24 and a guide of any of embodiments 25-38. Embodiment 40. An editing system comprising: a. a nucleic acid encoding the polypeptide of any of embodiments 1-24; and b. a guide of any of embodiments 25-38. Embodiment 41. An editing system comprising: a. a nucleic acid encoding the polypeptide of any of embodiments 1-24; and b. a nucleic acid encoding the guide of any of embodiments 25-29. Embodiment 42. The editing system of embodiment 40 or 41 wherein the nucleic acid encoding the polypeptide is an exogenous mRNA. Embodiment 43. The editing system of embodiment 40 or 41, wherein the nucleic acid encoding the polypeptide is a DNA. Embodiment 44. The editing system of any of embodiments 41-43, wherein the nucleic acid encoding the guide is an exogenous mRNA. Embodiment 45. The editing system any of embodiments 41-43, wherein the nucleic acid encoding the guide is a DNA. Embodiment 46. A composition comprising the editing system of any of embodiments 39-45 and an LNP. Embodiment 47. A viral vector comprising the editing system of embodiment 41. Embodiment 48. A method of editing a target nucleic acid, comprising administering a composition of embodiment 46 or a viral vector of embodiment 47 to a subject. Embodiment 49. A method of creating a double stranded break or a nick in a target nucleic acid, comprising administering a composition of embodiment 46 or a viral vector of embodiment 47 to a subject. Embodiment 50. A method of editing a target nucleic acid, comprising contacting a composition of embodiment 46 or a viral vector of embodiment 47 with a cell. Embodiment 51. A method of creating a double stranded break or a nick in a target nucleic acid, comprising contacting a composition of embodiment 46 or a viral vector of embodiment 47 with a cell. Embodiment 52. The composition of embodiment 45 or a viral vector of embodiment 46 for use in therapy. Embodiment 53. A polypeptide, comprising a region having at least 800, at least 900, at least 1000, or at least 1050 linked amino acids, wherein the amino acid sequence of the region has at least 85%, at least 90%, at least 95%, or 100% identity to an equal length portion of any of SEQ ID NOs: 4-23 or 231-323, optionally wherein the polypeptide is isolated. Embodiment 54. A polypeptide, comprising a region having at least 900, at least 1000, or at least 1100 linked amino acids, wherein the amino acid sequence of the region has at least 85%, at least 90%, at least 95%, or 100% identity to an equal length portion of SEQ ID NO: 6-23, 250-308; optionally wherein the polypeptide is isolated. Embodiment 55. A polypeptide, comprising a region having at least 900, at least 1000, at least 1100, at least 1200, or at least 1300 linked amino acids, wherein the amino acid sequence of the region has at least 85%, at least 90%, at least 95%, or 100% identity to an equal length portion of any of SEQ ID NOs: 10-23 or 266-308; optionally wherein the polypeptide is isolated. Embodiment 56. A polypeptide, comprising a region, wherein the amino acid sequence of the region has at least 85%, at least 90%, at least 95%, or 100% identity to any of SEQ ID NOs: 4-23 or 231- 323; optionally wherein the polypeptide is isolated. Embodiment 57. A polypeptide, wherein the amino acid sequence of the polypeptide has at least 85%, at least 90%, at least 95%, or 100% identity to any of SEQ ID NOs: 4-23 or 231-323; optionally wherein the polypeptide is isolated. Embodiment 58. The polypeptide of embodiment 57, wherein the amino acid sequence of the polypeptide has at least 85%, at least 90%, at least 95%, or 100% identity to any of SEQ ID NOs: 12, 13, 20, 241, 242, 245, 247, 260, 266, 267, 268, 271, 274, 275, 279, 282, 283, 284, 288, 289, 292, 297, 299, 300, 301, 302, 314, or 323. Embodiment 59. The polypeptide of any of embodiments 53-56, consisting of the region. Embodiment 60. The polypeptide of any of embodiments 53-56, comprising a heterologous domain. Embodiment 61. The polypeptide of embodiment 60, wherein the heterologous domain is selected from a transcriptional activator, a transcriptional repressor, a methyltransferase, a demethylase, a deaminase, an acetyltransferase, or a deacetylase. Embodiment 62. The polypeptide of any of embodiments 53-56 or 59-61, wherein the amino acid sequence of the region has at least 85%, at least 90%, at least 95%, or 100% identity to an equal length portion of any of SEQ ID NOs: 13, 241, 267, 275, 284, 289, or 314. Embodiment 63. The polypeptide of any of embodiments 53-62, wherein the polypeptide comprises a non-PI region and a PI domain, wherein the non-PI region of the polypeptide has an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 3186, 3188, 3190, 3192, 3194, 3196, or 3198. Embodiment 64. The polypeptide of any of embodiments 53-62, wherein the polypeptide comprises a non-PI region and a PI domain, wherein the non-PI region of the polypeptide has an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 3186 and the PI domain has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3187. Embodiment 65. The polypeptide of any of embodiments 53-62, wherein the polypeptide comprises a non-PI region and a PI domain, wherein the non-PI region of the polypeptide has an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 3188 and the PI domain has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3189. Embodiment 66. The polypeptide of any of embodiments 53-62, wherein the polypeptide comprises a non-PI region and a PI domain, wherein the non-PI region of the polypeptide has an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 3190 and the PI domain has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3191. Embodiment 67. The polypeptide of any of embodiments 53-62, wherein the polypeptide comprises a non-PI region and a PI domain, wherein the non-PI region of the polypeptide has an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 3192 and the PI domain has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3193. Embodiment 68. The polypeptide of any of embodiments 53-62, wherein the polypeptide comprises a non-PI region and a PI domain, wherein the non-PI region of the polypeptide has an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 3194 and the PI domain has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3195. Embodiment 69. The polypeptide of any of embodiments 53-62, wherein the polypeptide comprises a non-PI region and a PI domain, wherein the non-PI region of the polypeptide has an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 3196 and the PI domain has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3197. Embodiment 70. The polypeptide of any of embodiments 53-62, wherein the polypeptide comprises a non-PI region and a PI domain, wherein the non-PI region of the polypeptide has an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 3198 and the PI domain has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3199. Embodiment 71. The polypeptide of any of embodiments 53-70, wherein the polypeptide has exactly two catalytically active nuclease active sites. Embodiment 72. The polypeptide of any of embodiments 53-70, wherein the polypeptide has exactly one catalytically active nuclease active site. Embodiment 73. The polypeptide of any of embodiments 53-70, wherein the polypeptide has zero catalytically active nuclease active sites. Embodiment 74. The polypeptide of any of embodiments 53-73, comprising a nuclear localization signal. Embodiment 75. The polypeptide of embodiment 74, wherein the nuclear localization signal is at the N-terminus of the polypeptide. Embodiment 76. The polypeptide of embodiment 74, wherein the nuclear localization signal is at the C-terminus of the polypeptide. Embodiment 77. The polypeptide of embodiment 74, wherein the polypeptide comprises two nuclear localization signals, one at each of the N-terminus and the C-terminus of the polypeptide. Embodiment 78. A nucleic acid encoding the polypeptide of any of embodiments 53-77. Embodiment 79. The nucleic acid of embodiment 78, wherein the nucleic acid is a DNA. Embodiment 80. The nucleic acid of embodiment 78, wherein the nucleic acid is an mRNA comprising a coding region that encodes for the polypeptide of any of embodiments 53-77. Embodiment 81. The nucleic acid of embodiment 78, wherein the mRNA is an exogenous mRNA comprising a coding region that encodes for the polypeptide of any of embodiments 53-73. Embodiment 82. The mRNA of embodiment 80 or 81, comprising a 5’ cap structure. Embodiment 83. The mRNA of any of embodiments 80-82, comprising a 3’-poly(A) tail. Embodiment 84. The mRNA of any of embodiments 80-83, comprising a 5’-UTR. Embodiment 85. The mRNA of any of embodiments 80-84, comprising a 3’-UTR. Embodiment 86. The mRNA of embodiment 80 or 81, comprising a 5’-cap structure and 3’-poly(A) tail. Embodiment 87. The mRNA of any of embodiments 80-86, wherein the coding region is codon optimized for expression in a eukaryotic cell. Embodiment 88. The mRNA of embodiment 87, wherein the coding region has been codon optimized for expression in a mammalian cell. Embodiment 89. The mRNA of embodiment 88, wherein the mammalian cell is a human cell. Embodiment 90. The mRNA of any of embodiments 80-89, wherein each nucleobase of the mRNA is selected from adenine, guanine, cytosine, uracil, thymine, and N1-methylpseudouracil. Embodiment 91. The mRNA of any of embodiments 80-89, wherein each nucleobase of the mRNA is selected from adenine, guanine, cytosine, and N1-methylpseudouracil. Embodiment 92. The mRNA of any of embodiments 80-89, wherein each nucleobase of the mRNA is selected from adenine, guanine, cytosine, and uracil. Embodiment 93. A guide, wherein the guide comprises a protein-recognition region, wherein the protein-recognition region interacts with the polypeptide of any of embodiments 53-57. Embodiment 94. The guide of embodiment 93, wherein the guide is a dual guide. Embodiment 95. The guide of embodiment 93, wherein the guide is a single guide. Embodiment 96. A guide, consisting of an oligonucleotide according to the following formula: T1-D-X-S1a-B1-S1b-H1-S1b’-B2-S1a’-L1-S2-H2-S2’-L2-S3-H3-S3’-(L3-S4-H4-S4’)n-(L4-S5-H5-S5’)m-T2; wherein: ^ D consists of 17-25 linked nucleosides, wherein the nucleobase sequence of D is complementary to the nucleobase sequence of a target DNA; ^ X is absent, or consists of 1-2 linked nucleosides that are not complementary to the sequence of the target DNA; ^ each of T1and T2is absent or independently consists of 1-30 linked nucleosides; ^ S1aand S1a’ each consist of 6-10 linked nucleosides, wherein the nucleobase sequence of S1ais 100% complementary to the nucleobase sequence of S1a’; ^ S1band S1b’ each consist of 2-14 linked nucleosides, wherein the nucleobase sequence of S1bis 100% complementary to the nucleobase sequence of S1b’; ^ S2and S2’ each consist of 2-6 linked nucleosides, wherein the nucleobase sequence of S2is 100% complementary to the nucleobase sequence of S2’; ^ S3and S3’ each consist of 2-12 linked nucleosides, wherein the nucleobase sequence of S3is 100% complementary to the nucleobase sequence of S3’; ^ S4and S4’ each consist of 4-14 linked nucleosides, wherein the nucleobase sequence of S4is 100% complementary to the nucleobase sequence of S4’; ^ S5and S5’ each consist of 4-11 linked nucleosides, wherein the nucleobase sequence of S5is 100% complementary to the nucleobase sequence of S5’; ^ n is 0 or 1; ^ m is 0 or 1; ^ B1is absent, or consists of 1-3 linked nucleosides; ^ B2is absent or consists of 1-4 linked nucleosides; ^ wherein B1is not equal to B2unless both are absent; ^ each of H1, H2, H3, H4, and H5independently consists of 3-6 linked nucleosides; ^ L1consists of 2-3 linked nucleosides; ^ each of L2, L3, and L4independently consists of 0-10 linked nucleosides. Embodiment 97. The guide of embodiment 96, wherein the guide comprises a protein-recognition region that binds to the polypeptide of any of embodiments 53-77. Embodiment 98. The guide of any of embodiments 96-97, wherein T1is absent. Embodiment 99. The guide of any of embodiments 96-98, wherein T2is absent. Embodiment 100. The guide of any of embodiments 96-99, wherein T2consists of 2-6 linked nucleosides comprising a uracil nucleobase. Embodiment 101. The guide of any of embodiments 96-100, wherein X is absent. Embodiment 102. The guide of any of embodiments 96-101, wherein n is 1 and m is 0. Embodiment 103. The guide of any of embodiments 96-102, wherein n and m are both 1. Embodiment 104. The guide of any of embodiments 96-103, wherein the number of nucleosides in B2is greater than the number of nucleosides in B1. Embodiment 105. The guide of any of embodiments 96-104, wherein each L1nucleoside is an adenosine. Embodiment 106. The guide of any of embodiments 96-105, wherein the guide binds to polypeptide of any of embodiments 53-77. Embodiment 107. The guide of embodiment 96 or 97, wherein T1and X are absent; T2is absent or consists of 2-6 linked uridines; S1aand S1a’ each consist of 7 linked nucleosides; S1band S1b’ each consist of 3-9 linked nucleosides; S2and S2’ each consist of 3 linked nucleosides; S3and S3’ each consist of 4 linked nucleosides; S4and S4’ each consist of 5 linked nucleosides; S5and S5’, when present, each consist of 7-9 linked nucleosides; n is 1; m is 0 or 1; B1consists of 1 nucleoside; B2consists of 4 linked nucleosides; Each of H1,H2,H3and H4,when present, consist of 4 linked nucleosides; L1consists of 2 linked adenosine nucleosides; L2consists of 5 linked nucleosides; L3consists of 0 nucleosides; and L4, when present, consists of 4 linked nucleosides. Embodiment 108. The guide of embodiment 107, wherein S1band S1b’ each consist of 7 linked nucleosides. Embodiment 109. The guide of embodiment 107 or 108, wherein S5and S5’ each consists of 7 linked nucleosides. Embodiment 110. The guide of any of embodiments 107-109, wherein the nucleobase sequence of B2is AAAG. Embodiment 111. The guide of any of embodiments 107-108, wherein the nucleobase sequence of L2is AAACU. Embodiment 112. The guide of any of embodiments 107-111, wherein the nucleobase sequence of L3is UUUUAA. Embodiment 113. The guide of any of embodiments 107-112, wherein the nucleobase sequence of H2is GUCA. Embodiment 114. The guide of any of embodiments 107-113, wherein n is 1 and m is 0. Embodiment 115. The guide of any of embodiments 107-114, wherein n and m are both 1. Embodiment 116. The guide of embodiment 107, wherein S1band S1b’ each consist of 7 linked nucleosides; n is 1 and m is 0; the nucleobase sequence of B2is AAAG; the nucleobase sequence of L2is AAACU; the nucleobase sequence of L3is UUUUAA, and the nucleobase sequence of H2is GUCA. Embodiment 117. The guide of any of embodiments 107-116, wherein the guide comprises a region of at least 40, at least 50, at least 60, or at least 70 nucleosides, wherein the nucleobase sequence of the region has at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal length region of any of SEQ ID NOs: 131, 961-964, 1016-1020, 1052-1070, 1313-1314, or 2825. Embodiment 118. The guide of embodiment 117, wherein the nucleobase sequence of the region has 100% identity to an equal length region of any of SEQ ID NOs: 131, 961-964, 1016-1020, 1052- 1070, 1313-1314, or 2825. Embodiment 119. The guide of any of embodiments 107-118, wherein D consists of 20-23 nucleosides having a nucleobase sequence that is 100% complementary to the nucleobase sequence of a target DNA. Embodiment 120. The guide of embodiment 96 or 97, wherein T1and X are absent; T2is absent or consists of 6 linked uridines; S1aand S1a’ each consist of 9 linked nucleosides; S1band S1b’ each consist of 2-8 inked nucleosides; S2and S2’ each consist of 6 linked nucleosides; S3and S3’ each consist of 7 linked nucleosides; n and m are 0; B1consists of 3 nucleosides; B2consists of 4 linked nucleosides; H1consists of 4 linked nucleosides; H2consists of 5 linked nucleosides; H3consists of 4 linked nucleosides; L1consists of 2 linked adenosine nucleosides; and L2consists of 0 linked nucleosides. Embodiment 121. The guide of embodiment 120, wherein S1band S1b’ each consist of 4 linked nucleosides. Embodiment 122. The guide of any of embodiments 120-121, wherein L2consists of 0 linked nucleosides and T2consists of 6 linked uridines. Embodiment 123. The guide of any of embodiments 120-122, wherein the nucleobase sequence of B2is UAAC. Embodiment 124. The guide of any of embodiments 120-123, wherein the nucleobase sequence of L2is GAACUC. Embodiment 125. The guide of any of embodiments 120-124, wherein the nucleobase sequence of H2is UUUAU. Embodiment 126. The guide of embodiment 120, wherein S1band S1b’ each consist of 4 linked nucleosides; n and m are 0; the nucleobase sequence of B2is UACC; the nucleobase sequence of L2is GAACUC; and the nucleobase sequence of H2is UUUAU. Embodiment 127. The guide of any of embodiments 120-126, wherein the guide comprises a region having a nucleobase sequence that has at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NOs: 520, 965-968, 1071-1094, or 1315-1318, wherein the region comprises at least 40, at least 50, at least 60, or at least 70 nucleosides. Embodiment 128. The guide of embodiment 127, wherein the nucleobase sequence of the region has 100% identity to an equal length region of any of SEQ ID NOs: 520, 965-968, 1071-1094, or 1315- 1318. Embodiment 129. The guide of any of embodiments 120-127, wherein D consists of 20-23 nucleosides having a nucleobase sequence that is 100% complementary to the nucleobase sequence of a target DNA. Embodiment 130. The guide of embodiment 96 or 97, wherein T1and X are absent; T2is absent or consists of 1-7 linked nucleosides; S1aand S1a’ each consist of 7 linked nucleosides; S1band S1b’ each consist of 4-12 linked nucleosides; S2and S2’ each consist of 5 linked nucleosides; S3and S3’ each consist of 6 linked nucleosides; S4and S4’ each consist of 6 linked nucleosides; S5and S5’, when present, each consist of 7 linked nucleosides; n is 1; m is 0 or 1; B1consists of 1 nucleoside; B2consists of 3 linked nucleosides; Each of H1,H2,and H3consist of 4 linked nucleosides; H4and H5, when present, consist of 3 linked nucleosides; L1consists of 3 linked adenosine nucleosides; L2and L3consist of 0 linked nucleosides; L4, when present, consists of 1-7 linked nucleosides. Embodiment 131. The guide of embodiment 130, wherein S1band S1b’ each consist of 6 linked nucleosides. Embodiment 132. The guide of any of embodiments 130-131, wherein the nucleobase sequence of B2is GAG. Embodiment 133. The guide of any of embodiments 130-132, wherein the nucleobase sequence of H2is AUCC. Embodiment 134. The guide of embodiment 130, wherein S1band S1b’ each consist of 6 linked nucleosides; n is 1 and m is 0; the nucleobase sequence of B2is GAG; and the nucleobase sequence of H2is AUCC. Embodiment 135. The guide of any of embodiments 130-134, wherein the guide comprises a region having a nucleobase sequence that has at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NOs: 546, 973-976, 1021-1023, 1095-1126, or 1319- 1342, wherein the region comprises at least 40, at least 50, at least 60, or at least 70 nucleosides. Embodiment 136. The guide of embodiment 135, wherein the nucleobase sequence of the region has 100% identity to an equal length region of any of SEQ ID NOs: 546, 973-976, 1021-1023, 1095- 1126, or 1319-1342. Embodiment 137. The guide of any of embodiments 130-136, wherein D consists of 20-24 nucleosides having a nucleobase sequence that is 100% complementary to the nucleobase sequence of a target DNA. Embodiment 138. The guide of embodiment 96 or 97, wherein T1and X are absent; T2is absent or consists of 1-6 linked nucleosides; S1aand S1a’ each consist of 7 linked nucleosides; S1band S1b’ each consist of 4-12 inked nucleosides; S2and S2’ each consist of 4 linked nucleosides; S3and S3’ each consist of 4 linked nucleosides; S4and S4’ each consist of 5 linked nucleosides; S5and S5’, when present, each consist of 7 linked nucleosides; n is 1; m is 0 or 1; B1consists of 1 nucleoside; B2consists of 3 nucleosides; H1consists of 4 linked nucleosides; H2consists of 3 linked nucleosides; H3consists of 6 linked nucleosides; H4consists of 4 linked nucleosides; H5, when present, consist of 3 linked nucleosides; L1consists of 2 linked adenosine nucleosides; L2consists of 2 linked nucleosides; L3consists of 0 linked nucleosides; L4, when present, consists of 7 linked nucleosides. Embodiment 139. The guide of embodiment 138, wherein S1band S1b’ each consist of 10 linked nucleosides. Embodiment 140. The guide of any of embodiments 138-139, wherein n is 1 and m is 0. Embodiment 141. The guide of any of embodiments 138-140, wherein the nucleobase sequence of B2is GAG. Embodiment 142. The guide of any of embodiments 138-141, wherein the nucleobase sequence of L2is AA. Embodiment 143. The guide of any of embodiments 138-142, wherein the nucleobase sequence of H2is UAA. Embodiment 144. The guide of embodiment 138, wherein S1band S1b’ each consist of 10 linked nucleosides; n is 1 and m are 0; the nucleobase sequence of B2is GAG; the nucleobase sequence of L2is AA; and the nucleobase sequence of H2is UAA. Embodiment 145. The guide of any of embodiments 138-144, wherein the guide comprises a region having a nucleobase sequence that has at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NOs: 562, 985-989, 1027-1029, 1153-1182, or 1362- 1390, wherein the region comprises at least 40, at least 50, at least 60, or at least 70 nucleosides. Embodiment 146. The guide of embodiment 145, wherein the nucleobase sequence of the region has 100% identity to an equal length region of any of SEQ ID NOs: 562, 985-989, 1027-1029, 1153- 1182, or 1362-1390. Embodiment 147. The guide of any of embodiments 138-146, wherein D consists of 20-24 nucleosides having a nucleobase sequence that is 100% complementary to the nucelobase sequence of a target DNA. Embodiment 148. The guide of embodiment 96 or 97, wherein T1and X are absent; T2is absent or consists of 2-5 linked nucleosides; S1aand S1a’ each consist of 9 linked nucleosides; S1band S1b’ each consist of 2-10 linked nucleosides; S2and S2’ each consist of 4 linked nucleosides; S3and S3’ each consist of 9 linked nucleosides; n and m are 0; B1consists of 1 nucleoside; B1consists of 3 nucleosides; H1consists of 4 linked nucleosides; H2consists of 3 linked nucleosides; H3consists of 4 linked nucleosides; L1consists of 2 linked adenosine nucleosides; L2consists of 7 linked nucleosides. Embodiment 149. The guide of embodiment 148, wherein S1band S1b’ each consist of 6 linked nucleosides. Embodiment 150. The guide of any of embodiments 148-149, wherein the nucleobase sequence of B2is CUA. Embodiment 151. The guide of any of embodiments 148-150, wherein the nucleobase sequence of L2is GUGUUUA. Embodiment 152. The guide of any of embodiments 148-151, wherein the nucleobase sequence of H2is AAA. Embodiment 153. The guide of embodiment 148, wherein S1band S1b’ each consist of 10 linked nucleosides; n and m are 0; the nucleobase sequence of B2is CUA; the nucleobase sequence of L2is GUGUUUA; and the nucleobase sequence of H2is AAA. Embodiment 154. The guide of any of embodiments 148-153, wherein the guide comprises a region having a nucleobase sequence that has at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NOs: 593, 1006-1010, 1272-1292, 1394-1408, 2827- 2833, wherein the region comprises at least 40, at least 50, at least 60, or at least 70 nucleosides. Embodiment 155. The guide of embodiment 160, wherein the nucleobase sequence of the region has 100% identity to an equal length region of any of SEQ ID NOs: 593, 1006-1010, 1272-1292, 1394- 1408, 2827-2833. Embodiment 156. The guide of any of embodiments 148-155, wherein D consists of 20-24 nucleosides having a nucleobase sequence that is 100% complementary to the nucleobase sequence of a target DNA. Embodiment 157. The guide of embodiment 96 or 97, wherein T1and X are absent; T2is absent or consists of 3 linked nucleosides; S1aand S1btogether, and S1a’ and S1b’ together, each consist of 12-20 linked nucleosides; S2and S2’ each consist of 3 linked nucleosides; S3and S3’ each consist of 3 linked nucleosides; S3and S3’ each consist of 10-13 linked nucleosides; n is 1 and m is 0; B1consists of 0 nucleosides; B2consists of 0 nucleosides; H1,H2,H3, and H4consist of 4 linked nucleosides; L1consists of 2 linked adenosine nucleosides; L2consists of 2 linked nucleosides; and L3consists of 6 linked nucleosides. Embodiment 158. The guide of embodiment 157, wherein S1aand S1btogether, and S1a’ and S1b’ together, each consist of 15 linked nucleosides. Embodiment 159. The guide of any of embodiments 157-158, wherein L3consists of 3 linked nucleosides. Embodiment 160. The guide of any of embodiments 157-159, wherein S3and S3’ each consist of 13 linked nucleosides. Embodiment 161. The guide of any of embodiments 157-159, wherein the nucleobase sequence of L2is GU. Embodiment 162. The guide of any of embodiments 160-161, wherein the nucleobase sequence of H2is GAAA. Embodiment 163. The guide of embodiment 157, wherein S1aand S1btogether, and S1a’ and S1b’ together, each consist of 15 linked nucleosides; n is 1 and m is 0; the nucleobase sequence of L2is GU; and the nucleobase sequence of H2is GAAA. Embodiment 164. The guide of any of embodiments 157-163, wherein the guide comprises a region having a nucleobase sequence that has at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NOs: 568, 990-993, 1030-1034, 1183-1206, or 1391- 1393, wherein the region comprises at least 40, at least 50, at least 60, or at least 70 nucleosides. Embodiment 165. The guide of embodiment 164, wherein the nucleobase sequence of the region has 100% identity to an equal length region of any of SEQ ID NOs: 568, 990-993, 1030-1034, 1183- 1206, or 1391-1393. Embodiment 166. The guide of any of embodiments 158-165, wherein D consists of 20-24 nucleosides having a nucleobase sequence that is 100% complementary to the nucleobase sequence of a target DNA. Embodiment 167. A guide, consisting of an oligonucleotide according to the following formula: T1-D-X-S1a-B1-S1b-H1-S1b’-B2-S1a’-L1-S2-H2-S2’-L2-S3-H3-S3’-(L3-S4-H4-S4’)n-T2; wherein: D consists of 17-25 linked nucleosides, wherein the nucleobase sequence of D is complementary to the nucleobase sequence of a target DNA; X is absent, or consists of 1-2 linked nucleosides that are not complementary to the nucleobase sequence of the target DNA; each of T1and T2is absent or independently consists of 1-30 linked nucleosides; S1aand S1a’ each consist of 7 linked nucleosides, wherein the nucleobase sequence of S1ais 100% complementary to the nucleobase sequence of S1a’; S1band S1b’ each consist of 4-12 linked nucleosides, wherein the nucleobase sequence of S1bis 100% complementary to the nucleobase sequence of S1b’; S2and S2’ each consist of 6 linked nucleosides, wherein the nucleobase sequence of S2is 100% complementary to the nucleobase sequence of S2’; S3and S3’ each consist of 5 or 6 linked nucleosides, wherein the nucleobase sequence of S3is 100% complementary to the nucleobase sequence of S3’; S4and S4’, when present, each consist of 6 linked nucleosides, wherein the nucleobase sequence of S4is 100% complementary to the nucleobase sequence of S4’; n is 0 or 1; B1is consists of 1 nucleoside; B2consists of 3 linked nucleosides; each of H1, H2, H3, and H4independently consists of 3-4 linked nucleosides; L1consists of 17 linked nucleosides; L2consists of 0 linked nucleosides; and L3consists of 8 linked nucleosides. Embodiment 168. The guide of embodiment 167, wherein S1band S1b’ each consist of 8 linked nucleosides. Embodiment 169. The guide of any of embodiments 167-168, wherein the nucleobase sequence of B2is GAA. Embodiment 170. The guide of any of embodiments 167-169, wherein the nucleobase sequence of L2is AAAAAUUUAUUCAAAAC (SEQ ID NO: 78). Embodiment 171. The guide of any of embodiments 167-170, wherein the nucleobase sequence of H2is GAAA. Embodiment 172. The guide of embodiment 171, wherein S1band S1b’ each consist of 8 linked nucleosides; n and m are 0; the nucleobase sequence of B2is GAA; the nucleobase sequence of L2is AAAAAUUUAUUCAAAAC (SEQ ID NO: 78); and the nucleobase sequence of H2is GAAA. Embodiment 173. The guide of any of embodiments 167-172, wherein the guide comprises a region having a nucleobase sequence that has at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NOs: 554, 981-984, 1024-1026, 1127-1152, or 1343- 1361, wherein the region comprises at least 40, at least 50, at least 60, or at least 70 nucleosides. Embodiment 174. The guide of embodiment 173, wherein the nucleobase sequence of the region has 100% identity to an equal length region of any of SEQ ID NOs: 554, 981-984, 1024-1026, 1127- 1152, or 1343-1361. Embodiment 175. The guide of any of embodiments 167-174, wherein D consists of 20-24 nucleosides having a nucleobase sequence that is 100% complementary to the nucleobase sequence of a target DNA. Embodiment 176. A guide of any of embodiments 93-95, wherein the guide comprises a region having a nucleobase sequence that has at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NOs: 121-141, 510-602, 961-1430, 1436-2833 wherein the region comprises at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 nucleosides. Embodiment 177. A guide of any of embodiments 93-95, wherein the guide comprises a protein recognition region that has a nucleobase sequence that has at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NOs: 121-141, 510-602, 961- 1430, 1436-2833. Embodiment 178. The guide any of embodiments 93-177, wherein the guide comprises a modified oligonucleotide. Embodiment 179. The guide of any of embodiments 93-178, wherein the guide comprises at least one modified sugar moiety. Embodiment 180. The guide of embodiment 179, wherein the modified sugar moiety is selected from a 2’-OMe and a 2’-F. Embodiment 181. The guide of embodiment 179, wherein the modified oligonucleotide comprises a 2’- OMe or 2’-F sugar moiety within the first five nucleosides at the 5’ end of the guide or the last five nucleosides at the 3’ end of the guide. Embodiment 182. The guide of embodiment 179-181, wherein the guide comprises 2-32’-OMe sugar moieties at the 5’ end of the guide and 2-32’-OMe sugar moieties at the 3’-end of the guide. Embodiment 183. The guide of embodiment 182, wherein each non-2’-OMe sugar moiety of the guide comprises an unmodified RNA sugar moiety. Embodiment 184. The guide of any of embodiments 179-183, wherein the guide comprises a modified internucleoside linkage. Embodiment 185. The guide of embodiment 184, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage. Embodiment 186. The guide of embodiment 184 or 185, wherein the guide comprises at least one modified internucleoside linkage within the first five nucleosides at the 5’ end of the guide or the last five nucleosides at the 3’ end of the guide. Embodiment 187. The guide of embodiment 186, wherein the guide comprises 2-3 phosphorothioate internucleoside linkages at the 5’ end of the guide and 2-3 phosphorothioate internucleoside linkages at the 3’ end of the guide. Embodiment 188. The guide of embodiment 187, wherein each remaining internucleoside linkage is an unmodified phosphodiester internucleoside linkage. Embodiment 189. The guide of any of embodiments 184-188, wherein each internucleoside linkage of the guide is a standard length internucleoside linkage. Embodiment 190. The guide of any of embodiments 93-177, wherein the guide comprises an unmodified oligonucleotide. Embodiment 191. The guide of any of embodiments 93-190, wherein the guide comprises a target recognition region that is at least 90%, at least 95%, or 100% complementary to a target DNA sequence. Embodiment 192. The guide of embodiment 191, wherein the target recognition region comprises 17- 25, 19-24, 20-24, 20-23, 20-22, 21-22, 21-23, 21-24, 20, 21, 22, 23, or 24 linked nucleosides. Embodiment 193. An editing system comprising the polypeptide of any of embodiments 53-77 and a guide of any of embodiments 93-192. Embodiment 194. An editing system comprising: a nucleic acid of any of embodiments 78-92; and a guide of any of embodiments 93-192. Embodiment 195. An editing system comprising: a nucleic acid of any of embodiments 78-92; and a nucleic acid encoding the guide of any of embodiments 93-177 or 190. Embodiment 196. The editing system of embodiment 194 or 195 wherein the nucleic acid encoding the polypeptide is an exogenous mRNA. Embodiment 197. The editing system of embodiment 194 or 195, wherein the nucleic acid encoding the polypeptide is a DNA. Embodiment 198. The editing system any of embodiments 195-197, wherein the nucleic acid encoding the guide is a DNA. Embodiment 199. A composition comprising the editing system of any of embodiments 193-198 and an LNP. Embodiment 200. A viral vector comprising the editing system of embodiment 195. Embodiment 201. A method of editing a target nucleic acid, comprising administering a composition of embodiment 199 or a viral vector of embodiment 200 to a subject. Embodiment 202. A method of editing a target nucleic acid, comprising contacting a composition of embodiment 199 or a viral vector of embodiment 200 with a cell. Embodiment 203. A method of creating a double stranded break or a nick in a target nucleic acid, comprising contacting a composition of embodiment 199 or a viral vector of embodiment 200 with a cell. Embodiment 204. The composition of embodiment 199 or a viral vector of embodiment 200 for use in therapy Embodiments provided herein are directed to guided nucleic acid binding agents, editing systems comprising guided nucleic acid binding agents, and methods of use thereof. In certain embodiments, the guided nucleic acid binding agent comprises a Cas protein. In certain embodiments, the guided nucleic acid binding agent comprises a Cas enzyme. In certain embodiments, the guided nucleic acid binding agent comprises a Cas protein comprising a RuvC domain and an HNH domain. In certain embodiments, the guided nucleic acid binding agent comprises a class 2, type II Cas protein. In certain embodiments, the guided nucleic acid binding agent comprises a class 2, type II Cas endonuclease. In certain embodiments, the guided nucleic acid binding agent comprises a Cas protein comprising a RuvC domain and an HNH domain. In certain embodiments, the guided nucleic acid binding agent comprises a modified Cas protein with an inactivating mutation in one of the nuclease active sites (a “Cas nickase”). In certain embodiments, the guided nucleic acid binding agent comprises a modified Cas protein with an inactivating mutation in both nuclease active sites (a “dead Cas” or “dCas”). In certain embodiments, the protein is isolated. In certain embodiments, the protein is engineered. In certain embodiments, an editing system comprises a guided nucleic acid binding agent and a guide. In certain embodiments, a guide consists of a single oligonucleotide and is a single guide, comprising a target-recognition region and a protein-recognition region. In certain embodiments, the guide is a dual guide, wherein a first oligonucleotide comprises the target-recognition region and a first portion of a protein- recognition region, and a second oligonucleotide comprises the second portion of a protein-recognition region. In certain such embodiments, the first oligonucleotide is a “crRNA” and the second oligonucleotide is a “tracrRNA”. The target-recognition region is a region of an oligonucleotide (single guide or crRNA) that has a nucleobase sequence complementary to an equal length portion of a target sequence within the complementary strand of a target DNA. I. Certain Nucleic Acid Binding Agents A. Certain Polypeptides In certain embodiments, a guided nucleic binding agent is an isolated or engineered polypeptide. In certain embodiments, the polypeptide consists of the Cas protein, or is a fusion protein comprising a Cas protein. In certain embodiments, the Cas protein is a Cas enzyme. In alternative embodiments, the Cas protein is a dead Cas protein. In certain embodiments, the polypeptide that constitutes a Cas protein has one or more domains. In certain embodiments, a domain of the polypeptide is a RuvC domain, which is comprised of one or more subdomains such as a RuvCI, RuvCII, and / or RuvCIII subdomains, e.g., as described herein. In certain embodiments, a domain of the polypeptide is an HNH domain, e.g., as described herein. In certain embodiments, a domain of the polypeptide is a PAM-interacting (PI) domain, e.g., as described herein. In certain embodiments, the polypeptide comprises a RuvC domain and an HNH domain. In certain embodiments, the polypeptide comprises a RuvC domain, an HNH domain, and a PI domain. In certain embodiments, the polypeptide comprises a RuvC domain, an HNH domain, a PI domain, and a nuclear localization sequence (NLS). In certain embodiments, a RuvC domain is split into two or three noncontiguous subdomains, termed RuvCI, RuvCII, and RuvCIII. In certain embodiments, the HNH domain is part of a NUC lobe. In certain embodiments, the polypeptide further comprises a REC lobe. In certain embodiments, the REC lobe is made up of subdomains corresponding to three REC domains, RECI, RECII, and RECIII. In certain embodiments, RECI is split into two noncontiguous subdomains RECIa and RECIb. In certain embodiments, the polypeptide comprises a bridge helix domain. Representative sequences corresponding to these domains are described in the table below. Table 1 Predicted domain and subdomain structure of novel ION-Cas Proteins ION-Cas Domain / SEQ Subdomain Start Stop SeID IDquenceID site*site*NO: CDKTTGEMISINRAMWDYSMNFMELINSDEF TFKDELAQKKTKAVKLL IONCAS ADFEYEDLQEYYFSAPVKRMIWQTILVVREIE 0 GGKISCDKLFGVKAFTESEYNKICFEKTDFED NENKYAQLLGQYFDVISLAKTLYDQVVLNGIL C 1 2 3 4 5 6 7 NKKVMINTLFVVNGFKMRLASISTDNIIFNNA NQLILDETNQKILKEVIKYNRDLIKKNDTTVN HKNLTEKDLILLYDTFKDKLKNSIYIKGEDFSN 8 901 2 3 4 DENFFARLDESFLWKEDKSDKAKSDYILFADK DCTDKDYYEKYQTIYHLRKHLLGTTEKADAR FIYLALHHMLKYRGNFLYEGQKFTELDDFKII 5 6 7 8 9 0 KKDDDGFWIENTENAMFFKNRGLNDYHHAH DAYLACIIGLYIQKVYPGWKNELNFKKYRREF EKYYEEQKKYNTKSKKEIYISMLNRFTKNYEK * 3 for I In certain embodiments, the polypeptide comprises a region of at least 20, at least 25, or 30 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 85. In certain embodiments, the polypeptide comprises a comprises a region of at least 20, at least 25, or 30 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 85. In certain embodiments, the polypeptide comprises a region of at least 30, at least 40, at least 50, or 55 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 92. In certain embodiments, the polypeptide comprises a region of at least 30, at least 40, at least 50, or 55 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 92. In certain embodiments, the polypeptide comprises a region of at least 70, at least 80, at least 90, or at least 100 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 94. In certain embodiments, the polypeptide comprises a region of at least 70, at least 80, at least 90, or at least 100 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 94. In certain embodiments, the polypeptide comprises a region of at least 15, at least 16, at least 17, or 18 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 97. In certain embodiments, the polypeptide comprises a comprises a region of at least 15, at least 16, at least 17, or 18 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 97. In certain embodiments, the polypeptide comprises a region of at least 30, at least 40, at least 45, or 50 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 104. In certain embodiments, the polypeptide comprises a region of at least 30, at least 40, at least 45, or 50 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 104. In certain embodiments, the polypeptide comprises a region of at least 70, at least 80, at least 90, or at least 100 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 106. In certain embodiments, the polypeptide comprises a region of at least 70, at least 80, at least 90, or at least 100 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 106. In certain embodiments, the polypeptide comprises a region of at least 35, at least 40, at least 45, or 48 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 109. In certain embodiments, the polypeptide comprises a region of at least 35, at least 40, at least 45, or 48 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 109. In certain embodiments, the polypeptide comprises a region of at least 35, at least 40, at least 45, or 47 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 116. In certain embodiments, the polypeptide comprises a region of at least 35, at least 40, at least 45, or 47 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 116. In certain embodiments, the polypeptide comprises a region of at least 70, at least 80, at least 90, or at least 100 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 118. In certain embodiments, the polypeptide comprises a region of at least 70, at least 80, at least 90, or at least 100 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 118. In certain embodiments, the polypeptide comprises a RuvCI domain that has at least 70% sequence identity to SEQ ID NO: 85, SEQ ID NO: 97, or SEQ ID NO: 109. In certain embodiments, the polypeptide comprises a RuvCI domain that has at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 85, SEQ ID NO: 97, or SEQ ID NO: 109. In certain embodiments, the polypeptide comprises a RuvCII domain that has at least 70% sequence identity to SEQ ID NO: 92, SEQ ID NO: 104, or SEQ ID NO: 116. In certain embodiments, the polypeptide comprises a RuvCII domain that has at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 92, SEQ ID NO: 104, or SEQ ID NO: 116. In certain embodiments, the polypeptide comprises a RuvCIII domain that has at least 70% sequence identity to SEQ ID NO: 94, SEQ ID NO: 106, or SEQ ID NO: 118. In certain embodiments, the polypeptide comprises a RuvCIII domain that has at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 94, SEQ ID NO: 106, or SEQ ID NO: 118. In certain embodiments, the polypeptide comprises a RuvC domain comprised of subdomains having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 85, 92, and 94; or SEQ ID NOs: 97, 104, and 106; or SEQ ID NOs: 109, 116, and 118. In certain embodiments, the polypeptide comprises a region of at least 70, at least 80, at least 90, or at least 100 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 93. In certain embodiments, the polypeptide comprises a region of at least 70, at least 80, at least 90, or at least 100 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 93. In certain embodiments, the polypeptide comprises a region of at least 70, at least 80, at least 90, or at least 100 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 105. In certain embodiments, the polypeptide comprises a region of at least 70, at least 80, at least 90, or at least 100 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 105. In certain embodiments, the polypeptide comprises a region of at least 70, at least 80, at least 90, or at least 100 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 117. In certain embodiments, the polypeptide comprises a region of at least 70, at least 80, at least 90, or at least 100 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 117. In certain embodiments, the polypeptide comprises an HNH domain that has at least 70% sequence identity to any of SEQ ID NOs: 93, 105, or 117. In certain embodiments, the polypeptide comprises an HNH domain that has at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NO: 93, 105, or 117. In certain embodiments, the polypeptide comprises a region of at least 70, at least 75, or at least 80 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 87. In certain embodiments, the polypeptide comprises a region of at least 70, at least 75, or at least 80 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 87. In certain embodiments, the polypeptide comprises a region of at least 70, at least 75, or at least 80 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 99. In certain embodiments, the polypeptide comprises a region of at least 70, at least 75, or at least 80 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 99. In certain embodiments, the polypeptide comprises a region of at least 70, at least 75, or 77 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 111. In certain embodiments, the polypeptide comprises a region of at least 70, at least 75, or 77 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 111. In certain embodiments, the polypeptide comprises a region of at least 150, at least 175, or at least 200 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 89. In certain embodiments, the polypeptide comprises a region of at least 150, at least 175, or at least 200 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 89. In certain embodiments, the polypeptide comprises a region of at least 150, at least 175, or at least 200 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 101. In certain embodiments, the polypeptide comprises a region of at least 150, at least 175, or at least 200 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 101. In certain embodiments, the polypeptide comprises a region of at least 150, at least 175, or at least 200 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 113. In certain embodiments, the polypeptide comprises a region of at least 150, at least 175, or at least 200 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 113. In certain embodiments, the polypeptide comprises a region of at least 125, at least 150, or at least 175 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 88. In certain embodiments, the polypeptide comprises a region of at least 125, at least 150, or at least 175 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 88. In certain embodiments, the polypeptide comprises a region of at least 125, at least 150, or at least 175 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 100. In certain embodiments, the polypeptide comprises a region of at least 125, at least 150, or at least 175 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 100. In certain embodiments, the polypeptide comprises a region of at least 50, at least 55, or 60 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 112. In certain embodiments, the polypeptide comprises a region of at least 50, at least 55, or 60 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 112. In certain embodiments, the polypeptide comprises a region of at least 125, at least 150, or at least 175 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 90. In certain embodiments, the polypeptide comprises a region of at least 125, at least 150, or at least 175 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 90. In certain embodiments, the polypeptide comprises a region of at least 125, at least 150, or at least 175 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 102. In certain embodiments, the polypeptide comprises a region of at least 125, at least 150, or at least 175 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 102. In certain embodiments, the polypeptide comprises a region of at least 125, at least 150, or at least 175 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 114. In certain embodiments, the polypeptide comprises a region of at least 125, at least 150, or at least 175 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 114. In certain embodiments, the polypeptide comprises a RECIa domain that has at least 70% sequence identity to SEQ ID NO: 87, SEQ ID NO: 99, or SEQ ID NO: 111. In certain embodiments, the polypeptide comprises a RECIa domain that has at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 87, SEQ ID NO: 99, or SEQ ID NO: 111. In certain embodiments, the polypeptide comprises a RECIb domain that has at least 70% sequence identity to SEQ ID NO: 89, SEQ ID NO: 101, or SEQ ID NO: 113. In certain embodiments, the polypeptide comprises a RECIb domain that has at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 89, SEQ ID NO: 101, or SEQ ID NO: 113. In certain embodiments, the polypeptide comprises a RECII domain that has at least 70% sequence identity to SEQ ID NO: 88, SEQ ID NO: 100, or SEQ ID NO: 112. In certain embodiments, the polypeptide comprises a RECII domain that has at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 88, SEQ ID NO: 100, or SEQ ID NO: 112. In certain embodiments, the polypeptide comprises a RECIII domain that has at least 70% sequence identity to SEQ ID NO: 90, SEQ ID NO: 102, or SEQ ID NO: 114. In certain embodiments, the polypeptide comprises a RECIII domain that has at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 90, SEQ ID NO: 102, or SEQ ID NO: 114. In certain embodiments, the polypeptide comprises a region of at least 500, at least 550, at least 600, or 648 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 91. In certain embodiments, the polypeptide comprises a region of at least 500, at least 550, at least 600, or 648 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 91. In certain embodiments, the polypeptide comprises a region of at least 500, at least 550, at least 600, or 650 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 103. In certain embodiments, the polypeptide comprises a region of at least 500, at least 550, at least 600, or 650 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 103. In certain embodiments, the polypeptide comprises a region of at least 500, at least 550, at least 600, at least 650, or at least 656 amino acids having a sequence with at least 70% sequence identity to an equal- length portion of SEQ ID NO: 115. In certain embodiments, the polypeptide comprises a region of at least 500, at least 550, at least 600, at least 650, or at least 656 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal- length portion of SEQ ID NO: or 115. In certain embodiments, the polypeptide comprises a REC lobe that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 91, 103, or 115. In certain embodiments, the polypeptide comprises a region of at least 500, at least 550, at least 600, or 616 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 96. In certain embodiments, the polypeptide comprises a region of at least 500, at least 550, at least 600, or 616 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 96. In certain embodiments, the polypeptide comprises a region of at least 500, at least 550, at least 600, or at least 648 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 108. In certain embodiments, the polypeptide comprises a region of at least 500, at least 550, at least 600, or at least 648 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 108. In certain embodiments, the polypeptide comprises a region of at least 500, at least 550, at least 600, or at least 650 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 120. In certain embodiments, the polypeptide comprises a region of at least 500, at least 550, at least 600, or at least 648 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 120. In certain embodiments, the polypeptide comprises a NUC lobe that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 96, 108, or 120. In certain embodiments, the polypeptide comprises a region of at least 175, at least 200, at least 225, or 244 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 95. In certain embodiments, the polypeptide comprises at least 175, at least 200, at least 225, or 244 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 95. In certain embodiments, the polypeptide comprises a region of at least 175, at least 200, at least 225, or 242 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 107. In certain embodiments, the polypeptide comprises at least 175, at least 200, at least 225, or 242 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 107. In certain embodiments, the polypeptide comprises a region of at least 175, at least 200, at least 225, at least 250, or 278 amino acids having a sequence with at least 70% sequence identity to an equal-length portion of SEQ ID NO: 119. In certain embodiments, the polypeptide comprises at least 175, at least 200, at least 225, at least 250, or 278 amino acids having a sequence with at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal-length portion of SEQ ID NO: 119. In certain embodiments, the polypeptide comprises an PI domain that has at least 70% sequence identity to any of SEQ ID NOs: 95, 107, or 119. In certain embodiments, the polypeptide comprises a PI domain that has at least 45%, 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%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NOs: 95, 107, or 119. In certain embodiments, the polypeptide comprises a REC Lobe having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 91 and a NUC Lobe having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 96. In certain embodiments, the polypeptide comprises a REC Lobe having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 103 and a NUC Lobe having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 108. In certain embodiments, the polypeptide comprises a REC Lobe having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 115 and a NUC Lobe having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 120. In certain embodiments, the polypeptide comprises a REC Lobe having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 91; a NUC Lobe having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 96, and a PI domain having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 95. In certain embodiments, the polypeptide comprises a REC Lobe having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 103; a NUC Lobe having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 108, and a PI domain having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 107. In certain embodiments, the polypeptide comprises a REC Lobe having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 115; a NUC Lobe having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 120, and a PI domain having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 119. In certain embodiments, a polypeptide comprises: RuvCI-Bridge Helix-RECIa-RECII-RECIb- RECIII- RuvCII-HNH-RuvCIII-PI, in which each (sub)domain has at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to a corresponding sequence in Table 1. In certain embodiments, a polypeptide comprises: REC Lobe-NUC Lobe, in which each (sub)domain has at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to a corresponding sequence in Table 1. Table 2 Predicted non-PI region and PI domains of novel ION-Cas Proteins SEQ ION-Cas Region Start Stop ID ID i *i *SequenceID : 6 DRKVNRAIIGKQTIYGTRTKDNDEYIINKYSDIYSL DKKSCTELIKKLKDTPDKFLIYVNNREVYELIMRII DEYKDANNPFLAYKNEYGYILKNGKVPVKTLRY 7 8 9 MAQEYFLGLDLGTGSLGWAVTTMDYEIMRAHG KALWGVRLFESAKTAEERRTFRTGRRRLARRNW RIELLQEIFAEEIYKVDPGFYLRMKESRYTKEDKR 0 1

[0002] MNEQEYFLGLDMGTGSVGWAVTDTDYNLIKINR KKAWGSVLFETSEGAKKRRVNRCARRRLKRQKE RLSLLRELFEEEMQKVDKGFFLRMKESRYVYKDK 2 3

[0003] MKKILGLDLGTTSIGWAFVNEAENENEKSAIVKL GVRVIPLTTDEELDFEKGKPVSTNAERTLKRGMR RNLDRFQLRRKELIKVLKENHFIYDNTPLTETGKN 4 5

[0004] MSENYILGLDIGITSVGYGIIDYDTREVVDAGVRL FKEANVENNEGRRSKRGARRLKRRRRHRLQRVK KLLFDYHLIDENTELEIINPYEARVKGLYNVLTKA 6 7

[0005] MREQEYFLGFDIGTGSVGYAVTDLSYNLIKINRKY AWGSVLFDTSNGAEERRINRTNRRRYGREKERLA LLRELFEEEIYKIDRGFFIRLQESKFVFEDKKDENG 8 9 In certain embodiments, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3186. In certain embodiments, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3186 and a PI domain having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3187. In certain embodiments, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3186 and a PI domain having less than 75%, less than 60%, less than 50%, less than 40%, or less than 30% identity to SEQ ID NO: 3187. In certain embodiments, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3188. In certain embodiments, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3188 and a PI domain having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3189. In certain embodiments, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3188 and a PI domain having less than 75%, less than 60%, less than 50%, less than 40%, or less than 30% identity to SEQ ID NO: 3189. In certain embodiments, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3190. In certain embodiments, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3190 and a PI domain having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3191. In certain embodiments, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3190 and a PI domain having less than 75%, less than 60%, less than 50%, less than 40%, or less than 30% identity to SEQ ID NO: 3191. In certain embodiments, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3192. In certain embodiments, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3192 and a PI domain having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3193. In certain embodiments, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3192 and a PI domain having less than 75%, less than 60%, less than 50%, less than 40%, or less than 30% identity to SEQ ID NO: 3193. In certain embodiments, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3194. In certain embodiments, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3194 and a PI domain having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3195. In certain embodiments, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3194 and a PI domain having less than 75%, less than 60%, less than 50%, less than 40%, or less than 30% identity to SEQ ID NO: 3195. In certain embodiments, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3196. In certain embodiments, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3196 and a PI domain having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3197. In certain embodiments, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3196 and a PI domain having less than 75%, less than 60%, less than 50%, less than 40%, or less than 30% identity to SEQ ID NO: 3197. In certain embodiments, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3198. In certain embodiments, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3198 and a PI domain having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3199. In certain embodiments, the polypeptide comprises a non-PI region having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3198 and a PI domain having less than 75%, less than 60%, less than 50%, less than 40%, or less than 30% identity to SEQ ID NO: 3199. In certain embodiments, a polypeptide described above is a Cas protein. In certain embodiments, a polypeptide described above is a Cas enzyme. In certain embodiments, a polypeptide described above is a dead Cas protein. In certain embodiments, a polypeptide described above is a Cas fusion protein. B. Protein Engineering Methods of structure-guided protein engineering as well as protein engineering via directed evolution have been applied to a variety of Cas proteins (see Liu, et al., Trends in Biotechnology, 39(3):262-273, 2021). 1. Certain Heterologous Domains In certain embodiments, heterologous domain(s) from non-Cas proteins can be appended to a Cas protein to add a function (see, e.g. WO2014 / 152432; WO2016 / 063264; WO2022 / 162247; WO2022 / 140577; US2019 / 0233805), resulting in a Cas fusion protein. In certain embodiments, the Cas portion of the fusion protein is used to target the heterologous domain(s) to a DNA target of interest. In certain embodiments, the Cas portion of the fusion protein is a dead Cas protein. In certain such embodiments, the heterologous domains(s) are transcriptional activators or transcriptional repressors, or domains thereof. For example, in some cases, the heterologous domains(s) inhibit transcription (e.g., the heterologous domain is a transcriptional repressor, a polypeptide that functions via recruitment of transcription inhibitor proteins, a polypeptide that leads to modification of target DNA such as methylation, a polypeptide that leads to recruitment of a DNA modifier, a polypeptide that modulates histones associated with target DNA, or a polypeptide that recruits a histone modifier such as those that modify acetylation and / or methylation of histones). In some cases, the heterologous domain(s) increase transcription (e.g., the exogenous domain is a transcription activator, a polypeptide that acts via recruitment of transcription activator proteins, a polypeptide that leads to modification of target DNA such as demethylation, a polypeptide that leads to recruitment of a DNA modifier, a polypeptide that modulates histones associated with target DNA, or a polypeptide that recruits a histone modifier such as those that modify acetylation and / or methylation of histones). In certain embodiments, a Cas fusion protein includes an heterologous domain that has enzymatic activity that modifies a target nucleic acid (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity). In certain embodiments, the heterologous domain(s) modify a second polypeptide (e.g. a histone) associated with a target nucleic acid (e.g., polypeptides having methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity). Examples of polypeptides that can be used to increase transcription include, but are not limited to, transcriptional activators such as VP16, VP64, VP48, VP160, MyoD1, HSF1, RTA, SET7 / 9, or domains thereof; p65 subdomain (e.g., from NFkB), and activation domains of EDLL and / or TAL (e.g., for activity in plants); histone lysine methyltransferases such as SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1 or domains thereof; histone lysine demethylases such as JHDM2a / b, UTX, JMJD3 or domains thereof; histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, SRC1, ACTR, P160, CLOCK or domains thereof; and DNA demethylases such as TenElevenTranslocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, or domains thereof. Examples of polypeptides that can be used to decrease transcription include, but are not limited to, transcriptional repressor polypeptides such as the Krüppel associated box (KRAB or SKD) domains; KOX1 repression domains; the Mad mSIN3 interaction domain (SID); the ERF repressor domain (ERD), the SRDX repression domain (e.g., for repression in plants); histone lysine methyltransferases such as Pr-SET7 / 8, SUV4-20H1, RIZ1, or domains thereof; histone lysine demethylases such as JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASCI, JMJD2D, JARID1A / RBP2, JARID1B / PLU-I, JARID1C / SMCX, JARID1D / SMCY; histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRTI, SIRT2, HDAC11, or domains thereof; DNA methylases such as HhaI DNA m5c-methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMTI, CMT2 (plants), or domains thereof; and periphery recruitment elements such as Lamin A and Lamin B, or domains thereof. In certain embodiments, the heterologous domain has enzymatic activity that modifies the target nucleic acid. Examples of enzymatic activity that can be provided by the heterologous domain include but are not limited to: nuclease activity such as that provided by a restriction protein (e.g., Fokl nuclease), methyltransferase activity such as that provided by a methyltransferase (e.g., Hhal DNA m5c- methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants)); demethylase activity such as that provided by a demethylase (e.g., Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROSI, and the like), DNA repair activity, DNA damage activity, deamination activity such as that provided by a deaminase (e.g., a cytosine deaminase enzyme such as rat APOBEC1), dismutase integrase activity such as that provided by an integrase and / or resolvase (e.g., Gin invertase such as the hyperactive mutant of the Gin invertase, GinH106Y; human immunodeficiency virus type 1 integrase (IN); Tn3 resolvase), transposase activity, recombinase activity such as that provided by a recombinase (e.g., catalytic domain of Gin recombinase), polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity). In certain embodiments, the heterologous domain has enzymatic activity that modifies a protein associated with the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA) (e.g., a histone, an RNA binding protein, a DNA binding protein). Examples of enzymatic activity (that modifies a protein associated with a target nucleic acid) that can be provided by the heterologous domain include but are not limited to: methyltransferase activity such as that provided by a histone methyltransferase (HMT) (e.g., suppressor of variegation 3-9 homolog 1 (SUV39Hl, also known as KMT1A), euchromatic histone lysine methyltransferase 2 (G9A, also known as KMT1C and EHMT2), SUV39H2, ESET / SETDBI, SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, DOT1L, Pr-SET7 / 8, SUV4-20H1, EZH2, RIZ1), demethylase activity such as that provided by a histone demethylase (e.g., Lysine Demethylase 1A (KDM1A also known as LSD1), JHDM2a / b, JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARID1A / RBP2, JARID1B / PLU-1, JARID1C / SMCX, JARID1D / SMCY, UTX, JMJD3), acetyltransferase activity such as that provided by a histone acetylase transferase (e.g., catalytic core / fragment of the human acetyltransferase p300, GCN5, PCAF, CBP, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, HBO1 / MYST2, HMOF / MYST1, SRC1, ACTR, P160, CLOCK), deacetylase activity such as that provided by a histone deacetylase (e.g., HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRTI, SIRT2, HDACll), kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, and demyristoylation activity. Domains that have been appended to Cas proteins include FokI for highly specific genome editing (see, e.g. WO2014 / 152432), transcriptional repressor domains (e.g. KRAB; see, e.g., Alerasool, et al., Nature Methods, 1093-1096, 2020) and transcriptional activator domains (e.g. VP64 – see, e.g., WO2014 / 197748; p65; and RTA) for the regulation of gene expression, histone modification enzymes such as methyltransferases for epigenetic editing (see, e.g., WO2016 / 103233), and fluorescent proteins for genome imaging (see, e.g. Trends in Biotechnology, 39(3):262-273, 2021). One or more of these polypeptides can be appended to a Cas protein at either the N-terminus or the C-terminus of the Cas protein. In certain embodiments, multiple domains are appended to a Cas protein at the C-terminal, the N-terminal, or both (see, e.g. WO2019 / 204766). Additional repressor domains that can be appended to a Cas protein are provided in, for example, WO2022 / 140577; Alerasool, et al., Nature Methods, 1093-1096, 2020. For examples of some of the above polypeptides used in the context of fusions with Cas9, Zinc Finger, and / or TALE proteins (for site specific target nucleic modification, modulation of transcription, and / or target protein modification, e.g., histone modification), see, e.g.: Nomura et al,, J Am Chem Soc.2007 Jul 18;129(28):8676-7; Rivenbark et al., Epigenetics.2012 Apr;7(4):350-60; Nucleic Acids Res.2016 Jul 8;44(12):5615-28; Gilbert et. al., Cell.2013 Jul 18;154(2):442-51; Kearns et al., Nat Methods.2015 May;l2(5):401-3; Mendenhall et. al., Nat Biotechnol.2013 Dec;31(12):1133-6; Hilton et. al., Nat Biotechnol. 2015 May;33(5):510-7; Gordley et. al., Proc Natl Acad Sci US A.2009 Mar 31;106(13):5053-8; Akopian et. al., Proc Natl Acad Sci US A.2003 Jul 22;100(15):8688-91; Tan et., al., J Virol.2006 Feb;80(4):1939-48; Tan et. al., Proc Natl Acad Sci US A.2003 Oct 14;100(21):11997-2002; Papworth et. al., Proc Natl Acad Sci US A.2003 Feb 18;100(4):1621-6; Sanjana et. al., Nat Protoc.2012 Jan 5;7(1):171-92; Beerli et. al., Proc Natl Acad Sci USA.1998 Dec 8;95(25):14628-33; Snowden et. al., Curr Biol.2002 Dec 23;12(24):2159-66; Xu et.al., Xu et. al., Cell Discov.2016 May 3;2:16009; Komor et al., Nature.2016 Apr 20;533(7603):420-4; Chaikind et. al., Nucleic Acids Res.2016 Aug 11; Choudhury at. al., Oncotarget.2016 Jun 23; Du et. al., Cold Spring Harb Protoc.2016 Jan 4; Pham et. al., Methods Mol Biol.2016;1358:43-57; Balboa et al., Stem Cell Reports.2015 Sep 8;5(3):448-59; Hara et. al., Sci Rep.2015 Jun 9;5:11221; Piatek et. al., Plant Biotechnol J.2015 May;13(4):578-89; Hu et al., Nucleic Acids Res.2014 Apr;42(7):4375-90; Cheng et. al., Cell Res.2013 Oct;23(10):1163-71; Cheng et. al., Cell Res.2013 Oct;23(10):1163-71; and Maeder et. al., Nat Methods.2013 Oct;l0(l0):977-9. 2.Certain Nuclear Localization Sequences In certain embodiments, a Cas protein may include one or more nuclear localization sequences. A nuclear localization sequence is a short sequence of amino acids that promotes uptake of the protein into the nucleus of a cell. A variety of eukaryotic nuclear localization signal (NLS) sequences have been used to improve the nuclear uptake of proteins (see, e.g., Lu, et al. Cell Comm. and Signalling, 2021). An NLS may be located at the N-terminus and / or the C-terminus of a polypeptide, or it may be located at any point within the polypeptide sequence. A monopartite classical NLS has the sequence K (K / R) X (K / R), wherein K is lysine and X can be any amino acid. A bipartite NLS comprises two clusters of 2-3 positively charged amino acids separated by a region of 9-12 amino acids that comprises several prolines, with a consensus sequence of R / K(X)10-12KRXK, wherein R is arginine, K is lysine, and X is any amino acid. In certain embodiments, a Cas protein may comprise one or more nuclear localization sequences selected from the table below or a variant thereof. Table 3 Nuclear Localization Sequences Nuclear Localization Sequence SEQ ID NO: 3.Engineered Cas Variants with Altered PAM Specificity A Cas protein binds to target DNA at a sequence defined by the region of complementarity between the target-recognition region of the guide and the target DNA. Site-specific binding (and / or cleavage) of a double stranded target DNA for Cas proteins typically occurs at a site determined by both: (i) hybridization of the target-recognition region of the guide to the target DNA, and (ii) the presence of a short motif (referred to as the PAM, or protospacer adjacent motif) within the non-complementary strand of DNA. In certain embodiments, the PAM for a Cas protein is immediately 5’ of the protospacer sequence. In certain embodiments, the PAM for a Cas protein is immediately 3’ of the protospacer sequence. Methods for engineering the PAM interacting domain (PI domain) for Cas proteins, including SpCas9, SaCas9, StlCas9, FnCas9, AsCas12a, among others, have been previously described (see, e.g.,WO2016 / 141224; Table 1 in Liu, et al., Trends in Biotechnology, 39(3):262-273, 2021). A bacterial selection system can be used to optimize a Cas protein variant with a non-native PI domain configured to recognize a variant PAM. Bacteria are transfected with a plasmid encoding an inducible toxic gene as well as a plasmid encoding a Cas protein variant. After inducing the toxic gene, the only surviving bacteria are those in which the Cas protein variant cleaves the toxic gene. Using this method, a large library of Cas proteins with randomized mutations within the PI domain can be screened against a large randomized PAM library. For surviving clones, a bacterial-based site-depletion assay can then be used to profile the PAM specificities of the evolved Cas proteins. These methods have been previously used to identify PI domains with novel PAM recognition sequences for SpCas9, SaCas9, StlCas9 (Kleinstiver, Nature, 523(7561): 481-485, 2015); SaCas9 (Kleinstiver, Nature Biotech., 33(12):1293-1298, 2015). Similarly, a selection system using phage-assisted continuous evolution (PACE) has been used to identify Cas9 variants with expanded PAM recognition (Hu, et al., Nature, 556: 57-63, 2918; Miller, et al., Nature Biotech., 38: 471-481, 2020). Another method that has been previously used to engineer alternative PAM interacting domains that recognize a variant PAM is structure-guided protein engineering. Using this method, amino acid residues that form contacts with the target DNA are identified via crystal structures, and Cas protein variants containing mutations of these amino acids are then tested for activity. A variant of saCas9 with decreased off-target editing was identified using these methods (Tan, et al., PNAS, 116(42):20969-20976, 2019), as was a variant of AsCas12a (Kleinstiver, et al., Nature Biotech., 37(3): 276-282, 2019). This method was also used to identify a variant of FnCas9 with a broader PAM recognition sequence (Hirano, et al., Cell, 164:950- 961). A further method that has been used to generate Cas protein variants that recognize a variant PAM is to generate a domain-swapped chimera using direct substitution of the PI domain of a Cas protein with the PI domain of a closely related orthologous Cas protein. Early work showed that swapping the PI domain of SpCas9 with the PI domain of St3Cas9 to generate a variant of St3Cas9 that cleaved target DNA with the canonical SpCas95’-NGG-3’ PAM (Nishimasu, et al, Cell, 156(5):935-949, 2014). In a similar manner, several Cas9 variants that recognize variant PAMs were identified by swapping PI domains with closely related Cas proteins (Ma, et al., Nature Comm., 10:560, 2019). C. Certain Nucleic Acids Encoding Polypeptides 1. Certain Nucleic Acids In certain embodiments, provided herein are nucleic acids encoding polypeptides. In certain embodiments, the polypeptide is or is part of a guided nucleic acid binding agent. In certain embodiments, the polypeptide comprises or consists of a Cas protein. In certain embodiments, the polypeptide comprises or consists of an ION-Cas protein. In certain embodiments, the nucleic acid is a DNA. In certain embodiments, the nucleic acid is an RNA. In certain embodiments, the nucleic acid is an exogenous mRNA. In certain embodiments, an exogenous mRNA comprises a coding region (e.g., an open reading frame (ORF)) encoding a polypeptide sequence, a cap, and one or more non-coding regions. In certain embodiments, the exogenous mRNA comprises a cap, 5' UTR, 3' UTR, a coding region, a poly(A) tail, and optionally one or more introns. The mRNA may comprise nucleotides selected from adenosine, guanosine, cytosine, uridine, N1-methylpseudouridine, optionally selected from adenosine, guanosine, cytosine, and uridine. Naturally-occurring eukaryotic mRNA molecules can contain non-coding regions, including, but not limited to untranslated regions (UTR) at their 5 '-end (5' UTR) and / or at their 3 '-end (3' UTR), a 5'-cap structure and a 3'-poly(A) tail. Exogenous mRNA may be configured to include such regions, which facilitate cellular processing and translation. In certain embodiments, a formulation, e.g., an LNP may be configured to deliver an exogenous mRNA having an open reading frame encoding a polypeptide. In certain embodiments, provided are methods in which an exogenous mRNA is translated in a cell. An exogenous mRNA may comprise a repeat region of contiguous nucleosides such as adenosine nucleosides, e.g., a poly(A) tail. A poly(A) tail is a region that is 3’ of the 3' UTR that contains multiple, consecutive adenosine monophosphates. It is believed that a poly(A) tail may protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, and / or export of the mRNA from the nucleus, and in translation. A poly(A) tail may contain 10 to 300 contiguous adenosine nucleosides. For example, an exogenous mRNA may comprise at its 3’ terminus a repeat region of 50-100, 50-150, 50-200, 100-250, 120-160, or 200-300 adenosine monophosphates. 2. Capping Groups Eukaryotic mRNAs, as well as RNA viral genomes, include a 7-methylguanosine (m7G) cap at the 5′ end of the mRNA sequence, attached to the 5’-most mRNA nucleotide through a 5′,5′-triphosphate bridge (ppp) during mRNA in vitro transcription. The cap structure plays essential functions in mRNA translation by recruiting translation initiation factors, and different 5′ caps can be incorporated into naturally occurring mRNAs. Cap0 protects endogenous mRNA from nuclease attack and is also involved in nuclear export and translation initiation. Both Cap1 and Cap2 are two 5′ caps that contain additional methyl groups on the second or third ribonucleotide. The additional modification of Cap1 and Cap2 is believed to reduce immunogenicity compared to Cap0. An exogenous mRNA may comprise a specific capping group such as described herein or as known in the art. Examples of 5’ capping groups include: “Cap 0”: m7G(5')ppp(5')N; “Cap 1”: m7G(5')ppp(5')(2'OMeN); and “Cap 2”: m7G(5')ppp(5')(2'OMeN)(2'OMeN); in which m7G indicates a guanosine nucleoside methylated at its 7-position and having a free 3’-OH, (5') indicates a 5' point of attachment, p is a phosphate linkage, each N is independently a nucleoside, e.g. guanosine or adenosine, and (2'OMeN) is independently a 2’-O-methyl nucleoside, e.g., 2’-O-methylguanosine or 2’-O-methyladenosine. A first nucleoside adenosine following the cap may also be methylated at its N6 position. Specific capping groups include (m7(3′OMeG)(5′)ppp(5′)(2′OMeA)pG; 3'-O-Me-m7G(5')ppp(5')G (“ARCA” cap); G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; and m7G(5')ppp(5')(2'OMeN)pG (“CleanCap™” ), where m7(3′OMeG) indicates a guanosine nucleoside methylated at its 7-position and having a 3’-O-methyl. Commercial sources are available for some group (e.g., New England BioLabs, Ipswich, MA, and TriLink Biotechnologies, San Diego, CA).5'- Capping of exogenous mRNA may be completed post-transcriptionally, e.g., using Vaccinia Vims Capping Enzyme. Cap 1 structure may be generated using both Vaccinia Vims Capping Enzyme and a 2'-O methyl-transferase to generate. Cap 2 structure may be generated from the Cap 1 structure followed by the 2'-O-methylation of the 5'- third most nucleotide using a 2'-O methyl-transferase. Cap 3 structure may be generated from the Cap 2 structure followed by the 2'-O-methylation of the 5'-fourthmost nucleotide using a 2'-O methyl-transferase. Enzymes may be derived, for example, from a recombinant source. 3. Poly(A) Tail The 3'-poly(A) tail is a region of contiguous adenine nucleotides at the 3 '-end of the transcribed mRNA. In certain embodiments, the 3'-poly(A) tail comprises one to 400 adenine nucleotides. In certain embodiments, the 3’-poly(A) tail comprises unmodified adenine nucleosides linked by phosphodiester internucleoside linkages. In some embodiments, the exogenous mRNA comprises a stabilizing element. A stabilizing element may be a histone stem-loop. A stem-loop binding protein (SLBP), a 32 kDa protein has been identified. It is associated with the histone stem- loop at the 3 '-end of the histone messages in both the nucleus and the cytoplasm, and is believed to promote efficient 3 '-end processing of histone pre-mRNA and stimulation of translation. In some embodiments, the histone stem-loop sequence comprises a length of 15 to 45 nucleotides. In some embodiments, exogenous mRNA includes a coding region, at least one histone stem-loop, and optionally, a poly(A) sequence or polyadenylation signal. The poly(A) sequence or polyadenylation signal is believed to increase the expression of an encoded protein. The encoded protein, in some embodiments, is not a histone protein, a reporter protein (e.g. Luciferase, GFP, EGFP, b-Galactosidase, EGFP), or a marker or selection protein (e.g. alpha-Globin, Galactokinase and Xanthine:guanine phosphoribosyl transferase (GPT)). 4. Coding Region and Sequence Optimization A coding region may comprise a contiguous sequence beginning with a start codon (e.g., methionine (AUG)), and ending with a stop codon (e.g., UAA, UAG or UGA), and comprising one or more nucleotide sequences encoding amino acids. Generally, a coding region encodes a polypeptide which forms a protein in vivo upon administration to a subject. In certain embodiments, a coding region comprises selected nucleotide codons to improve one or more properties of the exogenous mRNA or of the encoded protein, and may comprise a codon optimized ORF. For example, a coding region of an exogenous mRNA sequence may be codon optimized. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; add or remove post translation modification sites in encoded protein (e.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or generally to reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art - non limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and / or proprietary methods. In certain embodiments, a coding region sequence is optimized using an optimization algorithm as provided herein or as known in the art. In certain embodiments, a coding region shares less than 95%, less than 90%, less than 80%, or less than 70% sequence identity to a naturally-occurring or wild-type sequence ORF (e.g., a naturally-occurring or wild-type mRNA sequence encoding a protein), or a range of values therebetween. In some embodiments, an exogenous mRNA may be one in which the levels of G / C are enhanced. The G / C-content of nucleic acid molecules (e.g., mRNA) may influence the stability of the RNA. RNA having an increased amount of guanine (G) and / or cytosine (C) residues may be more stable than RNA containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides. See, e.g., WO02 / 098443. The coding region comprises at least one start codon (an AUG nucleotide triplet) among its 5’-most nucleotides and at least one stop codon (an UAG, UAA, or UGA nucleotide triplet) among its 3’-most nucleotides. In certain embodiments, an exogenous mRNA includes 2 or more stop codons, e.g., 2-10 stop codons. Example codons for specific amino acids are known in the art. 5. Chemical Modifications For Exogenous mRNA The compositions of the present disclosure comprise, in some embodiments, an exogenous mRNA comprises modified nucleotides or nucleosides (those other than A, C, G, and U joined by only phosphodiester linkages; exclusive of 5’ cap). Such modified nucleotides and nucleosides can be naturally- occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. Such modifications can include those described herein with respect to oligonucleotides. In certain embodiments, a naturally-occurring modified nucleotide or nucleotide of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such naturally occurring modified nucleotides and nucleotides can be found, inter alia, in the widely recognized MODOMICS database. In certain embodiments, an exogenous mRNA comprises a modified nucleoside described in one or more of US application Nos. PCT / US2012 / 058519; PCT / US2013 / 075177; PCT / US2014 / 058897; PCT / US2014 / 058891; PCT / US2014 / 070413; PCT / US2015 / 36773; PCT / US2015 / 36759; PCT / US2015 / 36771; or PCT / IB 2017 / 051367 all of which are incorporated by reference herein. Hence, an exogenous mRNA can comprise unmodified nucleosides, modified nucleosides, or a combination thereof. In certain embodiments, each nucleoside in an exogenous mRNA is modified similarly to other nucleosides of the same type; for example, all uridine nucleosides are present as N1-methylpseudouridine nucleosides. A modification may provide reduced degradation and / or reduced immunogenicity compared to an RNA comprising only unmodified nucleosides. In certain embodiments, an exogenous mRNA comprises a modified nucleoside selected from 1- methylpseudouridine, 1-ethylpseudouridine, 5-methoxyuridine, 5-methylcytidine, and pseudouridine. In certain embodiments, an exogenous mRNA comprises a modified nucleoside selected from 5-methoxymethyl uridine, 5-methylthiouridine, 1-methoxymethyl pseudouridine, 5-methylcytidine, and 5-methoxycytidine. In certain embodiments, an exogenous mRNA comprises a modified nucleoside selected from a combination of two or more (e.g., 2, 3, 4) of any of the modified nucleobases of this paragraph. In certain embodiments, an exogenous mRNA comprises 1-methylpseudouridine at one or more, e.g. all uridine nucleosides. In certain embodiments, an exogenous mRNA comprises 1-methylpseudouridine at one or more, e.g., all uridine positions of the nucleic acid, and 5-methylcytidine at one or more, e.g., all cytidine positions of the nucleic acid. In certain embodiments, an exogenous mRNA comprises pseudouridine at one or more, e.g. all uridine nucleosides. In certain embodiments, an exogenous mRNA comprises pseudouridine at one or more, e.g., all uridine positions of the nucleic acid, and 5-methylcytidine at one or more, e.g., all cytidine positions of the nucleic acid. In certain embodiments, an exogenous mRNA comprises unmodified uridine at one or more, e.g., all uridine positions of the nucleic acid. In certain embodiments, all nucleotides of a particular type in an exogenous mRNA (or in a sequence region thereof) are modified nucleotides, wherein the modified type may be any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C. 6. Untranslated Regions (UTRs) In wild-type mRNA, certain regions of a nucleic acid may be transcribed into RNA but not translated. In exogenous mRNA as described herein, a 5' UTR may start at the transcription start site and continues to a start codon at the beginning of the coding region but does not include the start codon. The 3' UTR follows a stop codon may include a transcription termination signal. The 5' UTR and the 3' UTR do not encode protein (are non-coding regions). In certain embodiments, the presence of a UTR enhances the stability of the exogenous mRNA, or induces downregulation of the exogenous mRNA in undesirable sites or tissues. A variety of 5' UTR and 3' UTR sequences are known and available in the art. A 5' UTR is a region of an mRNA that is directly upstream (5') from the start codon (the first codon of an mRNA transcript translated by a ribosome). In certain embodiments, the 5' UTR may provide translation initiation, and / or form secondary structures which are involved in elongation factor binding. The 5' UTR may include an initiation sequence such as a Kozak sequence. The Kozak sequence, also called the Kozak consensus sequence, is believed to be involved in ribosomal initiation of translation. In certain embodiments, the Kozak sequence comprises AUGG. In certain embodiments, the Kozak sequence is GCCRCCAUGG (SEQ ID NO: 73) or CCRCCAUGG, where R is a purine nucleoside (adenosine or guanosine). In certain embodiments of the disclosure, a 5' UTR is an unmodified UTR, i.e., one found in nature. In another embodiment, a 5' UTR is a modified UTR, i.e., does not occur in nature. In certain embodiments, a modified UTR increases gene expression relative to an unmodified counterpart. Exemplary 5' UTRs include Xenopus laevis or human derived a-globin or b-globin (e.g., US Patent No.9012219), human cytochrome b- 245 a polypeptide, and hydroxysteroid (17b) dehydrogenase, and Tobacco etch virus (e.g., US9012219), CMV immediate-early 1 (IE1) gene (US2014 / 0206753, WO2013 / 185069), the sequence GGGAUCCUACC (SEQ ID NO: 74) (WO2014 / 144196), and a 5' UTR described in US Patent Application Publication No. 2010 / 0293625 or WO2015085318. In another embodiment, 5' UTR of a TOP gene is a 5' UTR of a TOP gene lacking the 5' TOP motif (the oligopyrimidine tract) (e.g., WO / 2015 / 101414, W02015 / 101415, WO / 2015 / 062738, WO2015 / 024667, WO2015 / 024667); 5' UTR element derived from ribosomal protein Large 32 (L32) gene (WO / 2015 / 101414, W02015 / 101415, WO / 2015 / 062738), 5' UTR element derived from the 5' UTR of an hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (WO2015 / 024667), or a 5' UTR element derived from the 5' UTR of ATP5A1 (WO2015 / 024667) can be used. In some embodiments, an internal ribosome entry site (IRES) is used instead of a 5' UTR. In general, an exogenous mRNA may include a UTR from any suitable gene. The naturally occurring UTR or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5' or 3' UTR may be inverted, shortened, or lengthened. A reference UTR, e.g., a naturally occurring UTR, may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping, or transposition of nucleotides or sequences thereof. In certain embodiments, the 3' UTR sequences may include adenosine- and uridine-containing repeats. Such AU rich sequences are believed to provide high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUASS nonamers, where S is adenosine or uridine. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class. It is believed that including the HuR specific binding sites into the 3' UTR will provide stabilization of the message in vivo. In certain embodiments, a 3' UTR includes a repeated ARE. Unmodified (natural) and modified (non-natural) 3' UTR sequences are known in the art. UTRs known in the art include globin UTRs, including Xenopus b-globin UTRs and human b-globin UTRs (9012219, US2011 / 0086907), a modified b-globin construct (US2012 / 0195936, WO2014 / 071963), a2- globin, al-globin, UTRs (WO2015 / 101415, WO2015 / 024667), CYBA (Ferizi et ah, 2015) and albumin (Thess et ah, 2015), bovine or human growth hormone (wild type or modified) (WO2013 / 185069, US2014 / 0206753, WO2014152774), rabbit b globin and hepatitis B virus (HBV), a-globin 3' UTR and Viral VEEV 3' UTR sequences, the sequence UUUGAAUU (WO2014 / 144196), human and mouse ribosomal protein, rps93'UTR (WO2015 / 101414), FIG4 (WO2015 / 101415), and human albumin 7 (W02015 / 101415). The untranslated region may also include an upregulation motif, e.g., a translation enhancer element (TEE). As a non-limiting example, the TEE may include those described in WO1999 / 024595, WO2012 / 009644, WO2009 / 075886, WO2007 / 025008, WO1999 / 024595, European Patent Publication No. EP2610341A1 and EP2610340A1, US Patent No. US6310197, US6849405, US7456273, US7183395, US Patent Publication No. US2009 / 0226470, US2011 / 0124100, US2007 / 0048776, US2009 / 0093049, or US2013 / 0177581 each of which is herein incorporated by reference in its entirety. In certain embodiments, the 5’ UTR or 3’ UTR may comprise one or more additional functional regions selected from an upregulation region or a ribosome binding region. In certain embodiments, the functional region is an upregulation region (e.g., TEE). In certain embodiments, the TEE is one known in the art, e.g., in US Application No.2009 / 0226470. In certain embodiments, an exogenous mRNA comprises a plurality of upregulation motifs which may be the same or different from each other and which number, e.g., 2, 3, 4, 5, or more. In certain embodiments, the exogenous mRNA comprises a ribosome binding region, e.g., an internal ribosome entry site (IRES). The IRES may be, for example, one described in US Patent No. US7468275 and International Patent Publication No. WO2001 / 055369, each of which is herein incorporated by reference in its entirety. In certain embodiments, the IRES is one known in the art, e.g., in WO2014 / 081507. In certain embodiments, an exogenous mRNA may comprise a double, triple or quadruple UTR such as a 5' UTR or 3' UTR. As used herein, a “double” UTR is one in which two copies of the same UTR are included contiguously or substantially contiguously. For example, an exogenous mRNA may comprise a double beta-globin 3' UTR as described in US Patent publication No.2010 / 0129877, which is incorporated herein by reference in its entirety. In certain embodiments, a 5’ UTR or 3’ UTR may comprise a repeated set of functional sequences, e.g., as AA, ABAB or AABB or ABCABC or variants thereof, in which each letter, A, B, and C represent a different functional region. The pattern may be repeated once, twice, or 3 or more times. Generally, any 5' UTR sequence and any 3' UTR sequence may be combined in a particular exogenous mRNA. Other non-coding sequences may also be used as regions or subregions within an exogenous mRNA. For example, introns or portions of introns sequences may be incorporated into regions of nucleic acid of the disclosure. In certain embodiments, inclusion of intronic sequences may increase protein production as well as nucleic acid levels. 7. Length In certain embodiments, an exogenous mRNA includes 200 to 3,000 nucleotides. For example, an exogenous mRNA may include 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides. Unless otherwise indicated, the exogenous mRNA comprises contiguous nucleosides. 8. Preparation In some embodiments, the RNA transcript is generated using a non-amplified, linearized DNA template in an in vitro transcription reaction to generate the RNA transcript. In some embodiments, the template DNA is isolated DNA. In some embodiments, the template DNA is cDNA. In some embodiments, the cDNA is formed by reverse transcription of a RNA polynucleotide. An in vitro transcription system typically comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase. The NTPs may be purchased from a supplier or may be synthesized as described herein according to methods known in the art. In vitro transcription of RNA is known in the art and is described in International Publication WO 2014 / 152027, which is incorporated by reference herein in its entirety. In certain embodiments, the exogenous mRNA is prepared in accordance with any one or more of the methods described in WO 2018 / 053209 and WO 2019 / 036682, each of which is incorporated by reference herein. 9. Purification Purification of the exogenous mRNA described herein may comprise steps including nucleic acid clean-up, quality assurance, and quality control. Clean-up may be performed by methods known in the arts such as, but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNATM oligo-T capture probes (EXIQON® Inc, Vedbaek, Denmark) or HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term “purified” when used in relation to exogenous mRNA refers to one that is separated from at least one contaminant. A “contaminant” is any substance that makes another unfit, impure or inferior. Thus, a purified nucleic acid (e.g., DNA and RNA) is present in a form or setting different from that in which it is found in nature, or a form or setting different from that which existed prior to subjecting it to a treatment or purification method. A quality assurance and / or quality control check may be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC. In some embodiments, the nucleic acids may be sequenced by methods including, but not limited to reverse-transcriptase-PCR. II. Certain Oligonucleotides In certain embodiments, provided herein are oligonucleotides, which consist of linked nucleosides. In certain embodiments, a single guide comprises an oligonucleotide. In certain embodiments, a dual guide comprises two oligonucleotides. Oligonucleotides may be unmodified oligonucleotides or may be modified oligonucleotides. Modified oligonucleotides comprise at least one modification relative to unmodified nucleic acids. That is, modified oligonucleotides comprise at least one modified nucleoside (comprising a modified sugar moiety and / or a modified nucleobase) and / or at least one modified internucleoside linkage. A. Certain Modified Nucleosides Modified nucleosides comprise a modified sugar moiety or a modified nucleobase or both a modifed sugar moiety and a modified nucleobase. 1. Certain Sugar Moieties In certain embodiments, modified sugar moieties are non-bicyclic modified sugar moieties. In certain embodiments, modified sugar moieties are bicyclic or tricyclic sugar moieties. In certain embodiments, modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of other types of modified sugar moieties. In certain embodiments, modified sugar moieties are non-bicyclic modified furanosyl sugar moieties comprising one or more substituent groups including, but not limited to, substituents at the 2′, 3′, 4′, and / or 5′ positions, as numbered below: In certain embodiments, the modified furanosyl sugar moiety is a ribosyl sugar moiety that is not an unmodified sugar moiety (i.e., an unmodified RNA or unmodified DNA moiety). In certain embodiments, the modified furanosyl sugar moiety is a xylosyl, lyxosyl, or arabinosyl sugar moiety. In certain embodiments, non-bicyclic modified sugar moieties are 2′-substituted sugar moieties and comprise a substituent group at the 2′-position. In certain embodiments one or more non-bridging substituent of non- bicyclic modified sugar moieties is branched. Examples of substituent groups suitable for the 2′-position of modified sugar moieties include but are not limited to: 2′-F, 2′-OCH3(“OMe” or “O-methyl”), and 2′- O(CH2)2OCH3(“MOE” or “O-methoxyethyl”). In certain embodiments, 2′-substituent groups are selected from: halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, C1-C10alkoxy, C1-C10substituted alkoxy, C1-C10alkyl, C1-C10substituted alkyl, S-alkyl, N(Rm)-alkyl, O-alkenyl, S-alkenyl, N(Rm)-alkenyl, O-alkynyl, S- alkynyl, N(Rm)-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2ON(Rm)(Rn) or OCH2C(=O)-N(Rm)(Rn), where each Rmand Rnis, independently, H, an amino protecting group, or substituted or unsubstituted C1-C10alkyl, O(CH2)2ON(CH3)2(“DMAOE”), or 2′- O(CH2)2O(CH2)2N(CH3)2(“DMAEOE”). Synthetic methods for some of these 2′-substituent groups may be found, e.g., in Cook et al., U.S.6,531,584; Cook et al., U.S.5,859,221; and Cook et al., U.S.6,005,087. Certain embodiments of these 2′-substituent groups may be further substituted with one or more substituent groups independently selected from: halo, cyano, ORa2, NO2, NH2, NHRa2, N(Ra2)2, C1-C6alkyl, C1-C6haloalkyl, C2-C6alkenyl, C2-C6alkynyl, C3-C10cycloalkyl, C6-C10aryl, heteroaryl, heterocyclyl, C1-C6alkylene-NH2, C1-C6alkylene-NHRa2, C1-C6alkylene-N(Ra2)2, C(O)Ra3, C(O)ORa3, C(O)NHRa3, C(O)N(C1-C4alkyl)Ra3, SRa3, S(O)2Ra3, S(O)Ra3, NHC(O)Ra3, N(C1-C4alkyl)C(O)Ra3, NHS(O)Ra3, N(C1-C4alkyl)S(O)Ra3, NHS(O)2Ra3, and N(C1-C4alkyl)S(O)2Ra3; each Ra2is independently selected from C2-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C3-C10cycloalkyl, C6-C10aryl, heteroaryl, and heterocyclyl; each Ra3is independently hydrogen, OH, C1-C6alkyl, C1-C6haloalkyl, C3-C10cycloalkyl, C6-C10aryl, heteroaryl, or heterocyclyl. In certain embodiments, a sugar moiety comprises two of the above substituents at the 2′-position. In certain embodiments, a sugar moiety comprises a 2′-fluoro and a second 2′-substituent. In certain embodiments, a 2′-substituted sugar moiety comprises a non-bridging 2′-substituent group selected from: F, NH2, N3, OCF3,OCH3, O(CH2)3NH2, CH2CH=CH2, OCH2CH=CH2, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(Rm)(Rn), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamide (OCH2C(=O)- N(Rm)(Rn)), where each Rmand Rnis, independently, H, an amino protecting group, or substituted or unsubstituted C1-C10alkyl. In certain embodiments, a 2′-substituted sugar moiety comprises a non-bridging 2′-substituent group selected from: F, OCF3,OCH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(CH3)2, O(CH2)2O(CH2)2N(CH3)2, O(CH2)2ON(CH3)2(“DMAOE”), O(CH2)2O(CH2)2N(CH3)2(“DMAEOE”), and OCH2C(=O)-N(H)CH3(“NMA”). In certain embodiments, a 2′-substituted sugar moiety comprises a 2′-substituent group selected from: F, OCH3, and OCH2CH2OCH3. In certain embodiments, modified furanosyl sugar moieties and nucleosides incorporating such modified furanosyl sugar moieties are further defined by stereochemical configuration. For example, a 2′- deoxyfuranosyl sugar moiety (i.e., 2′-(H)H furanosyl sugar moiety) may be in seven isomeric configurations other than the naturally occurring β-D-deoxyribosyl configuration. Such modified sugar moieties are described in, e.g., WO 2020 / 072991, incorporated by reference herein. A 2′-modified sugar moiety has an additional stereocenter at the 2′-position relative to a 2′-deoxyfuranosyl sugar moiety; therefore, such sugar moieties have a total of sixteen possible stereochemical configurations. Modified furanosyl sugar moieties described herein are in the β-D-ribosyl stereochemical configuration unless otherwise specified. In certain embodiments, non-bicyclic modified sugar moieties comprise a substituent group at the 4′- position. Examples of substituent groups suitable for the 4′-position of modified sugar moieties include, but are not limited to, alkoxy (e.g., methoxy), alkyl, and those described in Manoharan et al., WO 2015 / 106128. In certain embodiments, non-bicyclic modified sugar moieties comprise a substituent group at the 3′- position. Examples of substituent groups suitable for the 3′-position of modified sugar moieties include, but are not limited to, alkoxy (e.g., methoxy), alkyl (e.g., methyl, ethyl). In certain embodiments, non-bicyclic modified sugar moieties comprise a substituent group at the 5′- position. Examples of substituent groups suitable for the 5′-position of modified sugar moieties include, but are not limited to, vinyl, alkoxy (e.g., methoxy), alkynyl, allyl, and alkyl (e.g., methyl (R or S), ethyl (R or S)). In certain embodiments, non-bicyclic modified sugar moieties comprise more than one non-bridging sugar substituent, for example, 2′-F-5′-methyl sugar moieties, such as described in Migawa et al., US 2010 / 0190837, which is incorporated herein by reference, or alternative 2′- and 5′-modified sugar moieties as described in Rajeev et al., US 2013 / 0203836. Certain modified sugar moieties are bicyclic sugar moieties and comprise a substituent that bridges two atoms of the furanosyl ring to form a second ring. In certain embodiments, the bicyclic sugar moiety comprises a bridge between the 4′ and the 2′ furanose ring atoms. Examples of such 4′ to 2′ bridging sugar substituents include, but are not limited to: 4′-CH2-2′, 4′-(CH2)2-2′, 4′-(CH2)3-2′, 4′-CH2-O-2′ (“LNA”), 4′- CH2-S-2′, 4′-(CH2)2-O-2′ (“ENA”), 4′-CH(CH3)-O-2′ (referred to as “constrained ethyl” or “cEt” when in the S configuration), 4′-CH2-O-CH2-2′, 4′-CH2-N(R)-2′, 4′-CH(CH2OCH3)-O-2′ (“constrained MOE” or “cMOE”) and analogs thereof, 4′-C(CH3)(CH3)-O-2′ and analogs thereof, 4′-CH2-N(OCH3)-2′ and analogs thereof, 4′-CH2-O-N(CH3)-2′, 4′-CH2-C(H)(CH3)-2′, 4′-CH2-C(=CH2)-2′ and analogs thereof, 4′-C(RaRb)- N(R)-O-2′, 4′-C(RaRb)-O-N(R)-2′, 4′-CH2-O-N(R)-2′, and 4′-CH2-N(R)-O-2′, wherein each R, Ra, and Rbis, independently, H, a protecting group, or C1-C12alkyl. Representative U.S. patents that teach the preparation of such bicyclic sugar moieties include, but are not limited to: Imanishi et al., U.S.7,427,672; Swayze et al., U.S.7,741,457; Swayze et al., U.S.8,022,193; Seth et al., U.S.8,278,283; Prakash et al., U.S.8,278,425; and Seth et al., U.S.8,278,426. 2.Certain Modified Nucleobases In certain embodiments, modified oligonucleotides comprise one or more nucleoside comprising an unmodified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more nucleoside comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more nucleoside that does not comprise a nucleobase, referred to as an abasic nucleoside. In certain embodiments, modified oligonucleotides comprise one or more inosine nucleosides (i.e., nucleosides comprising a hypoxanthine nucleobase). An “unmodified nucleobase” is adenine (A), thymine (T), cytosine (C), uracil (U), or guanine (G). A modified nucleobase is a group of atoms other than unmodified A, T, C, U, or G capable of pairing with at least one other nucleobase. A 5-methylcytosine is an example of a modified nucleobase. A universal base is a modified nucleobase that can pair with any one of the five unmodified nucleobases. In certain embodiments, modified nucleobases are selected from: 5-substituted pyrimidines, 6- azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and O-6 substituted purines. In certain embodiments, modified nucleobases are selected from: 5-methylcytosine, 2- aminopropyladenine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N- methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5- propynyl (-C ^C-CH3) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), N1-methylpseudouracil, 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo (particularly 5-bromo), 5-trifluoromethyl, 5-halouracil, and 5- halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7- deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N- benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1,3-diazaphenoxazine-2-one, 1,3-diazaphenothiazine-2-one and 9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza- adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, Crooke, S.T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and those disclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S.T., Ed., CRC Press, 2008, 163- 166 and 442-443. Publications that teach the preparation of certain of the above noted modified nucleobases, as well as other modified nucleobases include without limitation, Rogers et al., U.S.5,134,066 ; Benner et al., U.S. 5,432,272; Matteucci et al., U.S.5,502,177 ; Froehler et al., U.S.5,594,121 ; and Cook et al., U.S.5,681,941. B. Certain Modified Internucleoside Linkages The naturally occurring internucleoside linkage of RNA and DNA is a 3' to 5' phosphodiester linkage. In certain embodiments, nucleosides of modified oligonucleotides may be linked together using one or more modified internucleoside linkages. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiesters, which contain a phosphodiester bond (also referred to as unmodified or naturally occurring linkages), phosphotriesters, methylphosphonates, phosphoramidates, phosphorothioates, phosphonoacetates (“PACE”), thiophosphonoacetates (“Thio-PACE”) and phosphorodithioates. Representative non-phosphorus containing internucleoside linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester, thionocarbamate (-O- C(=O)(NH)-S-); siloxane (-O-SiH2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Modified internucleoside linkages, compared to naturally occurring phosphodiester internucleoside linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. In certain embodiments, internucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. Methods of preparation of phosphorous-containing and non-phosphorous-containing internucleoside linkages are well known to those skilled in the art. In certain embodiments, a modified internucleoside linkage is any of those described in WO2021 / 030778, incorporated by reference herein. In certain embodiments, a modified internucleoside linkage comprises the formula: wherein such internucleoside linking group of a modified oligonucleotide: X is selected from O or S; R1is selected from H, C1-C6alkyl, and substituted C1-C6alkyl; and T is selected from SO2R2, C(=O)R3, and P(=O)R4R5, wherein: R2is selected from an aryl, a substituted aryl, a heterocycle, a substituted heterocycle, an aromatic heterocycle, a substituted aromatic heterocycle, a diazole, a substituted diazole, a C1-C6alkoxy, C1-C6alkyl, C1-C6alkenyl, C1-C6alkynyl, substituted C1-C6alkyl, substituted C1-C6alkenyl substituted C1-C6alkynyl, and a linker; R3is selected from an aryl, a substituted aryl, CH3, N(CH3)2, OCH3and a linker; R4is selected from OCH3, OH, C1-C6alkyl, substituted C1-C6alkyl and a linker; and R5is selected from OCH3, OH, C1-C6alkyl, and substituted C1-C6alkyl. In certain embodiments, a modified internucleoside linkage comprises a mesyl phosphoramidate linking group having a formula: . In modified internucleoside linkage is a neutral internucleoside linkage. Neutral without limitation, phosphotriesters, methylphosphonates, MMI (3'- CH2-N(CH3)-O-5'), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5'), formacetal (3'-O- CH2-O-5'), methoxypropyl (MOP), and thioformacetal (3'-S-CH2-O-5'). Further neutral internucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y.S. Sanghvi and P.D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral internucleoside linkages include nonionic linkages comprising mixed N, O, S and CH2component parts. In certain embodiments, a modified internucleoside linakge is a standard length internucleoside linkage, and is represented by Formula Z1, Z2, or Z3 below. As used herein, a “standard length internucleoside linkage” refers to an internucleoside linkage that has a structure represented byFormula Z1, Z2, or Z3: wherein independently for each internucleoside linking group of Formula Z1, Z2, or Z3: each X1is independently selected from O and S; X2is selected from O, NR1, CH2, and S; X3is selected from O, NR1, CH2, and S; L is absent, NR1, N(R1)SO2, –N=, O, C1-C6alkylene, or C1-C6heteroalkylene; each R1is independently selected from H, C1-C6alkyl, and substituted C1-C6alkyl, or two R1on the same atom together form =O; and R2is selected from -OH, -SH, C1-C22alkyl, substituted C1-C22alkyl, C2-C22alkenyl, substituted C2-C22alkenyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, heteroaryl, substituted heteroaryl, aryl, and substituted aryl; wherein when a group is substituted, it comprises one or more substituent groups selected from halo, -OH, -N(R1)2, -O-C1-C6alkyl, C1-C22alkyl, C2-C22alkenyl, cycloalkyl, heterocyclyl, heteroaryl, and aryl. In certain embodiments, a extended internucleoside linkage is an extended length internucleoside linkage. For example, in certain embodiments, an extended internucleoside linkage forms when two functional groups on two oligonucleotides react to link the two oligonucleotides together to form a single oligonucleotide comprising an extended internucleoside linakge. One such reaction is the click reaction between bicyclo[6.1.0]nonyne and an azide. Additional linkers suitable for joining two oligonucleotides with Click chemistry are described in “Click Chemistry for Biotechnology and Materials Science” Ed. Joerg Lahann, Wiley 2009. Further examples of linking chemistry include an inverse electron demand Diels-Alder reaction, e.g., as described in Argamunt et al., J. Org. Chem.2020, 85, 10, 6593–6604, Sarrett et al., Nat. Protocols 2021, 16, 3348–3381; Handula et al., Molecules, 2021, 26 (15), 4640, Wiessler et al., Int. J. Med. Sci.2010, 7 (1), 19–28; copper-catalyzed azide-alkyne cycloaddition (CuAAC) see, e.g., S. I. Presolski, et al., J. Am. Chem. Soc.2010, 132, 14570–14576; D. Soriano Del Amo, et al., J. Am. Chem. Soc., 2010, 132, 16893–16899; Staudinger reaction, see, e.g., Saxon and C. R. Bertozzi, Science, 2000, 287, 2007–2010; B. L. Nilsson, et al., Org. Lett., 2000, 2, 1939–1941, E. Saxon, et al., Org. Lett., 2000, 2, 2141–2143; formation of hydrazones and oximes, see, e.g., J. Y. Axup, et al., Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 16101– 16106; photoclick reactions, see, e.g., W. Song, et al., Angew. Chem., Int. Ed., 2008, 47, 2832–2835, A. Herner and Q. Lin, Top. Curr. Chem., 2016, 374, 1; strain-promoted alkyne-nitrone cycloaddition (SPANC) reactions, see, e.g., D. A. MacKenzie, et al., Curr. Opin. Chem. Biol., 2014, 21, 81–88; transition metal catalyzed cross coupling, see, e.g., M. Chalker, et al., J. Am. Chem. Soc., 2009, 131, 16346–16347; nucleophilic additions, in particular, of a thiol to a maleimide, see, e.g., Kang et al., Chem. Sci., 2021, 12, 13613-13647, Bernardim et al., Nat. Comm.2016, 7, 13128, Jain et al., Pharm. Res.2015, 32 (11), 3526- 3540. C. Certain Motifs In certain embodiments, guides (modified oligonucleotides) comprise one or more modified nucleoside(s) comprising a modified sugar moiety. In certain embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more modified internucleoside linkage. In such embodiments, the modified, unmodified, and differently modified sugar moieties, nucleobases, and / or internucleoside linkages of a modified oligonucleotide define a pattern or motif. In certain embodiments, the patterns of sugar moieties, nucleobases, and internucleoside linkages are each independent of one another. Thus, a modified oligonucleotide may be described by its sugar motif, nucleobase motif and / or internucleoside linkage motif (as used herein, nucleobase motif describes the modifications to the nucleobases independent of the sequence of nucleobases). 1. Certain Sugar Motifs In certain embodiments, oligonucleotides comprise one or more type of modified sugar and / or unmodified sugar moiety arranged along the oligonucleotide or region thereof in a defined pattern or sugar motif. In certain instances, such sugar motifs include but are not limited to any of the sugar modifications discussed herein. Generally, the guide comprises unmodified RNA nucleosides, and optionally one or more modified nucleosides as described herein. In certain embodiments, the guide comprises or consists of a modified oligonucleotide. In certain embodiments, the 5’-most nucleoside of a modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, the 3’-most nucleoside of a modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, at least 1, 2, 3, 4, or 5 of the 5’-most five nucleosides of a modified oligonucleotide comprise a modified sugar moiety. In certain embodiments, at least 1, 2, 3, 4, or 5 of the 3’-most five nucleosides of a modified oligonucleotide comprise a modified sugar moiety. In certain embodiments, the above modifications (sugar, nucleobase, internucleoside linkage) are incorporated into a modified oligonucleotide. In certain embodiments, modified oligonucleotides are characterized by their modification motifs and overall lengths as described herein, and such parameters are each independent of one another. Unless otherwise indicated, all modifications are independent of nucleobase sequence. In certain embodiments, a modified oligonucleotide is a guide. In certain embodiments, the guide has a sugar moiety, nucleobase, and / or internucleoside linkage motif described in any of the following references, each of which are hereby incorporated by reference: WO 2014 / 144761, WO 2015 / 026885, WO 2016 / 089433, WO 2016 / 100951, WO 2016 / 123230, WO 2016 / 164356, WO 2017 / 004261, WO 2017 / 004279, WO 2017 / 068377, WO 2017 / 136794, WO 2017 / 181107, WO 2017 / 214460, WO 2018 / 009822, WO 2018 / 057946, WO 2018 / 098383, WO 2018 / 107028, WO 2018 / 125964, WO 2019 / 084664, WO 2019 / 147275, WO 2019 / 147743, WO 2019 / 183000, WO 2019 / 237069, WO 2021 / 119006, WO 2021 / 119275, WO 2021 / 125840, WO 2021 / 207651 , WO 2021 / 207711, WO 2022 / 086846. 2. Certain Nucleobase Motifs In certain embodiments, oligonucleotides comprise modified and / or unmodified nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or motif. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases are modified. In certain embodiments, each purine or each pyrimidine is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, each thymine is modified. In certain embodiments, each uracil is modified. In certain embodiments, each cytosine is modified. In certain embodiments, some or all of the cytosine nucleobases in a modified oligonucleotide are 5-methyl cytosines. In certain embodiments, all of the cytosine nucleobases are 5-methyl cytosines and all of the other nucleobases of the modified oligonucleotide are unmodified nucleobases. In certain embodiments, modified oligonucleotides comprise a block of modified nucleobases. In certain such embodiments, the block is at the 3’-end of the oligonucleotide. In certain embodiments the block is within 3 nucleosides of the 3’-end of the oligonucleotide. In certain embodiments, the block is at the 5’- end of the oligonucleotide. In certain embodiments the block is within 3 nucleosides of the 5’-end of the oligonucleotide. In certain embodiments, the modified nucleobase is selected from pseudouracil, 2-6 diamino purine, 2-thiouracil, 4-thiouracil, 2-aminoadenine, 6-methyladenine, hypoxanthine, or 5-methylcytosine. 3. Certain Internucleoside Linkage Motifs In certain embodiments, oligonucleotides comprise modified and / or unmodified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or motif. In certain embodiments, each internucleoside linking group is a phosphodiester internucleoside linkage (P=O). In certain embodiments, each internucleoside linking group of a modified oligonucleotide is a phosphorothioate internucleoside linkage (P=S). In certain embodiments, each internucleoside linkage of a modified oligonucleotide is independently selected from a phosphorothioate internucleoside linkage and phosphodiester internucleoside linkage. In certain embodiments, each internucleoside linkage of a modified oligonucleotide is independently selected from a phosphorothioate internucleoside linkage, phosphodiester internucleoside linkage, and a phosphonoacetate internucleoside linkage. In certain embodiments, the 5’-most internucleoside linkage of a modified oligonucleotide is a modified internucleoside linkage. In certain embodiments, the 3’-most internucleoside linkage of the guide is a modified internucleoside linkage. In certain embodiments, at least 1, 2, 3, 4, or 5 of the 5’-most five internucleoside linkages of a modified oligonucleotide are modified internucleoside linkages. In certain embodiments, at least 1, 2, 3, 4, or 5 of the 3’-most five internucleoside linkages of a modified oligonucleotide are modified internucleoside linkages. In certain embodiments, the modified internucleoside linkage is a phosphorothioate internucleoside linkage. 4.Certain Guide Chemical Modification Motifs A guide can comprise any of the modifications commonly applied to modified oligonucleotides described herein, including modified sugar moieties, modified internucleoside linkages, and modified nucleobases. In certain embodiments, a guide is modified at the 3’-end, the 5’-end, or both the 3’-end and 5’- end. In certain embodiments, the three nucleosides at the 3’-end and the three nucleosides at the 5’-end are 2’-OMe nucleosides, and the remainder of the nucleosides of the guide are unmodified RNA nucleosides. In certain embodiments, each nucleoside within the 5’-most stem loop is an unmodified RNA nucleoside. In certain embodiments, In certain embodiments, each internucleoside linkage of the guide is an unmodified phosphodiester linkage. In certain embodiments, the guide comprises one or more modified internucleoside linkages. In certain embodiments, the guide comprises one or more phosphorothioate internucleoside linkages. In certain embodiments, the 3’-most one, two, three, four, or five internucleoside linkages are modified internucleoside linkages. In certain embodiments, the 5’-most one, two, three, four, or five internucleoside linkages are modified internucleoside linkages. In certain embodiments, the 3’-most and 5’-most one, two, three, four, or five internucleoside linkages are modified internucleoside linkages, and the remainder of the linkages are unmodified phosphodiester internucleoside linkages. In certain embodiments, the 3’-most one, two, three, four, or five internucleoside linkages are phosphorothioate internucleoside linkages. In certain embodiments, the 5’-most one, two, three, four, or five internucleoside linkages are phosphorothioate internucleoside linkages. In certain embodiments, the 3’-most and 5’-most one, two, three, four, or five internucleoside linkages are phosphorothioate internucleoside linkages, and the remainder of the linkages are unmodified phosphodiester internucleoside linkages. In certain embodiments, the three 3’-most and the three 5’-most internucleoside linkages are phosphorothioate internucleoside linkages, and the remainder of the linkages are unmodified phosphodiester internucleoside linkages. D. Certain Lengths In certain embodiments, oligonucleotides (including guides) can have any of a variety of ranges of lengths. In certain embodiments, oligonucleotides consist of X to Y linked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number nucleosides in the range. In certain such embodiments, X and Y are each independently selected from 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, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180 ; provided that X≤Y. For example, in certain embodiments, oligonucleotides consist of 50-180, 80-180, 90-180, 100-180, 110-180, 120-180, 130-180, 140-180, 150-180, 160-180, 160-170, 90-100,100-110, 110-120, 120-130, 130- 140, 140-150, 100-140, 110-140, 110-130, 120-140, or 120-150 linked nucleosides. In certain embodiments, a single guide oligonucleotide consists of 50-180, 80-180, 90-180, 100- 180, 110-180, 120-180, 130-180, 140-180, 150-180, 160-180, 160-170, 90-100,100-110, 110-120, 120-130, 130-140, 140-150, 100-140, 110-140, 110-130, 120-140, or 120-150 linked nucleosides. In certain embodiments, the target-recognition region of a guide consists of 15-30, 18-26, 18-24, 18-22, 20-26, 20-24, 20-22, 21-23, 22-23, 20, 21, 22, or 23 linked nucleosides. In certain embodiments, the protein-recognition region of a guide consists of 60-130, 60-120, 60-110, 60-100, 70-130, 70-120, 70-110, 70-100, 70-90, 70-80, 80-130, 80-120, 80-110, 80-100, 80-90, 90-130, 90-120, 90- 110, 90-100, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 linked nucleosides. In certain embodiments, the protein-recognition region of a guide represents a truncated version of a native guide for the corresponding Cas protein. In certain embodiments, the guide is truncated at the 3’ end. In certain embodiments, the guide is shortened by removing nucleotides from stem loop and / or hairpin structures, such as removing one base pair from a hairpin to shorten the hairpin. In certain embodiments, the truncated version is at least 5, at least 10, at least 15, or at least 20 nucleotides shorter than the native guide. In certain embodiments, the protein- recognition region of a guide represents a length-extended version of a native guide for the corresponding Cas protein. In certain embodiments, additional nucleotides are added at the 3’ end. In certain embodiments, additional nucleotides are inserted within loops and / or hairpins of the native guide. E. Nucleobase Sequence In certain embodiments, oligonucleotides (unmodified or modified oligonucleotides) are further described by their nucleobase sequence. In certain embodiments oligonucleotides have a nucleobase sequence that is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid. In certain such embodiments, a region of an oligonucleotide has a nucleobase sequence that is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid. In certain embodiments, the nucleobase sequence of a region or entire length of an oligonucleotide is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the second oligonucleotide or nucleic acid, such as a target nucleic acid. F. Secondary Structure In certain embodiments, oligonucleotides or portions thereof adopt a defined secondary structure. In certain embodiments, secondary structure is determined by Watson-Crick base pairing, Hoogsteen base pairing, and / or non-canonical base pairing interactions. The secondary structure of an oligonucleotide sequence can be predicted using standard software, such as the Vienna RNA package RNAfold (http: / / rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi; see Lorenz, et al., Algorithms for Molecular Biology, 6:126, 2011). In certain embodiments, an oligonucleotide may have more than one predicted secondary structure. A guide oligonucleotide has secondary structure, at least some portion of which is important in forming contacts with its cognate Cas protein. For example, the guide secondary structure features for binding to spCas9 have been described in detail based on both empirical evidence (see, e.g., Zhang, et al., ChemPlusChem, 2021 and Dong, et al., Curr. Opinion in Biotech., 2022) and the crystal structures of a guide- SpCas9-target DNA complex (see Nishimasu, et al., Cell, 2014). By adding a tetraloop to the duplex that forms between the native crRNA and native tracrRNA of SpCas9, the functions of the crRNA and tracrRNA are combined into a single guide RNA. The same tetraloop can be added to join the crRNA and tracrRNA of a novel type II Cas protein. A single guide for SpCas9 has conserved structural features within its protein- binding region, including the lower stem, the upper stem, the tetraloop, the bulge, the nexus region (a hairpin with a 5 nucleotide loop), and two hairpins at the 3’-end (Figure 6). The guide RNA for certain novel type II Cas proteins have been shown to have unique secondary structures with common features in the protein- binding region, including a duplex region (dual guides) or a stem loop (single guides) and one or more hairpins at the 3’ end. The 5’-most stem loop feature of a guide RNA is also known as the Repeat-Anti- repeat region (R-AR). In certain embodiments, a guide consists of an oligonucleotide that can be represented by the following formula: T1-D-X-S1a-B1-S1b-H1-S1b’-B2-S1a’-L1-S2-H2-S2’-L2-S3-H3-S3’-(L3-S4-H4-S4’)n-(L4-S5-H5- S5’)m-T2; wherein: ^ D consists of 15-25 linked nucleosides, wherein the nucleobase sequence of D is complementary to the nucleobase sequence of a target DNA; ^ X is absent, or consists of 1-2 linked nucleosides that are not complementary to the sequence of the target DNA; ^ each of T1 and T2 is absent or independently consists of 1-30 linked nucleosides; ^ S1aand S1a’ each consist of 6-12 linked nucleosides, wherein the nucleobase sequence of S1ais 100% complementary to the nucleobase sequence of S1a’; ^ S1band S1b’ each consist of 2-14 linked nucleosides, wherein the nucleobase sequence of S1bis 100% complementary to the nucleobase sequence of S1b’; ^ S2 and S2’ each consist of 2-6 linked nucleosides, wherein the nucleobase sequence of S2 is 100% complementary to the nucleobase sequence of S2’; ^ S3 and S3’ each consist of 2-12 linked nucleosides, wherein the nucleobase sequence of S3 is 100% complementary to the nucleobase sequence of S3’; ^ S4and S4’ each consist of 4-14 linked nucleosides, wherein the nucleobase sequence of S4is 100% complementary to the nucleobase sequence of S4’; ^ S5and S5’ each consist of 4-11 linked nucleosides, wherein the nucleobase sequence of S5is 100% complementary to the nucleobase sequence of S5’; ^ n is 0 or 1; ^ m is 0 or 1; ^ B1 consists of 0-3 linked nucleosides; ^ B2consists of 0-4 linked nucleosides; ^ wherein B1 is not equal to B2 unless both consist of 0 linked nucleosides; ^ each of H1, H2, H3, H4, and H5independently consists of 3-6 linked nucleosides; ^ L1 consists of 2-3 linked nucleosides; ^ each of L2, L3, and L4 independently consists of 0-10 linked nucleosides. ^ In certain embodiments, a guide specific for an ION-CAS protein is represented by the following formula: T1-D-X-S1a-B1-S1b-H1-S1b’-B2-S1a’-L1-S2-H2-S2’-L2-S3-H3-S3’-(L3-S4-H4-S4’)n-(L4-S5-H5-S5’)m- T2; ^ wherein each variable is as defined in the table below. IONC IONC IONC IONC IONC IONC 10 m d 24 L3 0 0 x x 0 0 0 0 6 3 x x S4 & '5 5 x x 6 6 5 5 10 13 x x Guide secondary structure features that are responsible for the binding of the guide to the Cas enzyme include having at least one duplex region at the 5’ end of the protein-recognition region (including one or more stem-loops for a single guide) and two or more additional stem-loop and / or hairpin regions. A bulge in the 5’-most stem-loop is conserved among guides for most type II Cas proteins; however, guides with diverse predicted secondary structures have also been reported (see, e.g., Alexander et al., CRISPR J., 6(3): 261-277, 2023). In certain embodiments, the activity of a guide is not driven by a specific nucleobase sequence, but rather is driven by the formation of a specific RNA secondary structure. The sequence that creates this secondary structure can be altered. One common method of altering the sequence while maintaining the three dimensional structure required for binding of the guide to its cognate Cas is to flip base-pairs within a stem or hairpin, such as in an “A-U” flip. In such embodiments, a natural A-U base pair is replaced with a U-A base pair, swapping the position of the “A” and the “U” in the guide sequence. This can particularly improve editing efficiencies when the original guide contains four consecutive Us, a putative pol-III terminator (see, e.g. Chen et al. Cell. (2013); 155(7): 1479-1491). Another method of altering the sequence within a stem or hairpin involves the replacement of less stable A-U or U-A base pairs with more stable C-G or G-C base pairs to increase overall hairpin stability (see, e.g. Riesenberg, et al., Nat. Commun., 2022). In certain embodiments a natural hairpin or stem-loop structure of the guide molecule is extended or replaced by an extended stem-loop. It has been demonstrated in certain cases that extension of the stem can enhance the assembly of the guide with the CRISPR-Cas protein (Chen et al. Cell. (2013); 155(7): 1479- 1491). In certain embodiments the stem of the stem loop is extended by at least 1, 2, 3, 4, 5 or more complementary base pairs (i.e. corresponding to the addition of 2, 4, 6, 8, 10 or more nucleotides in the guide). In certain embodiments, these are located at the end of the stem, adjacent to the loop of the stem loop. In certain embodiments, a natural hairpin or stem-loop structure of the guide is shortened. In certain such embodiments, the stem of the stem-loop is shortened by 1, 2, or 3 complementary base pairs (i.e. corresponding to the removal of 2, 4, or 6 nucleotides in the guide). It has also been demonstrated that, for SpCas9 guides, the loop region of the upper stem or hairpin 1 can be replaced by a longer loop comprising an aptamer. In certain embodiments, the aptamer binds to an effector protein. In certain embodiments, the aptamer binds to MS2, PP7, com, or boxB. In certain embodiments, an aptamer can be appended at the 3’ end of a guide, or it can be inserted into the guide as a new hairpin beyond the 3’-most hairpin of the native guide (see, e.g., Zalatan, et al, Cell, 2015). In certain embodiments, a guide can be modified such that it is activated only by an external chemical signal. In one such embodiment, a guide includes a 5’-extension, appended to the 5’-end of the target-recognition region of the guide, which forms a hairpin duplex with the target-recognition region. This 5’-extension also includes a theophylline aptamer. In this system, the addition of theophylline causes cleavage of the 5’-extension, and reveals the target-recognition region of the guide. In certain embodiments, a guide can be modified such that it can be deactivated by an external chemical signal. In one such embodiment, a guide includes a theophylline aptamer incorporated into the loop region of the first stem loop as well as a second theophylline aptamer incorporated into the loop of the second hairpin. The guide works for Cas9 cleavage of a target, but is cleaved when theophylline is added to the system. Various other external control systems for guides have been described (see, e.g., Zhang, et al., ChemPlusChem, 2021). C. Guide Chemical Modifications A guide can comprise any of the modifications commonly applied to modified oligonucleotides described herein, including modified sugar moieties, modified internucleoside linkages, and modified nucleobases. In certain embodiments, a guide is modified at the 3’-end, the 5’-end, or both the 3’-end and 5’- end. In certain embodiments, the three nucleosides at the 3’-end and the three nucleosides at the 5’-end are 2’-OMe nucleosides, and the remainder of the nucleosides of the guide are unmodified RNA nucleosides. In certain embodiments, each nucleoside within the 5’-most stem loop is an unmodified RNA nucleoside. In certain embodiments, In certain embodiments, each internucleoside linkage of the guide is an unmodified phosphodiester linkage. In certain embodiments, the guide comprises one or more modified internucleoside linkages. In certain embodiments, the guide comprises one or more phosphorothioate internucleoside linkages. In certain embodiments, the 3’-most one, two, three, four, or five internucleoside linkages are modified internucleoside linkages. In certain embodiments, the 5’-most one, two, three, four, or five internucleoside linkages are modified internucleoside linkages. In certain embodiments, the 3’-most and 5’-most one, two, three, four, or five internucleoside linkages are modified internucleoside linkages, and the remainder of the linkages are unmodified phosphodiester internucleoside linkages. In certain embodiments, the 3’-most one, two, three, four, or five internucleoside linkages are phosphorothioate internucleoside linkages. In certain embodiments, the 5’-most one, two, three, four, or five internucleoside linkages are phosphorothioate internucleoside linkages. In certain embodiments, the 3’-most and 5’-most one, two, three, four, or five internucleoside linkages are phosphorothioate internucleoside linkages, and the remainder of the linkages are unmodified phosphodiester internucleoside linkages. In certain embodiments, the three 3’-most and the three 5’-most internucleoside linkages are phosphorothioate internucleoside linkages, and the remainder of the linkages are unmodified phosphodiester internucleoside linkages. III. Certain Editing Systems In certain embodiments, an editing system comprises an guided nucleic acid binding agent and a guide. In certain embodiments, a guide consists of a single oligonucleotide and is a single guide. In certain embodiments, a guide consists of two oligonucleotides and is a dual guide. In certain embodiments, a guide comprises a target-recognition region and a protein-recognition region. In certain embodiments, the guide is a single guide consisting of an oligonucleotide that comprises a target-recognition region and a protein- recognition region. In certain embodiments, the guide is a dual guide, wherein a first oligonucleotide comprises the target-recognition region and a first component of a protein-recognition region, and a second oligonucleotide comprises the second component of a protein-recognition region. In certain such embodiments, the first oligonucleotide is a “crRNA” and the second oligonucleotide is a “tracrRNA”. In certain embodiments, the target-recognition region is a region of an oligonucleotide that has a sequence complementary to an equal length portion of a target sequence within the complementary strand of a target DNA In certain embodiments, an editing system comprises a guided nucleic acid binding agent and a guide. In certain embodiments, the guided nucleic acid agent comprises a Cas protein. In certain embodiments, the guided nucleic acid agent consists of a Cas protein. In certain embodiments, the Cas protein has an amino acid sequence of any of SEQ ID NOs: 4-23 or 231-323. In certain embodiments, the Cas protein is encoded by DNA having the nucleobase sequence of any of SEQ ID NOs: 24-43 or 324-416. In certain embodiments, the guide is an oligonucleotide. In certain embodiments, the guide consists of two oligonucleotides. In certain embodiments, the guide comprises a protein-recognition region having a nucleobase sequence selected from any of SEQ ID NOs: 121-141, 510-602, 961-1408, 2825, 2827-2833. In certain embodiments, an editing system comprises a guided nucleic acid agent comprising a guided nucleic acid protein and a guide, wherein the protein-recognition region of the guide has a nucleobase sequence that binds to the guided nucleic acid protein. In certain embodiments, the guided nucleic acid agent comprises an ION-Cas protein. In certain embodiments, the editing system comprises an ION-Cas protein having an amino acid sequence of SEQ ID NO: 12 and a guide comprising a protein-recognition region having a nucleobase sequence of SEQ ID NO: 129 or 130. In certain embodiments, the editing system comprises an ION-Cas protein having an amino acid sequence of SEQ ID NO: 13 and a guide comprising a protein-recognition region having a nucleobase sequence of SEQ ID NO: 131, 961-964, 1016-1020, 1052-1070, 1313-1314, or 2825. In certain embodiments, the editing system comprises an ION-Cas protein having an amino acid sequence of SEQ ID NO: 20 and a guide comprising a protein-recognition region having a nucleobase sequence of SEQ ID NO: 139. In certain embodiments, the editing system comprises an ION-Cas protein having an amino acid sequence of SEQ ID NO: 241 and a guide comprising a protein-recognition region having a nucleobase sequence of SEQ ID NO: 520, 965-968, 1071-1094, or 1315-1318. In certain embodiments, the editing system comprises an ION-Cas protein having an amino acid sequence of SEQ ID NO: 267 and a guide comprising a protein-recognition region having a nucleobase sequence of SEQ ID NO: 546, 973-976, 1021-1023, 1095-1126, or 1319-1342. In certain embodiments, the editing system comprises an ION-Cas protein having an amino acid sequence of SEQ ID NO: 275 and a guide comprising a protein- recognition region having a nucleobase sequence of SEQ ID NO: 554, 981-984, 1024-1026, 1127-1152, or 1343-1361. In certain embodiments, the editing system comprises an ION-Cas protein having an amino acid sequence of SEQ ID NO: 284 and a guide comprising a protein-recognition region having a nucleobase sequence of SEQ ID NO: 562, 985-989, 1027-1029, 1153-1182, or 1362-1390. In certain embodiments, the editing system comprises an ION-Cas protein having an amino acid sequence of SEQ ID NO: 289 and a guide comprising a protein-recognition region having a nucleobase sequence of SEQ ID NO: 568, 990-993, 1030-1034, 1183-1206, or 1392-1393. In certain embodiments, the editing system comprises an ION-Cas protein having an amino acid sequence of SEQ ID NO: 314 and a guide comprising a protein-recognition region having a nucleobase sequence of SEQ ID NO: 593, 1006-1010, 1272-1292, 1394-1408, 2827- 2833. In certain embodiments, the editing system comprises an ION-Cas protein having an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 12 and a guide comprising a protein-recognition region having a nucleobase sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 129 or 130 In certain embodiments, the editing system comprises an ION-Cas protein having an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 13 and a guide comprising a protein-recognition region having a nucleobase sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 131, 961-964, 1016-1020, 1052-1070, 1313-1314, or 2825. In certain embodiments, the editing system comprises an ION-Cas protein having an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 20 and a guide comprising a protein-recognition region having a nucleobase sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 141. In certain embodiments, the editing system comprises an ION-Cas protein having an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 241 and a guide comprising a protein-recognition region having a nucleobase sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of SEQ ID NO: 520, 965-968, 1071-1094, or 1315-1318. In certain embodiments, the editing system comprises an ION-Cas protein having an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 267 and a guide comprising a protein-recognition region having a nucleobase sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of SEQ ID NO: 546, 973-976, 1021-1023, 1095-1126, or 1319-1342. In certain embodiments, the editing system comprises an ION-Cas protein having an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 275 and a guide comprising a protein-recognition region having a nucleobase sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of SEQ ID NO: 554, 981-984, 1024-1026, 1127-1152, or 1343-1361. In certain embodiments, the editing system comprises an ION-Cas protein having an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 284 and a guide comprising a protein-recognition region having a nucleobase sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of SEQ ID NO: 562, 985-989, 1027-1029, 1153-1182, or 1362-1390. In certain embodiments, the editing system comprises an ION-Cas protein having an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 289 and a guide comprising a protein-recognition region having a nucleobase sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of SEQ ID NO: 568, 990-993, 1030-1034, 1183-1206, or 1392-1393. In certain embodiments, the editing system comprises an ION-Cas protein having an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 314 and a guide comprising a protein-recognition region having a nucleobase sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of SEQ ID NO: 593, 1006-1010, 1272-1292, 1394-1408, 2827-2833. IV. Certain Delivery Vehicles A. Lipid Nanoparticles ("LNP”) 1. Composition In certain embodiments, the LNP comprises a protonatable or cationic lipid, a neutral or zwitterionic lipid, a polymer-lipid, cholesterol or a derivative thereof, and optionally one or more excipients. The LNP at least partially encapsulates a cargo. In certain embodiments, the cargo comprises a nucleic acid, e.g., an exogenous mRNA. In certain embodiments, the cargo comprises a guide, e.g., an oligomeric agent. In certain embodiments, the cargo comprises an exogenous mRNA and a guide. In certain embodiments, the LNP is provided as a suspension in an aqueous medium. In certain embodiments, a pharmaceutical composition comprises an LNP at least partially encapsulating a cargo in an aqueous medium. In certain embodiments, the LNP comprises 30 to 70 mol % of a protonatable or cationic lipid; 5 to 30 mol % of a neutral or Zwitterionic lipid; 20 to 50 mol % of cholesterol or a derivative thereof; and 1 to 10 mol % of a polymer-lipid. In certain embodiments, the LNP comprises 40 to 60 mol % of a protonatable or cationic lipid; 5 to 15 mol % of a neutral or Zwitterionic lipid; 30 to 50 mol % of cholesterol or a derivative thereof; and 2 to 4 mol % of a polymer-lipid. The mol % of each of the protonatable or cationic lipid, a neutral or zwitterionic lipid, a polymer-lipid, cholesterol or a derivative thereof are determined together (“total lipid”) irrespective of any cargo or excipients. The LNP may be characterized by a molar ratio or mass ratio of a cargo (such as an exogenous mRNA or oligomeric agent) to total lipid. In certain embodiments, the lipid to cargo ratio is 20:1 to 1:1, 10:1 to 1:1, or 5:1 to 1:1 (mass:mass). The LNP composition may comprise one or more additional excipients, for example, a triglyceride, a surfactant, and / or a hydrophobic excipient such as a wax. Triglycerides include trimyristin (Dynasan 114), tripalmitin (Dynasan 116), or tristearin (Dynasan 118), Witeposol bases, glyceryl stearates (Imwitor 900), glyceryl behenates (Compritol 888 ATO), and glyceryl palmitostearates (Precirol ATO 5). Nonionic surfactants may include moieties such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters, nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers. Anionic surfactants include carboxylates, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. Specific surfactants include lecithin, Poloxamer 188, Poloxamer 407, Tyloxapol, Polysorbate 20, Polysorbate 60, Polysorbate 80, sodium cholate, sodium glycocholate, taurodeoxycholic acid sodium, butanol, butyric acid, cetylpyridinium chloride, sodium dodecyl sulfate, sodium oleate, polyvinyl alcohol, or Cremophor EL. Waxes include beeswax and cetyl palmitate. Other hydrophobic excipients include stearic acid, palmitic acid, behenic acid, Miglyol 812, and paraffin. a. Protonatable and Cationic Lipids In certain embodiments, provided is an LNP comprising a protonatable and / or cationic lipid. As used herein, a “protonatable lipid” is a lipid-comprising compound which is substantially protonated at or below physiological pH (e.g., pH 7-7.5, or pH 7.4). As used herein, a “cationic lipid” is a lipid-comprising compound that carries a nonexchangeable net positive charge. A lipid is generally a hydrophobic moiety, which may comprise be a hydrocarbon chain. It is believed that design of a protonatable and / or cationic lipid may enhance endosomal drug escape. Thus it is believed that, in vivo, delivery capacity of an LNP may benefit from electrostatic charge interactions with the endosome and subsequent LNP destabilization to release the cargo. It is believed that a protonatable lipid can aid delivery by either (i) protonating in mildly acidic environments and acquire positive charges and / or (ii) incorporating pH-labile groups that cleave or change conformation in a low pH environment. Such pH-sensitive lipids therefore help enhance the charge-based uptake of liposomes in target cells or trigger cargo release by destabilizing liposome membranes. A cationic lipid may be characterized by an inverted cubic or hexagonal liquid crystalline phase. Fusogenicity is a property which describes the ability of a lipid or multi-lipid construct to join with a lipid layer. See, e.g., Hashiba et al., Small Science, 3(1), 2023, 2200071; V. Gyanani, and R. Goswami, Pharmaceutics, 2023, 15(4): 1184. Lipid designs that are believed to impart fusogenic character to a lipid are (i) unsaturation in the lipid tails, (ii) branching in the lipid carbon chains, (iii) multi-tail lipids, e.g., 7C1 and G0-C14 lipids, see, e.g., Dahlman et al., Nat Nanotechnol.2014 Aug; 9(8): 648–655, and (iv) incorporation of polymer-lipids. It is believed that protonatable or cationic lipids comprising hydrocarbon chains including unsaturation may provide LNPs having higher fluidity. Example publications describing protonatable and / or cationic lipids include U.S, Patent Publication Nos.2006 / 0083780 and 2006 / 0240554; U.S. Pat. Nos. 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO 96 / 10390, the disclosures of each of which are herein incorporated by reference in their entirety. Example protonatable and / or cationic lipids include a protonatable or cationic head group, optionally include one or more linear hydrocarbon chains of 10-20 carbon atoms, and optionally one or more branched, optionally unsaturated, hydrocarbon chains of 12-30 carbon atoms, each of which is optionally interrupted by one or more functionalities selected from ester, ether, amine (e.g., tertiary amine), amide, carbonate, carbamate, urea, and disulfide. The protonatable or cationic lipid may comprise a branching moiety. In certain embodiments the branching moiety comprises a tertiary carbon atom, a quaternary carbon atom, a tertiary amine, a vicinal diol, an amide, a carbamate, or an acetal. In certain embodiments, the protonatable or cationic lipid is a protonatable lipid containing an amine (“protonatable amino lipid”), for example, a tertiary amine. It is believed that proton cycling of an amino group at disparate pH may provide additional stabilization of a negatively charged cargo (e.g., an exogenous mRNA), while promoting cargo release inside a low pH compartment in vivo (e.g., an endosome). In certain embodiments, the protonatable lipid has a pKa of the protonatable (e.g., amino) group in the range of about 4 to about 7. Such lipids will be largely surface neutralized at physiological pH. It is believed that this will reduce susceptibility to clearance. pKa measurements of lipids within lipid particles can be performed, for example, by using the fluorescent probe 2-(p-toluidino)-6-napthalene sulfonic acid (TNS), using methods described in Cullis et al., (1986) Chem Phys Lipids 40, 127-144. In certain embodiments, the protonatable or cationic lipid is DLin-MC3-DMA, ALC-0315, SM-102, or LP-01: b. Polymer-Lipids In certain embodiments provided is an LNP comprising a polymer-lipid. The polymer-lipid may be a polymer-functionalized lipid, in which the polymer and lipid (e.g., a hydrocarbon chain optionally interrupted by one or more intervening functionalities) are joined by covalent bonds with optional intervening atoms. The polymer-lipid optionally includes a branching moiety. The lipid may be, e.g., a hydrocarbon chain optionally interrupted by one or more intervening functionalities. The polymer-lipid generally includes an uncharged, hydrophilic moiety which is believed to limit aggregation, such as PEG, GMI, or ATTA, during formulation of an LNP. Thus, it is believed that a polymer-lipid may reduce aggregation when included in an LNP. In certain embodiments, the content of the polymer-lipid in the LNP is selected to reduce particle aggregation. Examples of polymer-lipids include polyethylene glycol (PEG)-modified lipids, monosialoganglioside GMI, and polyamide oligomers ("PAO") such as described in U.S. Pat. No.6,320,017. ATTA-lipids are described, e.g., in U.S. Patent No.6,320,017, and PEG-functionalized lipid are described, e.g., in U.S. Patent Nos. 5,820,873, 5,534,499 and 5,885,613. In certain embodiment, the polymer-lipid comprises one or more hydrocarbon chains that are interrupted by a biodegradable functional group, e.g., an ester. The polymer-lipid may comprise a branching moiety. In certain embodiments the branching moiety comprises a tertiary carbon atom, a quaternary carbon atom, a tertiary amine, a vicinal diol, an amide, a carbamate, or an acetal. The lipid and optional branching moiety is believed to influence associative strength of a polymer- lipid with the LNP. It is believed that at least three characteristics influence the rate of exchange: length of lipid chain, rigidity (e.g., as determined by saturation) of lipid chain, and size of the steric-barrier head group (see, e.g., U.S. Patent No.5,820,873). For some therapeutic applications it may be preferable for the PEG- functionalized lipid to be rapidly lost from the LNP in vivo, and thus the PEG-lipid would have relatively short lipid anchors. In other therapeutic applications it may be preferable for the nucleic acid-lipid particle to exhibit a longer plasma circulation lifetime and hence the PEG-lipid will possess relatively longer lipid anchors. It is believed that mPEG (mw2000)-diastearoylphosphatidylethanolamine (PEG-DSPE) will remain associated with an LNP for days in vivo. Other conjugates, such as PEG-CerC20 have similar staying capacity. PEG-CerC14, however, is believed to more rapidly exchange out of the formulation upon exposure to serum. In certain embodiments, the polymer-lipid is charge neutral. In certain embodiments, the polymer- lipid is Zwitterionic. The polymer may be a hydrophilic polymer, for example, a poly(ethylene glycol), also referred to as a poly(ethylene oxide) or PEG (“PEG-lipid”). In certain embodiments, PEG is an optionally substituted linear polymer of ethylene glycol or ethylene oxide. In certain embodiments, the PEG moiety is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In certain embodiments, the PEG moiety includes PEG copolymer such as PEG-polyurethane or PEG-polypropylene (see, e.g., J. Milton Harris, Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (1992)). In certain embodiments, the PEG moiety is a PEG homopolymer. In general, the length of the PEG chain may be represented by either a number indicating the number of repeating ethylene oxide units, or by a molecular weight in Daltons of the PEG portion of the polymer- lipid. In a particular example, PEG-DMG 2000 has the following structure: where 2000 is the approximate molecular weight in Daltons of the PEG portion of the molecule. The length of PEG chain may vary between individual lipids in a sample and thus may be expressed as an average number of repeating units or average molecular weight. In general, the length of the PEG chain may be represented by either a number indicating the number of repeating ethyl oxide units, or by a molecular weight in Daltons of the PEG portion of the polymer-lipid. c. Charge Neutral and Zwitterionic Lipids In certain embodiments, the LNP comprises a (charge) neutral or Zwitterionic lipid. The neutral or Zwitterionic lipid can generally be any lipid species which is uncharged or neutral zwitterionic form at physiological pH. Generally, the neutral or Zwitterionic lipid will include a polar head group and one or more (e.g., two) hydrophobic tail groups. Exemplary head groups include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and inositol. In certain embodiments, the neutral lipid comprises two hydrocarbon groups which are each optionally interrupted with a biodegradable moiety. Neutral or Zwitterionic lipids having a variety of acyl chain groups of varying chain length and degree of saturation are available or may be isolated or synthesized by well-known techniques. In certain embodiments, the neutral or Zwitterionic lipid comprises saturated fatty acids, or mono- or di-unsaturated fatty acids. Additionally, lipids having mixtures of saturated and unsaturated fatty acid chains can be used. In certain embodiments, a fatty acid is interrupted by a biodegradable moiety such as an ester. In certain embodiments, the neutral or Zwitterionic lipid comprises a phosphatidylcholine (PC), phosphatidylethanolamine (PE), glycerophospholipid, sphingophospholipid, sphingolipid, phosphono lipids, natural lecithins, or hydrogenated phospholipid. Such lipids include, for example diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. The selection of neutral or Zwitterionic lipid is generally guided by consideration of, e.g., LNP particle size and stability in circulation. In certain embodiments, the neutral or Zwitterionic lipid is a phospholipid. As used herein, a “phospholipid” refers to a lipid that includes a hydrophilic phosphate head group and one or more hydrophobic tail groups. In some embodiments, a phospholipid may facilitate fusion to a membrane. For example, a cationic phospholipid may interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane may allow delivery of the one or more components of the LNP, e.g., the cargo, through the membrane, e.g., into a cell. In certain embodiments, the neutral or Zwitterionic lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). d. Cholesterol and derivatives thereof Cholesterol is a ubiquitous structural membrane lipid and its role in a lipid composition depends on context. When combined with phospholipids with low gel-liquid crystalline phase transitions (Tm), cholesterol is believed to aid formation of a liquid-ordered phase which is characterized by increased bilayer thickness and membrane rigidity. It is believed that cholesterol and other linear and / or branched hydrocarbon- containing (e.g., low Tm) lipids combine such that the cross-sectional area of the lipid and cholesterol is lower than the sum of the individual cross-sectional areas. However, when combined with high Tm lipids, cholesterol is believed to increase membrane fluidity and provide narrower bilayers. In any case, cholesterol is believed to encourage a liquid-ordered phase. Additionally, cholesterol is believed to reduce the amount of surface-bound protein and improve circulation half-life. The amount of cholesterol in an LNP may be selected to match the in vivo membrane cholesterol content. In some embodiments, provided is an LNP comprising cholesterol. In certain embodiments, the cholesterol derivative is selected from β-sitosterol, β-sitosterol acetate, a β-sitosterol amino acid conjugate, fecosterol, ergosterol, 9,11-dehydroergosterol, campesterol, stigmasterol, brassicasterol, fucosterol, tomatidine, ursolic acid, daucosterol, cholesterol, 5-heptadecylresorcinol, cholesterol hemisuccinate, 6-keto-5α-hydroxycholesterol, 7α-hydroxycholesterol, 7β-hydroxycholesterol, 7- ketocholesterol, 7β,25-dihydroxycholesterol, 27-hydroxycholesterol, 25-hydroxycholesterol, 20α- hydroxycholesterol, 5α-cholestanol, 5β-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, 6-ketocholestanol, cholestery1-(4'-hydroxy)-butyl ether, 5α-cholestane, cholestenone, 5β-cholestanone, cholesteryl decanoate, vitamin D3, vitamin D2, calcipotriol, botulin, luperol, ursolic acid, oleanolic acid, DC-cholesterol, BHEM- cholesterol, cholesteryl oleate, or a combination thereof. See, e.g., Paunovska, K. et al., Adv Mater.2019 April; 31(14); Patel et al., Nat. Comm., (2020) 11:983; Ni et al., Nat. Comm.13, Article number: 4766 (2022); Kim et al., ACS Nano 2022, 16(9), 14792–14806. 2. LNP Formation The LNP can be formed by any method known in the art including, but not limited to, a continuous mixing method or a direct dilution process. In certain embodiments, provided is a method for preparing an LNP by a continuous mixing method. E.g., a process that includes providing an aqueous solution comprising a nucleic acid in a first reservoir, providing a lipid solution in a second reservoir, and mixing the aqueous solution with the lipid solution such that the organic lipid solution mixes with the aqueous solution so as to rapidly produce a LNP encapsulating the cargo. The lipid solution comprises a lower alcohol such as ethanol. This process and the apparatus for carrying this process are described in detail in U.S. Patent Publication No.2004 / 0142025, the disclosure of which is herein incorporated by reference in its entirety. By mixing the aqueous solution comprising a cargo with the organic lipid solution, the organic lipid solution undergoes a continuous stepwise dilution in the presence of the buffer solution (i.e., aqueous solution) to produce a lipid nanoparticle. In certain embodiments, provided is a method for preparing an LNP by a direct dilution process that includes forming a LNP solution and directly introducing the LNP solution into a collection vessel containing a controlled amount of dilution buffer. The collection vessel may include one or more elements configured to stir the contents of the collection vessel to facilitate dilution. In one aspect, the amount of dilution buffer present in the collection vessel is substantially equal to the volume of liposome solution introduced thereto. As a non-limiting example, a liposome solution in about 45% ethanol when introduced into the collection vessel containing an equal volume of dilution buffer will advantageously yield smaller particles. In certain embodiments, provided is a method for preparing an LNP by a direct dilution process in which a third reservoir containing dilution buffer is fluidly coupled to a second mixing region. In this embodiment, the LNP solution formed in a first mixing region is immediately and directly mixed with dilution buffer in the second mixing region. In preferred aspects, the second mixing region includes a T- connector arranged so that the LNP solution and the dilution buffer flows meet as opposing 180° flows; however, connectors providing shallower angles can be used. In one aspect, the flow rate of dilution buffer provided to the second mixing region is controlled to be substantially equal to the flow rate of liposome solution introduced thereto from the first mixing region. Such control of the dilution buffer flow rate may advantageously allow for small particle size formation at reduced concentrations. Processes and apparatuses for carrying out direct dilution processes are described in U.S. Patent Publication No.2007 / 0042031, the disclosure of which is herein incorporated by reference in its entirety. B. Viral Vectors In certain embodiments, a nucleic acid encoding an editing system, a guided nucleic acid binding agent, or a guide, may be introduced into a cell or organism through a vector. In certain embodiments In some cases, the vector is a plasmid, a minicircle, a CELiD, an adeno-associated virus (AAV) derived virion, or a lentivirus. Nonlimiting Disclosure and Incorporation by Reference Each of the literature and patent publications listed herein is incorporated by reference in its entirety. While certain compounds, compositions, and methods have been described herein with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references, GenBank accession numbers, ENSEMBL identifiers, and the like recited in the present application is incorporated herein by reference in its entirety. The sequence listing accompanying this filing identifies each nucleic acid sequence as either “RNA” or “DNA” as required; however, one of skill in the art will readily appreciate that designation of “RNA” or “DNA” to describe modified oligonucleotides is, in certain instances, arbitrary. For example, an oligonucleotide comprising a nucleoside comprising a 2’-OH sugar moiety and a thymine base could be described as a DNA having a modified sugar (i.e., 2’-OH in place of one 2’-H of DNA) or as an RNA having a modified base (i.e., thymine (5-methyl uracil) in place of an uracil of RNA); and certain nucleic acid compounds described herein comprise one or more nucleosides comprising modified sugar moieties having 2’-substituent(s) that are neither OH nor H. One of skill in the art will readily appreciate that labeling such nucleic acid compounds “RNA” or “DNA” does not alter or limit the description of such nucleic acid compounds. Herein, the description of compounds as having “the nucleobase sequence of” a SEQ ID NO. describes only the nucleobase sequence. Accordingly, absent additional description, such description of compounds by reference to a nucleobase sequence of a SEQ ID NO. does not limit sugar or internucleoside linkage modifications or presence or absence of additional substituents such as a conjugate group. Further, absent additional description, the nucleobases of a compound “having the nucleobase sequence of” a SEQ ID NO. include such compounds having modified forms of the identified nucleobases as described herein. Herein, sugar, internucleoside linkage, and nucleobase modifications may be indicated within a nucleotide or nucleobase sequence or may be indicated in text accompanying a sequence (e.g., in separate text that appears within or above or below a table of compounds). While effort has been made to accurately describe compounds in the accompanying sequence listing, should there be any discrepancies between a description in this specification and in the accompanying sequence listing, the description in the sequence listing is the accurate description. The compounds described herein include variations in which one or more atoms are replaced with a non-radioactive isotope or radioactive isotope of the indicated element. For example, compounds herein that comprise hydrogen atoms encompass all possible deuterium substitutions for each of the1H hydrogen atoms. Isotopic substitutions encompassed by the compounds herein include but are not limited to:2H or3H in place of1H,13C or14C in place of12C,15N in place of14N,17O or18O in place of16O, and33S,34S,35S, or36S in place of32S. In certain embodiments, non-radioactive isotopic substitutions may impart new properties on the oligomeric compound that are beneficial for use as a therapeutic or research tool. In certain embodiments, radioactive isotopic substitutions may make the compound suitable for research or diagnostic purposes such as imaging. EXAMPLES Example 1: Computational prediction and characterization of Cas proteins in HEK293 cells – expression and localization Metagenomes were assembled using publicly available datasets. CRISPR cassettes containing CRISPR array and nearby proteins were predicted by CRISPRFinder and CRISPRone and were classified into sub-classes and -types. For Class 2 type II CRISPR systems, protein domains were identified via Hidden Markov Models and sequences were aligned to Cas9 of Streptococcus pyogenes (SpCas9). In addition, tracrRNA were predicted by using the CRISPR array to predict the anti-repeat located at the 5’-end of tracrRNA and searching for rho-independent termination characterized by a GC-rich palindrome sequence followed by a run of U residues at the 3’-end of the tracrRNA.19 putative Cas proteins were identified. The corresponding Cas protein sequences and GeneArt (ThermoFisher) human codon optimized nucleic acid sequences are provided in the sequence listing, as indicated in the table below. The Cas protein DNA sequences were cloned into a pTwistCMVpuro vector by Twist Biosciences. In order to facilitate enzyme localization to nucleus, for each Cas protein, nucleoplasmin (SEQ ID NO: 3399) and c-Myc (SEQ ID NO: 3400) Nuclear Localization Signals (NLS) were appended at the N- and C- terminus, respectively. In addition, a HiBiT (Promega) tag was added to the protein C-terminus for expression detection. These additional elements were linked to the proteins via known protein linkers such as GGGS / (GS)3 / (GGGGS)3. The DNA sequences encoding the Cas proteins with NLS and HiBiT tag are provided in the sequence listing as indicated in the DNA sequences SEQ ID NO: 44-63 below. Table 4 Cas Proteins and SpCas9 Cas Protein + NLS + Cas Protein Cas Protein Cas Protein codon- i i d DNA HiBiT codon- IONCAS018 2242 62IONCAS019 2343 63The exp ress on an oca zat on o eac as prote n was t en eva uate n 93 cells.2 × 105HEK293 cells were transfected with 400 ng / well of codon-optimized DNA encoding putative Cas proteins using 4D Nucleofector (Lonza) and cells were seeded into a 96-well plate after transfection. After either 48 or 72 hours, cells were harvested. For each well, the HiBit level representing Cas protein expression was measured using the Nano-glo HiBit Lytic Detection System (Promega) per manufacturer’s protocol. Cas protein expression was normalized to cell count as measured by CellTiter-Glo Luminescent Cell Viability Assay (Promega). The expression of each Cas protein is presented as Luminescence of HiBit / Luminescence of CellTiter-Glo in the table below as the average of four measurements. Negative control “no program” groups were included in the analysis, consisting of cells that were not transfected. HiBit expression was detected in all samples, compared to no program negative controls. Some samples displayed high protein expression comparable to that of SpCas9, while some showed relatively low expression. Table 5 Expression of Cas proteins and SpCas9 in HEK293 cells, normalized to cell count Cas Protein Hibit r- IONCAS017 21 0.128 IONCAS018 22 0.195 Cellular locali zation of the Cas proteins was then examined by immunostaining. HeLa cells were seeded at a density of 1.5 × 104cells / well in an 8-well chamber slide and transfected with 100 ng / well of codon-optimized Cas protein expressing vectors with 0.4 µL BioT Transfection Reagent (Morganville Scientific). After 48 hours, cells were fixed in 4% formaldehyde and permeabilized with 0.1% Triton X-100 in PBS. After blocking the slide with 2% BSA in PBS at room temperature for 1 hour, slides were incubated overnight with mouse anti-HiBit antibody (Promega) at 4 °C overnight. The next day, slides were washed then incubated for 1 hour with Donkey anti-mouse secondary antibody AlexaFluor 555 (1:1000) combined with DAPI (1:1000). Slides were then washed and mounted with mounting medium. Images were taken using an EVOS cell imaging microscope. Cellular localization of Cas proteins is summarized in the table below. Control SpCas9 groups displayed nuclear localization as expected. Eight of the Cas proteins displayed nuclear localization, while the rest were predominantly localized to cytosol. Table 6 Cellular localization of Cas proteins and SpCas9 in HEK293 cells Cas protein ID Cas ProteinSEQ ID NOLocalization IONCAS016 20 Nucleus IONCAS017 21 Cytosol Example 2: Evaluation o f Cas protein activity in HEK293T nuclear extract Upon confirming expression in HEK293T cells, protein-recognition region sequences were designed. Sequences of protein-specific crRNA and tracrRNA were computationally predicted for each Cas protein and joined with a “gaaa” tetraloop to obtain the protein-recognition region of a putative guide, presented in the table below. The “gaaa” tetraloop between the putative crRNA and tracrRNA sequences is represented in lowercase letters. Cas proteins described above were screened for enzymatic activity using a cleavage assay in HEK293T cells. The assay measures cleavage of a synthesized tool DNA target in the nuclear extract of HEK293T cells which have been transfected to express a Cas protein and transcribe its corresponding guide. The first round of screening (Figure 1) was performed using a system consisting of a Cas protein and a guide comprising a general target-recognition region linked to a protein-recognition region. The second round of screening (Figure 2) was performed using a system consisting of a Cas protein and a guide comprising a target-recognition region that was optimized for that Cas protein linked to a protein-recognition region. Table 7 Sequences of protein-recognition regions predicted for the corresponding Cas proteins and SpCas9 Cas Protein- SEQ C P t i P t i . 1 2 3 4 C5gRNAGUUGUUGUUCCCGgaaaACGGUUACAACAAUAAGGUCCC125 GCCGCUGGCGGUGUACCUCAAAGCGAUAAUCCCACUUA AAGUGAAUACUUUAGGUGGGAUUUUUUUU 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 IONCAS018 22 C18gRNAGUUGUGAAUUGAUUUCgaaaGAAAUCAAUUCACAAUAAG141 GAUUAUUCCGUUGUGAAAACAUUUAAAGCGGUUCUUA CGAAUCGCUUUUUUU 1 8 2 In preliminary experiments, a target-recognition region known as a general spacer sequence (GSS) previously disclosed in Ran et al., Nature, 2015 was used in each guide. The GSS target-recognition region is shown in the table below. In addition, other target-recognition regions specific to each Cas protein were designed as shown in the table below. DNA sequences for each target-recognition region are included in the sequence listing (SEQ ID NO: 64-71). Table 8 Sequences of general and Cas-specific target-recognition regions used in guides Cas Target- Cas Protein ProteinSeq ID Cas protein screening using a general target-recognition region Guides with a general target-recognition region were designed by appending the general target- recognition region sequence (SEQ ID NO: 944) to the 5′-end of a protein-recognition sequence selected from the table above (SEQ ID NO: 121-141). A DNA template for in vitro synthesis of the guide RNA was constructed. The target-recognition sequence was appended 5′ of the Cas protein-recognition regions described above to create the guide, then a T7 promoter region (TAATACGACTCACTATA (SEQ ID NO: 72)) was appended to the 5′ of the target recognition sequence. Additionally, the four nucleobase AAGC was added at the 5′-end of the T7 promoter region to improve binding to the T7 promoter. The nucleobase sequences of the DNA templates used to transcribe guides listed in the table below are provided as SEQ ID NOs: 143-161, respectively. HiScribe® T7 High Yield RNA Synthesis Kit (New England Bioloabs) was used for T7 based in vitro transcription of the templates. Each DNA template in the sequence listing represents the top-strand sequence and has the same sequence as the RNA transcript at the end of transcription with the HiScribe® T7 High Yield RNA Synthesis Kit. Guide sequences are presented in the table below. For each guide sequence in the table below, the target-recognition region is represented by bold, underlined upper-case letters, and the protein-recognition region is represented by standard uppercase letters, with the tetraloop indicated in lowercase letters. The Cas protein that each guide was designed for is indicated in the table below by its Cas Protein ID and its corresponding SEQ ID NO. Table 9 Sequences of Guides with a general target-recognition region Cas Cas Protein Guide Guide P t iG id S 5′ 3′SEQ O. CACCGUCGCAAGACGACCACCGUAGCUCAGUCACCCCUG ACCUGCGGUUUUUU GGGACUCAACCAAGUCAUUCGUUUGAGAGCCUUGU SpCas9 1SpCas9GGGACUCAACCAAGUCAUUCGUUUUAGAGCUAGAAAUA g GCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAA 2690 AAGUGGCACCGAGUCGGUGCUUUU u es escr e n t e ta e a ove were use to test act v ty o t e r respect ve as prote ns n a cleavage assay. The DNA target used in the cleavage assay is a 156 bp double stranded DNA containing a general spacer (described above), followed by a PAM library. The DNA sequence (from 5’ to 3’) of the target is: ACACTCTTTCCCTACACGACGCTCTTCCGATCTCCAACTTCATCCACGTTCACGGGACTCAACC AAGTCATTCNNNNNNNCGGCAGACTTCTCCTCAGGAGTCAGATGCACCATGGTGTCTGTTAGAT CGGAAGAGCACACGTCTGAACTCCAGTC (SEQ ID NO: 230), wherein the underlined, bold nucleobases represent the general spacer (target-recognition region), and 7N (N = A, C, G, T) represents the PAM library. HEK293T cells, maintained in DMEM (10% FBS, 10 U / mL penicillin, 100 µg / mL streptomycin), were seeded in 10 cm dishes (Corning) at a density of 1.5 × 106cells / plate, and incubated for 24 hours. Cells were transfected with 5 µg / plate of pTwistCMV-puro vectors expressing Cas proteins listed in Table 4 herein above, using 20 µL BioT Transfection Reagent (Morganville Scientific). HEK293T nuclear extract was prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) according to manufacturer’s protocol. HiScribe T7 Quick High Yield RNA Synthesis kit (New England Biolabs) was used to transcribe guide RNA in vitro from the corresponding DNA templates described above. For each cleavage reaction, 5 µL of HEK293T nuclear extract expressing Cas protein and 200 nM in vitro-transcribed guide RNA were incubated at room temperature with 3 µL of cleavage buffer NEBuffer r3.1 (New England BioLabs) for 10 min to facilitate Cas-guide ribonucleoprotein (RNP) complex formation. Afterwards, 10 nM of DNA template was added to the reaction for overnight incubation at 37 °C, followed by the addition of proteinase K and RNAse to clean up the reaction. DNA templates were purified using DNA Clean & Concentrator-5 (Zymo Research). Digestion of DNA templates was analyzed and visualized using an Agilent TapeStation. As shown in Figure 1, Cas proteins IONCAS009 and IONCAS016 were found to have in vitro cleavage activity. To further examine the enzymatic activity of relatively highly expressed Cas proteins that did not display activity using the general spacer, an additional screen was performed using enzyme-specific target- recognition regions in place of the general spacer (target-recognition region) sequence. Cas protein screening using a Cas-specific target-recognition region Guides were designed by appending a Cas protein-specific target-recognition region sequence selected from Table 8 above (SEQ ID NO: 945-951) to the 5′-end of a protein-recognition sequence selected from the table above (SEQ ID NO: 121-141). A DNA template for in vitro synthesis of the guide RNA was constructed. The Cas-specific target- recognition sequence was appended 5′ of the Cas protein-recognition regions described above to create the guide, then a T7 promoter region (TAATACGACTCACTATA (SEQ ID NO: 72)) was appended to the 5′ of the target recognition sequence. In addition, since T7 strongly prefers to start with G and works best with GG, GG was added to the 5' of target-recognition regions that start with a nucleobase other than G. For target- recognition regions that start with G, one G was added to its 5'-end. Additionally, the four nucleobase AAGC was added at the 5'-end of the T7 promoter region to improve binding to the T7 polymerase. The nucleobase sequences of the DNA templates used to transcribe guides listed in the table below are provided as SEQ ID NOs: 162-174, respectively. HiScribe® T7 High Yield RNA Synthesis Kit (New England Bioloabs) was used for T7 based in vitro transcription of the templates. Each DNA template in the sequence listing represents the top-strand sequence and has the same sequence as the RNA transcript at the end of transcription with the HiScribe® T7 High Yield RNA Synthesis Kit. Guide sequences are presented in the table below. For each guide sequence in the table below, the target-recognition region is represented by bold, underlined upper-case letters, and the protein-recognition region is represented by standard uppercase letters, with the tetraloop indicated in lowercase letters. The Cas protein that each guide was designed for is indicated in the table below by its Cas Protein ID and its SEQ ID NO. Table 10 Sequences of Guides with a Cas protein-specific target-recognition regions Cas Cas Protein Guide Q O 1 2 3 C5s2- AUGUUUAUUUUGUGGCGCAGGUUGUUGUUCCCGGUUAUgaa aAUAAACGGUUACAACAAUAAGGUCCCGCCGCUGGCGGUGU 2694 C5gRNA-L ACCUCAAAGCGAUAAUCCCACUUAAAGUGAAUACUUUAGGU 5 6 7 8 9 0 1 2 3 The cleavage assay was repeated as described above, with the following changes: The guides used were composed of Cas-specific guides described in the Table above; and the DNA target used consisted of the Cas-specific target-recognition region appended to a 7-nt degenerate PAM sequence. When Cas-specific target-recognition regions were used, Cas proteins IONCAS008 and IONCAS013 showed in vitro cleavage activity in nuclear extract (Figure 2). Example 3: Identification of PAM sequences for Cas proteins The PAM sequence recognized by each Cas proteins that displayed in vitro enzymatic activity was determined through a dropout assay (Ran, FA et al. Nature.2015, 520, 186-191). In the dropout assay, the uncut fraction of the DNA templates was amplified via PCR reaction using i5 / i7 index primers from Integrated DNA Technologies (IDT). Amplicons were sequenced via Illumina MiSeq. For a given single-end MiSeq sequencing run, data was demultiplexed to generate FASTQ files using either MiSeq machine or Illumina bcl2fastq Conversion Software (https: / / support.illumina.com / sequencing / sequencing_software / bcl2fastq-conversion- software / downloads.html). A custom program was written to extract 7-mer PAM sequences located 3’ to a known spacer sequence from the FASTQ files. For each extracted PAM sequence in a sample, a count-per- million (CPM) value was calculated by dividing the number of reads containing the PAM sequence by the total number of reads of the sample. Relative PAM sequence depletion frequencies were calculated by dividing CPM values in a Cas protein treated sample by the corresponding CPM values in an untreated control sample. PAM sequence motifs at different depletion frequency thresholds were then generated using Python Logomaker software library available at https: / / logomaker.readthedocs.io / en / latest / (Tareen and Kinney 2019). The consensus PAM sequences of Cas proteins IONCAS008, IONCAS009, and IONCAS016 are summarized in the table below and shown in Figure 3. Table 11 Consensus PAM sequences of Cas proteins Cas IDCas Protein SEQ ID NO.PAM SequenceIn the table , or T; and “V” represents A, C, or G. Example 4: Evaluation of Cas protein cellular activity on gene targets in HEK293 cells The enzymatic activities of Cas proteins described above were evaluated in HEK293 cells, measuring editing in six genomic regions. Guides comprising a target-recognition region sequence designed for a genomic region linked to the 5'-end of a protein-recognition region sequence selected from the examples above were designed for each Cas protein. The guides were then evaluated in HEK293 cells using an in vitro cleavage assay. Six genomic regions that are widely-used in SpCas9 research were selected as targets for evaluation of cellular Cas protein activity. These regions are located within human genomic loci of AAVS1, EMX1, FANCF, HBB, HEKSite4, and HPRT. For each putative Cas protein and guide system, target-recognition regions were designed using CHOPCHOP v3 (Labun et al. Nucleic Acids Res, 2019) and Benchling software, and are presented in the table below. The Cas protein and gene target that each target-recognition region was designed for is indicated in the table below. DNA sequences for each target-recognition region are included in the sequence listing (SEQ ID NO: 175-199). Table 12 Sequences of gene-specific target-recognition regions for evaluating activity of Cas proteins and SpCas9 in HEK293 cells Cas Targ TargetCas ProteinProtein et- recognition Target-recognition region (5’ to SEQ ID ’ . C9sHP-2 UUCAUUUCAUCCGUGCUGAG 943 u es wee esg e y a g a as poe -spec c age-ecog o ego seque ce from the table above to the 5′-end of a protein-recognition sequence selected from the table above (SEQ ID NO: 121- 141). Guide sequences are presented in the table below. For each guide sequence, the target-recognition region is represented by bold, underlined upper-case letters, and the protein-recognition region is represented by standard uppercase letters, with the tetraloop indicated in lowercase letters. The Cas protein that each guide was designed for is indicated in the table below by its Cas Protein ID and its SEQ ID NO. The gene target that each guide was designed for is also indicated in the table below. DNA sequences for each guide are included in the sequence listing (SEQ ID NO: 200-229). Table 13 Sequences of Guides with gene-specific target-recognition regions Cas Cas get Protein Pro Guide Tar tein Guide ID Guide Sequence (5' to 3') SEQ O 7 8 9 0 1 2 3 4 IONCAS ACCUCCAAUGACUAGGGUGGGUUUGAGAGUAGU GUgaaaACACUACAAAGUUCAAAUA 1409 00913 C9gEMACGAUUUAUC GAAACUACCCGUCAGGGUGCUGCGGUUGCAGCUU 5 6 7 8 9 0 1 2 3 4 5 6 7 GACAUCGCAUAUUGCCCGCAUUGUGCGGAUAAAA AGAUCUGUUUGACAGGUCUUUUUU GGCACUGCGGCUGGAGGUGGGUUUUAGAGCUA 8 9 0 3 1 2 7 3 Plasmids expressing guides were designed by cloning the guide sequence into a U6 promoter-driven vector for transcription by RNA Polymerase III and ordered from Twist BioScience. The U6 promoter sequence (GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAA TTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATA ATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAA CTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCg; SEQ ID NO: 75) was appended to the 5′-end of the guide sequences, wherein the final g represented in lower case was removed for guides which have a 5′-GG. The transcription termination sequence (GTCTAGAGGTAC; SEQ ID NO: 76) was added 3′-end. 2 × 105HEK293 cells were transfected with 400 ng / well of codon-optimized DNA encoding the Cas protein and 400 ng / well of guide expressing plasmid using 4D Nucleofector (Lonza) and cells were seeded into 96-well plates after transfection. Two negative controls were included for analysis: cells transfected with SpCas9 / scrambled spgRNA, and non-transfected cells. After a 48 or 72-hour incubation period, genomic DNA was extracted from cells using DNeasy Blood & Tissue kits (Qiagen) following the manufacturer’s recommended protocol. Genomic PCRs were performed using primers spanning the target region, and libraries were constructed using i5 / i7 index primers from IDT. Amplicons were sequenced via MiSeq (Illumina). For a given paired-end MiSeq sequencing run, data was demultiplexed to generate FASTQ files as described above. The CRISPRessoBatch module of CRISPResso2 software (Clement et al. Nat Biotechnol, 2019) was used to process and analyze all sample sequencing data. A custom script was written to calculate editing percentages based on InDels only from CRISPResso2 analysis results. The gene editing quantification window was set to be ~40 nts long centered on a guide RNA sequence of ~20 nts, with ~10 nt upstream and ~10 nt downstream. To enable visualization alignments of low frequency InDels reads, the minimum threshold for report reads was set to 0.01% (default is 0.20%) and maximum reported alignments was set to 100 (default is 50). Results are presented as Percentage of InDels (%) summarized in the table below. “N.D.” indicates that no data was obtained. Table 14 % InDels of AAVS1 with Cas proteins and guides in HEK293 cells Target- Protein- Non- Cas Protein Guide recognition recognition Scramb transfe ol D Table 15 % InDels of EMX1 with Cas proteins and guides in HEK293 cells Target- Protein- Non- Scramb fe ol D SpCas9 1 SpgEM 2803 SpsEM 929 SpgRNA 142 73.70 0.03 0 IONCAS20 C16gEM 2804 C16sEM 929 C16gRNA 139 0.11 0.03 0 Table 16 % InDels of FANCF with Cas proteins and guides in HEK293 cells Target- Protein- Non- Cas Protein Guide recognition recognition Scramb transfe led ol D Table 17 % InDels of HBB with Cas proteins and guides in HEK293 cells Target- Protein- Non- Cas Protein Guide recognition recognition Scramb transfe ol D . . . . . . Table 18 % InDels of HEKSite4 with Cas proteins and guides in HEK293 cells Target- Protein- Non- Cas Protein Guide recognition recognition Scramb transfe re ionre ionled t d ol D Table 19 % InDels of HPRT with Cas proteins and guides in HEK293 cells Target- Protein- Non- Cas Protein Guide recognition recognition Scramb transfe ol D . . . Example 5: Computational prediction of Cas proteins and relevant guides Metagenomes were assembled using publicly available datasets. CRISPR cassettes containing CRISPR array and nearby proteins were predicted by CRISPRFinder and CRISPRone and were classified into sub-classes and -types. For Class 2 type II CRISPR systems, protein domains were identified via Hidden Markov Models and sequences were aligned to Cas9 of Streptococcus pyogenes (SpCas9). In addition, tracrRNA were predicted by using the CRISPR array to predict the anti-repeat located at the 5’-end of tracrRNA and searching for rho-independent termination characterized by a GC-rich palindrome sequence followed by a run of U residues at the 3’-end of the tracrRNA. After these analyses, 93 putative Cas proteins were identified. The corresponding Cas protein sequences and GeneArt (ThermoFisher) human codon optimized nucleic acid sequences are provided in the sequence listing, as indicated in the table below. The Cas protein DNA sequences were cloned into a pTwistCMVpuro vector by Twist Biosciences. In order to facilitate enzyme localization to nucleus, for each Cas protein, nucleoplasmin (SEQ ID NO: 3399) and c-Myc (SEQ ID NO: 3400) NLS were appended at the N- and C- terminus, respectively. In addition, a HiBiT (Promega) tag was added to the protein C-terminus for expression detection. These additional elements were linked to the proteins via known protein linkers such as GGGS / (GS)3 / (GGGGS)3. The DNA sequences encoding the Cas proteins with NLS and HiBiT tag are provided in the sequence listing, as indicated in the DNA sequences SEQ ID NO: 231-509 below. Table 20 Cas Proteins and SpCas9 Cas Protein codon- Cas Protein + NLS Cas Protein Cas Protein optimized DNA + HiBiT codon- IONCAS042252 345 438IONCAS043253 346 439 IONCAS077286 379 472IONCAS078287 380 473 IONCAS111320 413 506IONCAS112321 414 507Example 6: Eval uat on o Cas prote n act v ty n 93 nuc ear extract Protein-recognition region sequences for guides were designed. Sequences of protein-specific crRNA and tracrRNA were computationally predicted for each Cas protein and joined with a “gaaa” tetraloop to obtain the protein-recognition element of a putative guide, presented in the table below. The “gaaa” tetraloop between the putative crRNA and tracrRNA sequences is represented in lowercase nucleotides. Cas proteins described above were screened for enzymatic activity using a cleavage assay in HEK293T cells, as described herein above. The assay measures cleavage of a synthesized tool DNA target in the nuclear extract of HEK293T cells which have been transfected to express a Cas protein and transcribe its corresponding guide. Guides for each Cas protein comprising a target-recognition region linked to the 5'-end of a protein-recognition region were designed as described below Table 21 Sequences of protein-recognition regions predicted for the corresponding Cas proteins Cas Cas otein P Protein- SEQ Pr rotein reco nition Protein-reco nition re ion Se uence (5' to 3') ID O. 0 1 2 3 4 5 IONCAS GUUUUAGUACCCUCAUgaaaAUGAGGACCUACUAAAACAAGG 516 026237 C26gRNAGAAAUAUUCCCGAAAUCGAGACUUGUUUUCAAGUCUCUUUU UU 7 8 9 0 1 2 3 4 5 6 7 8 9 0 IONCAS25 C42gRNAGUUGUAGUUCCCUUCUgaaaUGAUAAGGUAGUACACCGCAAA 531 0422GCACUAGCGCCUUGCCAUUGUGGCAAGGCGCUAUCUUUUUU 2 3 4 5 6 7 8 9 0 1 2 3 4 5 IONCAS GUUUGAGAGUAGUGUAgaaaUACACUACGAGUUCAAAUAAAG 546 057267 C57gRNAAUUUAUCCAAAUCGUUGUGGAAACACAACAUCACAGUGUGU GGUGCAAAAGACUUGCUUUGCAAGUCUUUUUU 7 8 9 0 1 2 3 4 5 6 7 8 9 0 IONCAS GUUUGAGAACCUUGUAgaaaUACAAGGUGAGUUCAAAUAAGG 561 073282 C73gRNAAUUUAUCCGAAAUCGCCGAAAGGCAUGCUUGUUGCAUAUGA UAAAAAGACUUAAAUCUCGCAUUUGAGUCUUUUUU 2 3 4 5 6 7 8 9 0 1 2 3 4 5 IONCAS GUUUGAGAGUAGUGUAgaaaUACACUACGAGUUCAAAUAAAA 576 088297 C88gRNAAUUUUUUCAAAUCGUUCGGUAAAACCGUUCGCACAAGUGUU GUGCGUUGACCUUGCUUUUUAAGCAAGGUUUUU 7 8 9 0 1 2 3 4 5 6 7 8 9 0 IONCAS GUUGCGGAUUGGUCUCgaaaGCGACUCUUUUCGUAACAAGAA 591 103312 C103gRNAUGAAAAAUUCGCAGGCACUGCUCUACGGAGCAACCAGCCGU CAUGUCACGUACAGGGCGGCUUUUUU 2 3 4 5 6 7 8 9 0 1 2 Cas protein screening using a general target-recognition region Guides with a general target-recognition region were designed by adding the general target- recognition region sequence (SEQ ID NO: 944) to the 5′-end of a protein-recognition region sequence selected from the table above (SEQ ID NO: 510-602). A DNA template for in vitro synthesis of the guide RNA was constructed. The target-recognition sequence was appended 5′ of the Cas protein-recognition regions described above to create the guide, then a T7 promoter region (TAATACGACTCACTATA (SEQ ID NO: 72)) was appended to the 5′ of the target recognition sequence. Additionally, the four nucleotide AAGC was added at the 5′-end of the T7 promoter region to improve binding to the T7 promoter. The nucleobase sequences of the DNA templates used to transcribe guides C20g-C114g listed in the table below are provided as SEQ ID NOs: 603-695, respectively. HiScribe® T7 High Yield RNA Synthesis Kit (New England Biolabs) was used for T7 based in vitro transcription of the templates. Each DNA template in the sequence listing represents the top-strand sequence and has the same sequence as the RNA transcript at the end of transcription with the HiScribe® T7 High Yield RNA Synthesis Kit. Guide sequences are presented in the table below. For each guide sequence in the table below, the target-recognition region is represented by bold, underlined upper-case letters, and the protein-recognition region is represented by standard uppercase letters, with the tetraloop indicated in lowercase letters. The Cas protein that each guide was designed for is indicated in the table below by its Cas Protein ID and its corresponding SEQ ID NO. Table 22 Sequences of Guides with a general target-recognition region Cas Cas Protei Guide G id SEQ . 4 5 6 7 8 9 IONCA GGGACUCAACCAAGUCAUUCGUUUUAGUACCCUCAUgaaaA S026237 C26gUGAGGACCUACUAAAACAAGGGAAAUAUUCCCGAAAUCGA 2710 GACUUGUUUUCAAGUCUCUUUUUU 1 2 3 4 5 6 7 8 9 0 1 2 3 4 IONCA GGGACUCAACCAAGUCAUUCGUUGUAGUUCCCUUCUgaaaU S042252 C42gGAUAAGGUAGUACACCGCAAAGCACUAGCGCCUUGCCAUUG 2725 UGGCAAGGCGCUAUCUUUUUU 6 7 8 9 0 1 2 3 4 5 6 7 8 9 GGGACUCAACCAAGUCAUUCGUUUGAGAGUAGUGUAgaaaU IONCA ACACUACGAGUUCAAAUAAAGAUUUAUCCAA S057267 C57gAUCGUUGUG GAAACACAACAUCACAGUGUGUGGUGCAAAAGACUUGCUU 2740 1 2 3 4 5 6 7 8 9 0 1 2 3 GGGACUCAACCAAGUCAUUCGUCUUGAAUAGUAGUCgaaaG IONCA ACUACUAGCGUCAAGAUCAAACAAGACUUUUU S071281 C71gAGUCGAGU UUCACUUUUUGAGUCCGUAUUCGGACCAUUACAUAUGUACA 2754 5 6 7 8 9 0 1 2 3 4 5 6 7 GGGACUCAACCAAGUCAUUCGUUUGAGAGUAGUGUAgaaaU IONCA ACACUGCGAGUUCAAAUAACGUUAUCGCAACG S086295 C86gAAAUCGCCG UUUAUCGGUUGCACAGUGUGUGCUUAAAAAAUAGGCCUUA 2768 9 0 1 2 3 4 5 6 7 8 9 0 1 IONCA GGGACUCAACCAAGUCAUUCGCUGUGACUAAUCACGgaaaA S100309 C100gGAUAUAAAAUCAAAAUCAAAAUACAAUACGGGAAACCGUA 2782 AAUGGUUUUCUUUU 3 4 5 6 7 8 9 0 1 2 3 4 5 6 Cas proteins described above were screened for enzymatic activity in an in vitro cleavage assay wherein a synthesized DNA target was incubated with the nuclear extract of HEK293T cells that were transfected with pTwist-CMV-puro-Cas proteins, and their corresponding in vitro transcribed guide RNA. The DNA target is a 156 bp double stranded DNA containing a general spacer followed by a PAM library. The DNA sequence of the target is described above (SEQ ID NO: 230). HEK293T cells, maintained in DMEM (10% FBS, 10 U / mL penicillin, 100 µg / mL streptomycin), were seeded in 10 cm dishes (Corning) at a density of 1.5 × 106cells / plate, and incubated for 24 hours. Cells were transfected with 5 µg / plate of pTwistCMV-puro vectors expressing Cas proteins listed in Table 2 herein above, using 20 µL BioT Transfection Reagent (Morganville Scientific). HEK293T nuclear extract was prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) according to manufacturer’s protocol. HiScribe T7 Quick High Yield RNA Synthesis kit (New England Biolabs) was used to transcribe guide RNA in vitro from the corresponding DNA templates described above. For each cleavage reaction, 5 µL of HEK293T nuclear extract expressing Cas protein and 200 nM in vitro- transcribed guide RNA were incubated at room temperature with 3 µL of cleavage buffer NEBuffer r3.1 (New England BioLabs) for 10 min to facilitate Cas-guide ribonucleoprotein (RNP) complex formation. Afterwards, 10 nM of DNA template was added to the reaction for overnight incubation at 37 °C, followed by the addition of proteinase K and RNAse to clean up the reaction. DNA templates were purified using DNA Clean & Concentrator-5 (Zymo Research). Digestion of DNA templates was analyzed and visualized using an Agilent TapeStation. As shown in Figure 4a-4c, Cas proteins IONCAS030, IONCAS031, IONCAS034, IONCAS036, IONCAS050, IONCAS056, IONCAS057, IONCAS058, IONCAS061, IONCAS064, IONCAS065, IONCAS069, IONCAS073, IONCAS074, IONCAS075, IONCAS079, IONCAS080, IONCAS083, IONCAS088, IONCAS090, IONCAS091, IONCAS092, IONCAS093, IONCAS105, and IONCAS114 were found to have in vitro cleavage activity. Example 7: Identification of PAM sequences for Cas proteins The PAM sequences of Cas proteins that displayed in vitro enzymatic activity were determined through a dropout assay (Ran, FA et al. Nature.2015, 520, 186-191). In the dropout assay, the uncut fraction of the DNA templates was amplified via PCR reaction using i5 / i7 index primers from Integrated DNA Technologies (IDT). Amplicons were sequenced via Illumina MiSeq. For a given single-end MiSeq sequencing run, data was demultiplexed to generate FASTQ files using either MiSeq machine or Illumina bcl2fastq Conversion Software (https: / / support.illumina.com / sequencing / sequencing_software / bcl2fastq- conversion-software / downloads.html). A custom program was written to extract 7-mer PAM sequences located 3’ to a known spacer sequence from the FASTQ files. For each extracted PAM sequence in a sample, a count-per-million (CPM) value was calculated by dividing the number of reads containing the PAM sequence by the total number of reads of the sample. Relative PAM sequence depletion frequencies were calculated by dividing CPM values in a Cas protein treated sample by the corresponding CPM values in an untreated control sample. PAM sequence motifs at different depletion frequency thresholds were then generated using Python Logomaker software library available at https: / / logomaker.readthedocs.io / en / latest / (Tareen and Kinney 2019). The consensus PAM sequences of Cas proteins IONCAS030, IONCAS031, IONCAS034, IONCAS036, IONCAS050, IONCAS056, IONCAS057, IONCAS058, IONCAS061, IONCAS064, IONCAS065, IONCAS069, IONCAS073, IONCAS074, IONCAS075, IONCAS079, IONCAS080, IONCAS083, IONCAS088, IONCAS090, IONCAS091, IONCAS092, IONCAS093, IONCAS105, and IONCAS114 are summarized in the table below. Table 23 Consensus PAM sequences of Cas proteins Cas Protein ID Cas Protein SEQ ID NO. PAM Sequence IONCAS030241VGRRRA IONCAS114 323 CNNNRAA In the table above, “N” represents any nucleobase; “M” represents A or C; “R” represents A or G; “Y” repres B” represents G, T or C; “H” represents A, C, or T; and “V” represents A, C, or G. Example 8: Design and efficacy of guides in HEK293 cells Guides comprising a target-recognition region sequence linked to the 5'-end of a protein-recognition region sequence were designed for Cas proteins selected from the examples above. The guides were optimized by tuning a variety of parameters including target-recognition region length, length of the first stem-loop in the protein-recognition region, replacing A-U or U-A base pairs with C-G base pairs within predicted stems, replacement of loops and linkers with different sequences, and overall protein-recognition region length. The guides were then evaluated in HEK293 cells using the in vitro cleavage assay. Plasmids expressing the guides were designed by cloning the guide sequence into a U6 promoter- driven vector for transcription by RNA Polymerase III and ordered from Twist BioScience. The U6 promoter sequence (SEQ ID NO: 75) was appended to the 5′-end of the guide sequences, with the final 3′-G removed for guides which already have a 5′-GG. The transcription termination sequence (SEQ ID NO: 76) was added 3′-end. 2 × 105HEK293 cells were transfected with 400 ng / well of codon-optimized DNA encoding the Cas protein (indicated in the tables below) and 400 ng / well of guide-expressing plasmid (indicated in the tables below) using 4D Nucleofector (Lonza) and cells were seeded into 96-well plates after transfection. After a 48 or 72-hour incubation period, genomic DNA was extracted from cells using DNeasy Blood & Tissue kits (Qiagen) following the manufacturer’s recommended protocol. For the targets indicated in the table below, Genomic PCRs were performed using primers spanning the target region, and libraries were constructed using i5 / i7 index primers from IDT. Amplicons were sequenced via MiSeq (Illumina). For a given paired-end MiSeq sequencing run, data was demultiplexed to generate FASTQ files as described above. The CRISPRessoBatch module of CRISPResso2 software (Clement et al. Nat Biotechnol, 2019) was used to process and analyze all sample sequencing data, and InDel analysis was performed as described above. Target-Recognition Region Length Optimization Target-recognition region sequences with a length of 20-24 nt were constructed using PAM sequences determined for each Cas protein and target pair. The target-recognition region sequences can be found in the sequence listing as indicated in the table below (SEQ ID NO: 696-887). Guideswere designed by appending a target-recognition region sequence with a length of 20-24 nt to the 5′-end of a protein- recognition sequence selected from the examples above. Guides described in the tables below were constructed from the target-recognition regions identified by ID and SEQ ID NO in the column “Target-recognition Region” and the protein-recognition region identified by ID and SEQ ID NO in the column “Protein-recognition Region” In the tables below, the target that each guide was designed for is indicated in the columns labeled “Target”, and the length of the target- recognition region is indicated in the column labeled “Length”. The full sequences of the guides are found in the sequence listing as indicated in the column labeled “Guide”. Cas protein and guides were tested for in vitro activity in HEK293 cells, as described above, and results are presented in the tables below. For each Cas protein and guide system, the relative amount of InDels was calculated by dividing the %InDels of a target for a given Cas protein and guide system by the %InDels of that target for the Cas protein and guide system when the target-recognition region length is 20 nt. Results are presented in the tables below as “Relative Indels to 20 nt”. “N.D.” indicates that no data was obtained. Table 24 % InDels of targets with IONCAS009 protein and guides, varying target-recognition region length Guide Target-Recognition RegionProtein-RecognitionRegion Relativeels t Table 25 % InDels of targets with IONCAS030 protein and guides, varying target-recognition region length Protein- Guide Target-Recognition Region Recognition Region Relativeelst Table 26 % InDels of targets with IONCAS050 protein and guides, varying target-recognition region length Protein- Guide Target-Recognition Region RecognitionRelativeelst Table 27 % InDels of targets with IONCAS057 protein and guides, varying target-recognition region length Protein- Guide Target-Recognition Region Recognition Region Relativeelsnt Table 28 % InDels of targets with IONCAS064 protein and guides, varying target-recognition region length Guide Target-Recognition Region Protein-Recognition Region Relativeels t C64N3HBg1_23 1479 C64N3HBs1_23 766 23 C64gRNA 553 1.02 C64N3HBg1_24 1480 C64N3HBs1_24 767 24 C64gRNA 553 0.66 a e % InDels of targets with IONCAS065 protein and guides, varying target-recognition region length Protein- Guide Target-Recognition Region Recognition Re ion Relativeelst Table 30 % InDels of targets with IONCAS075 protein and guides, varying target-recognition region length Guide Target-Recognition Region Protein-Recognition Region Relativeels t C75FAg2_24 1505 C75FAs2_24 792 24 C75gRNA 563 0.77 C75HBg1 1506 C75HBs1 793 20 C75gRNA 563 1 Table 31 % InDels of targets with IONCAS080 protein and guides, varying target-recognition region length Protein- Guide Target-Recognition Region Recognition RiRelativeelst Table 32 % InDels of targets with IONCAS083 protein and guides, varying target-recognition region length Guide Target-Recognition Region Protein-Reco nition Re ion Relativeels t C83HBg2_21 1532 C83HBs2_21 819 21 C83gRNA 571 0.94 C83HBg2_22 1533 C83HBs2_22 820 22 C83gRNA 571 0.72 Table 33 % InDels of targets with IONCAS090 protein and guides, varying target-recognition region length Guide Target-Recognition Region Protein-Recognition Region Relativeels t Table 34 % InDels of targets with IONCAS092 protein and guides, varying target-recognition region length Guide Target-Recognition Region Protein-Rniti n R i n R l tiveels t C92FAg2_23 1559 C92FAs2_23 846 23 C92gRNA 580 0.53 C92FAg2_24 1560 C92FAs2_24 847 24 C92gRNA 580 0.35 Table 35 % InDels of targets with IONCAS105 protein and guides, varying target-recognition region length Guide Target-Recognition Region Protein-Recognition Region Relativeels t Table 36 % InDels of targets with IONCAS114 protein and guides, varying target-recognition region length Guide Target-Recognition Region Protein-Recognition Region RelativeTarget SEQ SEQ %InDels t Protein-Recognition Region Length Optimization for first stem-loop Protein-recognition region sequences were modified by varying the repeat:anti-repeat (R:AR) length by varying the length of the upper stem-loop in the 5′-most stem loop of the protein-recognition region, as predicted by ViennaFold software. The R:AR length is the number of nucleotides in the protein recognition region that are 5′ of the “gaaa” tetraloop, and isindicated in the column labeled “R:AR length” in the tables below. The complete protein-recognition region sequences associated with each R:AR modification are found in the sequence listing (SEQ ID NO: 961-1019) as indicated in the table below. Guides were then designed by appending a target-recognition region sequence selected from an example above to the 5′-end of a protein- recognition sequence with varying R:AR length. Guides described in the tables below were constructed from the target-recognition regions identified by ID and SEQ ID NO in the column “Target-recognition Region” and the protein-recognition region identified by ID and SEQ ID NO in the column “Protein-recognition Region” In the tables below, the target that each guide was designed for is indicated in the columns labeled “Target”, and the R:AR length of the protein-recognition region is indicated in the column labeled “R:AR Length”. The full sequences of the guides are found in the sequence listing (SEQ ID NO: 1601-1766) as indicated in the column labeled “Guide”. Cas protein and guides were tested for in vitro activity in HEK293 cells, as described above, and results are presented in the tables below. For each Cas protein and guide system, the relative amount of InDels was calculated by dividing the %InDels of a target for a given Cas protein and guide system by the %InDels of that target for the Cas protein and guide system with the original R:AR length (15 nt for IONCAS009, 16 nt for other Cas proteins). Results are presented in the tables below as “Relative InDels”. Table 37 % InDels of targets with IONCAS009 protein and guides, varying protein-recognition region R:AR length Target- Guide RecognitionProtein-Recognition RegionRelativeR i n I D ls R Table 38 % InDels of targets with IONCAS030 protein and guides, varying protein-recognition region R:AR length Target- Guide Reco nitionProtein-Reco nition Re ionveto 16 C30ASg1_RAR18 1615 C30ASs1 708C30g_R-AR18967 18 0.75C30ASg1_RAR20 1616 C30ASs1 708C30g_R-AR20968 20 0.39 Table 39 % InDels of targets with IONCAS050 protein and guides, varying protein-recognition region R:AR length Target- Guide Recognition Protein-Recognition Region Relative s R Table 40 % InDels of targets with IONCAS057 protein and guides, varying protein-recognition region R:AR length Target- Guide Recognition Protein-Recognition Region Relative Region InDels R Table 41 % InDels of targets with IONCAS064 protein and guides, varying protein-recognition region R:AR length Target- Guide RecognitionProtein-Recognition RegionRelatives R C64N3HBg1_RAR12 1658 C64N3HBs1 763 C64g_R-AR12 977 12 0.84 C64N3HBg1_RAR14 1659 C64N3HBs1 763 C64g_R-AR14 978 14 0.93 abe % InDels of targets with IONCAS065 protein and guides, varying protein-recognition region R:AR length Target- Guide Recognition Protein-Recognition Region Relative R i I Dls R Table 43 % InDels of targets with IONCAS075 protein and guides, varying protein-recognition region R:AR length Target- Guide ReconitionPr t in-R niti n R i nR l tives R C75ASg1_RAR18 1677 C75ASs1 783 C75g_R-AR18 988 18 1.31 C75ASg1_RAR20 1678 C75ASs1 783 C75g_R-AR20 989 20 0.89 Table 44 % InDels of targets with IONCAS080 protein and guides, varying protein-recognition region R:AR length GuideTarget-RecognitionProteiRelative Region n-Recognition RegionInDels R Table 45 % InDels of targets with IONCAS083 protein and guides, varying protein-recognition region R:AR length Target- Guide Recognition Protein-Recognition Region Relative Region InDels R Table 46 % InDels of targets with IONCAS090 protein and guides, varying protein-recognition region R:AR length Target- Guide RecognitionProtein-Recognition RegionRelatives R C90EMg2_RAR20 1720 C90EMs2 833C90g_R-AR201001 20 0.97C90HPg1_RAR12 1721 C90HPs1 838C90g_R-AR12998 12 0.56 Table 47 % InDels of targets with IONCAS092 protein and guides, varying protein-recognition region R:AR length Target- Guide Recognition Protein-Recognition Region Relative R i I Dls R Table 48 % InDels of targets with IONCAS105 protein and guides, varying protein-recognition region R:AR length Target- G id R iti P ti R iti R i Rltive s R C105ASg2_RAR14 1738 C105ASs2 858C105g_R-AR141007 14 1.10C105ASg2 1571 C105ASs2 858C105gRNA593 16 1 Table 49 % InDels of targets with IONCAS114 protein and guides, varying protein-recognition region R:AR length Target- Guide Recognition Protein-Recognition Region Relative s R Protein-Recognition Region Optimization: Second stem-loop Additional modifications to the protein-recognition regions were tested by modifying the sequence of the first linker (L1) and the second stem-loop (S2 and S2’ and H2) of the protein-recognition region of C9gRNA (SEQ ID NO: 131, described herein above). In the table below, specific modifications are indicated in the column labeled “Modification.” The complete protein-recognition region sequences associated with each SL-1 modification are found in the sequence listing (SEQ ID NO: 1016-1019) as indicated in the table below. Guides were then designed by appending a target-recognition region sequence selected from an example above to the 5′-end of a modified protein-recognition sequence. Guides described in the table below were constructed from the target-recognition regions identified by ID and SEQ ID NO in the column “Target-recognition Region” and the protein-recognition region identified by ID and SEQ ID NO in the column “Protein-recognition Region”. In the tables below, the target that each guide was designed for is indicated in the columns labeled “Target”, and the modification in the protein-recognition region is indicated in the column labeled “Modification”. The full sequences of the guides are found in the sequence listing (SEQ ID NO: 1767-1778) as indicated in the column labeled “Guide”. Cas protein and guides were tested for in vitro activity in HEK293 cells, as described above. For each IONAS009 protein and guide system, the percentage of insertions and deletions (% InDels) on each target is reported in the tables below. Table 50 % InDels of targets with IONCAS009 protein and guides, varying protein-recognition region sequence Target- Guide Recognition Protein-Recognition Region ls 4 1 C9gHE_SL1 _loopdel1772 C9sHE-2 700 C9g_SL1_loopdel 1017 loop deletion 0.157 Protein-Recognition Region Length Optimization Protein-recognition region sequences were modified by truncating the 3′-most stem-loop in the guide, or by removing base-pairs in the 3′-most stem-loop to effectively shorten the overall sequence length. The overall length of the protein-recognition region sequence is indicated in the column labeled “Length” in the tables below. The complete protein-recognition region sequences associated with each modification are found in the sequence listing (SEQ ID NO: 1020-1051) as indicated in the tables below. Additional target-recognition region sequences with a length of 20 nt were constructed using PAM sequences determined for each Cas protein and target pair. The target-recognition region sequences can be found in the sequence listing as indicated in the table below (SEQ ID NO: 888-890). Guides were then designed by appending a target-recognition region sequence to the 5′-end of a protein-recognition sequence with varying length. Guides described in the tables below were constructed from the target-recognition regions identified by ID and SEQ ID NO in the column “Target-recognition Region” and the protein-recognition region identified by ID and SEQ ID NO in the column “Protein-recognition Region”. In the tables below, the target that each guide was designed for is indicated in the columns labeled “Target”, and the length of the protein- recognition region sequence is indicated in the column labeled “Length”. The full sequences of the guides are found in the sequence listing (SEQ ID NO: 1409, 1413, 1417, 1856, 1860, 1868, 1872, 1889-1949) as indicated in the column labeled “Guide”. Cas protein and guides were tested for in vitro activity in HEK293 cells, as described above. For each Cas protein and guide system, the percentage of insertions and deletions (% InDels) on each target is reported in the tables below. Table 51 % InDels of targets with IONCAS009 protein and guides, varying protein-recognition region length GuideTarget-RecognitionRegion Protein-Recognition RegionTarget SEQ SEQ SEQ%ls Table 52 % InDels of targets with IONCAS057 protein and guides, varying protein-recognition region length GuideTarget-RecognitionRegion Protein-Recognition Region%s Table 53 % InDels of targets with IONCAS065 protein and guides, varying protein-recognition region length GuideTarget-RecognitionRegion Protein-Recognition Regions Table 54 % InDels of targets with IONCAS075 protein and guides, varying protein-recognition region length GuideTarget-RecognitionRegion Protein-Recognition RegionTarget SEQ SEQ%s Table 55 % InDels of targets with IONCAS080 protein and guides, varying protein-recognition region length GuideTarget-RecognitionRegion Protein-Recognition Region%ls0 7 8 3 3 5 6 7 3 0 Table 56 % InDels of targets with IONCAS083 protein and guides, varying protein-recognition region length GuideTarget-RecognitionRe ion Protein-Recognition Regionls0 0 9 C83FAg1-S3 1922 C83FAs1 813 C83g-S3 1038 82 35.73 C83FAg1-S4 1923 C83FAs1 813 C83g-S4 1039 80 30.04 2 2 1 1 9 Table 57 % InDels of targets with IONCAS090 protein and guides, varying protein-recognition region length GuideTarget-RecognitionRegion Protein-Recognition RegionT E E E%s Table 58 % InDels of targets with IONCAS092 protein and guides, varying protein-recognition region length GuideTarget-RecognitionRegion Protein-Recognition Regions Table 59 % InDels of targets with IONCAS114 protein and guides, varying protein-recognition region length GuideTarget-RecognitionRegion Protein-Recognition RegionTarget SEQ SEQ%s Protein-Recognition Region Sequence Optimization Protein-recognition region sequences were modified by swapping various base-pairs in the stems of predicted stem-loops (using ViennaFold), for example from A-U to G-C, or G-U wobble pair to G-C. The complete protein-recognition region sequences are found in the sequence listing (SEQ ID NO: 1053-1312) as indicated in the tables below. Guides were then designed by appending a target-recognition region sequence to the 5′- end of a protein-recognition sequence. Guides described in the tables below were constructed from the target-recognition regions identified by ID and SEQ ID NO in the column “Target-recognition Region” and the protein-recognition region identified by ID and SEQ ID NO in the column “Protein-recognition Region” In the tables below, the target that each guide was designed for is indicated in the columns labeled “Target”. The full sequences of the guides are found in the sequence listing (SEQ ID NO: 1950-2471) as indicated in the column labeled “Guide”. Cas protein and guides were tested for in vitro activity in HEK293 cells, as described above. For each Cas protein and guide system, the percentage of insertions and deletions (% InDels) on each target is reported in the tables below. The relative amount of InDels was also calculated by dividing the %InDels of a target for a given Cas protein and guide system by the %InDels of that target for the Cas protein and guide system with the original protein-recognition region. Results are presented in the tables below as “Relative InDels”. Table 60 % InDels of targets with IONCAS009 protein and guides, varying protein-recognition region sequence Protein- Target-Recognition Recognition GidR iR i%Relative ls C9HEg2-17 1985 C9sHE-2 700 C9g-17 1069 31.48 0.84 C9HEg2-18 1986 C9sHE-2 700 C9g-18 1070 24.20 0.64 a e % InDels of targets with IONCAS030 protein and guides, varying protein-recognition region sequence Protein- Target-Recognition Recognition % R l tive ls C30Asg2-6 2015 C30ASs2 713 C30g-6 1077 13.87 0.42 C30Asg2-7 2016 C30ASs2 713 C30g-7 1078 9.13 0.28 Table 62 % InDels of targets with IONCAS057 protein and guides, varying protein-recognition region sequence Protein- Target-Recognition Recognition ve ls C57FAg1-15 2047 C57FAs1 738 C57g-15 1109 21.86 0.51 C57FAg1-16 2048 C57FAs1 738 C57g-16 1110 2.26 0.05 C57HBg1-25 2089 C57HBs1 743 C57g-25 1119 50.73 1.07 C57HBg1-26 2090 C57HBs1 743 C57g-26 1120 39.95 0.84 Table 63 % InDels of targets with IONCAS065 protein and guides, varying protein-recognition region sequence Protein- Target-Recognition Recognition ve ls C65FAg1-26 2123 C65FAs1 768 C65g-26 1152 41.49 0.83 C65HEg1-0 1486 C65HEs1 773 C65gRNA 554 11.85 1 Table 64 % InDels of targets with IONCAS075 protein and guides, varying protein-recognition region sequence Protein- T t R iti R iti ve ls C75ASg1-6 2155 C75ASs1 783 C75g-6 1158 35.18 0.69 C75ASg1-7 2156 C75ASs1 783 C75g-7 1159 52.31 1.02 C75HBg1-18 2198 C75HBs1 793 C75g-18 1170 36.24 0.92 C75HBg1-19 2199 C75HBs1 793 C75g-19 1171 18.21 0.46 Table 65 % InDels of targets with IONCAS080 protein and guides, varying protein-recognition region sequence Protein- Target-Recognition Recognition ve ls C80N3FAg2-21 2230 C80N3FAs2 803 C80g-21 1202 13.47 0.40 C80N3FAg2-22 2231 C80N3FAs2 803 C80g-22 1203 28.11 0.84 Table 66 % InDels of targets with IONCAS083 protein and guides, varying protein-recognition region sequence Protein- ve ls C83FAg1-1 2260 C83FAs1 813 C83g-1 1207 24.43 0.84 C83FAg1-2 2261 C83FAs1 813 C83g-2 1208 33.96 1.17 Table 67 % InDels of targets with IONCAS090 protein and guides, varying protein-recognition region sequence Protein- Target-Recognition Recognition GidR iR i%Relative ls C90HPg2-13 2329 C90HPs2 889 C90g-13 1237 23.29 2.34 C90HPg2-14 2330 C90HPs2 889 C90g-14 1238 26.13 2.62 Table 68 % InDels of targets with IONCAS092 protein and guides, varying protein-recognition region sequence Protein- Target-Recognition Recognition ve ls C92HBg1-25 2362 C92HBs1 848 C92g-25 1270 7.37 0.92 C92HBg1-26 2363 C92HBs1 848 C92g-26 1271 17.29 2.15 Table 69 % InDels of targets with IONCAS105 protein and guides, varying protein-recognition region sequence Protein- T t R iti R iti ve ls C105ASg2-5 2395 C105ASs2 858 C105g-5 1277 46.77 0.93 C105ASg2-6 2396 C105ASs2 858 C105g-6 1278 44.99 0.89 Table 70 % InDels of targets with IONCAS114 protein and guides, varying protein-recognition region sequence Target-Recognition Protein- Guide RegionRecognition RegionTarget SEQ%Relative ls C114HPg1-15 2466 C114HPs1 883 C114g-15 1307 20.37 0.92 C114HPg1-16 2467 C114HPs1 883 C114g-16 1308 23.44 1.06 Example 9: Design and efficacy of modified guides in HepG2 cells, LNP delivery Modified guides comprising a target-recognition region sequence linked to the 5'-end of a protein- recognition region sequence were designed for Cas proteins selected from the examples above, then purchased from IDT. The guides were optimized by tuning the target-recognition region length, protein- recognition region length, and protein-recognition region sequence. The guides were then screened for enzymatic activity in an in vitro assay wherein HepG2 cells were incubated with an LNP formulation of a Cas protein and guide system. Guides were designed according to the sequences indicated in the tables below by their SEQ ID NO. and ordered from IDT. Each guide in the tables below has the following modifications: the nucleosides at positions 1 and 2 at the 5'-end are 2'-OMe sugar moieties wherein each nucleoside is linked to the next nucleoside with phosphorothioate internucleoside linkage; and the last two nucleosides at the 3'-end are 2'- OMe sugar moieties, wherein each nucleoside is linked to the preceding nucleoside by a phosphorothioate internucleoside linkage. Human HepG2 cells were split at the density of 20,000 cells per well in 96-well plates and cultured in DMEM medium with 10% FBS and 1% PenStrep. After 24 hours, culture media were changed. RNA / LNP complexes were generated by mixing RNA (Cas protein mRNA / guide at 1:2 ratio) and with a LNP formulation (Kazemian, et al., Mol. Pharmaceutics, 2022, 19, 1669-1689; Albertsen et al., Adv. Drug Deliv. Rev.2022, 118, 114416; Cullis et al., Nat. Rev. Drug Discov.2024, 23, 709-722.). After incubating for 15 minutes, the mixture was added to the culture medium at doses indicated in the tables below. Cells were harvested for InDel analysis after 72 hours, and InDel analysis was performed as described herein above. Target-Recognition Region Length Optimization a length of 20-25 nt were selected from the examples above or designed using PAM sequences determined for each Cas protein and target pair. The target-recognition region sequences can be found in the sequence listing as indicated in the tables below. Protein-recognition region sequences for each Cas protein were designed, truncating the UUUUUU (6U) sequence at the 3′-end. The protein-recognition region sequences are found in the sequence listing as indicated in the tables below. Guides were designed by appending a target-recognition region sequence with a length of 20-25 nt to the 5′-end of a protein-recognition sequence with or without a 3′-6U sequence. Guides described in the tables below were constructed from the target-recognition regions identified by ID and SEQ ID NO in the column “Target-recognition Region” and the protein-recognition region identified by ID and SEQ ID NO in the column “Protein-recognition Region”. In the tables below, the length of the target-recognition region sequence is indicated in the column labeled “Length”, and the presence of 6U at the 3'-end of the protein-recognition sequence is indicated in the column labeled “3'-end”. The nucleobase sequences of the guides and the fully annotated sequences describing the modifications on the guides are found in the sequence listing as indicated in the Guide columns labeled “SEQ ID NO.” and “Mod. SEQ ID No.”, respectively. Cas proteins and guides were tested for in vitro activity in HepG2 cells, as described above. For each Cas protein and guide system, the percentage of insertions and deletions (% InDels) on each target is reported in the tables below. Table 71 % InDels of EMX1 with IONCAS009 protein and guides, varying target-recognition region length and 3′-6U Guide Target-Recognition RegionProtein-recognitionRegion %InDelsng0 2 8 7 8 8 2 9 3 5 Table 72 % InDels of HEKSite4 with IONCAS009 protein and guides, varying target-recognition region length and 3′-6U Guide Target-Recognition RegionProtein-recognitionRegion %InDelsMod g 0 9 5 3 0 4 7 2 9 4 Table 73 % InDels of FANCF with IONCAS057 protein and guides, varying target-recognition region length and 3′-6U Guide Target-Recognition RegionProtein-recognitionRegion %InDelsng47 63 51 44 60160463 Table 74 % InDels of HBB with IONCAS057 protein and guides, varying target-recognition region length and 3′-6U Guide Target-Recognition RegionProtein-recognitionRegion %InDelsMod ng7 56 9 1 6616 Table 75 % InDels of AAVS1 with IONCAS030 protein and guides, varying target-recognition region length and 3′-6U Guide Target-Recognition RegionProtein-recognitionRegion %InDels6 g 03 21 33 21 09 05 16 24 80 02 75 44 Table 76 % InDels of AAVS1 with IONCAS065 protein and guides, varying target-recognition region length and 3′-6U Guide Target-Recognition RegionProtein-recognitionRegion %InDelsMod ng75 83 37 76 93 75 64 35 Table 77 % InDels of HEKSite4 with IONCAS065 protein and guides, varying target-recognition region length and 3′-6U Guide Target-Recognition RegionProtein-recognitionRegion %InDelsng0 46 46 3 3434 Table 78 % InDels of AAVS1 with IONCAS075 protein and guides, varying target-recognition region length and 3′-6U Guide Target-Recognition RegionProtein-recognitionRegion %InDelsMod ng4 4 6 3 39 4 74 4 49 51 41 21 88 71 68 41 Table 79 % InDels of FANCF with IONCAS080 protein and guides, varying target-recognition region length and 3′-6U Guide Target-Recognition RegionProtein-recognitionRegion %InDelsng615020 C80N3FAg2- 24NU1848 2919 C80N3FAs2_24 807 24 C80g-0 1030 -- 11.06 0.73 a e % InDels of HEKSite4 with IONCAS080 protein and guides, varying target-recognition region length and 3′-6U Guide Target-Recognition RegionProtein-recognitionRegion %InDelsM d ng92 07 29 17 14 12 Table 81 % InDels of FANCF with IONCAS083 protein and guides, varying target-recognition region length and 3′-6U Guide Target-Recognition RegionProtein-recognitionRegion %InDels6 g 30 02 51 95 37 37 76 35 54 58393127 C83FAg2-21NU 1861 2939C83FAs2-21U 913 21 C83g-NU 1035 -- 4.62 0.61C83FAs2-2724 ab e 8 % InDels of HPRT with IONCAS090 protein and guides, varying target-recognition region length and 3′-6U Guide Target-Recognition RegionProtein-recognitionRegion %InDelsM 6 g 05 27 47 23 08 33 51 28 08 60622022 378848 Table 83 % InDels of AAVS1 with IONCAS105 protein and guides, varying target-recognition region length and 3′-6U Guide Target-Recognition RegionProtein-recognitionRegion %InDelsMod SEQ SEQ ng71 05 78 30 23 01 62 24 Table 84 % InDels of HBB with IONCAS105 protein and guides, varying target-recognition region length and 3′-6U Guide Target-Recognition RegionProtein-recognitionRegion %InDels6 g 03 70 50 22 54 60 39 37 Protein-Recognition Region Sequence Optimization Protein-recognition region sequences were modified by swapping various base-pairs in the stem- loops, for example from A-U to C-G, or G-U to G-C. Selected protein-recognitions sequences are shown in the table below. All protein-recognition region sequences are found in the sequence listing as indicated in the tables below. Guides were then designed by appending a target-recognition region sequence to the 5′-end of a protein-recognition sequence. In the table below, the “gaaa” tetraloop is represented in lowercase letters. Table 85 Select protein-recognition region sequences designed for each Cas protein Cas Protein Cas Protein Protein- SEQ. ID SEQ ID NO. Recognition Protein-Recognition Region Sequence ID Region ID NO. 2 6 3 0 5 8 8 Guides described in the tables below were constructed from the target-recognition regions identified by ID and SEQ ID NO in the column “Target-recognition Region” and the protein-recognition region identified by ID and SEQ ID NO in the column “Protein-recognition Region”. In the tables below, the target that each guide was designed for is indicated in the columns labeled “Target”. The nucleobase sequences of the guides and the fully annotated sequences describing the modifications on the guides are found in the sequence listing as indicated in the Guide columns labeled “SEQ ID NO.” and “Mod. SEQ ID No.”, respectively. Cas proteins and guides were tested for in vitro activity in HepG2 cells, as described above. For each Cas protein and guide system, the percentage of insertions and deletions (% InDels) on each target is reported in the tables below. Table 86 % InDels of targets with IONCAS009 protein and guides, varying protein-recognition region sequence GuideTarget-RecognitionProtein-Recognition Region Region%InDelsng28 95 61 0642 39 12 52 Table 87 % InDels of targets with IONCAS030 protein and guides, varying protein-recognition region sequence GuideTarget-RecognitionProtein-Recognition Region Region%InDelsng10 09 19 06 09 21 90 32 66 59 Table 88 % InDels of targets with IONCAS057 protein and guides, varying protein-recognition region sequence GuideTarget-RecognitionProtein-Recognition Region Region%InDelsMod. ng05 86 50 54 37 00 57 14 77 55 54 71 51 25 98 95 39 37 23 60 88 99 49 68 44 86 04 93 02 04 69 50 49 33 87 58 C57FAg1-41 2515 3019 C57FAs1_23 741 C57g-41 1325 45.57 4.57 C57FAg1-42 2516 3020 C57FAs1_23 741 C57g-42 1326 42.18 4.83 21 74 37 12 95 08 69 05 02 24 82 53 81 79 50 00 18 32 Table 89 % InDels of targets with IONCAS065 protein and guides, varying protein-recognition region sequence GuideTarget-RecognitionProtein-Recognition Region Region%InDelsng1904 25 67 93 00 52 38 34 97 50 66 23 C65HEg1-40 2545 3049 C65HEs1_23 776 C65g-40 1353 35.79 5.01 C65HEg1-41 2546 3050 C65HEs1_23 776 C65g-41 1354 19.60 2.55 72 41 78 49 82 11 25 0560099312407879009230852679951856123875 Table 90 % InDels of targets with IONCAS075 protein and guides, varying protein-recognition region sequence GuideTarget-RecognitionProtein-Recognition Region Region%InDelsMod. ng57 20 24 34 35 79 78 39 71 34 88 82 03 70 06 47 57 79 67 32 18 81 44 64 19 48 74 02 58 51 35 22 866617 C75ASg2-48 2602 3108 C75ASs2-22U 906 C75g-48 1376 94.66 16.94 C75ASg2-49 2603 3109 C75ASs2-22U 906 C75g-49 1377 89.13 15.40 89 16 42 28 57 38 33 35 52 86 24 50 96 2863 Table 91 % InDels of targets with IONCAS080 protein and guides, varying protein-recognition region sequence GuideTarget-RecognitionProtein- Region Recognition Region%InDelsng7307 34 59 73380840 Table 92 % InDels of targets with IONCAS105 protein and guides, varying protein-recognition region sequence GuideTarget-RecognitionProtein-Recognition Region Region%InDelsMod. ng06 61 64 01 69 91 47 07 18 74 38 93 38 45 49 50 14 28 11 04 82 21 66 4153 06 77 53 16 47 20 77 90 27 96 C105HBg2-32 2654 3163 C105HBs2-22U 920 C105g-32 1397 69.35 3.99 C105HBg2-33 2655 3164 C105HBs2-22U 920 C105g-33 1398 63.78 4.07 04 39 99 26 47 93 55 05 41 54 34

[0006] Example 10: Design and efficacy of Cas fusion proteins and modified guides on epigenetic repression of PCSK9 in Hepa1-6 cells, LNP delivery Cas proteins described above were inserted into a a Cas fusion protein comprising a catalytically inactive Cas protein and a DNA demethylation domain. Modified guides comprising a target-recognition region sequence targeted to PCSK9 linked to the 5'-end of a protein-recognition region sequence were designed for the Cas fusion proteins and purchased from IDT. The Cas fusion protein and modified guide systems were tested in mouse Hepa1-6 cells for epigenetic repression of PCSK9. Design of Cas fusion proteins for DNA methylation Nuclease-dead versions of Cas proteins described herein above were designed by replacing the aspartic acid and histidine residues involved in nuclease activity with alanine. For Cas protein IONCAS009, the point mutations are D11A and H871A. For Cas protein IONCAS075 the point mutations are D11A and H868A. To impart DNA methylation activity, the C-terminal domain of Dnmtl3l (3A3L, SEQ ID NO: 958) was fused to the N-terminus of the catalytically-inactive Cas protein via a XTEN80 linker sequence (SEQ ID NO: 959) included to add flexibility and spacing. The 3A3L domain and XTEN80 linker were previously disclosed in International Patent Application No. WO 2023 / 215711. The sequences of the Cas fusion proteins are provided in the sequence listing, as indicated in the table below. The Cas fusion protein DNA sequences were cloned into a pmRVac vector from VectorBuilder for in vitro transcription (SEQ ID NO: 77). In order to facilitate enzyme localization to nucleus, nucleoplasmin (SEQ ID NO: 3399) and c-Myc (SEQ ID NO: 3400) NLS were appended at the N- and C- terminus of each Cas protein sequence, respectively. In addition, a HiBiT (Promega) tag was added to the protein C-terminus for expression detection. These additional elements were linked to the proteins via known protein linkers such as GGGS / (GS)3 / (GGGGS)3. A poly-A tail was added to the 3’-end of the DNA sequences encoding the Cas proteins with NLS and HiBiT tag. These DNA sequences are provided in the sequence listing, as indicated in SEQ ID NO: 956-957 below. Table 93 Sequences of Cas fusion proteins Cas Protein + NLS Cas Protein codon- Design and efficacy of Cas fusion protein and guide systems targeted to PCSK9 Target-recognition region sequences for PCSK9 were designed using PAM sequences determined for each Cas fusion protein and PCSK9. The target-recognition region sequences can be found in the sequence listing as indicated in the tables below. Guides were then designed by appending a target-recognition region sequence to the 5′-end of protein-recognition sequence C9g-NU (SEQ ID NO: 1052) for IONCAS009-d or C75g-46 (SEQ ID NO: 1375) for IONCAS075-d. A guide for positive control SpCas9 was designed as well. Each guide in the tables below has the following modifications: the nucleosides at positions 1 and 2 at the 5'-end are 2'-OMe sugar moieties wherein each nucleoside is linked to the next nucleoside with phosphorothioate internucleoside linkage; and the last two nucleosides at the 3'-end are 2'-OMe sugar moieties, wherein each nucleoside is linked to the preceding nucleoside by a phosphorothioate internucleoside linkage. Guides described in the tables below were constructed from the target-recognition regions identified by ID and SEQ ID NO in the column “Target-recognition Region” and the protein-recognition region identified by ID and SEQ ID NO in the column “Protein-recognition Region”. The nucleobase sequences of the guides and the fully annotated sequences describing the modifications on the guides are found in the sequence listing as indicated in the Guide columns labeled “SEQ ID NO.” and “Mod. SEQ ID No.”, respectively. Mouse Hepa1-6 cells were seeded at a density of 20,000 cells per well in a 96-well plate 2-4 hours prior to transfection. RNA / LNP complexes were generated by mixing 220 ng of Cas fusion protein mRNA and 440 ng of guide RNA with an LNP formulation. After incubating for 10 minutes at room temperature, the mixture was added to the culture medium. Samples were collected 3 days post transfection. RNA was extracted using RLT buffer (Thermo Fisher) and Pall AcroPrep Advance 96-well plates. PCSK9 RNA was quantified using quantitative PCR using TaqMan primer probe set Mm00463746_m1 (ThermoFisher). PCSK9 RNA levels were normalized to GAPDH RNA using TaqMan primer probe set Mm99999915_g1 (Thermofisher) or ACTB using TaqMan primer probe set Mm02619580_g1 (Thermofisher). PCSK9 RNA expression is presented in the tables below as percent PCSK9 RNA relative to the amount of PCSK9 RNA in untreated control cells (% UTC). Table 94 Effect of IONCAS009-d and guide systems on PCKS9 expression in Hepa1-6 cells, 3 days GuideTarget-RecognitionProtein- 9 C) C9PCSKg-2 3262 3323 C9PCSKs-2 3201 C9g-NU 1052 94 C9PCSKg-3 3263 3324 C9PCSKs-3 3202 C9g-NU 1052 91 Table 95 Effect of IONCAS075-d and guide systems on PCKS9 expression in Hepa1-6 cells, 3 days GuideTarget-RecognitionProtein- 9 C) C75PCSKg-12 3296 3357 C75PCSKs-12 3235 C75g-46 1375 76 C75PCSKg-13 3297 3358 C75PCSKs-13 3236 C75g-46 1375 105 Duration of Action of Cas fusion protein and guide systems targeted to PCSK9 Guides were selected from the activity screen above and tested for their activity over time in mouse Hepa1-6 cells. Mouse Hepa1-6 cells were seeded at a density of 20,000 cells per well in a 96-well plate 2-4 hours prior to transfection. RNA / LNP complexes were generated by mixing 220 ng of Cas fusion protein mRNA and 440 ng of guide RNA with an LNP formulation. SpCas9 and guide SpPCSKg were included as a control. After incubating for 10 minutes at room temperature, the mixture was added to the culture medium. Samples were collected at time points indicated in the tables below. Cells were split every 2-3 days to maintain optimal cell density. RNA was extracted using RLT buffer (Thermo Fisher) and Pall AcroPrep Advance 96-well plates. PCSK9 RNA was quantified using quantitative PCR using TaqMan primer probe set Mm00463746_m1 (ThermoFisher). PCSK9 RNA levels were normalized to GAPDH RNA using TaqMan primer probe set Mm99999915_g1 (Thermofisher) or ACTB using TaqMan primer probe set Mm02619580_g1 (Thermofisher). PCSK9 RNA expression is presented in the tables below as perc...

Claims

CLAIMS 1. A polypeptide, comprising a region having at least 800, at least 900, at least 1000, or at least 1050 linked amino acids, wherein the amino acid sequence of the region has at least 85%, at least 90%, at least 95%, or 100% identity to an equal length portion of any of SEQ ID NOs: 4-23 or 231-323, optionally wherein the polypeptide is isolated.

2. A polypeptide, comprising a region having at least 900, at least 1000, or at least 1100 linked amino acids, wherein the amino acid sequence of the region has at least 85%, at least 90%, at least 95%, or 100% identity to an equal length portion of SEQ ID NO: 6-23, 250-308; optionally wherein the polypeptide is isolated.

3. A polypeptide, comprising a region having at least 900, at least 1000, at least 1100, at least 1200, or at least 1300 linked amino acids, wherein the amino acid sequence of the region has at least 85%, at least 90%, at least 95%, or 100% identity to an equal length portion of any of SEQ ID NOs: 10-23 or 266-308; optionally wherein the polypeptide is isolated.

4. A polypeptide, comprising a region, wherein the amino acid sequence of the region has at least 85%, at least 90%, at least 95%, or 100% identity to any of SEQ ID NOs: 4-23 or 231-323; optionally wherein the polypeptide is isolated.

5. A polypeptide, wherein the amino acid sequence of the polypeptide has at least 85%, at least 90%, at least 95%, or 100% identity to any of SEQ ID NOs: 4-23 or 231-323; optionally wherein the polypeptide is isolated.

6. The polypeptide of claim 5, wherein the amino acid sequence of the polypeptide has at least 85%, at least 90%, at least 95%, or 100% identity to any of SEQ ID NOs: 12, 13, 20, 241, 242, 245, 247, 260, 266, 267, 268, 271, 274, 275, 279, 282, 283, 284, 288, 289, 292, 297, 299, 300, 301, 302, 314, or 323.

7. The polypeptide of any of claims 1-4, consisting of the region.

8. The polypeptide of any of claims 1-4, comprising a heterologous domain.

9. The polypeptide of claim 8, wherein the heterologous domain is selected from a transcriptional activator, a transcriptional repressor, a methyltransferase, a demethylase, a deaminase, an acetyltransferase, or a deacetylase.

10. The polypeptide of any of claims 1-4 or 7-9, wherein the amino acid sequence of the region has at least 85%, at least 90%, at least 95%, or 100% identity to an equal length portion of any of SEQ ID NOs: 13, 241, 267, 275, 284, 289, or 314.

11. The polypeptide of any of claims 1-10, wherein the polypeptide comprises a non-PI region and a PI domain, wherein the non-PI region of the polypeptide has an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 3186, 3188, 3190, 3192, 3194, 3196, or 3198.

12. The polypeptide of any of claims 1-10, wherein the polypeptide comprises a non-PI region and a PI domain, wherein the non-PI region of the polypeptide has an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 3186 and the PI domain has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3187.

13. The polypeptide of any of claims 1-10, wherein the polypeptide comprises a non-PI region and a PI domain, wherein the non-PI region of the polypeptide has an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 3188 and the PI domain has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3189.

14. The polypeptide of any of claims 1-10, wherein the polypeptide comprises a non-PI region and a PI domain, wherein the non-PI region of the polypeptide has an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 3190 and the PI domain has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3191.

15. The polypeptide of any of claims 1-10, wherein the polypeptide comprises a non-PI region and a PI domain, wherein the non-PI region of the polypeptide has an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 3192 and the PI domain has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3193.

16. The polypeptide of any of claims 1-10, wherein the polypeptide comprises a non-PI region and a PI domain, wherein the non-PI region of the polypeptide has an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 3194 and the PI domain has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3195.

17. The polypeptide of any of claims 1-10, wherein the polypeptide comprises a non-PI region and a PI domain, wherein the non-PI region of the polypeptide has an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 3196 and the PI domain has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3197.

18. The polypeptide of any of claims 1-10, wherein the polypeptide comprises a non-PI region and a PI domain, wherein the non-PI region of the polypeptide has an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 3198 and the PI domain has an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3199.

19. The polypeptide of any of claims 1-18, wherein the polypeptide has exactly two catalytically active nuclease active sites.

20. The polypeptide of any of claims 1-18, wherein the polypeptide has exactly one catalytically active nuclease active site.

21. The polypeptide of any of claims 1-18, wherein the polypeptide has zero catalytically active nuclease active sites.

22. The polypeptide of any of claims 1-21, comprising a nuclear localization signal.

23. The polypeptide of claim 22, wherein the nuclear localization signal is at the N-terminus of the polypeptide.

24. The polypeptide of claim 22, wherein the nuclear localization signal is at the C-terminus of the polypeptide.

25. The polypeptide of claim 22, wherein the polypeptide comprises two nuclear localization signals, one at each of the N-terminus and the C-terminus of the polypeptide.

26. A nucleic acid encoding the polypeptide of any of claims 1-25.

27. The nucleic acid of claim 26, wherein the nucleic acid is a DNA.

28. The nucleic acid of claim 26, wherein the nucleic acid is an mRNA comprising a coding region that encodes for the polypeptide of any of claims 1-25.

29. The nucleic acid of claim 26, wherein the mRNA is an exogenous mRNA comprising a coding region that encodes for the polypeptide of any of claims 1-21.

30. The mRNA of claim 28 or 29, comprising a 5’ cap structure.

31. The mRNA of any of claims 28-30, comprising a 3’-poly(A) tail.

32. The mRNA of any of claims 28-31, comprising a 5’-UTR.

33. The mRNA of any of claims 28-32, comprising a 3’-UTR.

34. The mRNA of claim 28 or 29, comprising a 5’-cap structure and 3’-poly(A) tail.

35. The mRNA of any of claims 28-34, wherein the coding region is codon optimized for expression in a eukaryotic cell.

36. The mRNA of claim 35, wherein the coding region has been codon optimized for expression in a mammalian cell.

37. The mRNA of claim 36, wherein the mammalian cell is a human cell.

38. The mRNA of any of claims 28-37, wherein each nucleobase of the mRNA is selected from adenine, guanine, cytosine, uracil, thymine, and N1-methylpseudouracil.

39. The mRNA of any of claims 28-37, wherein each nucleobase of the mRNA is selected from adenine, guanine, cytosine, and N1-methylpseudouracil.

40. The mRNA of any of claims 28-37, wherein each nucleobase of the mRNA is selected from adenine, guanine, cytosine, and uracil.

41. A guide, wherein the guide comprises a protein-recognition region, wherein the protein-recognition region interacts with the polypeptide of any of claims 1-25.

42. The guide of claim 41, wherein the guide is a dual guide.

43. The guide of claim 41, wherein the guide is a single guide.

44. A guide, consisting of an oligonucleotide according to the following formula: T1-D-X-S1a-B1-S1b-H1-S1b’-B2-S1a’-L1-S2-H2-S2’-L2-S3-H3-S3’-(L3-S4-H4-S4’)n-(L4-S5-H5-S5’)m-T2; wherein: ^ D consists of 17-25 linked nucleosides, wherein the nucleobase sequence of D is complementary to the nucleobase sequence of a target DNA; ^ X is absent, or consists of 1-2 linked nucleosides that are not complementary to the sequence of the target DNA; ^ each of T1and T2is absent or independently consists of 1-30 linked nucleosides; ^ S1aand S1a’ each consist of 6-10 linked nucleosides, wherein the nucleobase sequence of S1ais 100% complementary to the nucleobase sequence of S1a’; ^ S1band S1b’ each consist of 2-14 linked nucleosides, wherein the nucleobase sequence of S1bis 100% complementary to the nucleobase sequence of S1b’; ^ S2and S2’ each consist of 2-6 linked nucleosides, wherein the nucleobase sequence of S2is 100% complementary to the nucleobase sequence of S2’; ^ S3and S3’ each consist of 2-12 linked nucleosides, wherein the nucleobase sequence of S3is 100% complementary to the nucleobase sequence of S3’; ^ S4and S4’ each consist of 4-14 linked nucleosides, wherein the nucleobase sequence of S4is 100% complementary to the nucleobase sequence of S4’; ^ S5and S5’ each consist of 4-11 linked nucleosides, wherein the nucleobase sequence of S5is 100% complementary to the nucleobase sequence of S5’; ^ n is 0 or 1; ^ m is 0 or 1; ^ B1is absent, or consists of 1-3 linked nucleosides; ^ B2is absent or consists of 1-4 linked nucleosides; ^ wherein B1is not equal to B2unless both are absent; ^ each of H1, H2, H3, H4, and H5independently consists of 3-6 linked nucleosides; ^ L1consists of 2-3 linked nucleosides; ^ each of L2, L3, and L4independently consists of 0-10 linked nucleosides.

45. The guide of claim 44, wherein the guide comprises a protein-recognition region that binds to the polypeptide of any of claims 1-25.

46. The guide of any of claims 44-45, wherein T1is absent.

47. The guide of any of claims 44-46, wherein T2is absent.

48. The guide of any of claims 44-46, wherein T2consists of 2-6 linked nucleosides comprising a uracil nucleobase.

49. The guide of any of claims 44-48, wherein X is absent.

50. The guide of any of claims 44-49, wherein n is 1 and m is 0.

51. The guide of any of claims 44-50, wherein n and m are both 1.

52. The guide of any of claims 44-51, wherein the number of nucleosides in B2is greater than the number of nucleosides in B1.

53. The guide of any of claims 44-52, wherein each L1nucleoside is an adenosine.

54. The guide of any of claims 44-53, wherein the guide binds to polypeptide of any of claims 1-25.

55. The guide of claim 44 or 45, wherein T1and X are absent; T2is absent or consists of 2-6 linked uridines; S1aand S1a’ each consist of 7 linked nucleosides; S1band S1b’ each consist of 3-9 linked nucleosides; S2and S2’ each consist of 3 linked nucleosides; S3and S3’ each consist of 4 linked nucleosides; S4and S4’ each consist of 5 linked nucleosides; S5and S5’, when present, each consist of 7-9 linked nucleosides; n is 1; m is 0 or 1; B1consists of 1 nucleoside; B2consists of 4 linked nucleosides; ^ Each of H1,H2,H3and H4,when present, consist of 4 linked nucleosides; ^ L1consists of 2 linked adenosine nucleosides; ^ L2consists of 5 linked nucleosides; ^ L3consists of 0 nucleosides; and ^ L4, when present, consists of 4 linked nucleosides.

56. The guide of claim 55, wherein S1band S1b’ each consist of 7 linked nucleosides.

57. The guide of claim 55 or 56, wherein S5and S5’ each consists of 7 linked nucleosides.

58. The guide of any of claims 55-57, wherein the nucleobase sequence of B2is AAAG.

59. The guide of any of claims 55-58, wherein the nucleobase sequence of L2is AAACU.

60. The guide of any of claims 55-59, wherein the nucleobase sequence of L3is UUUUAA.

61. The guide of any of claims 55-60, wherein the nucleobase sequence of H2is GUCA.

62. The guide of any of claims 55-61, wherein n is 1 and m is 0.

63. The guide of any of claims 55-62, wherein n and m are both 1.

64. The guide of claim 55, wherein S1band S1b’ each consist of 7 linked nucleosides; n is 1 and m is 0; the nucleobase sequence of B2is AAAG; the nucleobase sequence of L2is AAACU; the nucleobase sequence of L3is UUUUAA, and the nucleobase sequence of H2is GUCA.

65. The guide of any of claims 55-64, wherein the guide comprises a region of at least 40, at least 50, at least 60, or at least 70 nucleosides, wherein the nucleobase sequence of the region has at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an equal length region of any of SEQ ID NOs: 131, 961-964, 1016-1020, 1052-1070, 1313-1314, or 2825.

66. The guide of claim 65, wherein the nucleobase sequence of the region has 100% identity to an equal length region of any of SEQ ID NOs: 131, 961-964, 1016-1020, 1052-1070, 1313-1314, or 2825.

67. The guide of any of claims 55-66, wherein D consists of 20-23 nucleosides having a nucleobase sequence that is 100% complementary to the nucleobase sequence of a target DNA.

68. The guide of claim 44 or 45, wherein T1and X are absent; T2is absent or consists of 6 linked uridines; S1aand S1a’ each consist of 9 linked nucleosides; S1band S1b’ each consist of 2-8 inked nucleosides; S2and S2’ each consist of 6 linked nucleosides; S3and S3’ each consist of 7 linked nucleosides; n and m are 0; B1consists of 3 nucleosides; B2consists of 4 linked nucleosides; ^ H1consists of 4 linked nucleosides; ^ H2consists of 5 linked nucleosides; ^ H3consists of 4 linked nucleosides; ^ L1consists of 2 linked adenosine nucleosides; and ^ L2consists of 0 linked nucleosides.

69. The guide of claim 68, wherein S1band S1b’ each consist of 4 linked nucleosides.

70. The guide of any of claims 68-69, wherein L2consists of 0 linked nucleosides and T2consists of 6 linked uridines.

71. The guide of any of claims 68-70, wherein the nucleobase sequence of B2is UAAC.

72. The guide of any of claims 68-71, wherein the nucleobase sequence of L2is GAACUC.

73. The guide of any of claims 68-72, wherein the nucleobase sequence of H2is UUUAU.

74. The guide of claim 68, wherein S1band S1b’ each consist of 4 linked nucleosides; n and m are 0; the nucleobase sequence of B2is UACC; the nucleobase sequence of L2is GAACUC; and the nucleobase sequence of H2is UUUAU.

75. The guide of any of claims 68-74, wherein the guide comprises a region having a nucleobase sequence that has at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NOs: 520, 965-968, 1071-1094, or 1315-1318, wherein the region comprises at least 40, at least 50, at least 60, or at least 70 nucleosides.

76. The guide of claim 75, wherein the nucleobase sequence of the region has 100% identity to an equal length region of any of SEQ ID NOs: 520, 965-968, 1071-1094, or 1315-1318.

77. The guide of any of claims 68-75, wherein D consists of 20-23 nucleosides having a nucleobase sequence that is 100% complementary to the nucleobase sequence of a target DNA.

78. The guide of claim 44 or 45, wherein T1and X are absent; T2is absent or consists of 1-7 linked nucleosides; S1aand S1a’ each consist of 7 linked nucleosides; S1band S1b’ each consist of 4-12 linked nucleosides; S2and S2’ each consist of 5 linked nucleosides; S3and S3’ each consist of 6 linked nucleosides; S4and S4’ each consist of 6 linked nucleosides; S5and S5’, when present, each consist of 7 linked nucleosides; n is 1; m is 0 or 1; B1consists of 1 nucleoside; B2consists of 3 linked nucleosides; ^ Each of H1,H2,and H3consist of 4 linked nucleosides; ^ H4and H5, when present, consist of 3 linked nucleosides; ^ L1consists of 3 linked adenosine nucleosides; ^ L2and L3consist of 0 linked nucleosides; ^ L4, when present, consists of 1-7 linked nucleosides.

79. The guide of claim 78, wherein S1band S1b’ each consist of 6 linked nucleosides.

80. The guide of any of claims 78-79, wherein the nucleobase sequence of B2is GAG.

81. The guide of any of claims 78-80, wherein the nucleobase sequence of H2is AUCC.

82. The guide of claim 78, wherein S1band S1b’ each consist of 6 linked nucleosides; n is 1 and m is 0; the nucleobase sequence of B2is GAG; and the nucleobase sequence of H2is AUCC.

83. The guide of any of claims 78-82, wherein the guide comprises a region having a nucleobase sequence that has at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NOs: 546, 973-976, 1021-1023, 1095-1126, or 1319-1342, wherein the region comprises at least 40, at least 50, at least 60, or at least 70 nucleosides.

84. The guide of claim 83, wherein the nucleobase sequence of the region has 100% identity to an equal length region of any of SEQ ID NOs: 546, 973-976, 1021-1023, 1095-1126, or 1319-1342.

85. The guide of any of claims 78-84, wherein D consists of 20-24 nucleosides having a nucleobase sequence that is 100% complementary to the nucleobase sequence of a target DNA.

86. The guide of claim 44 or 45, wherein T1and X are absent; T2is absent or consists of 1-6 linked nucleosides; S1aand S1a’ each consist of 7 linked nucleosides; S1band S1b’ each consist of 4-12 inked nucleosides; S2and S2’ each consist of 4 linked nucleosides; S3and S3’ each consist of 4 linked nucleosides; S4and S4’ each consist of 5 linked nucleosides; S5and S5’, when present, each consist of 7 linked nucleosides; n is 1; m is 0 or 1; B1consists of 1 nucleoside; B2consists of 3 nucleosides; ^ H1consists of 4 linked nucleosides; ^ H2consists of 3 linked nucleosides; ^ H3consists of 6 linked nucleosides; ^ H4consists of 4 linked nucleosides; ^ H5, when present, consist of 3 linked nucleosides; ^ L1consists of 2 linked adenosine nucleosides; ^ L2consists of 2 linked nucleosides; ^ L3consists of 0 linked nucleosides; ^ L4, when present, consists of 7 linked nucleosides.

87. The guide of claim 86, wherein S1band S1b’ each consist of 10 linked nucleosides.

88. The guide of any of claims 86-87, wherein n is 1 and m is 0.

89. The guide of any of claims 86-88, wherein the nucleobase sequence of B2is GAG.

90. The guide of any of claims 86-89, wherein the nucleobase sequence of L2is AA.

91. The guide of any of claims 86-90, wherein the nucleobase sequence of H2is UAA.

92. The guide of claim 86, wherein S1band S1b’ each consist of 10 linked nucleosides; n is 1 and m are 0; the nucleobase sequence of B2is GAG; the nucleobase sequence of L2is AA; and the nucleobase sequence of H2is UAA.

93. The guide of any of claims 86-92, wherein the guide comprises a region having a nucleobase sequence that has at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NOs: 562, 985-989, 1027-1029, 1153-1182, or 1362-1390, wherein the region comprises at least 40, at least 50, at least 60, or at least 70 nucleosides.

94. The guide of claim 93, wherein the nucleobase sequence of the region has 100% identity to an equal length region of any of SEQ ID NOs: 562, 985-989, 1027-1029, 1153-1182, or 1362-1390.

95. The guide of any of claims 86-94, wherein D consists of 20-24 nucleosides having a nucleobase sequence that is 100% complementary to the nucleobase sequence of a target DNA.

96. The guide of claim 44 or 45, wherein T1and X are absent; T2is absent or consists of 2-5 linked nucleosides; S1aand S1a’ each consist of 9 linked nucleosides; S1band S1b’ each consist of 2-10 linked nucleosides; S2and S2’ each consist of 4 linked nucleosides; S3and S3’ each consist of 9 linked nucleosides; n and m are 0; B1consists of 1 nucleoside; B1consists of 3 nucleosides; H1consists of 4 linked nucleosides; H2consists of 3 linked nucleosides; H3consists of 4 linked nucleosides; L1consists of 2 linked adenosine nucleosides; L2consists of 7 linked nucleosides.

97. The guide of claim 96, wherein S1band S1b’ each consist of 6 linked nucleosides.

98. The guide of any of claims 96-97, wherein the nucleobase sequence of B2is CUA.

99. The guide of any of claims 96-98, wherein the nucleobase sequence of L2is GUGUUUA.

100. The guide of any of claims 96-99, wherein the nucleobase sequence of H2is AAA.

101. The guide of claim 96, wherein S1band S1b’ each consist of 10 linked nucleosides; n and m are 0; the nucleobase sequence of B2is CUA; the nucleobase sequence of L2is GUGUUUA; and the nucleobase sequence of H2is AAA.

102. The guide of any of claims 96-101, wherein the guide comprises a region having a nucleobase sequence that has at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to any of SEQ ID NOs: 593, 1006-1010, 1272-1292, 1394-1408, 2827-2833, wherein the region comprises at least 40, at least 50, at least 60, or at least 70 nucleosides.

103. The guide of claim 102, wherein the nucleobase sequence of the region has 100% identity to an equal length region of any of SEQ ID NOs: 593, 1006-1010, 1272-1292, 1394-1408, 2827-2833.

104. The guide of any of claims 96-103, wherein D consists of 20-24 nucleosides having a nucleobase sequence that is 100% complementary to the nucleobase sequence of a target DNA.

105. The guide of claim 44 or 45, wherein T1and X are absent; T2is absent or consists of 3 linked nucleosides; S1aand S1btogether, and S1a’ and S1b’ together, each consist of 12-20 linked nucleosides; S2and S2’ each consist of 3 linked nucleosides; S3and S3’ each consist of 3 linked nucleosides; S3and S3’ each consist of 10-13 linked nucleosides; n is 1 and m is 0; B1consists of 0 nucleosides; B2consists of 0 nucleosides; ^ H1,H2,H3, and H4consist of 4 linked nucleosides; ^ L1consists of 2 linked adenosine nucleosides; ^ L2consists of 2 linked nucleosides; and ^ L3consists of 6 linked nucleosides.

106. The guide of claim 105, wherein S1aand S1btogether, and S1a’ and S1b’ together, each consist of 15 linked nucleosides.

107. The guide of any of claims 105-106, wherein L3consists of 3 linked nucleosides.

108. The guide of any of claims 105-107, wherein S3and S3’ each consist of 13 linked nucleosides.

109. The guide of any of claims 105-107, wherein the nucleobase sequence of L2is GU.

110. The guide of any of claims 108-109, wherein the nucleobase sequence of H2is GAAA.

111. The guide of claim 105, wherein S1aand S1btogether, and S1a’ and S1b’ together, each consist of 15 linked nucleosides; n is 1 and m is 0; the nucleobase sequence of L2is GU; and the nucleobase sequence of H2is GAAA.

112. The guide of any of claims 105-111, wherein the guide comprises a region having a nucleobase sequence that has at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NOs: 568, 990-993, 1030-1034, 1183-1206, or 1391-1393, wherein the region comprises at least 40, at least 50, at least 60, or at least 70 nucleosides.

113. The guide of claim 112, wherein the nucleobase sequence of the region has 100% identity to an equal length region of any of SEQ ID NOs: 568, 990-993, 1030-1034, 1183-1206, or 1391-1393.

114. The guide of any of claims 106-113, wherein D consists of 20-24 nucleosides having a nucleobase sequence that is 100% complementary to the nucleobase sequence of a target DNA.

115. A guide, consisting of an oligonucleotide according to the following formula: T1-D-X-S1a-B1-S1b-H1-S1b’-B2-S1a’-L1-S2-H2-S2’-L2-S3-H3-S3’-(L3-S4-H4-S4’)n-T2; wherein: ^ D consists of 17-25 linked nucleosides, wherein the nucleobase sequence of D is complementary to the nucleobase sequence of a target DNA; ^ X is absent, or consists of 1-2 linked nucleosides that are not complementary to the nucleobase sequence of the target DNA; ^ each of T1and T2is absent or independently consists of 1-30 linked nucleosides; ^ S1aand S1a’ each consist of 7 linked nucleosides, wherein the nucleobase sequence of S1ais 100% complementary to the nucleobase sequence of S1a’; ^ S1band S1b’ each consist of 4-12 linked nucleosides, wherein the nucleobase sequence of S1bis 100% complementary to the nucleobase sequence of S1b’; ^ S2and S2’ each consist of 6 linked nucleosides, wherein the nucleobase sequence of S2is 100% complementary to the nucleobase sequence of S2’; ^ S3and S3’ each consist of 5 or 6 linked nucleosides, wherein the nucleobase sequence of S3is 100% complementary to the nucleobase sequence of S3’; ^ S4and S4’, when present, each consist of 6 linked nucleosides, wherein the nucleobase sequence of S4is 100% complementary to the nucleobase sequence of S4’; ^ n is 0 or 1; ^ B1is consists of 1 nucleoside; ^ B2consists of 3 linked nucleosides; ^ each of H1, H2, H3, and H4independently consists of 3-4 linked nucleosides; ^ L1consists of 17 linked nucleosides; ^ L2consists of 0 linked nucleosides; and ^ L3consists of 8 linked nucleosides.

116. The guide of claim 115, wherein S1band S1b’ each consist of 8 linked nucleosides.

117. The guide of any of claims 115-116, wherein the nucleobase sequence of B2is GAA.

118. The guide of any of claims 115-117, wherein the nucleobase sequence of L2is AAAAAUUUAUUCAAAAC (SEQ ID NO: 78).

119. The guide of any of claims 115-118, wherein the nucleobase sequence of H2is GAAA.

120. The guide of claim 119, wherein S1band S1b’ each consist of 8 linked nucleosides; n and m are 0; the nucleobase sequence of B2is GAA; the nucleobase sequence of L2is AAAAAUUUAUUCAAAAC (SEQ ID NO: 78); and the nucleobase sequence of H2is GAAA.

121. The guide of any of claims 115-120, wherein the guide comprises a region having a nucleobase sequence that has at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NOs: 554, 981-984, 1024-1026, 1127-1152, or 1343-1361, wherein the region comprises at least 40, at least 50, at least 60, or at least 70 nucleosides.

122. The guide of claim 121, wherein the nucleobase sequence of the region has 100% identity to an equal length region of any of SEQ ID NOs: 554, 981-984, 1024-1026, 1127-1152, or 1343-1361.

123. The guide of any of claims 115-122, wherein D consists of 20-24 nucleosides having a nucleobase sequence that is 100% complementary to the nucleobase sequence of a target DNA.

124. A guide of any of claims 41-43, wherein the guide comprises a region having a nucleobase sequence that has at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NOs: 121-141, 510-602, 961-1430, 1436-2833 wherein the region comprises at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 nucleosides.

125. A guide of any of claims 41-43, wherein the guide comprises a protein recognition region that has a nucleobase sequence that has at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NOs: 121-141, 510-602, 961-1430, 1436-2833.

126. The guide any of claims 41-125, wherein the guide comprises a modified oligonucleotide.

127. The guide of any of claims 41-126, wherein the guide comprises at least one modified sugar moiety.

128. The guide of claim 127, wherein the modified sugar moiety is selected from a 2’-OMe and a 2’-F.

129. The guide of claim 127, wherein the modified oligonucleotide comprises a 2’-OMe or 2’-F sugar moiety within the first five nucleosides at the 5’ end of the guide or the last five nucleosides at the 3’ end of the guide.

130. The guide of claim 127-129, wherein the guide comprises 2-32’-OMe sugar moieties at the 5’ end of the guide and 2-32’-OMe sugar moieties at the 3’-end of the guide.

131. The guide of claim 130, wherein each non-2’-OMe sugar moiety of the guide comprises an unmodified RNA sugar moiety.

132. The guide of any of claims 127-131, wherein the guide comprises a modified internucleoside linkage.

133. The guide of claim 132, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

134. The guide of claim 132 or 133, wherein the guide comprises at least one modified internucleoside linkage within the first five nucleosides at the 5’ end of the guide or the last five nucleosides at the 3’ end of the guide.

135. The guide of claim 134, wherein the guide comprises 2-3 phosphorothioate internucleoside linkages at the 5’ end of the guide and 2-3 phosphorothioate internucleoside linkages at the 3’ end of the guide.

136. The guide of claim 135, wherein each remaining internucleoside linkage is an unmodified phosphodiester internucleoside linkage.

137. The guide of any of claims 132-136, wherein each internucleoside linkage of the guide is a standard length internucleoside linkage.

138. The guide of any of claims 41-125, wherein the guide comprises an unmodified oligonucleotide.

139. The guide of any of claims 41-138, wherein the guide comprises a target recognition region that is at least 90%, at least 95%, or 100% complementary to a target DNA sequence.

140. The guide of claim 139, wherein the target recognition region comprises 17-25, 19-24, 20-24, 20-23, 20-22, 21-22, 21-23, 21-24, 20, 21, 22, 23, or 24 linked nucleosides.

141. An editing system comprising the polypeptide of any of claims 1-25 and a guide of any of claims 41- 139.

142. An editing system comprising: a nucleic acid of any of claims 26-40; and a guide of any of claims 41-140.

143. An editing system comprising: a nucleic acid of any of claims 26-40; and a nucleic acid encoding the guide of any of claims 41-125 or 138.

144. The editing system of claim 142 or 143 wherein the nucleic acid encoding the polypeptide is an exogenous mRNA.

145. The editing system of claim 142 or 143, wherein the nucleic acid encoding the polypeptide is a DNA.

146. The editing system any of claims 143-145, wherein the nucleic acid encoding the guide is a DNA.

147. A composition comprising the editing system of any of claims 141-146 and an LNP.

148. A viral vector comprising the editing system of claim 143.

149. A method of editing a target nucleic acid, comprising administering a composition of claim 146 or a viral vector of claim 148 to a subject.

150. A method of editing a target nucleic acid, comprising contacting a composition of claim 147 or a viral vector of claim 148 with a cell.

151. A method of creating a double stranded break or a nick in a target nucleic acid, comprising contacting a composition of claim 147 or a viral vector of claim 148 with a cell.

152. The composition of claim 147 or a viral vector of claim 148 for use in therapy.

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