WO2025072763A2 - Compositions and methods for precision editing with cas dimers - Google Patents

Abstract

Disclosed herein are systems, composition and methods of modifying a target double-stranded DNA (dsDNA) molecule in a cell. In general, systems, compositions, and methods comprise a compact, dimerizing CRISPR-associated (Cas) protein, an RNA-dependent DNA polymerase, and one or more guide nucleic acids, or uses thereof.

Description

COMPOSITIONS AND METHODS FOR PRECISION EDITING WITH CAS DIMERS

CROSS-REFERENCE TO RELATED APPLICATIONS

[1] The present application claims priority to U.S. Provisional Application 63/586,887, filed September 29, 2023; U.S. Provisional Application 63/649,164, filed May 17, 2024; U.S. Provisional Application 63/685,933, filed August 22, 2024, the contents each of which are incorporated herein by reference in their entireties.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[2] The contents of the electronic sequence listing (MABI_048_03WO_SeqList_ST26.xml; Size: 224,764 bytes; and Date of Creation: September 27, 2024) are herein incorporated by reference in its entirety.

BACKGROUND

[3] While gene disruption with CRISPR based technology is now a mature technique, precision editing of one or a few nucleotides in the human genome remains a major challenge. Therefore, there is a need for the development of systems that are capable of introducing specific nucleotide sequence modifications at atarget site with high specificity and efficiency.

SUMMARY

[4] Disclosed herein are compositions, systems and methods for precision editing of DNA. Precision editing involves very precise modification of a target DNA sequence. In some instances, precision editing involves modifying only one or a few nucleotides of a target sequence in a genome. In some instances, precision editing involves inserting or deleting a short nucleotide sequence. In some instances, precision editing involves replacing a short nucleotide sequence with a desired nucleotide sequence. The desired nucleotide sequence is encoded on a template RNA (retRNA) that serves as a template for an RNA dependent DNA polymerase (RDDP), e.g., a reverse transcriptase.

[5] Precision editing systems described herein generally comprise a compact Cas protein that homodimerizes, and a guide nucleic acid that guides the dimer to atarget sequence in a DNA molecule. In some embodiments, each monomer of the dimer is linked to an RDDP. In some embodiments, the dimer cuts both strands of DNA leaving staggered ends with 5’ overhangs. In some embodiments, precision editing systems described herein comprise two template RNAs (retRNAs), each containing an intended edit or desired nucleotide sequence. A template RNA is designed to hybridize to single stranded DNA created at a cut site. The linked RDDPs polymerize a new strand of DNA at each 3’ end of the cut DNA. An exemplary system is shown in FIG. 1 A. Endogenous repair proteins, including exonucleases and single stranded break repair proteins, may be useful for repairing the DNA cut site, see FIG. 2. In some embodiments, systems comprise DNA repair proteins to enhance DNA repair and further reduce unintended edits. Fusion of DNA repair proteins to the dimerizing Cas protein may enhance repair relative to a Cas protein that doesn’t dimerize, effectively recruiting twice as much repair protein, see, e.g., FIGs. 3 and 4. Without being bound by theory, it is contemplated that polymerization at both cut ends may reduce the chances of byproducts (unintended edits), even though the dimer produces a double stranded break.

[6] In some embodiments, precision editing systems comprise a homodimerizing effector protein, an RDDP, and two guide RNAs that hybridize to opposite strands of a DNA molecule at some distance apart from each other (e.g., 10-10,000 bp). The effector proteins form ribonucleoprotein (RNP) complexes with the two guide RNAs at these two different sites where they cleave the dsDNA, generating single stranded DNA (ssDNA) flaps at both sites that extend towards each other as shown in FIG. 8. These systems also comprise two retRNAs, each of which binds to a portion of one of the ssDNA flaps. The retRNAs each have a template sequence for generating a sequence of interest while the region between the two cut sites is deleted. Such systems may be referred to as dual-cut dual-flap systems.

[7] In some aspects, the present disclosure provides systems for modifying a target strand (TS) and/or a non target strand (NTS) of a double stranded DNA (dsDNA) molecule, the system comprising: a) an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein forms a dimer with itself in a cell; b) an RNA-directed DNA polymerase (RDDP) or a nucleic acid encoding the RDDP; c) a guide RNA or nucleic acid encoding the guide RNA, wherein the guide RNA comprises: i) a first region comprising a scaffold sequence, and ii) a second region comprising a spacer sequence that hybridizes to a target sequence of the TS, wherein the first region is located 5’ of the second region; d) a TS template RNA (retRNA) or nucleic acid encoding the TS retRNA, wherein the TS retRNA comprises: i) a TS primer binding sequence (PBS) that hybridizes to the TS, and ii) a TS template sequence; and e) a NTS retRNA or nucleic acid encoding the NTS retRNA, wherein the NTS retRNA comprises: i) a NTS PBS that hybridizes to the NTS, and ii) a NTS template sequence. In some embodiments, the effector protein cuts the TS and the NTS to generate staggered ends, thereby generating single stranded DNA with a free 3 ’ end on the TS (TS ssDNA) and single stranded DNA with a free 3’ end on the NTS (NTS ssDNA). In some embodiments, the TS PBS hybridizes to at least a portion of the TS ssDNA, and the NTS PBS hybridizes to at least a portion of the NTS ssDNA. In some embodiments, the RDDP polymerizes a new strand of ssDNA on the TS that is complementary to the TS template sequence, and a new strand of ssDNA on the NTS that is complementary to the NTS template sequence. In some embodiments, the system comprises a DNA repair protein. In some embodiments, the DNA repair protein is covalently linked to the effector protein. In some embodiments, the DNA repair protein is covalently linked to the RDDP. In some embodiments, the RDDP is covalently linked to the effector protein. In some embodiments, the guide RNA is covalently linked to the retRNA. In some embodiments, the TS template sequence is located 5’ of the TS PBS and the NTS template is located 5 ’ of the NTS PBS, optionally wherein the 3 ’ end of the PBS is linked to the 5’ end of the template sequence. In some embodiments, the TS retRNA, NTS retRNA, or each of the TS retRNA and NTS retRNA comprise a protein localization sequence, and the RDDP comprises or is covalently linked to a peptide that binds the protein localization sequence, the protein localization sequence thereby localizing the RDDP to the retRNA. In some embodiments, the TS template sequence and/or NTS template sequence comprises a difference of at least one nucleotide relative to an otherwise identical sequence in the dsDNA molecule (providing the interchangeability of thymines and uracils). In some embodiments, the effector protein is a Type V Cas protein. In some embodiments, the length of the effector protein is 400 to 500, 400 to 600, 400 to 700, 400 to 800, or 400-900 linked amino acids. In some embodiments, the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, the amino acid sequence of the effector protein is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, the effector protein comprises an amino acid substitution selected from TABLE 1.1 relative to SEQ ID NO: 1. In some embodiments, the scaffold sequence comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 83, 84, and 85. In some embodiments, the target sequence is a sequence present in a eukaryotic organism, optionally wherein the eukaryotic organism is a mammal, optionally wherein the mammal is a human. In some embodiments, the RDDP comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 2-82. In some embodiments, the systems comprise an expression vector, wherein the expression vector comprises any combination of: the nucleic acid encoding the effector protein; the nucleic acid encoding the RDDP; the nucleic acid encoding the guide RNA; and the nucleic acids encoding the TS retRNA and NTS retRNA. In some embodiments, the expression vector is an adeno-associated viral (AAV) vector, optionally wherein the AAV vector is an scAAV vector. In some embodiments, the systems comprise a lipid or lipid nanoparticle. In some embodiments, the nucleic acid encoding the effector protein or the nucleic acid encoding the RDDP comprises a messenger RNA.

[8] In some aspects, the present disclosure provides compositions for modifying a target strand (TS) and/or a non target strand (NTS) of a double stranded DNA (dsDNA) molecule, the composition comprising: a) an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein forms a dimer with itself in a cell; b) an RNA-directed DNA polymerase (RDDP) or a nucleic acid encoding the RDDP; c) a guide RNA or nucleic acid encoding the guide RNA, wherein the guide RNA comprises: i) a first region comprising a scaffold sequence, and ii) a second region comprising a spacer sequence that hybridizes to a target sequence of the TS, wherein the first region is located 5 ’ of the second region; d) a TS template RNA (retRNA) or nucleic acid encoding the TS retRNA, wherein the TS retRNA comprises: i) a TS primer binding sequence (PBS) that hybridizes to the TS, and ii) a TS template sequence; and e) a NTS retRNA or nucleic acid encoding the NTS retRNA, wherein the NTS retRNA comprises: i) a NTS PBS that hybridizes to the NTS, and ii) a NTS template sequence. In some embodiments, the compositions comprise a lipid nanoparticle (LNP), wherein the nucleic acid encoding the effector protein comprises an mRNA, wherein the LNP contains the mRNA, optionally wherein the mRNA comprises the nucleic acid encoding the RDDP, optionally wherein the LNP comprises the guide RNA, the TS retRNA, the NTS retRNA, or a combination thereof. In some embodiments, at least one of the guide RNA, the TS retRNA, and the NTS retRNA comprises a chemical modification.

[9] In some aspects, the present disclosure provides pharmaceutical compositions comprising the composition described herein, and a pharmaceutically acceptable excipient.

[10] In some aspects, the present disclosure provides expression vectors for modifying a target strand (TS) and/or a non target strand (NTS) of a double stranded DNA (dsDNA) molecule, the expression vector comprising: a) a first nucleotide sequence encoding an effector protein, wherein the effector protein forms a dimer with itself in a cell; b) a second nucleotide sequence encoding a RDDP; c) a third nucleotide sequence encoding a guide RNA, wherein the guide RNA comprises i) a first region comprising a scaffold sequence, and ii) a second region comprising a spacer sequence that hybridizes to a target sequence of the TS, wherein the first region is located 5 ’ of the second region; d) a fourth nucleotide sequence encoding a TS retRNA, wherein the TS retRNA comprises: i) a TS primer binding sequence (PBS) that hybridizes to the TS, and ii) a TS template sequence; and e) a fifth nucleotide sequence encoding a NTS retRNA, wherein the NTS retRNA comprises: i) a NTS PBS that hybridizes to the NTS, and ii) a NTS template sequence. In some embodiments, the expression vector is an AAV vector.

[11] In some aspects, the present disclosure provides systems for deleting a region of a double stranded DNA (dsDNA) molecule, the system comprising: a) an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein forms a dimer with itself in a cell; b) an RNA-directed DNA polymerase (RDDP) or a nucleic acid encoding the RDDP; c) a first guide RNA (gRNA) or a nucleic acid encoding the first gRNA, wherein the first gRNA comprises: i) a first scaffold sequence, and ii) a first spacer sequence that hybridizes to a first target sequence on a first strand of the dsDNA molecule, wherein the first scaffold sequence is located 5’ of the first spacer sequence, and wherein the effector protein and the first gRNA form a first RNP complex that cleaves the dsDNA molecule to form a first single stranded DNA (ssDNA) flap from the first strand of the dsDNA molecule; d) a second gRNA or a nucleic acid encoding the second gRNA, wherein the second gRNA comprises: i) a second scaffold sequence, and ii) a second spacer sequence that hybridizes to a second target sequence on a second strand of the dsDNA molecule, wherein the second scaffold sequence is located 5 ’ of the second spacer sequence, and wherein the effector protein and the second gRNA form a second RNP complex that cleaves the dsDNA molecule to form a second ssDNA flap from the second strand of the dsDNA molecule; e) a first template RNA (retRNA) or a nucleic acid encoding the first retRNA, wherein the first retRNA comprises: i) a first primer binding sequence (PBS) that hybridizes to at least a portion of the first ssDNA flap, and ii) a first template sequence; and f) a second retRNA or a nucleic acid encoding the second retRNA, wherein the second retRNA comprises: i) a second PBS that hybridizes to at least a portion of the second ssDNA flap, and ii) a second template sequence. In some embodiments, the nucleic acid encoding the effector protein, the RDDP, the gRNAs, and the retRNAs are combined in a single AAV vector. In some embodiments, the first spacer sequence and the second spacer sequence hybridize to the first target sequence and the second target sequence of the dsDNA molecule respectively, wherein the first target sequence and the second target sequence are 10 to 10,000 base pairs apart. In some embodiments, the system comprises a BREX27 peptide or a nucleic acid encoding the BREX27 peptide, optionally wherein the BREX27 peptide comprises an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 112.

[12] In some aspects, the present disclosure provides systems for modifying a target strand (TS) of a double stranded DNA (dsDNA) molecule, the system comprising: a) an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein forms a dimer with itself in a cell; b) an RNA-directed DNA polymerase (RDDP) or a nucleic acid encoding the RDDP; c) a guide RNA (gRNA) or a nucleic acid encoding the gRNA, wherein the gRNA comprises: i) a first region comprising a scaffold sequence, and ii) a second region comprising a spacer sequence that hybridizes to a target sequence of the TS of the dsDNA molecule, wherein the first region is located 5’ of the second region; and wherein the effector protein and the gRNA form a RNP complex that produces a double stranded break; and d) a TS template RNA (retRNA) or a nucleic acid encoding the TS retRNA, wherein the TS retRNA comprises: i) a TS primer binding sequence (PBS) that hybridizes to the TS of the dsDNA molecule, and ii) a TS template sequence. In some embodiments, the effector protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, the effector protein comprises at least one amino acid substitution relative to SEQ ID NO: 1, wherein the amino acid substitution is D220R. In some embodiments, the effector protein is linked to RDDP to form a fusion protein. In some embodiments, the fusion protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 114-123. In some embodiments, the fusion protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 115, 117, 119, 122, and 123, wherein the amino acid sequence comprises a P2A peptide that results in cleavage of the fusion protein at the site of the P2A. In some embodiments, the gRNA comprises a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 124 or 125. In some embodiments, the nucleic acid sequences encoding the effector protein, the RDDP, the gRNA, and the TS retRNAs are combined in a single AAV vector. In some embodiments, a nucleic acid sequence encoding the effector protein and a nucleic acid sequence encoding the RDDP is linked by a nucleic acid sequence encoding a P2A peptide.

[13] In some aspects, the present disclosure provides compositions comprising an engineered guide nucleic acid or a nucleic acid encoding the same, wherein the guide nucleic acid comprises or consists of a nucleotide sequence according to SEQ ID NO: 129. In some embodiments, the present disclosure provides compositions comprising an engineered guide nucleic acid or a nucleic acid encoding the same, wherein the guide nucleic acid comprises or consists of a nucleotide sequence selected from SEQ ID NO: 130, SEQ ID NO: 131, and a combination thereof. In some embodiments, SEQ ID NO: 131 is located 5’ of SEQ ID NO: 130. In some embodiments, the nucleic acid encoding the engineered guide nucleic acid is located in a AAV vector. In some embodiments, the composition comprises an effector protein or a nucleic acid encoding the same, wherein the effector protein comprises an amino acid sequence that is at least is 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1.

[14] In some aspects, the present disclosure provides precision editing systems comprising a fused RNA, wherein the fused RNA comprises: a) a guide RNA (gRNA) comprising from 5’ to 3’: a tracrRNA sequence, a repeat sequence, and a spacer sequence; and b) a template RNA (retRNA), wherein the retRNA is covalently linked to the 5’ end of the gRNA. In some embodiments, the fused RNA comprises one or more linker nucleotides between: i) the gRNA and the template RNA; ii) the tracrRNA sequence and the repeat sequence; iii) the repeat sequence and the spacer sequence; or a combination thereof. In some embodiments, the systems comprise an effector protein or nucleic acid encoding the same, and an RNA-directed DNA polymerase (RDDP) or a nucleic acid encoding the RDDP.

[15] In some aspects, the present disclosure provides compositions comprising an engineered RDDP or a nucleic acid encoding the same, wherein the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 15 and comprises the amino acid substitutions of D12R, N24R, N72R, N114K, and N195R; wherein the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 56 and comprises the amino acid substitutions of N24R, N72R, N114K, and N195R; or wherein the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 65 and comprises the amino acid substitutions of E12R, N24R, N72R, N114K, and N195R.

[16] In some aspects, the present disclosure provides cells modified by the systems described herein, the compositions described herein, or the expression vectors described herein. In some embodiments, the cell is a eukaryotic cell, optionally wherein the eukaryotic cell is a mammalian cell, optionally wherein the mammalian cell is a human cell.

[17] In some aspects, the present disclosure provides methods of modifying a dsDNA molecule, the method comprising contacting a target nucleic acid with the systems described herein, the compositions described herein, or the expression vectors described herein.

DESCRIPTION OF FIGURES

[18] FIG. 1A shows an exemplary precision editing system with a dimerized effector protein, RDDPs optionally linked to the effector protein, a guide RNA that localizes the effector protein to a double stranded DNA target nucleic acid, and template RNAs (retRNA) that hybridize to both strands of target DNA. Dotted lines indicated that the retRNAs are optionally circularized (5’ and 3’ ends covalently linked).

[19] FIG. IB shows an enlargement of the TS retRNA shown in FIG. 1A. The NTS in FIG. 1A could be similarly labeled.

[20] FIG. 2 shows how target dsDNA may be repaired after it is modified by a precision editing system described herein. The target dsDNA is cut and new DNA complementary to the template sequence is synthesized at the 3’ ends of the cut dsDNA. 5’ overhangs of the cut dsDNA are removed by exogenous or endogenous exonucleases. The newly synthesized single stranded DNA ends anneal to the free ends of the cut dsDNA. The 3’ ends of the cut dsDNA not used in the annealing reaction can be further processed by endogenous flap endonucleases.

[21] FIG. 3 shows peptides fused to each monomer of a dimerized compact Cas protein (“dimer”). Such peptides may have DNA repair modulating activity.

[22] FIG. 4 shows non-limiting examples of DNA repair peptides that may be fused to one or both monomers of a dimerized compact Cas protein, e.g., recruiting peptides that can recruit endogenous DNA repair proteins, single stranded break (SSB) repair proteins, and exonucleases.

[23] FIG. 5 illustrates the editing level of five RDDP candidates and the wild-type M-MLV RDDP against different targets in HEK293T cells.

[24] FIG. 6A - FIG. 6B illustrate the editing levels of RDDP candidates and MMLV RDDP against gene target sites. FIG. 6A shows editing levels in HEK target sites. FIG. 6B shows editing levels in FANCF target sites.

[25] FIG. 7A - FIG. 7B illustrate editing levels of variant RDDP candidates comprising arginine and lysine mutations in FANCF (FIG. 7A) and HEK FIG. 7B) target sites.

[26] FIG. 8 illustrates an exemplary dual-cut dual-flap system for precise deletion of a region of dsDNA.

[27] FIG. 9 illustrates an exemplary precision editing system using a Type V Cas nuclease that creates double stranded breaks with the target strand being extended by the RT. [28] FIG. 10 illustrates part of an exemplary plasmid encoding a gRNA and a retRNA that are not fused (split) and an exemplary plasmid encoding a fused RNA (gretRNA) comprising a gRNA fused to a retRNA.

[29] FIG. 11 shows the results of an experiment testing the ability of various CasM.265466-RDDP fusion proteins to generate precise edits, as assessed with a fluorescent reporter in mammalian cells.

DETAILED DESCRIPTION

[30] 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 disclosure.

[31] 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, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.

Definitions

[32] Unless otherwise indicated, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise indicated or obvious from context, the following terms have the following meanings:

[33] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.

[34] Any reference to “or” herein is intended to encompass “and/or” unless otherwise stated. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.

[35] Use of the term “including” as well as other forms, such as “includes” and “included,” is not limiting.

[36] As used herein, the term “comprise” and its grammatical equivalents specifies the presence of stated features, integers, steps, operations, elements, and/or components, but does not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

[37] As used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number and numbers +/- 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.

[38] The terms “% identical,” “% identity,” and “percent identity,” or grammatical equivalents thereof, with reference to an amino acid sequence or nucleotide sequence, refer to the percent of residues that are identical between respective positions of two sequences when the two sequences are aligned for maximum sequence identity. The % identity is calculated by dividing the total number of the aligned residues by the number of the residues that are identical between the respective positions of the at least two sequences and multiplying by 100. Generally, computer programs can be employed for such calculations. Illustrative programs that compare and align pairs of sequences, include ALIGN (Myers and Miller, Comput Appl Biosci. 1988 Mar;4(l): 11-7), FASTA (Pearson and Lipman, Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444-8; Pearson, Methods Enzymol. 1990;183:63-98) and gapped BLAST (Altschul et al., Nucleic Acids Res. 1997 Sep l;25(17):3389-40), BLASTP, BLASTN, or GCG (Devereux et al., Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1): 387-95). To determine sequence identity, sequences can be aligned using various convenient methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.), available over the world wide web at sites including ncbi.nlm.nili.gov/BLAST, ebi.ac.uk/Tools/msa/tcoffee/, ebi.ac.uk/Tools/msa/muscle/, mafft.cbrc.jp/alignment/software/. See, e.g., Altschul et al. (1990), J. Mol. Bioi. 215:403-10.

[39] The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[40] “Binding” as used herein (e.g. with reference to an RNA-binding domain of a polypeptide, binding to a target nucleic acid, and the like) refers to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid; between a CAS polypeptide/guide RNA complex and a target nucleic acid; and the like). While in a state of non-covalent interaction, the macromolecules are said to be “associated” or “interacting” or “binding” (e.g., when a molecule X is said to interact with a molecule Y, it is meant the molecule X binds to molecule Y in a non-covalent manner). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), but some portions of a binding interaction may be sequencespecific. Binding interactions are generally characterized by a dissociation constant (KD) of less than KF⁶ M, less than IO^-7 M, less than IO^-8 M, less than IO^-9 M, less than IO ¹⁰ M, less than 10 ¹¹ M, less than 10 ¹²M, less than 10 ¹³ M, less than 10 ¹⁴ M, or less than 10 ¹⁵ M. “Affinity” refers to the strength of binding, increased binding affinity being correlated with a lower KD.

[41] By “binding domain” it is meant a protein or nucleic acid domain that is able to bind non- covalently to another molecule. A binding domain can bind to, for example, a DNA molecule (a DNA- binding domain), an RNA molecule (an RNA-binding domain) and/or a protein molecule (a proteinbinding domain). In the case of a protein having a protein-binding domain, it can in some cases bind to itself (to form homodimers, homotrimers, etc.) and/or it can bind to one or more regions of a different protein or proteins.

[42] The term, “catalytically inactive effector protein,” as used herein, refers to an effector protein that is modified relative to a naturally-occurring effector protein to have a reduced or eliminated catalytic activity relative to that of the naturally-occurring effector protein, but retains its ability to interact with a guide nucleic acid. The catalytic activity that is reduced or eliminated is often a nuclease activity but can be nickase activity. The naturally-occurring effector protein may be a wild-type protein. In some embodiments, the catalytically inactive effector protein is referred to as a catalytically inactive variant of an effector protein, e.g., a Cas effector protein.

[43] The term, “cis cleavage,” as used herein, refers to cleavage (hydrolysis of a phosphodiester bond) of a target nucleic acid by a complex of an effector protein and a guide nucleic acid, wherein at least a portion of the guide nucleic acid is hybridized to at least a portion of the target nucleic acid. Cleavage may occur within or directly adjacent to the portion of the target nucleic acid that is hybridized to the portion of the guide nucleic acid.

[44] The terms, “cleave,” “cleaving,” and “cleavage,” as used herein, with reference to a nucleic acid molecule or nuclease activity of an effector protein, refer to the hydrolysis of a phosphodiester bond of a nucleic acid molecule that results in breakage of that bond. The result of this breakage can be a nick (hydrolysis of a single phosphodiester bond on one side of a double-stranded molecule), single strand break (hydrolysis of a single phosphodiester bond on a single -stranded molecule) or double strand break (hydrolysis of two phosphodiester bonds on both sides of a double-stranded molecule) depending upon whether the nucleic acid molecule is single-stranded (e.g., ssDNA or ssRNA) or double -stranded (e.g., dsDNA) and the type of nuclease activity being catalyzed by the effector protein.

[45] The terms, “complementary” and “complementarity,” as used herein, with reference to a nucleic acid molecule or nucleotide sequence, refer to the characteristic of a polynucleotide having nucleotides that base pair with their Watson-Crick counterparts (C with G; or A with T) in a reference nucleic acid. For example, when every nucleotide in a polynucleotide forms a base pair with a reference nucleic acid, that polynucleotide is said to be 100% complementary to the reference nucleic acid. In a double stranded DNA or RNA sequence, the upper (sense) strand sequence is in general, understood as going in the direction from its 5'- to 3 '-end, and the complementary sequence is thus understood as the sequence of the lower (antisense) strand in the same direction as the upper strand. Following the same logic, the reverse sequence is understood as the sequence of the upper strand in the direction from its 3'- to its 5 '-end, while the ‘reverse complement’ sequence or the ‘reverse complementary’ sequence is understood as the sequence of the lower strand in the direction of its 5'- to its 3 '-end. Each nucleotide in a double stranded DNA or RNA molecule that is paired with its Watson-Crick counterpart called its complementary nucleotide.

[46] The term, “conservative substitution” as used herein refers to the replacement of one amino acid for another such that the replacement takes place within a family of amino acids that are related in their side chains. Conversely, the term “non-conservative substitution” as used herein refers to the replacement of one amino acid residue for another that does not have a related side chain. Genetically encoded amino acids can be divided into four families having related side chains: (1) acidic (negatively charged): Asp (D), Glu (G); (2) basic (positively charged): Lys (K), Arg (R), His (H); (3) non-polar (hydrophobic): Cys (C), Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Met (M), Trp (W), Gly (G), Tyr (Y), with non-polar also being subdivided into: (i) strongly hydrophobic: Ala (A), Vai (V), Leu (L), He (I), Met (M), Phe (F); and (ii) moderately hydrophobic: Gly (G), Pro (P), Cys (C), Tyr (Y), Trp (W); and (4) uncharged polar: Asn (N), Gin (Q), Ser (S), Thr (T). Amino acids may be related by aliphatic side chains: Gly (G), Ala (A), Vai (V), Leu (L), He (I), Ser (S), Thr (T), with Ser (S) and Thr (T) optionally being grouped separately as aliphatic -hydroxyl. Amino acids may be related by aromatic side chains: Phe (F), Tyr (Y), Trp (W). Amino acids may be related by amide side chains: Asn (N), Glu (Q). Amino acids may be related by sulfur-containing side chains: Cys (C) and Met (M).

[47] The term, “cleavage assay,” as used herein, refers to an assay designed to visualize, quantitate or identify cleavage of a nucleic acid. In some embodiments, the cleavage activity may be cis-cleavage activity.

[48] The term, “clustered regularly interspaced short palindromic repeats (CRISPR),” as used herein, refers to a segment of DNA found in the genomes of certain prokaryotic organisms, including some bacteria and archaea, that includes repeated short sequences of nucleotides interspersed at regular intervals between unique sequences of nucleotides derived from another organism.

[49] The term, “donor nucleic acid,” as used herein, refers to a nucleic acid that is (designed or intended to be) incorporated into a target nucleic acid or target sequence.

[50] The term, “% editing efficiency,” as used herein, refers to the percent of target nucleic acids in a sample or population of cells exhibiting an edited target nucleic acid. Editing efficiency may also be referred to as % editing level or % edited. There are multiple approaches to evaluate % editing efficiency, including, but not limited to, next generation sequence and real time PCR.

[51] The term, “target nucleic acid,” as used herein, refers to a nucleic acid that is selected as the nucleic acid for modification, binding, hybridization or any other activity of or interaction with a nucleic acid, protein, polypeptide, or peptide described herein. A target nucleic acid may comprise RNA, DNA, or a combination thereof. A target nucleic acid may be single-stranded (e.g., singlestranded RNA or single -stranded DNA) or double-stranded (e.g., double-stranded DNA).

[52] The term, “target sequence,” as used herein, when used in reference to a target nucleic acid, refers to a sequence of nucleotides found within a target nucleic acid. Such a sequence of nucleotides can, for example, hybridize to a respective length portion of a guide nucleic acid. Hybridization of the guide nucleic acid to the target sequence may bring an effector protein into contact with the target nucleic acid.

[53] A nucleotide sequence that “encodes” a particular polypeptide or protein, is a nucleotide sequence that is transcribed into mRNA (in the case of DNA) and/or is translated (in the case of mRNA) into a polypeptide. [54] The term “transgene” as used herein refers to a nucleotide sequence that is inserted into a cell for expression of said nucleotide sequence in the cell. A transgene is meant to include (1) a nucleotide sequence that is not naturally found in the cell (e.g., a heterologous nucleotide sequence); (2) a nucleotide sequence that is a mutant form of a nucleotide sequence naturally found in the cell into which it has been introduced; (3) a nucleotide sequence that serves to add additional copies of the same (e.g., exogenous or homologous) or a similar nucleotide sequence naturally occurring in the cell into which it has been introduced; or (4) a silent naturally occurring or homologous nucleotide sequence whose expression is induced in the cell into which it has been introduced. A donor nucleic acid can comprise a transgene . The cell in which transgene expression occurs can be a target cell, such as a host cell.

[55] The term, “functional fragment,” as used herein, refers to a fragment of a protein that retains some function relative to the entire protein. Non-limiting examples of functions are nucleic acid binding, protein binding, nuclease activity, nickase activity, deaminase activity, demethylase activity, or acetylation activity. A functional fragment may be a recognized functional domain, e.g., a catalytic domain such as, but not limited to, a RuvC domain.

[56] The terms, “fusion effector protein,” and “fusion protein,” may be used interchangeably herein and refer to a protein comprising at least two heterologous polypeptides. Often a fusion effector protein comprises an effector protein and a fusion partner protein. In general, the fusion partner protein is not an effector protein. Examples of fusion partner proteins are provided herein.

[57] The terms “fusion partner protein” or “fusion partner,” as used herein, refer to a protein, polypeptide or peptide that is fused, or linked via a linker, to an effector protein. The fusion partner generally imparts some function to the fusion protein that is not provided by the effector protein.

[58] The term, “effector protein,” as used herein, refers to a polypeptide that non-covalently binds to a guide nucleic acid to form a complex that contacts a target nucleic acid, wherein at least a portion of the guide nucleic acid hybridizes to a target sequence of the target nucleic acid. A complex between an effector protein and a guide nucleic acid can include multiple effector proteins or a single effector protein. In some embodiments, the effector protein modifies the target nucleic acid when the complex contacts the target nucleic acid. In some embodiments, the effector protein does not modify the target nucleic acid, but it is fused to a fusion partner protein that modifies the target nucleic acid when the complex contacts the target nucleic acid. A non-limiting example of an effector protein modifying a target nucleic acid is cleaving of a phosphodiester bond of the target nucleic acid. Additional examples of modifications an effector protein can make to target nucleic acids are described herein and throughout.

[59] The term “DNA repair protein” refers to a protein that is involved in the repair of DNA damage. These proteins maintain the integrity of the genome by correcting various types of DNA damage, such as breaks, mismatches, and other abnormalities that can occur due to environmental factors, metabolic by-products, or replication errors.

[60] The term “single -stranded break (SSB) repair protein” refers to a protein that is involved in the repair of single -stranded breaks in DNA, which are essentially nicks in one strand of the DNA double helix.

[61] The term “exonuclease” refers to a protein that cleaves nucleotides one at a time from an end (either the 5’ or the 3’ end) of a polynucleotide chain, such as DNA or RNA.

[62] The term, “genetic disease,” as used herein, refers to a disease, disorder, condition, or syndrome associated with or caused by one or more mutations in the DNA of an organism having the genetic disease.

[63] The term, “guide nucleic acid,” as used herein, refers to at least one nucleic acid comprising: a first nucleotide sequence that complexes to an effector protein on either the 5’ or 3’ terminus and the first nucleotide sequence can be fused to a second nucleotide sequence that hybridizes to a target nucleic acid. The first sequence may be referred to herein as a repeat sequence or guide sequence. The second sequence may be referred to herein as a spacer sequence. A guide nucleic acid may be referred to interchangeably with the term, “guide RNA” (gRNA). It is understood that guide nucleic acids may comprise DNA, RNA, or a combination thereof (e.g., RNA with a thymine base). Guide nucleic acids may include a chemically modified nucleobase or phosphate backbone.

[64] The term, “extended guide RNA (rtgRNA),” as used herein refers to a single nucleic acid molecule comprising (not necessarily in the following order) ( 1) a guide RNA comprising (a) a protein binding sequence and (b) a spacer sequence; (2) optionally, a linker; and (3) a template RNA (retRNA) comprising (a) a primer binding sequence and (b) a template sequence. In some embodiments, the orientation of the rtgRNA from 5’ to 3’ is: guide nucleic acid, optional linker, and template RNA. In some embodiments, the orientation of the rtgRNA from 5’ to 3’ is: template RNA, linker, and guide RNA. It is understood that extended guide RNAs may comprise DNA, RNA, or a combination thereof (e.g., RNA with a thymine base).

[65] The term “template RNA (retRNA)” as used herein, refers to a nucleic acid comprising: a primer binding sequence and a template sequence. It is understood that template RNAs may comprise DNA, RNA, or a combination thereof (e.g., RNA with a thymine base). In some embodiments, the template RNA is linked to a guide RNA via a linker sequence to form an rtgRNA.

[66] The term, “template sequence,” as used herein, refers to a portion of a retRNA that contains a desired nucleotide modification relative to a target sequence or portion thereof. By way of non-limiting example, the desired edit may comprise one or more nucleotide insertions, deletions or substitutions relative to a target sequence or portion thereof. In some embodiments, it is identical to, complementary to, or reverse complementary to a target sequence or portion thereof. In some embodiments, the template sequence is complementary to a sequence of the target nucleic acid that is adjacent to a nick site of a target site to be edited, with the exception that it includes a desired edit. The template sequence (also referred in some embodiments as the RT template (RTT)) can be complementary to at least a portion of the target sequence with the exception of at least one nucleotide.

[67] The terms, “primer binding sequence (PBS),” as used herein, refer to a portion of a retRNA and serves to bind to a primer sequence of the target nucleic acid. In some embodiments, the primer binding sequence binds to a primer sequence in the target nucleic acid that is formed after the target nucleic acid is cleaved by an effector protein. In some embodiments, the primer binding sequence is linked to the 3’ end of a retRNA. In some embodiments, the primer binding sequence is located at the 5’ end of a retRNA.

[68] “Primer sequence” as used herein refers to a portion of the target nucleic acid that is capable of hybridizing with the primer binding sequence portion of a retRNA that is generated after cleavage of the target nucleic acid by an effector protein described herein.

[69] The term “handle sequence,” as used herein, refers to a sequence that binds non-covalently with an effector protein. A handle sequence may also be referred to herein as a “scaffold sequence”. In some embodiments, the handle sequence comprises all, or a portion of, a repeat sequence. In general, a single guide nucleic acid, also referred to as a single guide RNA (sgRNA), comprises a handle sequence that is capable of being non-covalently bound by an effector protein. The nucleotide sequence of a handle sequence may contain a portion of a tracrRNA, but generally does not comprise a sequence that hybridizes to a repeat sequence, also referred to as a repeat hybridization sequence.

[70] The term “trans-activating RNA (tracrRNA),” as used herein, refers to a nucleic acid that comprises a first sequence that is capable of being non-covalently bound by an effector protein, and a second sequence that hybridizes to a portion of a crRNA, which may be referred to as a repeat hybridization sequence.

[71] The terms, “CRISPR RNA” or “crRNA,” as used herein, refer to a type of guide nucleic acid, wherein the nucleic acid is RNA comprising a first sequence, often referred to herein as a spacer sequence, that hybridizes to a target sequence of a target nucleic acid, and a second sequence, often referred to herein as a repeat sequence or guide sequence, that interacts with an effector protein. In some embodiments, the second sequence is bound by the effector protein. In some embodiments, the second sequence hybridizes to a portion of a tracrRNA, wherein the tracrRNA forms a complex with the effector protein.

[72] The term, “extension,” as used herein refers to additional nucleotides added to a nucleic acid, RNA, or DNA, or additional amino acids added to a peptide, polypeptide, or protein. Extensions may be processed during the formation of the guide RNA. In some embodiments, the extension comprises or consists of a template RNA.

[73] By "hybridizable" or "complementary" or "substantially complementary" it is meant that a nucleic acid (e.g. RNA, DNA) comprises a sequence of nucleotides that enables it to noncovalently bind, i.e. form Watson-Crick base pairs and/or G/U base pairs, "anneal", or "hybridize," to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength. Standard Watson-Crick base-pairing includes: adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C) [DNA, RNA] . In addition, for hybridization between two RNA molecules (e.g. , dsRNA), and for hybridization of a DNA molecule with an RNA molecule (e.g., when a DNA target nucleic acid base pairs with a guide RNA, etc.): guanine (G) can also base pair with uracil (U). For example, G/U base-pairing is at least partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti -codon base-pairing with codons in mRNA. Thus, in the context of this disclosure, a guanine (G) (e.g., of dsRNA duplex of a guide RNA molecule; of a guide RNA base pairing with a target nucleic acid, etc.) is considered complementary to both a uracil (U) and to an adenine (A). For example, when a G/U base-pair can be made at a given nucleotide position of a dsRNA duplex of a guide RNA molecule, the position is not considered to be non-complementary, but is instead considered to be complementary.

[74] The conditions of temperature and ionic strength determine the "stringency" of the hybridization. Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. The conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. For hybridizations between nucleic acids with short stretches of complementarity (e.g., complementarity over 35 or less, 30 or less, 25 or less, 22 or less, 20 or less, or 18 or less nucleotides) the position of mismatches can become important (see Sambrook et al., supra, 11.7-11.8). Typically, the length for a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides ormore, or 30 nucleotides or more). Temperature, wash solution salt concentration, and other conditions may be adjusted as necessary according to factors such as length of the region of complementation and the degree of complementation.

[75] While hybridization typically occurs between two nucleotide sequences that are complementary, mismatches between bases are possible. It is understood that two nucleotide sequences need not be 100% complementary to be specifically hybridizable, or for hybridization to occur. Moreover, a nucleotide sequence may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a bulge, a loop structure or hairpin structure, etc.). A polynucleotide can comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which it will hybridize. For example, an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method. Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), e.g., using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482- 489), and the like.

[76] The conditions appropriate for hybridization between two nucleotide sequences depend on the length of the sequence and the degree of complementarity, variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. For hybridizations between nucleic acids with short stretches of complementarity (e.g., complementarity over 35 or less, 30 or less, 25 or less, 22 or less, 20 or less, or 18 or less nucleotides) the position of mismatches can become important (see Sambrook et al., supra, 11.7-11.8). Typically, the length for a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more). Temperature, wash solution salt concentration, and other conditions may be adjusted as necessary according to factors such as length of the region of complementation and the degree of complementation. Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein; and Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). The conditions of temperature and ionic strength determine the “stringency” of the hybridization.

[77] The term, “heterologous,” as used herein, with reference to at least two different polypeptide sequences, means that the two different polypeptide sequences are not found similarly connected to one another in a native nucleic acid or protein. A protein that is heterologous to the effector protein is a protein that is not covalently linked via an amide bond to the effector protein in nature. In some embodiments, a heterologous protein is not encoded by a species that encodes the effector protein. A guide nucleic acid may comprise a first sequence and a second sequence, wherein the first sequence and the second sequence are not found covalently linked via a phosphodiester bond in nature. Thus, the first sequence is considered to be heterologous with the second sequence, and the guide nucleic acid may be referred to as a heterologous guide nucleic acid.

[78] The term “linked” as used herein in reference to an amino acid or nucleic acid sequence refers to any covalent mechanism by which two amino acid sequences or nucleic acid sequences are connected to each other in sequence. For example, in some embodiments, two sequences are linked directly together by a covalent bond (e.g., an amide bond or phosphodiester bond). In some embodiments, two sequences are linked together by a peptide or nucleic acid linker.

[79] The term, “linked amino acids” as used herein, refers to at least two amino acids linked by an amide bond or a peptide bond.

[80] The term, “linker,” as used herein, refers to an amino acid sequence or nucleic acid sequence that links a first polypeptide to a second polypeptide or a first nucleic acid to a second nucleic acid.

[81] The term, “modified target nucleic acid,” as used herein, refers to a target nucleic acid, wherein the target nucleic acid has undergone a modification, for example, after contact with an effector protein. In some embodiments, the modification is an alteration in the sequence of the target nucleic acid. In some embodiments, the modified target nucleic acid comprises an insertion, deletion, or replacement of one or more nucleotides compared to the unmodified target nucleic acid.

[82] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein, refer to a polymeric form of amino acids. A polypeptide may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. Accordingly, polypeptides as described herein may comprise one or more mutations, one or more sequence modifications, or both. A peptide generally has a length of 100 or fewer linked amino acids.

[83] A polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence identity can be determined in a number of different ways.

[84] A DNA sequence that "encodes" a particular RNA is a DNA nucleotide sequence that is transcribed into RNA. A DNA polynucleotide may encode an RNA (mRNA) that is translated into protein (and therefore the DNA and the mRNA both encode the protein), or a DNA polynucleotide may encode an RNA that is not translated into protein (e.g. tRNA, rRNA, microRNA (miRNA), a "non-coding" RNA (ncRNA), a guide RNA, etc.).

[85] The term, "regulatory element," as used herein, refers to a transcriptional and/or translational control sequence, such as a promoter, enhancer, polyadenylation signal, terminator, protein degradation signal, and the like, that provide for and/or regulate transcription of a non-coding sequence (e.g., guide RNA) or a coding sequence (e.g., RNA-guided endonuclease and the like) and/or regulate translation of an encoded polypeptide. [86] The term, “promoter” or “promoter sequence,” is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (3’ direction) coding or non-coding sequence. A transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase, can also be found in a promoter region. Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes. Various promoters, including inducible promoters, may be used to drive expression by the various vectors of the present disclosure.

[87] The term, “protospacer adjacent motif (PAM),” as used herein, refers to a nucleotide sequence found in a target nucleic acid that directs an effector protein to modify the target nucleic acid at a specific location. In some embodiments, a PAM is required for a complex of an effector protein and a guide nucleic acid to hybridize to and modify the target nucleic acid. In some embodiments, the complex does not require a PAM to modify the target nucleic acid. One example of a PAM sequences is NTTN, where N can be any nucleic acid.

[88] The term “RNA-dependent DNA polymerase (RDDP),” as used herein, refers to a DNA polymerase that uses a single-stranded RNA as a template for the synthesis of a complementary DNA strand.

[89] The term, “RuvC” domain as used herein refers to a region of an effector protein that is capable of cleaving a target nucleic acid, and in certain instances, of processing a pre-crRNA. In some embodiments, the RuvC domain is located near the C-terminus of the effector protein. A single RuvC domain may comprise RuvC subdomains, for example a RuvCI subdomain, a RuvCII subdomain and a RuvCIII subdomain. The term “RuvC” domain can also refer to a “RuvC-like” domain. Various RuvC-like domains are known in the art and are easily identified using online tools such as InterPro (https://www.ebi.ac.uk/interpro/). For example, a RuvC-like domain may be a domain which shares homology with a region of TnpB proteins of the IS605 and other related families of transposons.

[90] The term, “nickase” as used herein refers to an enzyme that possess catalytic activity for single stranded nucleic acid cleavage of a double stranded nucleic acid. A nickase cleaves a phosphodiester bond between two nucleotides of only one strand of dsDNA.

[91] The terms, “nuclease” and “endonuclease” are used interchangeably herein to mean an enzyme which possesses catalytic activity for nucleic acid cleavage. The term nuclease may include enzymes with nickase activity. “Nuclease” and “endonuclease” are used interchangeably herein to mean an enzyme which possesses catalytic activity for nucleic acid cleavage (e.g., ribonuclease activity (ribonucleic acid cleavage), deoxyribonuclease activity (deoxyribonucleic acid cleavage), etc.).

[92] The term, “nuclease activity,” is used to refer to catalytic activity that results in nucleic acid cleavage (e.g., ribonuclease activity (ribonucleic acid cleavage), or deoxyribonuclease activity (deoxyribonucleic acid cleavage), etc.).

[93] The term “naturally-occurring,” “unmodified,” or “wild type” as used herein as applied to a nucleic acid, a polypeptide, a cell, or an organism, refers to a nucleic acid, polypeptide, cell, or organism that is found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism that can be isolated from a source in nature is naturally occurring.

[94] The terms, “non-naturally occurring” and “engineered,” as used herein, are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid, refer to a molecule, such as but not limited to, a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid refers to a modification of that molecule (e.g., chemical modification, nucleotide sequence, or amino acid sequence) that is not present in the naturally molecule. The terms, when referring to a composition or system described herein, refer to a composition or system having at least one component that is not naturally associated with the other components of the composition or system. By way of a non-limiting example, a composition may include an effector protein and a guide nucleic acid that do not naturally occur together. Conversely, and as a non-limiting further clarifying example, an effector protein or guide nucleic acid that is “natural,” “naturally-occurring,” or “found in nature” includes an effector protein and a guide nucleic acid from a cell or organism that have not been genetically modified by the hand of man.

[95] The term, “variant,” is intended to mean a form or version of a protein that differs from the wild-type protein. A variant may have a different function or activity relative to the wild-type protein.

[96] The term, “sequence modification,” as used herein refers to a modification of one or more nucleic acid residues of a nucleotide sequence or one or more amino acid residue of an amino acid sequence, such as chemical modification of one or more nucleobases; or chemical modifications to the phosphate backbone, a nucleotide, a nucleobase, or a nucleoside. Such modifications can be made to an effector protein amino acid sequence or guide nucleic acid nucleotide sequence, or any sequence disclosed herein (e.g., a nucleic acid encoding an effector protein or a nucleic acid that, when transcribed, produces a guide nucleic acid). Methods of modifying a nucleic acid or amino acid sequence are known. One of ordinary skill in the art will appreciate that the sequence modification(s) may be located at any position(s) of a nucleic acid such that the function of the nucleic acid, protein, composition or system is not substantially decreased. Nucleic acids provided herein can be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, which is generally termed in vitro-transcription, cloning, enzymatic, or chemical cleavage, etc. In some embodiments, the nucleic acids provided herein are not uniformly modified along the entire length of the molecule. Different nucleotide modifications and/or backbone structures can exist at various positions within the nucleic acid.

[97] The term, “nucleic acid expression vector,” as used herein, refers to a plasmid that can be used to express a nucleic acid of interest. [98] The term, “nuclear localization signal (NLS),” as used herein, refers to an entity (e.g., peptide) that facilitates localization of a nucleic acid, protein, or small molecule to the nucleus, when present in a cell that contains a nuclear compartment.

[99] A person of ordinary skill in the art would appreciate that referring to a “nucleotide(s)”, and/or “nucleoside(s)”, in the context of a nucleic acid molecule having multiple residues, is interchangeable and describe the sugar and base of the residue contained in the nucleic acid molecule. Similarly, a skilled artisan could understand that linked nucleotides and/or linked nucleosides, as used in the context of a nucleic acid having multiple linked residues, are interchangeable and describe linked sugars and bases of residues contained in a nucleic acid molecule. When referring to a “nucleobase(s)”, or linked nucleobase, as used in the context of a nucleic acid molecule, it can be understood as describing the base of the residue contained in the nucleic acid molecule, for example, the base of a nucleotide, nucleosides, or linked nucleotides or linked nucleosides. A person of ordinary skill in the art when referring to nucleotides, nucleosides, and/or nucleobases would also understand the differences between RNA and DNA (generally the exchange of uridine for thymidine or vice versa) and the presence of nucleoside analogs, such as modified uridines, do not contribute to differences in identity or complementarity among polynucleotides as long as the relevant nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for all of thymidine, uridine, or modified uridine; another example is cytosine and 5- methylcytosine, both of which have guanosine or modified guanosine as a complement). Thus, for example, the sequence 5'- AXG where X is any modified uridine, such as pseudouridine, Nl-methyl pseudouridine, or 5- methoxyuridine, is considered 100% identical to AUG in that both are perfectly complementary to the same sequence (5' -CAU).

[100] Thus, the term "recombinant" polypeptide does not necessarily refer to a polypeptide whose amino acid sequence does not naturally occur. Instead, a "recombinant" polypeptide is encoded by a recombinant non-naturally occurring DNA sequence, but the amino acid sequence of the polypeptide can be naturally occurring ("wild type") or non-naturally occurring (e.g., a variant, a mutant, etc.). A recombinant polypeptide is the product of a process run by a human or machine.

[101] A “vector” or “expression vector” is a replicon, such as plasmid, phage, virus, artificial chromosome, or cosmid, to which another DNA segment, i.e., an "insert", may be attached so as to bring about the replication of the attached segment in a cell.

[102] The term “viral vector,” as used herein, refers to a nucleic acid to be delivered into a host cell via a recombinantly produced virus or viral particle.

[103] The term, “nuclease domain”, as used herein, refers to a polypeptide sequence or domain within a nuclease which possesses the catalytic activity for nucleic acid cleavage. A cleavage domain can be contained in a single polypeptide chain or cleavage activity can result from the association of two (or more) polypeptides. A single nuclease domain may consist of more than one isolated stretch of amino acids within a given polypeptide.

[104] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[105] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[106] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[107] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure . Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

Introduction

[108] In some embodiments, the present disclosure provides systems and methods comprising: a) at least one of a polypeptide and/or a nucleic acid encoding the polypeptide; and b) at least one of a guide nucleic acid and/or a DNA molecule encoding the guide nucleic acid, and uses thereof, wherein the polypeptide and the guide nucleic acid form a complex that binds a target nucleic acid.

[109] Polypeptides described herein may bind and, optionally, cleave nucleic acids in a sequencespecific manner. Polypeptides described herein may bind a target region of a target nucleic acid and cleave the target nucleic acid within the target region or at a position adjacent to the target region. In some embodiments, a polypeptide is activated when it binds a target region of a target nucleic acid to cleave a region of the target nucleic acid that is near, but not adjacent to the target region. A polypeptide may be an effector protein, such as a CRISPR-associated (Cas) protein, which may be coupled to a guide nucleic acid that imparts activity or sequence selectivity to the polypeptide. In general, guide nucleic acids comprise a first sequence that is at least partially complementary to a target nucleic acid, which may be referred to as a spacer sequence. In some embodiments, compositions, systems, and methods comprising effector proteins and guide nucleic acids can further comprise a second sequence, at least a portion of which interacts with the polypeptide. In some embodiments, the second sequence comprises a sequence that is similar or identical to a portion of a tracrRNA sequence, a CRISPR repeat sequence, or a combination thereof. In some embodiments, the guide nucleic acid does not comprise a tracrRNA.

[HO] Effector proteins disclosed herein may cleave nucleic acids, including single stranded RNA (ssRNA), double stranded DNA (dsDNA), and single-stranded DNA (ssDNA). Polypeptides disclosed herein may provide cis cleavage activity, trans cleavage activity, nickase activity, or a combination thereof. Cis cleavage activity is cleavage of a target nucleic acid that is hybridized to a guide nucleic acid (crRNA or sgRNA), wherein cleavage occurs within or directly adjacent to the region of the target nucleic acid that is hybridized to the guide nucleic acid. Nickase activity is the selective cleavage of one strand of a dsDNA molecule.

[Hl] The compositions, systems and methods described herein are non-naturally occurring. In some embodiments, compositions, systems and methods comprise a guide nucleic acid or a use thereof. In some embodiments, compositions, systems and methods comprise an engineered polypeptide or a use thereof. In general, compositions and systems described herein are not found in nature. In some embodiments, compositions, methods and systems described herein comprise at least one non- naturally occurring component. For example, disclosed compositions, methods and systems may comprise a guide nucleic acid, wherein the sequence of the guide nucleic acid is different or modified from that of a naturally-occurring guide nucleic acid. In some embodiments, compositions, systems and methods comprise at least two components that do not naturally occur together. For example, disclosed compositions, methods and systems may comprise a guide nucleic acid comprising a repeat region and a spacer region which do not naturally occur together. Also, by way of non-limiting example, disclosed compositions, methods and systems may comprise a guide nucleic acid and an effector protein that do not naturally occur together. Conversely, and for clarity, an effector protein or guide nucleic acid that is “natural,” “naturally-occurring,” or “found in nature” includes effector proteins and guide nucleic acids from cells or organisms that have not been genetically modified by a human or machine.

[112] In some embodiments, the guide nucleic acid comprises a non-natural nucleotide sequence. In some embodiments, the non-natural nucleotide sequence is a nucleotide sequence that is not found in nature. The non-natural nucleotide sequence may comprise a portion of a naturally-occurring sequence, wherein the portion of the naturally-occurring sequence is not present in nature absent the remainder of the naturally-occurring sequence. In some embodiments, the guide nucleic acid comprises two naturally-occurring sequences arranged in an order or proximity that is not observed in nature. In some embodiments, compositions and systems comprise a ribonucleotide complex comprising an effector protein and a guide nucleic acid that do not occur together in nature . Engineered guide nucleic acids may comprise a first sequence and a second sequence that do not occur naturally together. For example, a guide nucleic acid may comprise a sequence of a naturally-occurring repeat region and a spacer region that is complementary to a naturally-occurring eukaryotic sequence. The guide nucleic acid may comprise a sequence of a repeat region that occurs naturally in an organism and a spacer region that does not occur naturally in that organism. A guide nucleic acid may comprise a first sequence that occurs in a first organism and a second sequence that occurs in a second organism, wherein the first organism and the second organism are different. The guide nucleic acid may comprise a third sequence disposed at a 3 ’ or 5 ’ end of the guide nucleic acid, or between the first and second sequences of the guide nucleic acid. In some embodiments, a guide nucleic acid is a crRNA, wherein the crRNA comprises a repeat sequence and a spacer sequence that is complementary to a eukaryotic target sequence. In some embodiments, a guide nucleic acid may comprise a repeat sequence, an intermediary sequence, and a spacer sequence coupled by one or more linker sequences. In some embodiments, the guide nucleic acid comprises two heterologous sequences arranged in an order or proximity that is not observed in nature. Therefore, compositions and systems described herein are not naturally occurring.

Precision Editing Systems

[113] In some embodiments, the present disclosure provides compositions, systems, and methods for precision editing. The present disclosure provides each of the components for such precision editing systems (e.g., effector protein, RDDP, guide RNA and retRNA) as well as systems comprising the individual components. In some embodiments, the effector protein and the RDDP are provided in the form of a fusion protein. In some embodiments, the effector protein and the RDDP are provided as two separate proteins. In some embodiments, the effector protein (or fusion protein comprising an effector protein) forms a complex with a guide RNA, a retRNA, or a combination thereof.

[114] In some embodiments, compositions and systems comprise an aptamer and an aptamer binding moiety (also simply referred to as a moiety). In some embodiments, the aptamer binding moiety is an aptamer binding peptide/protein. In some embodiments, compositions and systems comprise a moiety that that binds an RNA-aptamer. This moiety may be fused to an RDDP and the RNA-aptamer may be linked to the gRNA or template RNA, or a combination thereof. Thus, the moiety may serve to deliver the RDDP to the target sequence and thus, the RDDP need not be linked to the effector protein. By way of non-limiting example, in some embodiments, compositions and systems comprise an MS2 protein localization sequence. In some embodiments, a guide RNA comprises an MS2 protein localization sequence. In some embodiments, the template RNA comprises an MS2 protein localization sequence. The MS2 protein localization sequence may be located at the 5’ or 3’ terminus of the template RNA. In some embodiments, the RDDP is fused to an MS2 coat protein (or protein that is capable of binding the MS2 protein localization sequence), thereby localizing the RDDP to the MS2 protein localization sequence and therefore, the effector protein, template RNA and/or target nucleic acid. Additional examples of such localizing systems are described by Chen et al., FEBS J. (2013) 280:3734-3754. In some embodiments, an RDDP is linked to an effector protein. In such embodiments, the RDDP may not be fused to an MS2 coat protein and the template RNA may not comprise an MS2 protein localization sequence.

[115] In some embodiments, compositions and systems described herein comprise a compact Cas protein that homodimerizes, and a guide nucleic acid that guides the dimer to a target sequence in a DNA molecule. In some embodiments, each monomer of the dimer is linked to an RDDP. In some embodiments, the dimer cuts both strands of DNA leaving staggered ends with 5’ overhangs. See e.g., FIG.1A. In some embodiments, compositions and systems described herein further comprise a target strand (TS) template RNA (retRNA) and a non-target strand (NTS) retRNA. Each template RNA is designed to hybridize to single stranded DNA created at the cut site. The linked RDDPs polymerize a new strand of DNA at each 3’ end of the cut DNA. See e.g., FIG.1B. System components shown in FIG. 1A and FIG.1B are exemplary and non-limiting.

[116] In some embodiments, the present disclosure provides compositions, systems, and methods for modifying a target strand (TS) and/or a non target strand (NTS) of a double stranded DNA (dsDNA) molecule. In general, a target strand of a dsDNA molecule is the strand of DNA to which a guide nucleic acid hybridizes. Typically, the spacer sequence of the guide nucleic acid hybridizes to a target sequence on the target strand. In general, the guide nucleic acid does not hybridize to the non-target strand. In some embodiments, the compositions and systems comprise (a) an effector protein; (b) an RNA-directed DNA polymerase (RDDP); (c) a guide RNA that hybridizes to a target sequence of the TS; (d) a TS template RNA (retRNA) that hybridizes to the TS; and (e) a NTS retRNA that hybridizes to the NTS. See e.g., FIG.1A. In some embodiments, the effector protein cuts the TS and the NTS to generate staggered ends, thereby generating a single stranded DNA with a free 3’ end on the TS (TS ssDNA) and a single stranded DNA with a free 3’ end on the NTS (NTS ssDNA). See e.g., FIG.1A and FIG.1B.

[117] Endogenous repair proteins, including exonucleases and single stranded break repair proteins, may be useful for repairing a DNA cut site generated by compositions, systems, and methods described herein. See e.g., FIG. 2. In some embodiments, compositions and systems described herein comprise a DNA repair protein to enhance DNA repair and further reduce unintended edits. In some embodiments, the DNA repair protein is covalently linked to an RDDP. [118] In some embodiments, an effector protein of a composition, system, or method described herein forms a dimer with itself in a cell. See e.g., FIG.3. In some embodiments, the dimer may be a homodimer or a heterodimer. In some embodiments, the presence of the inducer stimulates dimerization of the two effectors within the cytoplasm and makes it thermodynamically worthwhile for the dimerized effector to localize to the nucleus. Fusion of DNA repair proteins to a dimerizing Cas protein may enhance repair relative to a Cas protein that does not dimerize, effectively recruiting twice as much repair protein. See e.g., FIG. 4. Without being bound by theory, it is contemplated that polymerization at both ends reduces any chance of byproducts (unintended edits), even though the dimer produces a double stranded break.

[119] In some embodiments, compositions and systems described herein comprise a homodimerizing effector protein, an RDDP, and two different guide RNAs that hybridize to opposite strands of a dsDNA molecule at some distance apart from each other. In some embodiments, the distance is about 10 bp to about 10,000 bp. In some embodiments, the first target sequence and the second target sequence are greater than 10,000 base pairs apart. The effector proteins form ribonucleoprotein (RNP) complexes with the two guide RNAs at these two different sites respectively where they cleave the dsDNA, generating single stranded DNA (ssDNA) flaps at both sites that extend towards each other as shown in FIG. 8. These systems also comprise two retRNAs, each of which has a primer binding sequence (PBS) that binds to a portion of one of the ssDNA flaps. The retRNAs each have a template sequence for generating a sequence of interest while the region between the two cut sites is deleted.

[120] In some embodiments, the present disclosure provides compositions, systems, and methods for deleting or inserting a region of a double stranded DNA (dsDNA) molecule. In some embodiments, compositions and systems described herein comprise (a) an effector protein, wherein the effector protein forms a dimer with itself in a cell; (b) an RNA-directed DNA polymerase (RDDP); (c) a first guide RNA (gRNA), wherein the first gRNA comprises (i) a first scaffold sequence, and (ii) a first spacer sequence that hybridizes to a first target sequence on a first strand of the dsDNA molecule, wherein the first scaffold sequence is located 5’ of the first spacer sequence, and wherein the effector protein and the first gRNA form a first RNP complex that cleaves the dsDNA molecule to form a first single stranded DNA (ssDNA) flap from the first strand of the dsDNA molecule; (d) a second gRNA, wherein the second gRNA comprises (i) a second scaffold sequence, and (ii) a second spacer sequence that hybridizes to a second target sequence on a second strand of the dsDNA molecule, wherein the second scaffold sequence is located 5 ’ of the second spacer sequence, and wherein the effector protein and the second gRNA form a second RNP complex that cleaves the dsDNA molecule to form a second ssDNA flap from the second strand of the dsDNA molecule; (e) a first template RNA (retRNA), wherein the first retRNA comprises (i) a first primer binding sequence (PBS) that hybridizes to at least a portion of the first ssDNA flap, and (ii) a first template sequence; and (f) a second retRNA, wherein the second retRNA comprises (i) a second PBS that hybridizes to at least a portion of the second ssDNA flap, and (ii) a second template sequence. See e.g., FIG. 8. In some embodiments, the first spacer sequence and the second spacer sequence hybridize to a first target sequence and a second target sequence of the dsDNA molecule respectively. In some embodiments, the first target sequence and the second target sequence are 10 to 10,000 base pairs apart. In some embodiments, the first target sequence and the second target sequence are greater than 10,000 base pairs apart. Methods may comprise modifying a target nucleic acid with such a composition or system.

[121] In some embodiments, compositions and systems described herein comprise a homodimerizing effector protein (e.g., a Type V Cas nuclease) that produces a double stranded break, an RDDP, a guide nucleic acid that guides the dimer to a target sequence of a dsDNA molecule, and a template RNA designed to hybridize to a single stranded DNA from the target strand (TS) to extend the TS. See e.g. FIG. 9. Precision editing with a nuclease and TS extension may be different from standard RT editing with a non-target strand (NTS) nickase and NTS extension. In some embodiments, the TS extension with Type V Cas proteins allow editing of the seed region and/or PAM of the TS. In some embodiments, editing of the seed region and/or PAM of the TS prevents subsequent rounds of targeting, thereby reducing the amount of imprecise indels. Non-limiting examples of Type V proteins for such compositions, systems, and methods include CasM.265466.

[122] In some embodiments, the present disclosure provides compositions, systems, and methods for modifying a target strand (TS) of a double stranded DNA (dsDNA) molecule. In some embodiments, compositions and systems described herein comprise (a) an effector protein, wherein the effector protein forms a dimer with itself in a cell; (b) an RNA-directed DNA polymerase (RDDP); (c) a guide RNA, wherein the guide RNA comprises (i) a first region comprising a scaffold sequence, and (ii) a second region comprising a spacer sequence that hybridizes to a target sequence of the TS, wherein the first region is located 5’ of the second region; and wherein the effector protein and the gRNA form a RNP complex that produces a double stranded break; and a TS template RNA (retRNA), wherein the TS retRNA comprises a TS primer binding sequence (PBS) that hybridizes to the TS, and a TS template sequence. See e.g., FIG. 9. In some embodiments, the effector protein is a Type V Cas protein. Nonlimiting examples of Type V Cas proteins for such systems include CasM.265466.

Effector Proteins

[123] Provided herein, are compositions, systems, and methods comprising an effector protein or a nucleic acid encoding the same, and uses thereof. In general, the effector protein is a CRISPR associated (Cas) protein. In some embodiments, the effector protein is not a Type II Cas protein. In some embodiments, the effector protein is not a Cas9 protein. In some embodiments, the effector protein is a Type V protein. In some embodiments, the Type V effector protein comprises an RuvC domain and does not comprise an HNH domain. In some embodiments, the effector protein homodimerizes. In some embodiments, the effector protein is a compact effector protein, e.g., about 400 to about 700 amino acids in size. In preferred embodiments, the effector protein is a compact, Type V protein that forms a homodimer.

[124] In some embodiments, compositions and systems described herein comprise an effector protein that is similar to a naturally occurring effector protein. The effector protein may lack a portion of the naturally occurring effector protein. The effector protein may comprise a mutation relative to the naturally-occurring effector protein, wherein the mutation is not found in nature. The effector protein may also comprise at least one additional amino acid relative to the naturally-occurring effector protein. For example, the effector protein may comprise an addition of a nuclear localization signal relative to the natural occurring effector protein. In certain embodiments, a nucleotide sequence encoding the effector protein is codon optimized (e.g., for expression in a eukaryotic cell) relative to the naturally occurring sequence.

[125] An effector protein may be brought into proximity of a target nucleic acid in the presence of a guide nucleic acid when the guide nucleic acid includes a nucleotide sequence that is complementary with a target sequence in the target nucleic acid. The ability of an effector protein to modify a target nucleic acid may be dependent upon the effector protein being bound to a guide nucleic acid and the guide nucleic acid being hybridized to a target nucleic acid. An effector protein may also recognize a protospacer adjacent motif (PAM) sequence present in the target nucleic acid, which may direct the modification activity of the effector protein. An effector protein may modify a target nucleic acid by cis cleavage or trans cleavage. The modification of the target nucleic acid generated by an effector protein may, as a non-limiting example, result in modulation of the expression of the target nucleic acid (e.g., increasing or decreasing expression of the nucleic acid) or modulation of the activity of a translation product of the target nucleic acid (e.g. , inactivation of a protein binding to an RNA molecule or hybridization).

[126] An effector protein may be a CRISPR-associated (“Cas”) protein. An effector protein may function as a single protein, including a single protein that is capable of binding to a guide nucleic acid and modifying a target nucleic acid. Alternatively, an effector protein may function as part of a multiprotein complex, including, for example, a complex having two or more effector proteins, including two or more of the same effector proteins (e.g., dimer or multimer). An effector protein, when functioning in a multiprotein complex, may have only one functional activity (e.g., binding to a guide nucleic acid), while other effector proteins present in the multiprotein complex are capable of the other functional activity (e.g., modifying a target nucleic acid). An effector protein may be a modified effector protein having increased modification activity and/or increased substrate binding activity (e. g. , substrate selectivity, specificity, and/or affinity) . Alternatively, or in addition, an effector protein may be a catalytically inactive effector protein having reduced modification activity or no modification activity. Accordingly, an effector protein as used herein encompasses a modified polypeptide that does not have nuclease activity. [127] In certain embodiments, effector proteins described herein comprise one or more functional domains. Effector protein functional domains can include a protospacer adjacent motif (PAM)- interacting domain, an oligonucleotide-interacting domain, one or more recognition domains, a nontarget strand interacting domain, and a RuvC domain. A PAM interacting domain can be a target strand PAM interacting domain (TPID) or a non-target strand PAM interacting domain (NTPID). In some embodiments, a PAM interacting domain, such as a TPID or a NTPID, on an effector protein describes a region of an effector protein that interacts with target nucleic acid. In some embodiments, the effector proteins comprise a RuvC domain. In some embodiments, a RuvC domain comprises with substrate binding activity, catalytic activity, or both. In some embodiments, the RuvC domain may be defined by a single, contiguous sequence, or a set of RuvC subdomains that are not contiguous with respect to the primary amino acid sequence of the protein. An effector protein of the present disclosure may include multiple RuvC subdomains, which may combine to generate a RuvC domain with substrate binding or catalytic activity. For example, an effector protein may include three RuvC subdomains (RuvC-I, RuvC-II, and RuvC-III) that are not contiguous with respect to the primary amino acid sequence of the effector protein, but form a RuvC domain once the protein is produced and folds. In some embodiments, effector proteins comprise one or more recognition domain (REC domain) with a binding affinity for a guide nucleic acid or for a guide nucleic acid-target nucleic acid heteroduplex. An effector protein may comprise a zinc finger domain. In some embodiments, the effector protein does not comprise an HNH domain.

[128] An effector protein may be small, which may be beneficial for nucleic acid detection or editing

(for example, the effector protein may be less likely to adsorb to a surface or another biological species due to its small size). The smaller nature of these effector proteins may allow for them to be more easily packaged and delivered with higher efficiency in the context of genome editing and more readily incorporated as a reagent in an assay. In some embodiments, the length of the effector protein is at least 300 linked amino acid residues. In some embodiments, the length of the effector protein is at least 325 linked amino acid residues. In some embodiments, the length of the effector protein is at least 350 linked amino acid residues. In some embodiments, the length of the effector protein is at least 375 linked amino acid residues. In some embodiments, the length of the effector protein is at least 400 linked amino acid residues. In some embodiments, the length of the effector protein is at least 425 linked amino acid residues. In some embodiments, the length of the effector protein is at least 450 linked amino acid residues. In some embodiments, the length of the effector protein is not greater than 700 linked amino acid residues. In some embodiments, the length of the effector protein is not greater than 750 linked amino acid residues. In some embodiments, the length of the effector protein is not greater than 800 linked amino acid residues. In some embodiments, the length of the effector protein is not greater than 850 linked amino acid residues. In some embodiments, the length of the effector protein is not greater than 900 linked amino acid residues. In some embodiments, the length of the effector protein is about 350 to about 450 linked amino acid residues. In some embodiments, the length of the effector protein is about 375 to about 475 linked amino acid residues. In some embodiments, the length of the effector protein is about 400 to about 450 linked amino acid residues. In some embodiments, the length of the effector protein is about 400 to about 500 linked amino acid residues. In some embodiments, the length of the effector protein is about 350 to about 400, about 400 to about 450, about 450 to about 550, about 400 to about 420, about 420 to about 440, about 440 to about 460, about 460 to about 480, about 480 to about 500, about 500 to about 520, about 520 to about 540, about 540 to about 560, about 560 to about 580, about 580 to about 600, about 600 to about 620, about 620 to about 640, about 640 to about 660, about 660 to about 680, about 680 to about 700 linked amino acids. In some embodiments, the length of the effector protein is at least 200, at least 225, at least 250, at least 275 at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, at least 500, at least 525, at least 550, at least 575, at least 600, at least 625, at least 650, at least 675, at least 700, at least 725, at least 750, at least 775 linked amino acids. In some embodiments, the length of the effector protein is about 700 to about 800 linked amino acid residues.

[129] TABLE 1 provides exemplary amino acid sequences (e.g., SEQ ID NO: 1, 86 or 87) of effector proteins, guide sequences, and PAM sequences that may be useful in the compositions, systems and methods described herein.

[130] In some embodiments, systems and compositions comprise an effector protein and a guide nucleic acid, wherein the effector protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1, and the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence:

ACAGCUUAUUUGGAAGCUGAAAUGUGAGGUUUAUAACACUCACAAGAAUCCUGAAA AAGGAUGCCAAAC (SEQ ID NO: 85). In some embodiments, systems and compositions comprise an effector protein and a guide nucleic acid, wherein the effector protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1, and the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence: ACAGCUUAUCUGGAAGCUGAAAUGUGAGGUUUAUAACACUCACAAGAAGCCUGAAAA AGGCUGCCAGAC (SEQ ID NO: 129). In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 124 or 125.

[131] In some embodiments, effector proteins differ from SEQ ID NO: 1, 86 or 87 by one or more amino acids. In some embodiments, effector proteins differ from SEQ ID NO: 1 by 1 amino acid, 2 amino acids, 3 amino acids, 4, amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids or 10 amino acids. In some embodiments up to 15 amino acids are modified. In some embodiments up to 20 amino acids are modified. In some embodiments up to 25 amino acids are modified.

[132] In some embodiments, the modifications are conservative substitutions relative to SEQ ID NO: 1, 86 or 87. In some embodiments, the amino acids that differ from the effector proteins are nonconservative substitutions relative to SEQ ID NO: 1, 86 or 87. In some embodiments, a mutation may affect the catalytic activity of the effector protein and results in a catalytically reduced or catalytically inactive mutant. In some embodiments, a mutation can result in the effector protein having nickase activity or increased nickase activity. In some embodiments, a mutation can result in the effector protein having reduced or no nuclease activity but gaining nickase activity.

[133] In certain embodiments, compositions, systems and methods described herein provided herein comprise an effector protein, wherein the amino acid sequence of the effector protein comprises at least about 200, at least about 220, at least about 240, at least about 260, at least about 280, at least about 300, at least about 320, at least about 340, at least about 360, at least about 380, or at least about 400 contiguous amino acids of SEQ ID NO: 1, 86 or 87.

Modulation of Effector protein activity

[134] The effector proteins of the present disclosure may have nuclease activity, nickase activity, or no cleavage activity on target nucleic acids. In some embodiments, the effector protein of the present disclosure has a combination of the above activities on a target nucleic acid. In some embodiments, the cleavage activity of the effector proteins is modulated between nuclease activity and nickase activity on the target nucleic acid. In some embodiments, the cleavage activity of the effector protein is modulated between nickase activity and no cleavage activity on the target nucleic acid. In some embodiments, the cleavage activity of the effector protein is modulated between nuclease activity and no cleavage activity on the target nucleic acid. In some embodiments, the cleavage activity of the effector protein is modulated between nuclease activity, nickase activity, and no cleavage activity on the target nucleic acid. In some embodiments, the effector protein has nuclease activity. In some embodiments the effector protein has nickase activity.

[135] In some embodiments, the cleavage activity of the effector protein on the target nucleic acid is modulated based on the length of the spacer sequence of a guide nucleic acid. In some embodiments, the spacer length confers nuclease activity to the effector protein on the target nucleic acid. In some embodiments, the spacer length confers nickase activity to the effector protein on the target nucleic acid. In some embodiments, the spacer length confers no cleavage activity to the effector protein on the target nucleic acid. In some embodiments, the spacer length is 10-20 nucleotides. In some embodiments, the spacer length is 11-17 nucleotides. In some embodiments, the spacer length is 14- 16 nucleotides. In some embodiments, the spacer length is 10, 12, 13, 14, 15, 16 or 17 nucleotides. In some embodiments, the spacer length is 15 nucleotides. Protospacer Adjacent Motif (PAM)

[136] Effector proteins of the present disclosure, dimers thereof, and multimeric complexes thereof, may cleave or nick a target nucleic acid within or near a protospacer adjacent motif (PAM) sequence of the target nucleic acid. In some embodiments, cleavage occurs within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides of a 5’ or 3’ terminus of a PAM sequence. In some embodiments, a target nucleic acid may comprise a PAM sequence adjacent to a target sequence that is complementary to a guide nucleic acid spacer region. PAMs in compositions, systems, and methods herein are further described throughout the application. In some embodiments the PAM is 5’-NNTN-3’, wherein N can be any nucleic acid. In some embodiments, the PAM is 5’- TNTR-3’, wherein N can be any nucleic acid and R is selected from A and G. In some embodiments, the PAM is TNTG, wherein N can be any nucleic acid. Non-limiting examples of 5’-TNTR-3’ PAM sequences are disclosed in TABLE 1. In some embodiments, the effector protein recognizes a PAM sequence as shown in TABLE 1, wherein: N represents any nucleotide; K represents A, C, or G; Y represents C or T; and R represents A or G. In some embodiments, the effector protein recognizes a PAM sequence comprising any of the following nucleotide sequences as set forth in TABLE 1. In some embodiments, the PAM is selected from: AATA, AATC, AATG, AATT, ACTA, ACTC, ACTG, ACTT, AGTA, AGTG, AGTC, AGTG, AGTT, ATTA, ATTC, ATTG, ATTT, CATA, CATC, CATG, CATT, CCTA, CCTC, CCTG, CCTT, CGTA, CGTC, CGTG, CGTT, CTTA, CTTC, CTTG, CTTT, GATA, GATC, GATG, GATT, GCTA, GCTC, GCTG, GCTT, GGTA, GGTC, GGTG, GGTT, GTTA, GTTC, GTTG, GTTT, TATA, TATC, TATG, TATT, TCTA, TCTC, TCTG, TCTT, TGTA, TGTC, TGTG, TGTT, TTTA, TTTC, TTTG, and TTTT.

RNA-dependent DNA polymerases (RDDP)

[137] In some embodiments, the present disclosure provides for systems, compositions, and methods comprising an RNA-dependent DNA polymerase (RDDP) or a use thereof. Herein, RDDPs are enzymes that are capable of generating a DNA polynucleotide from an RNA template polynucleotide. In some embodiments, the RDDP is a reverse transcriptase. Herein, the term RDDP encompasses functional domains thereof. In some embodiments, the functional domain is a polymerase domain, an RNAse domain, or a combination thereof.

[138] In some embodiments, the RDDP comprises less than 800 amino acids. In some embodiments, the RDDP comprises less than 800, less than 700, less than 600, less than 500, less than 400, less than 300 amino acids. In some embodiments, the RDDP comprises at least 200, at least 220, at least 240, at least 260, at least 280, or at least 300 amino acids. In some embodiments, the RDDP comprises at least 277 amino acids. In some embodiments, the RDDP comprises 200 to 800 amino acids. In some embodiments, the RDDP comprises 277 to 800 amino acids. In some embodiments, the RDDP comprises 300 to 800, 400 to 800, 500 to 800, or 600 to 800 amino acids. In some embodiments, the RDDP comprises 250 to 750, 250 to 700, 250 to 650, 250 to 600, 250 to 550, 250 to 500, 250 to 450, 250 to 400, 250 to 350, 275 to 750, 275 to 700, 275 to 650, 275 to 600, 275 to 550, 275 to 500, 275 to 450, 275 to 400, 275 to 350, 300 to 750, 300 to 700, 300 to 650, 300 to 600, 300 to 550, 300 to 500, 300 to 450, 300 to 400, or 300 to 350 amino acids.

[139] In some embodiments, the length of the RDDP is less than 800 amino acids. In some embodiments, the length of the RDDP is less than 800, less than 700, less than 600, less than 500, less than 400, less than 300 linked amino acids. In some embodiments, the length of the RDDP is at least 200, at least 220, at least 240, at least 260, at least 280, or at least 300 linked amino acids. In some embodiments, the length of the RDDP is at least 277 linked amino acids. In some embodiments, the length of the RDDP is 200 to 800 linked amino acids. In some embodiments, the length of the RDDP is 277 to 800 linked amino acids. In some embodiments, the length of the RDDP is 300 to 800, 400 to 800, 500 to 800, or 600 to 800 linked amino acids. In some embodiments, the length of the RDDP is 250 to 750, 250 to 700, 250 to 650, 250 to 600, 250 to 550, 250 to 500, 250 to 450, 250 to 400, 250 to 350, 275 to 750, 275 to 700, 275 to 650, 275 to 600, 275 to 550, 275 to 500, 275 to 450, 275 to 400, 275 to 350, 300 to 750, 300 to 700, 300 to 650, 300 to 600, 300 to 550, 300 to 500, 300 to 450, 300 to 400, or 300 to 350 linked amino acids.

[140] In some embodiments, the RDDP, when used in combination with an effector protein described herein, demonstrates an editing efficiency of at least 1%. In some embodiments, the RDDP, when used in combination with an effector protein described herein, demonstrates an editing efficiency of at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or at least 10%. In some embodiments, the RDDP, when used in combination with an effector protein described herein, demonstrates an editing efficiency that is greater than the editing efficiency observed with a Moloney murine leukemia virus (M-MLV) reverse transcriptase (e.g., SEQ ID NO: 81, 82, or 111). In some embodiments, the RDDP, when used in combination with an effector protein described herein, demonstrates an editing efficiency that is at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5- fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or at least 10-fold greater than the editing efficiency observed with an M-MLV reverse transcriptase.

[141] Exemplary RDDP sequences are provided in TABLE 2 below. In some embodiments, the RDDP comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one of SEQ ID NOs: 2-82 and 111. In some embodiments, the RDDP comprises or consists of an amino sequence selected from SEQ ID NOs: 2-82 and 111.

[142] In some embodiments, RDDPs comprise at least 200, at least 225, at least 250, at least 275 at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, at least 500, at least 525, at least 550, at least 575, at least 600, at least 625, at least 650, at least 675, at least 700, at least 725, at least 750, at least 775 contiguous amino acids of a sequence selected from SEQ ID NOs: 2-82 and 111. Engineered Proteins

[143] In some embodiments, systems, compositions and methods disclosed herein comprise an engineered protein or a use thereof. An engineered protein may comprise a modified form of a wild type counterpart protein (e.g., an effector protein and/or RDDP). The modified form of the wild type counterpart may comprise an amino acid change (e.g., deletion, insertion, or substitution) that reduces the nucleic acid-cleaving activity of the effector protein relative to the wild type counterpart. For example, a nuclease domain (e.g., RuvC domain) of an effector protein may be deleted or mutated relative to a wild type counterpart effector protein so that it is no longer functional or comprises reduced nuclease activity. The modified form of the effector protein may have less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid-cleaving activity of the wild-type counterpart. Engineered effector proteins may have no substantial nucleic acid-cleaving activity. An engineered effector protein may be enzymatically inactive or “dead,” also referred to in some embodiments as a dead protein or a dCas protein. An engineered effector protein may bind to a guide nucleic acid and/or a target nucleic acid but not cleave the target nucleic acid. An enzymatically inactive effector protein may comprise an enzymatically inactive domain (e.g., inactive nuclease domain). Enzymatically inactive may refer to an activity less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, or less than 10% activity compared to the wild-type counterpart. A dead protein may associate with a guide nucleic acid to activate or repress transcription of a target nucleic acid. In some embodiments, the enzymatically inactive protein is fused to a fusion partner protein that confers an alternative activity to an effector protein activity. Such fusion proteins are described herein and throughout. By way of non-limiting example, an alternative activity may be a transcriptional activation, transcription repression, deaminase activity, transposase activity, and recombinase activity. In some embodiments, activity (e.g., nuclease activity) of effector proteins and/or compositions described herein can be measured relative to a WT effector protein or compositions containing the same in a cleavage assay.

[144] In some embodiments, any of the protein sequences herein may be codon optimized. In some embodiments, effector protein described herein are encoded by a codon optimized nucleic acid. In some embodiments, a nucleic acid sequence encoding an effector protein described herein, is codon optimized. This type of optimization can entail a mutation of an effector protein encoding nucleotide sequence to mimic the codon preferences of the intended host organism or cell while encoding the same polypeptide. Thus, the codons can be changed, but the encoded protein remains unchanged. For example, if the intended target cell was a human cell, a human codon- optimized effector proteinencoding nucleotide sequence could be used. As another non-limiting example, if the intended host cell were a mouse cell, then a mouse codon-optimized effector protein - encoding nucleotide sequence could be generated. As another non-limiting example, if the intended host cell were a eukaryotic cell, then a eukaryote codon-optimized effector protein nucleotide sequence could be generated. As another non-limiting example, if the intended host cell were a prokaryotic cell, then a prokaryote codon- optimized effector protein -encoding nucleotide sequence could be generated. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.or.jp/codon. Accordingly, in some embodiments, effector proteins described herein may be codon optimized for expression in a specific cell, for example, a bacterial cell, a plant cell, a eukaryotic cell, an animal cell, a mammalian cell, or a human cell. In some embodiments, the effector protein is codon optimized for a human cell.

[145] It is understood that when describing coding sequences of polypeptides described herein, said coding sequences do not necessarily require a codon encoding a N-terminal Methionine (M) or a Valine (V) as described for the effector proteins described herein. One skilled in the art would understand that a start codon could be replaced or substituted with a start codon that encodes for an amino acid residue sufficient for initiating translation in a host cell. In some embodiments, when a modifying heterologous peptide, such as a fusion partner protein, protein tag or NLS, is located at the N terminus of the effector protein, a start codon for the heterologous peptide serves as a start codon for the effector protein as well. Thus, the natural start codon encoding an amino acid residue sufficient for initiating translation (e.g. , Methionine (M) or a Valine (V)) of the effector protein may be removed or absent.

[146] In some embodiments, the RDDP and/or effector proteins described herein comprise one or more amino acid substitutions as compared to a naturally occurring RDDP and/or effector protein. In some embodiments, the amino acid substitution is a conservative amino acid substitution. In general, a conservative amino acid substitution is the substitution of an amino acid residue for another amino acid residue with similar chemical properties (e.g., size, charge, or polarity). Conservative substitutions may be made by exchanging an amino acid from one of the groups listed below (group 1 to 6) for another amino acid of the same group.

[147] Amino acid residues may be divided into groups based on common side chain properties, as follows: (group 1) hydrophobic: norleucine, methionine (Met), Alanine (Ala), Valine (Vai), Leucine (Leu), Isoleucine (He); (group 2) neutral hydrophilic: Cysteine (Cys), Serine (Ser), Threonine (Thr), Asparagine (Asn), Glutamine (Gin); (group 3) acidic: Aspartic acid (Asp), Glutamic acid (Glu); (group 4) basic: Histidine (His), Lysine (Lys), Arginine (Arg); (group 5) residues that influence chain orientation: Glycine (Gly), Proline (Pro); and (group 6) aromatic: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe). The substitution of one amino acid with another amino acid in a same group listed above may be considered a conservative amino acid substitution. The substitution of one amino acid with another amino acid in a different group listed above may be considered a nonconservative amino acid substitution.

[148] In some embodiments, an effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 1, wherein the amino acid substitution is at a position selected from K58, 180, T84, K105, N193, C202, S209, G210, A218, D220, E225, C246, N286, M295, M298, A306, Y315, Q360, and a combination thereof. In some embodiments, the polypeptide comprises an amino acid sequence that is 100% identical to SEQ ID NO: 1, with the exception of at least one amino acid substitution relative to SEQ ID NO: 1, wherein the amino acid substitution is a position selected from K58, 180, T84, K105, N193, C202, S209, G210, A218, D220, E225, C246, N286, M295, M298, A306, Y315, Q360, and a combination thereof. In some embodiments, the amino acid substitution is selected from K58X, I80X, T84X, K105X, N193X, C202X, S209X, G210X, A218X, D220X, E225X, C246X, N286X, M295X, M298X, A306X, Y315X, and Q360X, wherein X is selected from R, K, and H.

[149] In some embodiments, the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 1, wherein the amino acid substitution is selected from I80R, T84R, K105R, C202R, G210R, A218R, D220R, E225R, C246R, Q360R, I80K, T84K, G210K, N193K, C202K, A218K, D220K, E225K, C246K, N286K, A306K, Q360K, I80H, T84H, K105H, G210H, C202H, A218H, D220H, E225H, C246H, Q360H, K58W, S209F, M295W, M298L, Y315M, D220R/A306K and D220R/K250N and a combination thereof. In some embodiments, the polypeptide comprises an amino acid sequence that is 100% identical to SEQ ID NO: 1, with the exception of at least one amino acid substitution relative to SEQ ID NO: 1, wherein the amino acid substitution is selected from I80R, T84R, K105R, C202R, G210R, A218R, D220R, E225R, C246R, Q360R, I80K, T84K, G210K, N193K, C202K, A218K, D220K, E225K, C246K, N286K, A306K, Q360K, I80H, T84H, K105H, G210H, C202H, A218H, D220H, E225H, C246H, Q360H, K58W, S209F, M295W, M298L, Y315M, D220R/A306K and D220R/K250N and a combination thereof. In some aspects, these engineered effector proteins demonstrate enhanced nuclease activity relative to the wild-type effector protein.

[150] In some embodiments, the effector protein is an engineered effector protein and comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 1, wherein the amino acid substitution is selected from D237A, D418A, D418N, E335A, and E335Q, and a combination thereof. In some embodiments, the polypeptide comprises an amino acid sequence that is 100% identical to SEQ ID NO: 1, with the exception of at least one amino acid substitution relative to SEQ ID NO: 1, wherein the amino acid substitution is selected from D237A, D418A, D418N, E335A, and E335Q, and a combination thereof. In some aspects, these engineered effector proteins demonstrate reduced or abolished nuclease activity relative to the wild-type effector protein. TABLE 1.1 provides the exemplary amino acid alterations relative to SEQ ID NO: 1 useful in compositions, systems, and methods described herein. [151] Engineered effector proteins may provide enhanced catalytic activity (e.g. , nuclease or nickase activity) as compared to a naturally occurring nuclease or nickase. Engineered effector proteins may provide enhance nucleic acid binding activity, e.g., enhanced binding of a guide nucleic acid and/or target nucleic acid, and/or may demonstrate a stronger affinity for a target nucleic acid sequence. Without wishing to be bound by theory, substation of positively charged amino acids is thought to increase the interaction between the effector proteins and/or RDDPs and the negatively charged target nucleic acid sequences.

[152] In some embodiments, the RDDP is an engineered RDDP. Exemplary engineered RDDPs are provided in TABLE 3 below. For each engineered RDDP, the parental RDDP is listed followed by the amino acid mutations in parentheses. For example, 2691319 (D12R-D72R-N195R) refers to an engineered RDDP based on 2691319 (SEQ ID NO: 13) and comprising the mutations D12R, D72R, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that it at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one of SEQ ID NOs: 71- 80, wherein the effector protein comprises a corresponding amino acid substitution as described in TABLE 3. In some embodiments, the engineered RDDP comprises or consists of an amino sequence selected from SEQ ID NOs: 71-80.

[153] In some embodiments, the engineered RDDP comprises an amino acid sequence that it at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one of SEQ ID NOs: 132- 152. In some embodiments, the engineered RDDP comprises or consists of an amino sequence selected from SEQ ID NOs: 132-152. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 13 and comprises the amino acid substitutions of D12R, N24R, D72R, N114K, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 15 and comprises the amino acid substitutions of D12R, N24R, N72R, N114K, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 50 and comprises the amino acid substitutions of D12R, N24R, D72R, N114K, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 51 and comprises the amino acid substitutions of D12R, N24R, N72R, N114K, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 52 and comprises the amino acid substitutions of E12R, N24R, N72R, N114K, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 53 and comprises the amino acid substitutions of E12R, N24R, N72R, N114K, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 54 and comprises the amino acid substitutions of D12R, N24R, N72R, N114K, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 55 and comprises the amino acid substitutions of E12R, N24R, S72R, andN195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 56 and comprises the amino acid substitutions ofN24R, N72R, N114K, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 57 and comprises the amino acid substitutions of D12R, N24R, N72R, N114K, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 58 and comprises the amino acid substitutions of E12R, N24R, N72R, N114K, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 59 and comprises the amino acid substitutions of D12R, N24R, N72R, N114K, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 60 and comprises the amino acid substitutions of D9R, N21R, N69R, G11 IK, and N192R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 61 and comprises the amino acid substitutions of N12R, N24R, N72R, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 62 and comprises the amino acid substitutions of E12R, N24R, N72R, N114K, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 63 and comprises the amino acid substitutions of D12R, N24R, N72R, G114K, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 64 and comprises the amino acid substitutions of D38R, N50R, N98R, N140K, and N221R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 65 and comprises the amino acid substitutions of E12R, N24R, N72R, N114K, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 66 and comprises the amino acid substitutions ofN12R, N24R, D72R, N114K, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 67 and comprises the amino acid substitutions of K12R, N24R, D72R, N114K, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 68 and comprises the amino acid substitutions of N12R, N24R, D72R, N114K, and N195R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 69 and comprises the amino acid substitutions of D34R, N46R, N94R, D136K, and N217R. In some embodiments, the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 70 and comprises the amino acid substitutions ofN12R, N24R, D72R, N114K, and N195R.

[154] In some embodiments, the present disclosure provides composition comprising an engineered RDDP described herein or a nucleic acid encoding the same.

Fusion proteins

[155] In some embodiments, the present disclosure provides a fusion protein comprising an effector protein described herein and an RDDP described herein. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, an effector protein and an RDDP. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, an RDDP and an effector protein.

[156] In some embodiments, the fusion protein described herein comprises an effector protein comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NO: 1 and an RDDP comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 2-82 and 111. In some embodiments, the fusion protein described herein comprises an effector protein comprising or consisting of an amino acid sequence of SEQ ID NO: 1 and an RDDP comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 2-82 and 111.

[157] In some embodiments, the fusion protein described herein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 114-123, 153-159. In some embodiments, the fusion protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 115, 117, 119, 122, 123, and 153-159, wherein the amino acid sequence comprises a P2A peptide that results in cleavage of the fusion protein at the site of the P2A.

Tethers

[158] In some embodiments the effector protein is complexed with all or part of a biological tether or a protein localization sequence. In some embodiments, the RDDP is bound to the biological tether or protein localization sequence. In some embodiments the guide RNA is bound to an aptamer that is recognized by the biological tether protein. Non-limiting examples of a biological tether or protein localization sequence is MS2, Csy4 or lambda N protein.

[159] In some embodiments, the effector proteins are complexed with a biological tether, and comprises all or part of (e.g., DNA binding domain from) the MS2 coat protein, endoribonuclease Csy4, or the lambda N protein. These proteins can be used to recruit RNA molecules containing a specific stem-loop structure to a locale specified by the Type V effector protein guide RNA targeting sequences. For example, a Type V effector protein variant fused to MS2 coat protein, endoribonuclease Csy4, or lambda N can be used to recruit a long non-coding RNA (IncRNA) such as XIST or HOTAIR; see, e.g., Keryer-Bibens et al., Biol. Cell 100: 125-138 (2008), that is linked to the Csy4, MS2 or lambda N binding sequence. Alternatively, the Csy4, MS2 or lambda N protein binding sequence can be linked to another protein, e.g., as described in Keryer-Bibens et al., supra, and the protein can be targeted to the dCpfl variant binding site using the methods and compositions described herein. In some embodiments, the Csy4 is catalytically inactive.

[160] In some embodiments, an RDDP is bound to an MCP (MS2 aptamer binding protein). In some embodiments, an MCP comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to

ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQKRKYTIK VEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIA ANSGIY (SEQ ID NO: 113).

[161] In some embodiments, a fusion protein comprises a Brex27 peptide that inhibits non- homologous end joining (NHEJ). In some embodiments, a Brex27 peptide comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to ALDFLSRLPLPPPVSPICTFVSPAAQKAFQPPRSCG (SEQ ID NO: 112).

Synthesis, Isolation and Assaying

[162] RDDPs, effector proteins and fusion proteins of the present disclosure of the present disclosure may be synthesized, using any suitable method. Additionally, nucleic acids, including mRNA encoding effector proteins and fusion proteins of the present disclosure may be synthesized using suitable methods. RDDPs, effector proteins and fusion proteins of the present disclosure may be produced in vitro or by eukaryotic cells or by prokaryotic cells. Effector proteins can be further processed by unfolding, e.g. heat denaturation, dithiothreitol reduction, etc. and may be further refolded, using any suitable method.

[163] Methods of generating and assaying the RDDPs, effector proteins and fusion proteins described herein are well known to one of skill in the art. Examples of such methods are described in the Examples provided herein. Any of a variety of methods can be used to generate an effector protein disclosed herein. Such methods include, but are not limited to, site-directed mutagenesis, random mutagenesis, combinatorial libraries, and other mutagenesis methods described herein (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999); Gillman et al., Directed Evolution Library Creation: Methods and Protocols (Methods in Molecular Biology) Springer, 2^nd ed (2014)). One non-limiting example of a method for preparing an effector protein is to express recombinant nucleic acids encoding the effector protein in a suitable microbial organism, such as a bacterial cell, a yeast cell, or other suitable cell, using methods well known in the art.

[164] In some embodiments, an RDDP, effector protein, and/or fusion protein provided herein is an isolated effector protein. In some embodiments, an RDDP, effector protein, and/or fusion protein described herein can be isolated and purified for use in compositions, systems, and/or methods described herein. Methods described here can include the step of isolating effector proteins described herein. An isolated an RDDP, effector protein, and/or fusion protein provided herein can be isolated by a variety of methods well-known in the art, for example, recombinant expression systems, precipitation, gel filtration, ion-exchange, reverse-phase and affinity chromatography, and the like. Other well-known methods are described in Deutscher et al., Guide to Protein Purification: Methods in Enzymology, Vol. 182, (Academic Press, (1990)). Alternatively, the isolated polypeptides of the present disclosure can be obtained using well-known recombinant methods (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999)). The methods and conditions for biochemical purification of a polypeptide described herein can be chosen by those skilled in the art, and purification monitored, for example, by a functional assay.

[165] RDDPs, effector proteins, and/or fusion proteins disclosed herein may be covalently linked or attached to a tag, e.g., a purification tag. A purification tag, as used herein, can be an amino acid sequence which can attach or bind with high affinity to a separation substrate and assist in isolating the protein of interest from its environment, which can be its biological source, such as a cell lysate. Attachment of the purification tag can be at the N or C terminus of the effector protein. Furthermore, an amino acid sequence recognized by a protease or a nucleic acid encoding for an amino acid sequence recognized by a protease, such as TEV protease or the HRV3C protease can be inserted between the purification tag and the effector protein, such that biochemical cleavage of the sequence with the protease after initial purification liberates the purification tag. Purification and/or isolation can be through high performance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. Examples of purification tags are as described herein.

Guide Nucleic Acids

[166] The compositions, systems, and methods of the present disclosure may comprise a guide nucleic acid or a use thereof. Unless otherwise indicated, compositions, systems and methods comprising guide nucleic acids or uses thereof, as described herein and throughout, include DNA molecules, such as expression vectors, that encode a guide nucleic acid. Accordingly, compositions, systems, and methods of the present disclosure comprise a guide nucleic acid or a nucleotide sequence encoding the guide nucleic acid.

[167] In general, guide nucleic acids, including guide RNA (gRNA), comprise a nucleotide sequence. Such nucleotide sequence may be described as a nucleotide sequence of either DNA or RNA. Due to WIPO Standard ST.26, the Us are being represented as Ts in RNA in the Sequence Listing provided herein. However, no matter the form the sequence is described, it is readily understood that such nucleotide sequences can be revised to be RNA or DNA, as needed, for describing a sequence within a guide nucleic acid itself or the sequence that encodes a guide nucleic acid. Similarly, disclosure of the nucleotide sequences described herein also discloses a complementary nucleotide sequence, a reverse nucleotide sequence, and the reverse complement nucleotide sequence, any one of which can be a nucleotide sequence for use in a guide nucleic acid. In some embodiments, a guide nucleic acid sequence(s) comprises one or more nucleotide alterations at one or more positions in any one of the sequences described herein. Alterations can include a nucleotide substitution, or a deletion, or an insertion.

[168] A guide nucleic acid of the present disclosure may comprise one or more of the following: a) an RNA nucleobase; b) a DNA nucleobase; c) a modified nucleobase; d) a modified sugar; and e) a modified backbone. Modified nucleobases, sugars and backbones are described in greater detail herein. A guide nucleic acid may be chemically synthesized or recombinantly produced by any suitable methods. Guide nucleic acids and portions thereof may be found in or identified from a CRISPR array present in the genome of a host organism or cell.

[169] In general, a guide nucleic acid is a nucleic acid molecule, at least a portion of which may be bound by an effector protein, thereby forming a ribonucleoprotein complex (RNP). Another portion of the guide nucleic acid molecule can comprise a spacer region which is complementary to at least a portion of the target nucleic acid sequence. In some embodiments, the guide nucleic acid imparts activity or sequence selectivity to the effector protein. When complexed with an effector protein, guide nucleic acids can bring the effector protein into proximity of a target nucleic acid. The guide nucleic acid spacer region may hybridize to a target nucleic acid or a portion thereof. In some embodiments, when a guide nucleic acid and an effector protein form an RNP, at least a portion of the RNP binds spacer region, recognizes, and/or hybridizes to a target nucleic acid. Those skilled in the art in reading the below specific examples of guide nucleic acids as used in RNPs described herein, will understand that in some embodiments, a RNP can hybridize, via the spacer region, to one or more target sequences in a target nucleic acid, thereby allowing the RNP to modify and/or recognize a target nucleic acid or sequence contained therein.

[170] In some embodiments, a guide nucleic acid comprises a first region that is capable of being non-covalently bound by an effector protein and a second region that hybridizes to a target nucleic acid. The first region may comprise or be referred to as a protein binding sequence. In some embodiments, the first region is located 5’ to the second region. In some embodiments, the second region is located 5’ to the first region. In some embodiments, the first region and second region are linked either by a covalent bond (e.g., a phosphodiester bond) or linker (e.g., one or more nucleotides). The protein binding sequence may comprise a repeat sequence, an intermediary sequence, a handle sequence, or a combination thereof. In some embodiments, the first region comprises a repeat sequence. In some embodiments, the first region comprises an intermediary sequence and a repeat sequence. In some embodiments, the first region comprises a handle sequence. In some embodiments, an effector protein binds to at least a portion of the first region. In some embodiments, the second region comprises a spacer sequence, wherein the spacer sequence can interact in a sequence-specific manner with (e.g. , has complementarity with, or can hybridize to a target sequence in) a target nucleic acid.

[171] A guide nucleic acid, as well as any components thereof (e.g., spacer region, repeat region, linker) may comprise one or more deoxyribonucleotides, ribonucleotides, biochemically or chemically modified nucleotides (e.g., one or more sequence modifications as described herein), and any combinations thereof. A guide nucleic acid may comprise a naturally occurring sequence. A guide nucleic acid may comprise a non-naturally occurring sequence, wherein the sequence of the guide nucleic acid, or any portion thereof, may be different from the sequence of a naturally occurring nucleic acid. The guide nucleic acid may be chemically synthesized or recombinantly produced. Guide nucleic acids and portions thereof may be found in or identified from a CRISPR array present in the genome of a host organism or cell.

[172] Modifications can further include changing of nucleic acids described herein (e.g., engineered guide nucleic acids) to provide the nucleic acid with a new or enhanced feature, such as improved stability. Such modifications of a nucleic acid include a nucleobase base modification, a backbone modification, a sugar modification, a phosphorothioate intemucleotide linkage, or combinations thereof. In some embodiments, the modifications can be of one or more nucleotides, nucleosides, or nucleobases in a nucleic acid. In some embodiments, uridines can be exchanged for pseudouridines (e.g., IN-Methyl-Pseudouridine). In some embodiments, all uridines can be exchanged for IN-Methyl- Pseudouridine. In this application, U can represent uracil or IN-Methyl -Pseudouridine.

[173] In some embodiments, the guide nucleic acid comprises a protein binding sequence that is bound by an effector protein. The protein binding sequence may also be referred to a scaffold. In some embodiments, the guide nucleic acid or scaffold thereof comprises a hairpin or stem-loop structure that is recognized by the effector protein. The hairpin or stem -loop structure may comprise a repeat region. In some embodiments, the guide nucleic acid comprises at least a portion of a tracrRNA sequence. In nature, a tracrRNA is a guide nucleic acid that has trans activating activity on a target nucleic acid through a repeat hybridization sequence. In some embodiments, guide nucleic acids of the instant disclosure do not comprise a repeat hybridization sequence. In some embodiments, guide nucleic acids of the instant disclosure do not comprise atracrRNA.

[174] In some embodiments, the guide nucleic acid comprises a repeat region that interacts with the effector protein. The term, “repeat region” may be used interchangeably herein with the term, “repeat sequence.” In some embodiments, an effector protein interacts with a repeat region. In some embodiments, an effector protein does not interact with a repeat region. Typically, the repeat region is adjacent to the spacer region. In certain embodiments, the repeat region is followed by the spacer region in the 5’ to 3’ direction. Exemplary repeat region sequences for exemplary effector proteins provided herein are shown in TABLE 1.

[175] In some embodiments, the guide nucleic acid comprises a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 124 or 125.

Spacer Sequences

[176] In general, guide nucleic acids comprise a spacer sequence that hybridizes with a target sequence of a target nucleic acid. The term, “spacer sequence,” refers to a region of the guide nucleic acid that hybridizes to a target sequence of a target nucleic acid. The terms “spacer sequence,” “targeting sequence” and “spacer region” are used interchangeably herein and throughout. The spacer sequence may comprise a sequence that is complementary with a target sequence of a target nucleic acid. In some embodiments, the spacer sequence is complementary to the target sequence on the target strand of a dsDNA molecule. In some embodiments, the spacer sequence is complementary to the target sequence on the non-target strand of a dsDNA molecule. The spacer sequence can function to direct the guide nucleic acid to the target nucleic acid for detection and/or modification of the target nucleic acid. The spacer sequence may be complementary to a target sequence that is adjacent to a PAM that is recognizable by an effector protein of interest.

[177] The spacer sequence of a guide nucleic acid is generally complementary to a target sequence of a target nucleic acid. It is understood that the spacer sequence need not be 100% complementary to that of a target sequence of a target nucleic acid to hybridize or hybridize specifically to the target sequence. However, there is sufficient complementarity between the spacer sequence and the target sequence in order for the spacer sequence to hybridize to the target sequence. The spacer sequence of a guide nucleic acid may be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to a target sequence of a target nucleic acid.

[178] In some embodiments, the spacer sequence is 15-50 linked nucleotides in length. In some embodiments, the spacer sequence is 15-26, 15-24, 15-22, 15-20, 15-18, 16-28, 16-26, 16-24, 16-22, 16-20, 16-18, 17-26, 17-24, 17-22, 17-20, 17-18, 18-26, 18-24, or 18-22 linked nucleotides in length. In some embodiments, the spacer sequence is 18-24 linked nucleotides in length. In some embodiments, the spacer sequence is at least 15 linked nucleotides in length. In some embodiments, the spacer sequence is at least 16, 18, 20, or 22 linked nucleotides in length. In some embodiments, the spacer sequence comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the spacer sequence is at least 17 linked nucleotides in length. In some embodiments, the spacer sequence is at least 18 linked nucleotides in length. In some embodiments, the spacer region is at least 20 linked nucleotides in length. In some embodiments, the spacer sequence is at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a target sequence of the target nucleic acid. In some embodiments, the spacer sequence is 100% complementary to the target sequence of the target nucleic acid. In some embodiments, the spacer sequence comprises at least 15 contiguous nucleotides that are complementary to the target nucleic acid. It is understood that the sequence of a spacer sequence need not be 100% complementary to that of a target sequence of a target nucleic acid to hybridize or hybridize specifically to the target sequence. The spacer sequence may comprise at least one nucleotide that is not complementary to the corresponding nucleotide of the target sequence. In some embodiments the spacer sequence is less than 100% complementary to the target sequence, but still can bind to the target sequence. In some embodiments the spacer sequence is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the target sequence.

[179] In some embodiments, a guide nucleic acid, a spacer region thereof or a spacer sequence thereof, comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides that are complementary to a eukaryotic sequence. Such a eukaryotic sequence is a sequence of nucleotides that is present in a host eukaryotic cell. Such a sequence of nucleotides is distinguished from nucleotide sequences present in other host cells, such as prokaryotic cells, or viruses. By way of non-limiting example, said sequences present in a eukaryotic cell can be located in a gene, an exon, an intron, or a non-coding (e.g., promoter or enhancer) region. In some embodiments, a linker is present between the spacer and repeat sequences.

[180] In some embodiments, a spacer sequence is adjacent to a repeat sequence. In some embodiments, a spacer sequence follows a repeat sequence in a 5’ to 3’ direction. In some embodiments, a spacer sequence precedes a repeat sequence in a 5’ to 3’ direction. In some embodiments, the spacer sequence(s) and the repeat sequence(s) of the guide nucleic acid are present within the same molecule. In some embodiments, the spacer(s) and repeat sequence(s) are linked directly to one another. In some embodiments, a linker is present between the spacer(s) and repeat sequences. Linkers may be any suitable linker. In some embodiments, the spacer sequence(s) and the repeat sequence(s) of the guide nucleic acid are present in separate molecules, which are joined to one another by base pairing interactions.

Nucleic acid linkers

[181] In some embodiments, a guide nucleic acid for use with compositions, systems, and methods described herein comprises one or more linkers, or a nucleic acid encoding one or more linkers. In some embodiments, the guide nucleic acid comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten linkers. In some embodiments, the guide nucleic acid comprises one, two, three, four, five, six, seven, eight, nine, or ten linkers. In some embodiments, the guide nucleic acid comprises two or more linkers. In some embodiments, at least two or more linkers are the same. In some embodiments, at least two or more linkers are not same.

[182] In some embodiments, a linker comprises one to ten, one to seven, one to five, one to three, two to ten, two to eight, two to six, two to four, three to ten, three to seven, three to five, four to ten, four to eight, four to six, five to ten, five to seven, six to ten, six to eight, seven to ten, or eight to ten linked nucleotides. In some embodiments, the linker comprises one, two, three, four, five, six, seven, eight, nine, or ten linked nucleotides. In some embodiments, a linker comprises a nucleotide sequence of 5 GAAA-3’ (SEQ ID NO: 41).

[183] In some embodiments, a guide nucleic acid comprises one or more linkers connecting one or more repeat sequences. In some embodiments, the guide nucleic acid comprises one or more linkers connecting one or more repeat sequences and one or more spacer sequences. In some embodiments, the guide nucleic acid comprises at least two repeat sequences connected by a linker.

Repeat Sequences

[184] Guide nucleic acids described herein may comprise one or more repeat sequences. In some embodiments, a repeat sequence comprises a nucleotide sequence that is not complementary to a target sequence of a target nucleic acid. In some embodiments, a repeat sequence comprises a nucleotide sequence that may interact with an effector protein. In some embodiments, a repeat sequence includes a nucleotide sequence that is capable of forming a guide nucleic acid-effector protein complex (e.g., a RNP complex). In some embodiments, the repeat sequence may also be referred to as a “proteinbinding segment.”

[185] In some embodiments, a repeat sequence is adjacent to a spacer sequence. In some embodiments, a repeat sequence is followed by a spacer sequence in the 5’ to 3’ direction. In some embodiments, a repeat sequence is adjacent to an intermediary sequence. In some embodiments, a repeat sequence is 3’ to an intermediary sequence. In some embodiments, an intermediary sequence is followed by a repeat sequence, which is followed by a spacer sequence in the 5’ to 3’ direction. In some embodiments, a repeat sequence is linked to a spacer sequence and/or an intermediary sequence. In some embodiments, a guide nucleic acid comprises a repeat sequence linked to a spacer sequence, which may be a direct link or by any suitable linker, examples of which are described herein.

[186] In some embodiments, the spacer sequence(s) and the repeat sequence(s) of the guide nucleic acid are present within the same polynucleotide molecule. In some embodiments, a spacer sequence is adjacent to a repeat sequence. In some embodiments, a spacer sequence follows a repeat sequence in a 5’ to 3’ direction. In some embodiments, a spacer sequence precedes a repeat sequence in a 5’ to 3’ direction. In some embodiments, the spacer(s) and repeat sequence(s) are linked directly to one another. In some embodiments, a linker is present between the spacer(s) and repeat sequence(s). Linkers may be any suitable linker. In some embodiments, the spacer sequence(s) and the repeat sequence(s) of the guide nucleic acid are present in separate polynucleotide molecules, which are joined to one another by base pairing interactions.

[187] In some embodiments, the repeat sequence comprises two sequences that are complementary to each other and hybridize to form a double stranded RNA duplex (dsRNA duplex). In some embodiments, the two sequences are not directly linked and hybridize to form a stem loop structure. In some embodiments, the dsRNA duplex comprises 5, 10, 15, 20 or 25 base pairs (bp). In some embodiments, not all nucleotides of the dsRNA duplex are paired, and therefore the duplex forming sequence may include a bulge. In some embodiments, the repeat sequence comprises a hairpin or stemloop structure, optionally at the 5’ portion of the repeat sequence. In some embodiments, a strand of the stem portion comprises a sequence and the other strand of the stem portion comprises a sequence that is, at least partially, complementary. In some embodiments, such sequences may have 65% to 100% complementarity (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementarity). In some embodiments, a guide nucleic acid comprises nucleotide sequence that when involved in hybridization events may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g. , a bulge, a loop structure or hairpin structure, etc.). Exemplary repeat sequences are provided in TABLE 1.

Intermediary Sequences

[188] Guide nucleic acids described herein may comprise one or more intermediary sequences. In general, an intermediary sequence used in the present disclosure is not transactivated or transactivating. An intermediary sequence may also be referred to as an intermediary RNA, although it may comprise deoxyribonucleotides instead of or in addition to ribonucleotides, and/or modified bases. In general, the intermediary sequence non-covalently binds to an effector protein. In some embodiments, the intermediary sequence forms a secondary structure, for example in a cell, and an effector protein binds the secondary structure.

[189] The terms, “intermediary RNA,” “intermediary RNA sequence,” and “intermediary sequence” as used herein, may refer to a nucleotide sequence in a handle sequence, wherein the intermediary RNA sequence is capable of, at least partially, being non-covalently bound to an effector protein to form a complex (e.g., an RNP complex). An intermediary RNA sequence is not a transactivating nucleic acid in systems, methods, and compositions described herein.

[190] In some embodiments, a length of the intermediary sequence is at least 30, 50, 70, 90, 110, 130, 150, 170, 190, or 210 linked nucleotides. In some embodiments, the length of the intermediary sequence is not greater than 30, 50, 70, 90, 110, 130, 150, 170, 190, or 210 linked nucleotides. In some embodiments, the length of the intermediary sequence is about 30 to about 210, about 60 to about 210, about 90 to about 210, about 120 to about 210, about 150 to about 210, about 180 to about 210, about 30 to about 180, about 60 to about 180, about 90 to about 180, about 120 to about 180, or about 150 to about 180 linked nucleotides.

[191] An intermediary sequence may also comprise or form a secondary structure (e.g., one or more hairpin loops) that facilitates the binding of an effector protein to a guide nucleic acid and/or modification activity of an effector protein on a target nucleic acid (e.g., a hairpin region). An intermediary sequence may comprise from 5’ to 3’, a 5’ region, a hairpin region, and a 3’ region. In some embodiments, the 5’ region may hybridize to the 3’ region. In some embodiments, the 5’ region of the intermediary sequence does not hybridize to the 3’ region.

[192] In some embodiments, the hairpin region may comprise a first sequence, a second sequence that is reverse complementary to the first sequence, and a stem-loop linking the first sequence and the second sequence. In some embodiments, an intermediary sequence comprises a stem-loop structure comprising a stem region and a loop region. In some embodiments, the stem region is 4 to 8 linked nucleotides in length. In some embodiments, the stem region is 5 to 6 linked nucleotides in length. In some embodiments, the stem region is 4 to 5 linked nucleotides in length. In some embodiments, an intermediary sequence comprises a pseudoknot (e.g., a secondary structure comprising a stem at least partially hybridized to a second stem or half-stem secondary structure). An effector protein may interact with an intermediary sequence comprising a single stem region or multiple stem regions. In some embodiments, the nucleotide sequences of the multiple stem regions are identical to one another. In some embodiments, the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others. In some embodiments, an intermediary sequence comprises 1, 2, 3, 4, 5 or more stem regions. Exemplary intermediary sequences are provided in TABLE 1.

Handle Sequences

[193] In some embodiments, compositions, systems and methods described herein comprise the nucleic acid, wherein the nucleic acid comprises a handle sequence. In some embodiments, the handle sequence comprises an intermediary sequence. In some embodiments, the intermediary sequence is at the 3 ’-end of the handle sequence. In some embodiments, the intermediary sequence is at the 5’- end of the handle sequence. In some embodiments, the handle sequence further comprises one or more of linkers and repeat sequences. In some embodiments, the linker comprises a sequence of 5’-GAAA-3.’ In some embodiments, the intermediary sequence is 5’ to the repeat sequence. In some embodiments, the intermediary sequence is 5’ to the linker. In some embodiments, the intermediary sequence is 3’ to the repeat sequence. In some embodiments, the intermediary sequence is 3’ to the linker. In some embodiments, the repeat sequence is 3’ to the linker. In some embodiments, the repeat sequence is 5’ to the linker.

[194] In some embodiments, a sgRNA may include a handle sequence having a hairpin region, as well as a linker and a repeat sequence. The sgRNA having a handle sequence can have a hairpin region positioned 3’ of the linker and/or repeat sequence. The sgRNA having a handle sequence can have a hairpin region positioned 5’ of the linker and/or repeat sequence. The hairpin region may include a first sequence, a second sequence that is reverse complementary to the first sequence, and a stem-loop linking the first sequence and the second sequence.

[195] In some embodiments, an effector protein may recognize a secondary structure of a handle sequence. In some embodiments, at least a portion of the handle sequence interacts with an effector protein described herein. Accordingly, in some embodiments, at least a portion of the intermediary sequence interacts with the effector protein described herein. In some embodiments, both, at least a portion of the intermediary sequence and at least a portion of the repeat sequence, interacts with the effector protein. In general, the handle sequence is capable of interacting (e.g., non-covalent binding) with any one of the effector proteins described herein.

[196] The term, “handle sequence,” as used herein, may refer to a sequence of nucleotides in a single guide RNA (sgRNA), that is: 1) capable of being non-covalently bound by an effector protein and 2) connects the portion of the sgRNA capable of being non-covalently bound by an effector protein to a nucleotide sequence that is hybridizable to a target nucleic acid. In general, the handle sequence comprises an intermediary RNA sequence, that is capable of being non-covalently bound by an effector protein. In some embodiments, the handle sequence further comprises a repeat sequence. In such embodiments, the intermediary RNA sequence or a combination of the intermediary RNA and the repeat sequence is capable of being non-covalently bound by an effector protein.

[197] In some embodiments, the handle sequence of an sgRNA comprises a stem-loop structure comprising a stem region and a loop region. In some embodiments, the stem region is 4 to 8 linked nucleotides in length. In some embodiments, the stem region is 5 to 6 linked nucleotides in length. In some embodiments, the stem region is 4 to 5 linked nucleotides in length. In some embodiments, the sgRNA comprises a pseudoknot (e.g., a secondary structure comprising a stem at least partially hybridized to a second stem or half-stem secondary structure). An effector protein may recognize an sgRNA comprising multiple stem regions. In some embodiments, the nucleotide sequences of the multiple stem regions are identical to one another. In some embodiments, the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others. In some embodiments, the sgRNA comprises at least 2, at least 3, at least 4, or at least 5 stem regions.

[198] A handle sequence may include deoxyribonucleosides, ribonucleosides, chemically modified nucleosides, or any combination thereof. In some embodiments, a length of the handle sequence is at least 30, 50, 70, 90, 110, 130, 150, 170, 190, or 210 linked nucleotides. In some embodiments, a length of the handle sequence is not greater than 30, 50, 70, 90, 110, 130, 150, 170, 190, or 210 linked nucleotides. In some embodiments, the length of the handle sequence is about 30 to about 210, about 60 to about 210, about 90 to about 210, about 120 to about 210, about 150 to about 210, about 180 to about 210, about 30 to about 180, about 60 to about 180, about 90 to about 180, about 120 to about 180, or about 150 to about 180 linked nucleotides.

[199] In some embodiments, the length of a handle sequence in an sgRNA is not greater than 50, 56, 66, 67, 68, 69, 70, 71, 72, 73, 95, or 105 linked nucleotides. In some embodiments, the length of a handle sequence in an sgRNA is about 30 to about 120 linked nucleotides. In some embodiments, the length of a handle sequence in an sgRNA is about 50 to about 105, about 50 to about 95, about 50 to about 73, about 50 to about 71, about 50 to about 70, or about 50 to about 69 linked nucleotides. In some embodiments, the length of a handle sequence in an sgRNA is 56 to 105 linked nucleotides, from 56 to 105 linked nucleotides, 66 to 105 linked nucleotides, 67 to 105 linked nucleotides, 68 to 105 linked nucleotides, 69 to 105 linked nucleotides, 70 to 105 linked nucleotides, 71 to 105 linked nucleotides, 72 to 105 linked nucleotides, 73 to 105 linked nucleotides, or 95 to 105 linked nucleotides. In some embodiments, the length of a handle sequence in an sgRNA is 40 to 70 nucleotides. In some embodiments, the length of a handle sequence in an sgRNA is 50, 56, 66, 67, 68, 69, 70, 71, 72, 73, 95, or 105 linked nucleotides. Exemplary handle sequences are provided in TABLE 1.

Single Guide Nucleic Acid Systems

[200] In some embodiments, compositions, systems and methods described herein comprise a single guide nucleic acid system comprising a guide nucleic acid or a nucleotide sequence encoding the guide nucleic acid. In some embodiments, a first region (FR) of the guide nucleic acid non-covalently interacts with the one or more polypeptides described herein. In some embodiments, a second region (SR) of the guide nucleic acid hybridizes with a target sequence of the target nucleic acid. In the single nucleic acid system, the guide nucleic acid forms a complex with the effector protein, and the effector protein is not transactivated by the guide nucleic acid. In other words, activity of effector protein does not require binding to a second or intermediary guide nucleic acid molecule. Exemplary guide nucleic acids for a single nucleic acid system are crRNAs or sgRNAs. crRNA

[201] In some embodiments, a single guide nucleic acid comprises a crRNA. In some embodiments, the guide nucleic acid is the crRNA. In general, a crRNA comprises a first region (FR) and a second region (SR), wherein the FR of the crRNA comprises a repeat sequence, and the SR of the crRNA comprises a spacer sequence. In some embodiments, the FR consists of a repeat sequence. In some embodiments, the spacer sequence follows the repeat sequence in a 5’ to 3’ direction. In some embodiments, the spacer sequence precedes the repeat sequence in a 5’ to 3’ direction. In some embodiments, the repeat sequence and the spacer sequences are directly connected to each other (e.g., covalent bond (phosphodiester bond)). In some embodiments, the repeat sequence and the spacer sequence are connected by a linker (e.g., one or more nucleotides). [202] In some embodiments, a crRNA is useful as a single guide nucleic acid system for compositions, methods, and systems described herein or as part of a single guide nucleic acid system for compositions, methods, and systems described herein. In such embodiments, a single guide nucleic acid system comprises a guide nucleic acid comprising a crRNA wherein, a repeat sequence of a crRNA is capable of causing a crRNA to interact with an effector protein. In some embodiments, a single guide nucleic acid system comprises a guide nucleic acid comprising a crRNA linked to another nucleotide sequence that is capable of being non-covalently bound by an effector protein. In some embodiments, a crRNA is sufficient to form complex with an effector protein (e.g., to form an RNP) through the repeat sequence and direct the effector protein to a target nucleic acid sequence through the spacer sequence.

[203] A crRNA may include deoxyribonucleosides, ribonucleosides, chemically modified nucleosides, or any combination thereof. In some embodiments, a crRNA comprises about: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 linked nucleotides. In some embodiments, a crRNA comprises at least: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 linked nucleotides. In some embodiments, the length of the crRNA is about 20 to about 120 linked nucleotides. In some embodiments, the length of a crRNA is about 20 to about 100, about 30 to about 100, about 40 to about 100, about 40 to about 90, about 40 to about 80, about 40 to about 70, about 40 to about 60, about 40 to about 50, about 50 to about 90, about 50 to about 80, about 50 to about 70, or about 50 to about 60 linked nucleotides. In some embodiments, the length of a crRNA is about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70 or about 75 linked nucleotides. sgRNA

[204] In some embodiments, a guide nucleic acid comprises a single guide RNA (sgRNA). In some embodiments, the guide nucleic acid is an sgRNA. In some embodiments, an sgRNA can have two or more linked guide nucleic acid components (e.g., an intermediary RNA sequence, a repeat sequence, a spacer sequence, and optionally a linker). In some embodiments, an sgRNA comprises a handle sequence, wherein the handle sequence comprises an intermediary sequence, a repeat sequence, and optionally a linker sequence. The combination of a spacer sequence (e.g., a nucleotide sequence that hybridizes to a target sequence in a target nucleic acid) with a handle sequence may be referred to herein as a single guide RNA (sgRNA), wherein the spacer sequence and the handle sequence are covalently linked. In some embodiments, the spacer sequence and handle sequence are linked by a phosphodiester bond. In some embodiments, the spacer sequence and handle sequence are linked by one or more linked nucleotides. In some embodiments, a guide nucleic acid may comprise a spacer sequence, a repeat sequence, or handle sequence, or a combination thereof. In some embodiments, the handle sequence may comprise a portion of, or all of, a repeat sequence. In general, an sgRNA comprises a first region (FR) and a second region (SR), wherein the FR comprises a handle sequence and the SR comprises a spacer sequence.

[205] In some embodiments, the compositions comprise a guide RNA and an effector protein without a tracrRNA (e.g. , a single nucleic acid system), wherein the guide RNA is an sgRNA. An sgRNA may include deoxyribonucleosides, ribonucleosides, chemically modified nucleosides, or any combination thereof. An sgRNA may also include a nucleotide sequence that forms a secondary structure (e.g., one or more hairpin loops) that facilitates the binding of an effector protein to the sgRNA and/or modification activity of an effector protein on a target nucleic acid (e.g., a hairpin region). Such a sequence can be contained within a handle sequence as described herein.

[206] In some embodiments, an sgRNA comprises one or more of a handle sequence, an intermediary sequence, a crRNA, a repeat sequence, a spacer sequence, a linker, or combinations thereof. For example, an sgRNA comprises a handle sequence and a spacer sequence; an intermediary sequence and a crRNA; an intermediary sequence, a repeat sequence and a spacer sequence; and the like.

[207] In some embodiments, an sgRNA comprises an intermediary sequence and an crRNA. In some embodiments, an intermediary sequence is 5’ to a crRNA in an sgRNA. In some embodiments, an sgRNA comprises a linked intermediary sequence and crRNA. In some embodiments, an intermediary sequence and a crRNA are linked in an sgRNA directly (e.g., covalently linked, such as through a phosphodiester bond). In some embodiments, an intermediary sequence and a crRNA are linked in an sgRNA by any suitable linker, examples of which are provided herein.

[208] In some embodiments, an sgRNA comprises a handle sequence and a spacer sequence. In some embodiments, a handle sequence is 5’ to a spacer sequence in an sgRNA. In some embodiments, an sgRNA comprises a linked handle sequence and spacer sequence. In some embodiments, a handle sequence and a spacer sequence are linked in an sgRNA directly (e.g., covalently linked, such as through a phosphodiester bond) In some embodiments, a handle sequence and a spacer sequence are linked in an sgRNA by any suitable linker, examples of which are provided herein.

[209] In some embodiments, an sgRNA comprises an intermediary sequence, a repeat sequence, and a spacer sequence. In some embodiments, an intermediary sequence is 5’ to a repeat sequence in an sgRNA. In some embodiments, an sgRNA comprises a linked intermediary sequence and repeat sequence. In some embodiments, an intermediary sequence and a repeat sequence are linked in an sgRNA directly (e.g., covalently linked, such as through a phosphodiester bond). In some embodiments, an intermediary sequence and a repeat sequence are linked in an sgRNA by any suitable linker, examples of which are provided herein. In some embodiments, a repeat sequence is 5’ to a spacer sequence in an sgRNA. In some embodiments, a sgRNA comprises a linked repeat sequence and spacer sequence. In some embodiments, a repeat sequence and a spacer sequence are linked in an sgRNA directly (e.g, covalently linked, such as through a phosphodiester bond) In some embodiments, a repeat sequence and a spacer sequence are linked in an sgRNA by any suitable linker, examples of which are provided herein.

[210] An exemplary handle sequence in an sgRNA may comprise, from 5’ to 3’, a 5’ region, a hairpin region, and a 3’ region. In some embodiments, the 5’ region may hybridize to the 3’ region. In some embodiments, the 5’ region does not hybridize to the 3’ region. In some embodiments, the 3’ region is covalently linked to a spacer sequence (e.g., through a phosphodiester bond). In some embodiments, the 5’ region is covalently linked to a spacer sequence (e.g., through a phosphodiester bond).

[2H] In some embodiments, compositions and systems described herein comprise an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 1, 86, and 87; and a guide nucleic acid that comprises an sgRNA. In some embodiments, the sgRNA comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 124 and 125. In some embodiments, the sgRNA consists of a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 124 and 125.

Dual Nucleic Acid Systems

[212] In some embodiments, compositions, systems and methods described herein comprise a dual nucleic acid system comprising a crRNA or a nucleotide sequence encoding the crRNA, a tracrRNA or a nucleotide sequence encoding the tracrRNA, and one or more effector proteins or a nucleotide sequence encoding the one or more effector proteins, wherein the crRNA and the tracrRNA are separate, unlinked molecules, wherein a repeat hybridization region of the tracrRNA is capable of hybridizing with an equal length portion of the crRNA to form a tracrRNA-crRNA duplex, wherein the equal length portion of the crRNA does not include a spacer sequence of the crRNA, and wherein the spacer sequence is capable of hybridizing to a target sequence of the target nucleic acid. In the dual nucleic acid system having a complex of the guide nucleic acid, tracrRNA, and the effector protein, the effector protein is transactivated by the tracrRNA. In other words, in a dual nucleic acid system, activity of the effector protein requires binding to a tracrRNA molecule.

[213] In some embodiments, a repeat hybridization sequence is at the 3’ end of a tracrRNA. In some embodiments, a repeat hybridization sequence may have a length of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, or about 20 linked nucleotides. In some embodiments, the length of the repeat hybridization sequence is 1 to 20 linked nucleotides.

[214] In some embodiments, systems, compositions, and methods comprise a crRNA or a use thereof. In general, a crRNA comprises a first region (FR) and a second region (SR), wherein the FR of the crRNA comprises a repeat sequence, and the SR of the crRNA comprises a spacer sequence. In some embodiments, the repeat sequence and the spacer sequences are directly connected to each other (e.g., covalent bond (phosphodiester bond)). In some embodiments, the repeat sequence and the spacer sequence are connected by a linker.

[215] In some embodiments, systems, compositions, and methods comprise a tracrRNA or a use thereof. In some embodiments, systems, compositions, and methods do not comprise a tracrRNA or a use thereof. A tracrRNA and/or tracrRNA-crRNA duplex may form a secondary structure that facilitates the binding of an effector protein to a tracrRNA or a tracrRNA-crRNA. In some embodiments, the secondary structure modifies activity of the effector protein on a target nucleic acid. In some embodiments, the secondary structure comprises a stem-loop structure comprising a stem region and a loop region. In some embodiments, the stem region is 4 to 8 linked nucleotides in length. In some embodiments, the stem region is 5 to 6 linked nucleotides in length. In some embodiments, the stem region is 4 to 5 linked nucleotides in length. In some embodiments, the secondary structure comprises a pseudoknot (e.g., a secondary structure comprising a stem at least partially hybridized to a second stem or half-stem secondary structure). An effector protein may recognize a secondary structure comprising multiple stem regions. In some embodiments, nucleotide sequences of the multiple stem regions are identical to one another. In some embodiments, the nucleotide sequences of at least one of the multiple stem regions is not identical to those of the others. In some embodiments, the secondary structure comprises at least two, at least three, at least four, or at least five stem regions. In some embodiments, the secondary structure comprises one or more loops. In some embodiments, the secondary structure comprises at least one, at least two, at least three, at least four, or at least five loops.

Template RNA

[216] In some embodiments, compositions, systems and methods described herein comprise a template RNA (retRNA), wherein the template RNA comprises a primer binding sequence and a template sequence. In some embodiments, the primer binding sequence hybridizes to a primer sequence on the non-target strand of the target dsDNA molecule. In some embodiments, the primer binding sequence hybridizes to a primer sequence on the target strand of the target dsDNA molecule. In some embodiments, the spacer sequence is complementary to the target sequence on the target strand of the dsDNA molecule, and the primer binding sequence and/or the template sequence is complementary to a primer sequence on the non-target strand of the target dsDNA molecule. In some embodiments, the spacer sequence is complementary to the target sequence on the non-target strand of the dsDNA molecule, and the primer binding sequence and/or the template sequence is complementary to a primer sequence on the target strand of the target dsDNA molecule.

[217] In some embodiments, the primer binding sequence is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides long. In some embodiments the template sequence is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides long. In some embodiments, at least a portion of the PBS is complimentary to at least a portion of the target nucleic acid sequence that is 5’ of the nucleotide at position 13 relative to the PAM sequence. Such embodiments are particularly useful when used in a system comprising a CasM.265466 effector protein.

[218] The template sequence may comprise one or more nucleotides having a different nucleobase than that of a nucleotide at the corresponding position in the target nucleic acid when a spacer sequence of the guide RNA and the target sequence are aligned for maximum identity. The one or more nucleotides may be contiguous. The one or more nucleotides may not be contiguous. The one or more nucleotides may each independently be selected from guanine, adenine, cytosine and thymine.

[219] In some embodiments, the retRNA comprises a secondary structure that can be bound by a peptide or protein that is part of or linked to the RDDP. In some embodiments, the secondary structure comprises an aptamer. In some embodiments, the aptamer is an MS2 aptamer (See Said et al (November 2009). “In vivo expression and purification of aptamer-tagged small RNA regulators”. Nucleic Acids Research. 37 (20): el33; and Johansson et al (1997). “RNA recognition by the MS2 phage coat protein”. Seminars in Virology. 8 (3): 176-185).

Target Nucleic Acids and Samples

[220] In general, systems, compositions and methods disclosed herein are useful for modifying a target nucleic acid, wherein the target nucleic acid comprises double stranded DNA (dsDNA). In general, dsDNA comprises a target strand (TS) and a non-target strand (NTS), wherein the guide RNA hybridizes to the target strand. The portion of the target strand to which the guide RNA hybridizes may be referred to as the target sequence of the target strand. The portion of the non-target strand that is complementary to the target sequence of the target strand may be referred to as the target sequence of the non-target strand. Herein, the term target sequence alone may be used to refer to the target sequence of the target strand and/or the target sequence of the non-target strand.

[221] In some embodiments, where a target strand comprises a target sequence, at least a portion of the guide nucleic acid is complementary to the target sequence on the target strand. In some embodiments, where the target nucleic acid is a double stranded nucleic acid comprising a target strand and a non-target strand, and wherein the target strand comprises a target sequence, at least a portion of the guide nucleic acid is complementary to the target sequence on the target strand. In some embodiments, a target nucleic acid comprises a protospacer adjacent motif (PAM) as described herein that is located on the non-target strand. Such a PAM described herein, in some embodiments, is adjacent (e.g., within 1, 2, 3, 4, 5, 10, 20, 25 nucleotides) to the 5’ or 3’ end of the target sequence on the non-target strand of the double stranded DNA molecule. In certain embodiments, such a PAM described herein is directly adjacent to the 5’or 3’ end of a target sequence on the non-target strand of the double stranded DNA molecule. In certain embodiments, such a PAM described herein is directly adjacent to the 5’ end of a target sequence on the non-target strand of the double stranded DNA molecule.

[222] In some embodiments, an effector protein described herein, or a multimeric complex (e.g., dimer) thereof, recognizes a PAM on a target nucleic acid. In some embodiments, the PAM comprises a PAM sequence set forth in TABLE 1.

[223] An effector protein or fusion protein of the present disclosure may cleave or nick a target nucleic acid within or near a protospacer adjacent motif (PAM) sequence of the target nucleic acid. In some embodiments, cleavage occurs within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 nucleotides of a 5’ or 3’ terminus of a PAM sequence. A target nucleic acid may comprise a PAM sequence adjacent to a sequence that is complementary to a guide nucleic acid spacer region.

[224] In some embodiments, the guide nucleic acid can bind to a target sequence, wherein the target sequence is a nucleotide sequence found in a eukaryotic organism. The nucleotide sequence found in a eukaryotic organism may be at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, or at least 50 nucleotides. The target sequence may be found in a gene associated with a disease. In some embodiments, the gene is selected from TABLE 4. In some embodiments, the target sequence may be found in a bacterium, a virus, a parasite, a protozoon, a fungus or other agents responsible for a disease.

[225] In some embodiments, the target sequence is within an exon of any one of the genes set forth in TABLE 4. In some embodiments, the target sequence spans the junction of two exons. In some embodiments, the target sequence is located within about 1 to about 300 nucleotides, about 10 to about 250, about 20 to about 200, about 30 to about 150, about 40 to about 100, or about 50 nucleotides of the 5’ untranslated region (UTR). In some embodiments, the target sequence is located within about 1 to about 300 nucleotides, about 10 to about 250, about 20 to about 200, about 30 to about 150, about 40 to about 100, or about 50 nucleotides of the 3’ UTR.

In some embodiments, the target sequence is at least partially within a targeted exon within any one of the genes set forth in TABLE 4. A targeted exon can mean any portion within, contiguous with, or adjacent to a specified exon of interest can be targeted by the compositions, systems, and methods described herein.

[226] In some embodiments, the target nucleic acid as described in the methods herein does not initially comprise a PAM sequence. However, any target nucleic acid of interest may be generated using the methods described herein to comprise a PAM sequence, and thus be a PAM target nucleic acid. A PAM target nucleic acid, as used herein, refers to a target nucleic acid that has been amplified to insert a PAM sequence that is recognized by an effector protein system. In some embodiments, systems, compositions, and methods disclosed herein may be utilized to create an integration site for an integrase (e.g., attB or attP for a large serine recombinase). [227] In some embodiments, the target nucleic acid is in a cell. In some embodiments, the cell is a single-cell eukaryotic organism; a plant cell an algal cell; a fungal cell; an animal cell; a cell an invertebrate animal; a cell a vertebrate animal such as fish, amphibian, reptile, bird, and mammal; or a cell a mammal such as a human, a non-human primate, an ungulate, a feline, a bovine, an ovine, and a caprine. In some embodiments, the cell is a eukaryotic cell. In exemplary embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a plant cell.

Mutations

[228] In some embodiments, target nucleic acids comprise a mutation. In some embodiments, a composition, system or method described herein can be used to modify a target nucleic acid comprising a mutation such that the mutation is modified to be a wild-type nucleotide or nucleotide sequence.

[229] A mutation may be in an open reading frame of a target nucleic acid. A mutation may result in the insertion of at least one amino acid in a protein encoded by the target nucleic acid. A mutation may result in the deletion of at least one amino acid in a protein encoded by the target nucleic acid. A mutation may result in the substitution of at least one amino acid in a protein encoded by the target nucleic acid. A mutation that results in the deletion, insertion, or substitution of one or more amino acids of a protein encoded by the target nucleic acid may result in misfolding of a protein encoded by the target nucleic acid. A mutation may result in a premature stop codon, thereby resulting in a truncation of the encoded protein.

[230] In some embodiments, a mutation comprises a point mutation or single nucleotide polymorphism (SNP), a chromosomal mutation, a copy number mutation, or any combination thereof. A point mutation optionally comprises a substitution, insertion, or deletion.

[231] In some embodiments, target nucleic acids comprise a mutation, wherein the mutation is a SNP. The single nucleotide mutation or SNP may be associated with a phenotype of the sample or a phenotype of the organism from which the sample was taken. The SNP, in some embodiments, is associated with altered phenotype from wild type phenotype. In some embodiments, a single nucleotide mutation, SNP, or deletion described herein is associated with a disease, such as a genetic disease. The SNP may be a synonymous substitution or a nonsynonymous substitution. The nonsynonymous substitution may be a missense substitution or a nonsense point mutation. The synonymous substitution may be a silent substitution. The mutation may be a deletion of one or more nucleotides. Often, the single nucleotide mutation, SNP, or deletion is associated with a disease such as cancer or a genetic disorder. The mutation, such as a single nucleotide mutation, a SNP, or a deletion, may be encoded in the sequence of a target nucleic acid from the germline of an organism or may be encoded in a target nucleic acid from a diseased cell, such as a cancer cell.

[232] In some embodiments, the target nucleic acid comprises a mutation associated with a disease. Exemplary diseases are provided in TABLE 5. In some examples, a mutation associated with a disease refers to a mutation whose presence in a subject indicates that the subject is susceptible to or suffers from, a disease, disorder, condition, or syndrome. In some examples, a mutation associated with a disease refers to a mutation which causes, contributes to the development of, or indicates the existence of the disease, disorder, condition, or syndrome. A mutation associated with a disease may also refer to any mutation which generates transcription or translation products at an abnormal level, or in an abnormal form, in cells affected by a disease relative to a control without the disease. In some examples, a mutation associated with a disease refers to a mutation whose presence in a subject indicates that the subject is susceptible to, or suffers from, a disease, disorder, or pathological state. In some embodiments, a mutation associated with a disease, comprises the co-occurrence of a mutation and the phenotype of a disease. The mutation may occur in a gene, wherein transcription or translation products from the gene occur at a significantly abnormal level or in an abnormal form in a cell or subject harboring the mutation as compared to a non-disease control subject not having the mutation.

Chemical Modifications

[233] Polypeptides (e.g., effector proteins) and nucleic acids (e.g., engineered guide nucleic acids) described herein can be further modified as described throughout and as further described herein. Examples are modifications of interest that do not alter primary sequence, including chemical derivatization of polypeptides, e.g., acylation, acetylation, carboxylation, amidation, etc. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences that have phosphorylated amino acid residues, e.g. phosphotyrosine, phosphoserine, or phosphothreonine.

[234] Modifications disclosed herein can also include modification of described polypeptides and/or engineered guide nucleic acids through any suitable method, such as molecular biological techniques and/or synthetic chemistry, to improve their resistance to proteolytic degradation, to change the target sequence specificity, to optimize solubility properties, to alter protein activity (e.g., transcription modulatory activity, enzymatic activity, etc.) or to render them more suitable. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g. D- amino acids or non-naturally occurring synthetic amino acids. D-amino acids may be substituted for some or all of the amino acid residues. Modifications can also include modifications with non-naturally occurring unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.

[235] Modifications can further include the introduction of various groups to polypeptides and/or engineered guide nucleic acids described herein. For example, groups can be introduced during synthesis or during expression of a polypeptide (e.g., an effector protein), which allow for linking to other molecules or to a surface. Thus, e.g., cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like.

[236] Modifications can further include modification of nucleic acids described herein (e.g., engineered guide nucleic acids) to provide the nucleic acid with a new or enhanced feature, such as improved stability. Such modifications of a nucleic acid include a base modification, a backbone modification, a sugar modification, or combinations thereof, of one or more nucleotides, nucleosides, or nucleobases in a nucleic acid.

[237] In some embodiments, nucleic acids (e.g., engineered guide nucleic acids) described herein comprise one or more modifications comprising: 2’0-methyl modified nucleotides, 2’ Fluoro modified nucleotides; locked nucleic acid (LNA) modified nucleotides; peptide nucleic acid (PNA) modified nucleotides; nucleotides with phosphorothioate linkages; a 5’ cap (e.g., a 7-methylguanylate cap (m7G)), phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3’-alkylene phosphonates, 5’-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3’- amino phosphoramidate and aminoalkyl phosphoramidates, phosphorodiamidates, thionophosphor amidates, thionoalkylphosphonates , thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3’-5’ linkages, 2’-5’ linked analogs of these, and those having inverted polarity wherein one or more intemucleotide linkages is a 3’ to 3’, 5’ to 5’ or 2’ to 2’ linkage; phosphorothioate and/or heteroatom intemucleoside linkages, such as -CH2-NH-O-CH2-, -CH2- N(CH3)-O-CH2- (known as a methylene (methylimino) or MMI backbone), -CH2-O-N(CH3)-CH2-, -CH2-N(CH3)- N(CH3)-CH2- and -O-N(CH3)-CH2-CH2- (wherein the native phosphodiester intemucleotide linkage is represented as -0-P(=0)(0H)-0-CH2-); morpholino linkages (formed in part from the sugar portion of a nucleoside); morpholino backbones; phosphorodiamidate or other non- phosphodiester intemucleoside linkages; siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; other backbone modifications having mixed N, O, S and CH2 component parts; and combinations thereof.

Vectors and Multiplexed Expression Vectors

[238] In some embodiments, compositions and systems provided herein comprise a vector system encoding a polypeptide (e.g., an effector protein, an RDDP, and/or a fusion protein thereof) and/or a guide nucleic acid described herein. In some embodiments, compositions and systems provided herein comprise a vector system encoding a guide nucleic acid (e.g., crRNA) described herein. In some embodiments, compositions and systems provided herein comprise a multi-vector system encoding an effector protein, an RDDP, and/or a fusion protein thereof and a guide nucleic acid described herein, wherein the guide nucleic acid and the effector protein, the RDDP, and/or the fusion protein thereof are encoded by the same or different vectors. In some embodiments, the guide and the engineered effector protein are encoded by different vectors of the system. In some embodiments, a nucleic acid encoding a polypeptide (e.g. , an effector protein, an RDDP, and/or a fusion protein thereof) comprises an expression vector. In some embodiments, an expression vector encoding a fusion protein comprises a nucleic acid sequence encoding a P2A peptide between the sequences encoding an effector protein and an RDDP. In some embodiments, an expression vector encoding a fusion protein does not comprise a nucleic acid sequence encoding a P2A peptide between the sequences encoding an effector protein and an RDDP. In such embodiments, the RDDP is fused to the effector protein. The RDDP may not be fused to an MS2 coat protein and the template RNA may not comprise an MS2 protein localization sequence. In some embodiments, a nucleic acid encoding a polypeptide is a messenger RNA. In some embodiments, an expression vector comprises or encodes an engineered guide nucleic acid. In some embodiments, the expression vector encodes the crRNA.

[239] In some embodiments, an expression vector may encode one or more of any system components, including but not limited to effector proteins, an RDDP, and/or a fusion protein thereof, guide nucleic acids, template nucleic acids as described herein. In some embodiments, an expression vector may encode 1, 2, 3, 4 or more of any system components. For example, an expression vector may encode two or more guide nucleic acids, wherein each guide nucleic acid comprises a different sequence. An expression vector may comprise the nucleic acid encoding an effector protein and a guide nucleic acid. An expression vector may encode an effector protein, a guide nucleic acid, and a template nucleic acid. In some embodiments, an expression vector comprises or encodes an effector protein, an RDDP, and/or a fusion protein thereof, a guide nucleic acid, a TS retRNA and a NTS retRNA described herein. In some embodiments, the expression vector comprises a polynucleotide encoding an effector protein that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of the sequences in TABLE 1. In some embodiments, the expression vector comprises a polynucleotide encoding an RDDP that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of the sequences in TABLEs 2-3. In some embodiments, the expression vector comprises a polynucleotide encoding a fusion protein that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of the sequences in TABLE 7. In some embodiments, the expression vector comprises a polynucleotide encoding a guide nucleic acid that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of the sequences in TABLEs 1 and 8. In some embodiments, the expression vector comprises a polynucleotide encoding a template nucleic acid that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of the sequences in TABLE 8.

[240] In some embodiments, a vector can comprise or encode one or more regulatory elements. Regulatory elements can refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate transcription of a non-coding sequence or a coding sequence and/or regulate translation of an encoded polypeptide. In some embodiments, a vector can comprise or encode for one or more additional elements, such as, for example, replication origins, antibiotic resistance (or a nucleic acid encoding the same), a tag (or a nucleic acid encoding the same), selectable markers, and the like.

[241] Vectors described herein can encode a promoter - a regulatory region on a nucleic acid, such as a DNA sequence, capable of initiating transcription of a downstream (3' direction) coding or noncoding sequence. As used herein, a promoter can be bound at its 3' terminus to a nucleic acid the expression or transcription of which is desired, and extends upstream (5' direction) to include bases or elements necessary to initiate transcription or induce expression, which could be measured at a detectable level. A promoter can comprise a nucleotide sequence, referred to herein as a “promoter sequence”. A promoter sequence can include a transcription initiation site, and one or more protein binding domains responsible for the binding of transcription machinery, such as RNA polymerase. When eukaryotic promoters are used, such promoters can contain “TATA” boxes and “CAT” boxes. Various promoters, including inducible promoters, may be used to drive expression, i.e., transcriptional activation, of the nucleic acid of interest. Accordingly, in some embodiments, the nucleic acid of interest can be operably linked to a promoter.

[242] Promotors can be any suitable type of promoter envisioned for the compositions, systems, and methods described herein. Examples include constitutively active promoters (e.g, CMV promoter), inducible promoters (e.g., heat shock promoter, tetracycline -regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc.), spatially restricted and/or temporally restricted promoters (e.g., a tissue specific promoter, a cell type specific promoter, etc.), etc. Suitable promoters include, but are not limited to: SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6), an enhanced U6 promoter, and a human Hl promoter (Hl). By transcriptional activation, it is intended that transcription will be increased above basal levels in the target cell by 10 fold, by 100 fold, or by 1000 fold, or more. In addition, vectors used for providing a nucleic acid that, when transcribed, produces a guide nucleic acid and/or a nucleic acid that encodes an effector protein to a cell may include nucleic acid sequences that encode for selectable markers in the target cells, so as to identify cells that have taken up the guide nucleic acid and/or an effector protein.

[243] In general, vectors provided herein comprise at least one promotor or a combination of promoters driving expression or transcription of one or more genome editing tools described herein. In some embodiments, the viral vector comprises a nucleotide sequence of a promoter. In some embodiments, the viral vector comprises two promoters. In some embodiments, the viral vector comprises three promoters. In some embodiments, the length of the promoter is less than about 500, less than about 400, or less than about 300 linked nucleotides. In some embodiments, the length of the promoter is at least 100 linked nucleotides. Non-limiting examples of promoters include CMV, 7SK, EFla, RPBSA, hPGK, EFS, SV40, PGK1, Ubc, human beta actin promoter, CAG, TRE, UAS, Ac5, Polyhedrin, CaMKIIa, GALI, Hl, TEF1, GDS, ADH1, CaMV35S, Ubi, U6, MNDU3, MSCV, MND and CAG.

[244] In some embodiments, the promoter is an inducible promoter that only drives expression of its corresponding gene when a signal is present, e.g. , a hormone, a small molecule, a peptide . Non-limiting examples of inducible promoters are the T7 RNA polymerase promoter, the T3 RNA polymerase promoter, the Isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter, a lactose induced promoter, a heat shock promoter, a tetracycline-regulated promoter (tetracycline-inducible or tetracycline-repressible), a steroid regulated promoter, a metal-regulated promoter, and an estrogen receptor-regulated promoter. In some embodiments, the promoter is an activation-inducible promoter, such as a CD69 promoter, as described further in Kulemzin et al., (2019), BMC Med Genomics, 12:44. In some embodiments, the promoter for expressing effector protein is a muscle-specific promoter. In some embodiments, the muscle -specific promoter comprises Ck8e, SPC5-12, or Desmin promoter sequence. In some embodiments, the promoter for expressing effector protein is a ubiquitous promoter. In some embodiments, the ubiquitous promoter comprises MND or CAG promoter sequence.

[245] In some embodiments, an effector protein (or a nucleic acid encoding same) and/or a guide nucleic acid (or a nucleic acid that, when transcribed, produces same) are co-administered with a donor nucleic acid. Coadministration can be contact with a target nucleic acid, administered to a cell, such as a host cell, or administered as method of nucleic acid detection, editing, and/or treatment as described herein, in a single vehicle, such as a single expression vector. In certain embodiments, an effector protein (or a nucleic acid encoding same) and/or a guide nucleic acid (or a nucleic acid that, when transcribed, produces same) are not co-administered with donor nucleic acid in a single vehicle. In certain embodiments, an effector protein (or a nucleic acid encoding same), a guide nucleic acid (or a nucleic acid that, when transcribed, produces same), and/or donor nucleic acid are administered in one or more or two or more vehicles, such as one or more, or two or more expression vectors.

Viral Vectors

[246] An expression vector can be a viral vector. In some embodiments, a viral vector comprises a nucleic acid to be delivered into a host cell via a recombinantly produced virus or viral particle. The nucleic acid may be single-stranded or double stranded, linear or circular, segmented or nonsegmented. The nucleic acid may comprise DNA, RNA, or a combination thereof. In some embodiments, the expression vector is an adeno-associated viral vector. There are a variety of viral vectors that are associated with various types of viruses, including but not limited to retroviruses (e.g., lentiviruses and y-retroviruses), adenoviruses, arenaviruses, alphaviruses, adeno-associated viruses (AAVs), baculoviruses, vaccinia viruses, herpes simplex viruses and poxviruses. A viral vector provided herein can be derived from or based on any such virus. Often the viral vectors provided herein are an adeno-associated viral vector (AAV vector). Generally, an AAV vector has two inverted terminal repeats (ITRs). According, in some embodiments, the viral vector provided herein comprises two inverted terminal repeats of AAV. The DNA sequence in between the ITRs of an AAV vector provided herein may be referred to herein as the sequence encoding the genome editing tools or a transgene. These genome editing tools can include, but are not limited to, an effector protein, effector protein modifications (e.g., nuclear localization signal (NLS), polyA tail), guide nucleic acid(s), respective promoter(s), and a donor nucleic acid, or combinations thereof. In some embodiments, a nuclear localization signal comprises an entity (e.g., peptide) that facilitates localization of a nucleic acid, protein, or small molecule to the nucleus, when present in a cell that contains a nuclear compartment.

[247] In general, viral vectors provided herein comprise at least one promotor or a combination of promoters driving expression or transcription of one or more genome editing tools described herein. In some embodiments, the length of the promoter is less than about 500, less than about 400, or less than about 300 linked nucleotides. In some embodiments, the length of the promoter is at least 100 linked nucleotides. Non-limiting examples of promoters include CMV, EFla, RPBSA, hPGK, EFS, SV40, PGK1, Ubc, human beta actin promoter, CAG, TRE, UAS, Ac5, Polyhedrin, CaMKIIa, GALI, Hl, TEF1, GDS, ADH1, CaMV35S, Ubi, U6, MNDU3, and MSCV. In some embodiments, the promoter is an inducible promoter that only drives expression of its corresponding gene when a signal is present, e.g., a hormone, a small molecule, a peptide. Non-limiting examples of inducible promoters are the T7 RNA polymerase promoter, the T3 RNA polymerase promoter, the Isopropyl-beta-D- thiogalactopyranoside (IPTG)-regulated promoter, a lactose induced promoter, a heat shock promoter, a tetracycline-regulated promoter (tetracycline -inducible or tetracycline-repressible), a steroid regulated promoter, a metal-regulated promoter, and an estrogen receptor-regulated promoter. In some embodiments, the promoter is an activation-inducible promoter, such as a CD69 promoter, as described further in Kulemzin et al., (2019), BMC Med Genomics, 12:44.

[248] In some embodiments, the coding region of the AAV vector forms an intramolecular doublestranded DNA template thereby generating an AAV vector that is a self-complementary AAV (scAAV) vector. In general, the sequence encoding the genome editing tools of an scAAV vector has a length of about 2 kb to about 3 kb. The scAAV vector can comprise nucleotide sequences encoding an effector protein, providing guide nucleic acids described herein, and a donor nucleic acid described herein. In some embodiments, the AAV vector provided herein is a self-inactivating AAV vector.

[249] In some embodiments, an AAV vector provided herein comprises a modification, such as an insertion, deletion, chemical alteration, or synthetic modification, relative to a wild-type AAV vector. [250] In some embodiments, the viral particle that delivers the viral vector described herein is an AAV. AAVs are characterized by their serotype. Non-limiting examples of AAV serotypes are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, scAAV, AAV-rhlO, chimeric or hybrid AAV, or any combination, derivative, or variant thereof.

Producing AA V Particles

[251] The AAV particles described herein can be referred to as recombinant AAV (rAAV). Often, rAAV particles are generated by transfecting AAV producing cells with an AAV-containing plasmid carrying the sequence encoding the genome editing tools, a plasmid that carries viral encoding regions, i.e., Rep and Cap gene regions; and a plasmid that provides the helper genes such as E1A, E1B, E2A, E4ORF6 and VA. In some embodiments, the AAV producing cells are mammalian cells. In some embodiments, host cells for rAAV viral particle production are mammalian cells. In some embodiments, a mammalian cell for rAAV viral particle production is a COS cell, a HEK293T cell, a HeLa cell, a KB cell, a derivative thereof, or a combination thereof. In some embodiments, rAAV virus particles can be produced in the mammalian cell culture system by providing the rAAV plasmid to the mammalian cell. In some embodiments, producing rAAV virus particles in a mammalian cell can comprise transfecting vectors that express the rep protein, the capsid protein, and the gene-of- interest expression construct flanked by the ITR sequence on the 5’ and 3’ ends. Methods of such processes are provided in, for example, Naso et al., BioDrugs, 2017 Aug;31 (4): 317-334 and Benskey et al., (2019), Methods Mol Biol., 1937:3-26, each of which is incorporated by reference in their entireties.

[252] In some embodiments, rAAV is produced in a non-mammalian cell. In some embodiments, rAAV is produced in an insect cell. In some embodiments, an insect cell for producing rAAV viral particles comprises a Sf9 cell. In some embodiments, production of rAAV virus particles in insect cells can comprise baculovirus. In some embodiments, production of rAAV virus particles in insect cells can comprise infecting the insect cells with three recombinant baculoviruses, one carrying the cap gene, one carrying the rep gene, and one carrying the gene-of-interest expression construct enclosed by an ITR on both the 5’ and 3’ end. In some embodiments, rAAV virus particles are produced by the One Bac system. In some embodiments, rAAV virus particles can be produced by the Two Bac system. In some embodiments, in the Two Bac system, the rep gene and the cap gene of the AAV is integrated into one baculovirus virus genome, and the ITR sequence and the gene-of-interest expression construct is integrated into another baculovirus virus genome. In some embodiments, in the One Bac system, an insect cell line that expresses both the rep protein and the capsid protein is established and infected with a baculovirus virus integrated with the ITR sequence and the gene-of-interest expression construct. Details of such processes are provided in, for example, Smith et. al., (1983), Mol. Cell. Biol., 3(12):2156-65; Urabe et al., (2002), Hum. Gene. Then, 1;13(16): 1935-43; and Benskey etal., (2019), Methods Mol Biol., 1937:3-26, each of which is incorporated by reference in its entirety. Lipid particles

[253] In some embodiments, compositions and systems provided herein comprise a lipid particle. In some embodiments, a lipid particle is a lipid nanoparticle (LNP). In some embodiments, a lipid or a lipid nanoparticle can encapsulate an expression vector. In some embodiments, a lipid or a lipid nanoparticle can encapsulate the effector protein, the sgRNA or crRNA, the nucleic acid encoding the effector protein and/or the DNA molecule encoding the sgRNA or crRNA. LNPs are effective for delivery of nucleic acids. Beneficial properties of LNP include ease of manufacture, low cytotoxicity and immunogenicity, high efficiency of nucleic acid encapsulation and cell transfection, multi-dosing capabilities and flexibility of design (Kulkami et al., (2018) Nucleic Acid Therapeutics, 28(3): 146- 157). In some embodiments, a method can comprise contacting a cell with an expression vector. In some embodiments, contacting can comprise electroporation, lipofection, or lipid nanoparticle (LNP) delivery of an expression vector.

Pharmaceutical Compositions and Modes of Administration

[254] Disclosed herein, in some aspects, are pharmaceutical compositions for modifying a target nucleic acid in a cell or a subject, comprising any one of the effector proteins, engineered effector proteins, fusion effector proteins, or guide nucleic acids as described herein and any combination thereof. Also disclosed herein, in some aspects, are pharmaceutical compositions comprising a nucleic acid encoding any one of the effector proteins, engineered effector proteins, fusion effector proteins, or guide nucleic acids as described herein and any combination thereof. In some embodiments, pharmaceutical compositions comprise a plurality of guide nucleic acids. Pharmaceutical compositions may be used to modify a target nucleic acid or the expression thereof in a cell in vitro, in vivo or ex vivo.

[255] In some embodiments, pharmaceutical compositions comprise one or more nucleic acids encoding an effector protein, fusion effector protein, fusion partner, a guide nucleic acid, or a combination thereof; and a pharmaceutically acceptable carrier or diluent. The effector protein, fusion effector protein, fusion partner protein, or combination thereof may be any one of those described herein. The one or more nucleic acids may comprise a plasmid. The one or more nucleic acids may comprise a nucleic acid expression vector. The one or more nucleic acids may comprise a viral vectol. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the vector is an adeno-associated viral (AAV) vector. In some embodiments, compositions, including pharmaceutical compositions, comprise a viral vector encoding a fusion effector protein and a guide nucleic acid, wherein at least a portion of the guide nucleic acid binds to the effector protein of the fusion effector protein.

[256] In some embodiments, pharmaceutical compositions comprise a virus comprising a viral vector encoding a fusion effector protein, an effector protein, a fusion partner, a guide nucleic acid, or a combination thereof; and a pharmaceutically acceptable carrier or diluent. The virus may be a lenti virus. The virus may be an adenovirus. The virus may be a non-replicating virus. The virus may be an adeno-associated virus (AAV). The viral vector may be a retroviral vector. Retroviral vectors may include gamma-retroviral vectors such as vectors derived from the Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV) or the Murine Stem cell Virus (MSCV) genome. Retroviral vectors may include lentiviral vectors such as those derived from the human immunodeficiency virus (HIV) genome. In some embodiments, the viral vector is a chimeric viral vector, comprising viral portions from two or more viruses. In some embodiments, the viral vector is a recombinant viral vector.

[257] In some embodiments, the viral vector is an AAV. The AAV may be any AAV known in the art. In some embodiments, the viral vector corresponds to a virus of a specific serotype. In some examples, the serotype is selected from an AAV1 serotype, an AAV2 serotype, AAV3 serotype, an AAV4 serotype, AAV5 serotype, an AAV6 serotype, AAV7 serotype, an AAV8 serotype, an AAV9 serotype, an AAV10 serotype, an AAV11 serotype, and an AAV12 serotype. In some embodiments the AAV vector is a recombinant vector, a hybrid AAV vector, a chimeric AAV vector, a self- complementary AAV (scAAV) vector, a single -stranded AAV or any combination thereof. scAAV genomes are generally known in the art and contain both DNA strands which can anneal together to form double -stranded DNA.

[258] In some embodiments, methods of producing delivery vectors herein comprise packaging a nucleic acid, or transgene, encoding an effector protein and a nucleic acid that, when transcribed, produces a guide nucleic acid, or a combination thereof, into an AAV vector. In some embodiments, methods of producing the delivery vector comprises, (a) contacting a cell with at least one nucleic acid, or transgene that, when transcribed, produces a guide nucleic acid; at least one nucleic acid that encodes: (i) a Replication (Rep) gene; and (ii) a Capsid (Cap) gene that encodes an AAV capsid protein; (b) expressing the AAV capsid protein in the cell; (c) assembling an AAV particle; and (d) packaging a Cas effector encoding nucleic acid into the AAV particle, thereby generating an AAV delivery vector. In some embodiments, promoters, stuffer sequences, and any combination thereof may be packaged in the AAV vector. In some embodiments, the AAV vector comprises a sequence encoding a guide nucleic acid. In some embodiments, the guide nucleic acid comprises a crRNA. In some embodiments, the guide nucleic acid is a crRNA. In some embodiments, the guide nucleic acid comprises a sgRNA. In some embodiments, the guide nucleic acid is a sgRNA. In some examples, the AAV vector can package 1, 2, 3, 4, or 5 nucleotide sequences encoding guide nucleic acids or copies thereof. In some examples, the AAV vector packages 1 or 2 nucleotide sequences encoding guide nucleic acids or copies thereof. In some embodiments, the AAV vector packages a nucleotide sequence encoding a first guide nucleic acid and a nucleotide sequence encoding a second guide nucleic acid, wherein the first guide nucleic acid and the second guide nucleic acid are the same. In some embodiments, the AAV vector packages a nucleotide sequence encoding a first guide nucleic acid and a nucleotide sequence encoding a second guide nucleic acid, wherein the first guide nucleic acid and the second guide nucleic acid are different. In some embodiments, the AAV vector comprises inverted terminal repeats, e.g., a 5’ inverted terminal repeat and a 3’ inverted terminal repeat. In some embodiments, the inverted terminal repeat comprises inverted terminal repeats from AAV. In some embodiments, the inverted terminal repeat comprises inverted terminal repeats of ssAAV vector or scAAV vector. In some embodiments, the AAV vector comprises a mutated inverted terminal repeat that lacks a terminal resolution site.

[259] In some embodiments, a hybrid AAV vector is produced by transcapsidation, e.g., packaging an inverted terminal repeat (ITR) from a first serotype into a capsid of a second serotype, wherein the first and second serotypes may be not the same. In some examples, the Rep gene and ITR from a first AAV serotype (e.g., AAV2) may be used in a capsid from a second AAV serotype (e.g., AAV9), wherein the first and second AAV serotypes may be not the same . As a non-limiting example, a hybrid AAV serotype comprising the AAV2 ITRs and AAV9 capsid protein may be indicated AAV2/9. In some examples, the hybrid AAV delivery vector comprises an AAV2/1, AAV2/2, AAV 2/4, AAV2/5, AAV2/8, or AAV2/9 vector.

[260] In some embodiments, the AAV vector may be a chimeric AAV vector. In some embodiments, the chimeric AAV vector comprises an exogenous amino acid or an amino acid substitution, or capsid proteins from two or more serotypes. In some examples, a chimeric AAV vector may be genetically engineered to increase transduction efficiency, selectivity, or a combination thereof.

[261] In some examples, the delivery vector may be a eukaryotic vector, a prokaryotic vector (e.g., a bacterial vector) a viral vector, or any combination thereof. In some embodiments, the delivery vehicle may be a non-viral vector. In some embodiments, the delivery vehicle may be a plasmid. In some embodiments, the plasmid comprises DNA. In some embodiments, the plasmid comprises RNA. In some examples, the plasmid comprises circular double-stranded DNA. In some examples, the plasmid may be linear. In some examples, the plasmid comprises one or more genes of interest and one or more regulatory elements. In some examples, the plasmid comprises a bacterial backbone containing an origin of replication and an antibiotic resistance gene or other selectable marker for plasmid amplification in bacteria. In some examples, the plasmid may be a minicircle plasmid. In some examples, the plasmid contains one or more genes that provide a selective marker to induce a target cell to retain the plasmid. In some examples, the plasmid may be formulated for delivery through injection by a needle carrying syringe. In some examples, the plasmid may be formulated for delivery via electroporation. In some examples, the plasmids may be engineered through synthetic or other suitable means known in the art. For example, in some embodiments, the genetic elements may be assembled by restriction digest of the desired genetic sequence from a donor plasmid or organism to produce ends of the DNA which may then be readily ligated to another genetic sequence.

[262] In some embodiments, the vector is a non-viral vector, and a physical method or a chemical method is employed for delivery into the somatic cell. Exemplary physical methods include electroporation, gene gun, sonoporation, magnetofection, or hydrodynamic delivery. Exemplary chemical methods include delivery of the recombinant polynucleotide via liposomes such as, cationic lipids or neutral lipids; dendrimers; nanoparticles; or cell-penetrating peptides.

[263] In some embodiments, a fusion effector protein as described herein is inserted into a vector. In some embodiments, the vector comprises a transgene which comprises a nucleotide sequence of one or more promoters, enhancers, ribosome binding sites, RNA splice sites, polyadenylation sites, a replication origin, and/or transcriptional terminator sequences.

[264] In some embodiments, the AAV vector comprises a self-processing array system for guide nucleic acid. Such a self-processing array system refers to a system for multiplexing, stringing together multiple guide nucleic acids under the control of a single promoter. In general, plasmids and vectors described herein comprise at least one promoter. In some embodiments, the promoters are constitutive promoters. In other embodiments, the promoters are inducible promoters. In additional embodiments, the promoters are prokaryotic promoters (e.g., drive expression of a gene in a prokaryotic cell). In some embodiments, the promoters are eukaryotic promoters, (e.g., drive expression of a gene in a eukaryotic cell). Exemplary promoters include, but are not limited to, CMV, EFla, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, polyhedron, CaMKIIa, GALI-10, TEF1, GDS, ADH1, CaMV35S, Ubi, Hl, U6, CaMV35S, SV40, CMV, 7SK, and HSV TK promoter. In some embodiments, the promoter is CMV. In some embodiments, the promoter is EFla. In some embodiments, the promoter is U6. In some embodiments, the promote is Hl. In some embodiments, the promoter is 7SK. In some embodiments, the promoter is ubiquitin. In some embodiments, vectors are bicistronic or polycistronic vector (e.g., having or involving two or more loci responsible for generating a protein) having an internal ribosome entry site (IRES) is for translation initiation in a capindependent manner.

[265] In some embodiments, the AAV vector comprises a promoter for expressing effector proteins. In some embodiments, the promoter for expressing effector protein is a site-specific promoter. In some embodiments, the promoter for expressing effector protein is a muscle-specific promoter. In some embodiments, the muscle-specific promoter comprises Ck8e, SPC5-12, or Desmin promoter sequence. In some embodiments, the promoter for expressing effector protein is a ubiquitous promoter. In some embodiments, the ubiquitous promoter comprises MND or CAG promoter sequence.

[266] In some embodiments, the AAV vector comprises a stuffer sequence. A stuffer sequence can refer to a non-coding sequence of nucleotides that adjusts the length of the viral genome when inserted into a vector to increase packaging efficiency, increase overall viral titer during production, increase transfection efficacy, increase transfection efficiency, and/or decrease vector toxicity. In some embodiments, the stuffer sequence comprises 5’ untranslated region, 3’ untranslated region or combination thereof. In some embodiments, a stuffer sequence serves no other functional purpose than to increase the length of the viral genome. In some embodiments, a stuffer sequence may increase the length of the viral genome as well as have other functional elements [267] In some embodiments, the 3 ’-untranslated region comprises a nucleotide sequence of an intron. In some embodiments, the 3 ’-untranslated region comprises one or more sequence elements, such as an intron sequence or an enhancer sequence. In some embodiments, the 3 '-untranslated region comprises an enhancer. In some embodiments, vectors comprise an enhancer. Enhancers are nucleotide sequences that have the effect of enhancing promoter activity. In some embodiments, enhancers augment transcription regardless of the orientation of their sequence . In some embodiments, enhancers activate transcription from a distance of several kilo basepairs. Furthermore, enhancers are located optionally upstream or downstream of a gene region to be transcribed, and/or located within the gene, to activate the transcription. Exemplary enhancers include, but are not limited to, WPRE; CMV enhancers; the R-U5' segment in LTRofHTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; the intron sequence between exons 2 and 3 of rabbit P-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981); and the genome region of human growth hormone (J Immunol., Vol. 155(3), p. 1286-95, 1995). In some embodiments, the enhancer is WPRE.

[268] In some embodiments, the AAV vector comprises one or more polyadenylation (poly A) signal sequences. In some embodiments, the polyadenlyation signal sequence comprises hGH poly A signal sequence. In some embodiments, the polyadenlyation signal sequence comprises sv40 poly A signal sequence.

[269] Pharmaceutical compositions described herein may comprise a salt. In some embodiments, the salt is a sodium salt. In some embodiments, the salt is a potassium salt. In some embodiments, the salt is a magnesium salt. In some embodiments, the salt is NaCl. In some embodiments, the salt is KNOs. In some embodiments, the salt is Mg²⁺ SOy².

[270] Non-limiting examples of pharmaceutically acceptable carriers and diluents suitable for the pharmaceutical compositions disclosed herein include buffers (e.g., neutral buffered saline, phosphate buffered saline); carbohydrates (e.g., glucose, mannose, sucrose, dextran, mannitol); polypeptides or amino acids (e.g., glycine); antioxidants; chelating agents (e.g., EDTA, glutathione); adjuvants (e.g., aluminum hydroxide); surfactants (Polysorbate 80, Polysorbate 20, or Pluronic F68); glycerol; sorbitol; mannitol; polyethyleneglycol; and preservatives.

[271] In some embodiments, pharmaceutical compositions are in the form of a solution (e.g. , a liquid) . The solution may be formulated for injection, e.g., intravenous or subcutaneous injection. In some embodiments, the pH of the solution is about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9. In some embodiments, the pH is 7 to 7.5, 7.5 to 8, 8 to 8.5, 8.5 to 9, or 7 to 8.5. In some embodiments, the pH of the solution is less than 7. In some embodiments, the pH is greater than 7.

[272] In some embodiments, pharmaceutical compositions comprise an: effector protein, fusion effector protein, fusion partner, a guide nucleic acid, or a combination thereof; and a pharmaceutically acceptable carrier or diluent. In some embodiments, pharmaceutical compositions comprise one or more nucleic acids encoding an: effector protein, fusion effector protein, fusion partner, a guide nucleic acid, or a combination thereof; and a pharmaceutically acceptable carrier or diluent.

[273] In some embodiments, guide nucleic acid can be a plurality of guide nucleic acids. In some embodiments, the effector protein comprises a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of the effector sequences of TABLE 1. In some embodiments, the guide nucleic acid comprises a nucleotide sequence of any one of the guide sequences of TABLE 1 or any combination thereof. In some embodiments, the PAM nucleic acid comprises a nucleotide sequence of any one of the PAM sequences of TABLE 1 or any combination thereof.

[274] In some embodiments, the methods of this invention include using an inhibitor of DNA synthesis. In some embodiments the inhibitor is an inhibitor of replication fork regression. In some embodiments the inhibitor is an inhibitor of single strand break repair. In some embodiments the inhibitor is an inhibitor of double-strand break repair. In some embodiments the inhibitor is an inhibitor of non-homologous enjoining. In some embodiments the inhibitor is an inhibitor of RAD51. In some embodiments the inhibitor is AZD7648.

Methods of Editing

[275] Methods of editing described herein may be employed to generate a genetically modified cell. The cell may be a eukaryotic cell (e.g. , a mammalian cell) or a prokaryotic cell (e.g. , an archaeal cell). The cell may be derived from a multicellular organism and cultured as a unicellular entity. The cell may comprise a heritable genetic modification, such that progeny cells derived therefrom comprise the heritable genetic mutation. The cell may be progeny of a genetically modified cell comprising a genetic modification of the genetically modified parent cell. A genetically modified cell may comprise a deletion, insertion, mutation, or non-native sequence relative to a wild-type version of the cell or the organism from which the cell was derived.

[276] Methods may comprise contacting a cell or a subject with a fusion protein, effector protein, or partner protein disclosed herein, or any combination thereof. Methods may comprise contacting a cell with a nucleic acid encoding the fusion protein, effector protein, or partner protein, or combination thereof. The nucleic acid may be an expression vector. The nucleic acid may comprise a messenger RNA. Methods may comprise contacting a cell or a subject with an extended guide RNA or a portion thereof. Methods may comprise contacting a cell or a subject with a nucleic acid (e.g. , DNA) encoding the extended guide RNA, or a portion thereof.

[277] In some embodiments, the present disclosure provides a mammalian cell comprising a system described herein, e.g. , an RDDP described herein (or a fusion protein comprising the same), an effector protein described herein (or a fusion protein comprising the same), and/or an rtgRNA. In some embodiments, the present disclosure provides a mammalian cell comprising one or more polynucleotides encoding one or more components of a system described herein, or one or more vectors comprising the same, e.g., a polynucleotide encoding an RDDP described herein (or a fusion protein comprising the same) or vector comprising the same, a polynucleotide encoding an effector protein described herein (or a fusion protein comprising the same) or a vector comprising the same, and/or a polynucleotide encoding an rtgRNA or a vector comprising the same.

[278] Methods of the disclosure may be performed in a subject. Compositions of the disclosure may be administered to a subject. A subject may be a human. A subject may be a mammal (e.g. , rat, mouse, cow, dog, pig, sheep, horse). A subject may be a vertebrate or an invertebrate. A subject may be a laboratory animal. A subject may be a patient. A subject may be at risk of developing, suffering from, or displaying symptoms a disease or disorder as set forth in herein. The subject may have a mutation associated with a gene described herein. The subject may display symptoms associated with a mutation of a gene described herein. In some embodiments, a mutation comprises a point mutation or single nucleotide polymorphism (SNP), a chromosomal mutation, a copy number mutation, or any combination thereof. A point mutation optionally comprises a substitution, insertion, or deletion. In some embodiments, a mutation comprises a chromosomal mutation. A chromosomal mutation can comprise an inversion, a deletion, a duplication, or a translocation. In some embodiments, a mutation comprises a copy number variation. A copy number variation can comprise a gene amplification or an expanding trinucleotide repeat. In some embodiments, mutations may be as described herein.

[279] Methods of the disclosure may be performed in a cell. A cell may be in vitro. A cell may be in vivo. A cell may be ex vivo. A cell may be an isolated cell. A cell may be a cell inside of an organism. A cell may be an organism. A cell may be a cell in a cell culture. A cell may be one of a collection of cells. A cell may be a mammalian cell or derived from a mammalian cell. A cell may be a rodent cell or derived from a rodent cell. A cell may be a human cell or derived from a human cell. A cell may be a eukaryotic cell or derived from a eukaryotic cell. A cell may be a pluripotent stem cell. A cell may be a plant cell or derived from a plant cell. A cell may be an animal cell or derived from an animal cell. A cell may be an invertebrate cell or derived from an invertebrate cell. A cell may be a vertebrate cell or derived from a vertebrate cell.

[280] A cell may be from a specific organ or tissue. Non-limiting examples of organs and tissues from which a cell may be obtained or in which a cell may be located include: muscle, adipose, bone, adrenal gland, pituitary gland, thyroid gland, pancreas, testes, ovaries, uterus, heart, lung, aorta, smooth vasculature, endometrium, brain, neurons, spinal cord, kidney, liver, esophagus, stomach, intestine, colon, bladder, and spleen.

[281] The tissue may be the subject’s blood, bone marrow, or cord blood. The tissue may be heterologous donor blood, cord blood, or bone marrow. The tissue may be allogenic blood, cord blood, or bone marrow. In some embodiments, the cell is a stem cell. Non-limiting examples of stem cells are hematopoietic stem cells, muscle stem cells (also referred to as myoblasts or muscle progenitor cells), and pluripotent stem cells (including induced pluripotent stem cells). In some embodiments, the cell is cell derived or differentiated from a pluripotent stem cell. In some embodiments, the cell is a hepatocyte.

[282] In some embodiments, the cell is an immune cell. Non-limiting examples of immune cells are lymphocytes (T cells, B cells, and NK cells), neutrophils, and monocytes/ macrophages.

Methods of Treating a Disease

[283] Compositions, systems and methods disclosed herein may be useful for treating a disease in a subject by modifying a target nucleic acid associated with a gene or expression of a gene related to the disease. In some embodiments, methods comprise administering a composition or cell described herein to a subject. By way of non-limiting example, the disease may be a cancer, an ophthalmological disorder, a neurological disorder, a neurodegenerative disease, a blood disorder, or a metabolic disorder, or a combination thereof. The disease may be an inherited disorder, also referred to as a genetic disorder. The disease may be the result of an infection or associated with an infection. Unless otherwise specified, the term, “disease,” includes disorders and syndromes.

[284] In some embodiments, methods of treating a disease in a subject comprise administering the components of a system described herein. Components of a system may be administered sequentially. Components of a system may be administered simultaneously. Components of a system may be administered in any order. In some embodiments, methods of treating a disease in a subject comprise administering a composition described herein. In some embodiments, methods of treating a disease in a subject comprise administering a pharmaceutical composition described herein. In some embodiments, methods of treating a disease in a subject comprise administering an expression vector (e.g., an AAV vector) described herein.

[285] Systems, compositions and methods described herein may be used to treat, prevent, or inhibit a disease or syndrome in a subject. In some embodiments, the disease is a liver disease, a lung disease, an eye disease, or a muscle disease. Exemplary diseases and syndromes include, but are not limited to, the diseases and syndromes listed in TABLE 5.

[286] In some embodiments, systems, compositions and methods modify at least one gene associated with the disease or the expression thereof. In some embodiments, the disease is Alzheimer’s disease, and the gene is selected from APT, BACE-1, PSD95, MAPT, PSENI, PSEN2, APOE, TARDBP, and APOEe4. In some embodiments, the disease is dementia and the gene is TARDBP. In some embodiments, the disease is Pick’s disease and the gene is TARDBP. In some embodiments, the disease is Parkinson’s disease, and the gene is selected from SNCA, GDNF, wA I.RRK2. In some embodiments, the disease comprises Centronuclear myopathy, and the gene is DNM2. In some embodiments, the disease is Huntington's disease, and the gene is HTT. In some embodiments, the disease is Alpha-1 antitrypsin deficiency (AATD) and the gene is SERPINA1. In some embodiments, the disease is amyotrophic lateral sclerosis (ALS), and the gene is selected from SOD1, FUS, C9ORF72, ATXN2, TARDBP, and CHCHD10. In some embodiments, the disease comprises Alexander Disease and the gene is GFAP. In some embodiments, the disease comprises anaplastic large cell lymphoma and the gene is CD30. In some embodiments, the disease comprises Angelman Syndrome and the gene is UBE3A. In some embodiments, the disease comprises calcific aortic stenosis and the gene is Apo(a). In some embodiments, the disease comprises CD3Z-associated primary T-cell immunodeficiency and the gene is CD3Z or CD247. In some embodiments, the disease comprises CD 18 deficiency, and the gene is ITGB2. In some embodiments, the disease comprises CD40L deficiency, and the gene is CD40L. In some embodiments, the disease comprises CNS trauma and the gene is VEGF. In some embodiments, the disease comprises coronary heart disease and the gene is selected from FGA, FGB, and FGG. In some embodiments, the disease comprises MECP2 Duplication syndrome and Rett syndrome and the gene is MECP2. In some embodiments, the disease comprises a bleeding disorder (coagulation) and the gene is FXI. In some embodiments, the disease comprises fragile X syndrome and the gene is FMRI . In some embodiments, the disease comprises Fuchs comeal dystrophy and the gene is selected from ZEB1, SLC4A11, and L0XHD1. In some embodiments, the disease comprises GM2 -Gangliosidoses (e.g., Tay Sachs Disease, Sandhoff disease) and the gene is selected from HEXA and HEXB. In some embodiments, the disease comprises Hearing loss disorders and the gene is DFNA36. In some embodiments, the disease is Pompe disease, including infantile onset Pompe disease (IOPD) and late onset Pompe disease (LOPD) and the gene is GAA. In some embodiments, the disease is Retinitis pigmentosa and the gene is selected from PDE6B, RHO, RP1, RP2, RPGR, PRPH2, IMPDH1, PRPF31, CRB1, PRPF8, TULP1, CA4, HPRPF3, ABCA4, EYS, CERKL, FSCN2, TOPORS, SNRNP200, PRCD, NR2E3, MERTK, USH2A, PR0M1, KLHL7, CNGBI, TTC8, ARL6, DHDDS, BEST1, LRAT, SPARA7, CRX, CLRN1, RPE65, and WDR19. In some embodiments, the disease comprises Leber Congenital Amaurosis Type 10 and the gene is CEP290. In some embodiments, the disease is cardiovascular disease and/or lipodystrophies and the gene is selected from XBCG5, ABCG8, AGP, ANGPTL3, APOCHI, APOE, APOA1, APOL1, ARH, CDKN2B, CFB, CXCL12, FXI, FXII GATA-4, MIA3, MKL2, MTHFD1L, MYH7, NKX2-5, NOTCH1, PKK, PCSK9, PSRCI, SMAD3, and TTR. In some embodiments, the disease comprises acromegaly and the gene is GHR. In some embodiments, the disease comprises acute myeloid leukemia and the gene is CD22. In some embodiments, the disease is diabetes, and the gene is selected from GCGR and ANGPTL7. In some embodiments, the disease is NAFLD/NASH, and the gene is selected from DGAT2 and PNPLA3. In some embodiments, the disease is cancer, and the gene is selected from STATS, YAP1, FOXP3, AR (Prostate cancer), mA IRI '4 (multiple myeloma). In some embodiments, the disease is cystic fibrosis, and the gene is CFTR. In some embodiments, the disease is facioscapulohumeral muscular dystrophy (FSHD) and the gene is DUX4. In some embodiments, the disease comprises angioedema and the gene is PKK. In some embodiments, the disease comprises thalassemia and the gene is TMPRSS6. In some embodiments, the disease comprises achondroplasia and the gene is FGFR3. In some embodiments, the disease comprises Cri du chat syndrome and the gene is CTNND2. In some embodiments, the disease comprises sickle cell anemia and the gene is Beta globin (HBB) gene. In some embodiments, the disease comprises Alagille Syndrome and the gene is selected from JAG1 and N0TCH2. In some embodiments, the disease comprises Charcot Marie Tooth disease and the gene is selected from PMP22 andMFN2. In some embodiments, the disease comprises Crouzon syndrome and the gene is selected from YGFR2, FGFR3, and FGFR3. In some embodiments, the disease comprises Dravet Syndrome and the gene is selected from SCN1A and SCN2A. In some embodiments, the disease comprises Emery-Dreifuss syndrome, and the gene is selected from EMD, LMNA, SYNE1, SYNE2, FHL1, and TMEM43. In some embodiments, the disease comprises Factor V Leiden thrombophilia and the gene is F5. In some embodiments, the disease comprises Fanconi anemia, and the gene is selected from FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, FANCN, FANCP, FANCS, RAD51C, and XPF. In some embodiments, the disease comprises Familial Creutzfeld-Jakob disease and the gene is PRNP. In some embodiments, the disease comprises Familial Mediterranean Fever and the gene is MEFV. In some embodiments, the di’ease comprises Friedreich's ataxia and the gene is FXN. In some embodiments, the disease comprises Gaucher disease, and the gene is GBA. In some embodiments, the disease comprises human papilloma virus (HPV) infection and the gene is HPV E7. In some embodiments, the disease comprises hemochromatosis and the gene is HFE, optionally comprising a C282Y mutation. In some embodiments, the disease comprises Hemophilia A and the gene is FVIII. In some embodiments, the disease comprises histiocytosis and the gene is CD1. In some embodiments, the disease comprises immunodeficiency 17 and the gene is CD3D. In some embodiments, the disease comprises immunodeficiency 13 and the gene is CD4. In some embodiments, the disease comprises Common Variable Immunodeficiency and the gene is selected from CD19 and CD81. In some embodiments, the disease comprises Joubert syndrome, and the gene is selected from INPP5E, TMEM216, AHI1, NPHP1, CEP 290, TMEM67, RPGRIP1L, ARL13B, CC2D2A, 0FD1, TMEM138, TCTN3, ZNF423, wA AMRC . In some embodiments, the disease comprises leukocyte adhesion deficiency and the gene is CD18. In some embodiments, the disease comprises Li-Fraumeni syndrome and the gene is TP53. In some embodiments, the disease comprises lymphoproliferative syndrome and the gene is CD27. In some embodiments, the disease comprises Lynch syndrome and the gene is selected from MSH2, MLH1, MSH6, PMS2, PMS1, TGFBR2, and MLH3. In some embodiments, the disease comprises mantle cell lymphoma and the gene is CD5. In some embodiments, the disease comprises Marfan syndrome and the gene is FBN1. In some embodiments, the disease comprises mastocytosis and the gene is CD2. In some embodiments, the disease comprises methylmalonic acidemia and the gene is selected from MMAA, MMAB, and MUT. In some embodiments, the disease is mycosis fungoides and the gene is CD7. In some embodiments, the disease comprises neurofibromatosis and the gene is selected from NF1 and NF2. In some embodiments, the disease comprises osteogenesis imperfecta and the gene is selected from COL1A1, COL1A2, and IFITM5. In some embodiments, the disease is nonsmall cell lung cancer, and the gene is selected from KRAS, EGFR, ALK, METexl4, BRAF V600E, R0S1, RET, mA NTRK. In some embodiments, the disease comprises Peutz-Jeghers syndrome and the gene is STK11. In some embodiments, the disease comprises polycystic kidney disease and the gene is selected from PKD1 and PKD2. In some embodiments, the disease comprises Severe Combined Immune Deficiency and the gene is selected from IL7R RAG1, and JAK3. In some embodiments, the disease comprises PRKAG2 cardiac syndrome, and the gene is PRKAG2. In some embodiments, the disease comprises spinocerebellar ataxia and the gene is selected from ATXN1, ATXN2, ATXN3, PLEKHG4, SPTBN2, CACNA1A, ATXN7, ATXN8OS, ATXN10, TTBK2, PPP2R2B, KCNC3, PRKCG, ITPR1, TBP, KCND3, and FGF14. In some embodiments, the disease comprises Usher Syndrome and the gene is selected from MY07A, USH1C, CDH23, PCDH15, USH1G, USH2A, GPR98, DFNB31, and CLRN1. In some embodiments, the disease comprises von Willebrand disease, and the gene is VWF In some embodiments, the disease comprises Waardenburg syndrome, and the gene is selected from PAX3, MITF, WS2B, WS2C, SNAI2, EDNRB, EDN3, and SOXIO. In some embodiments, the disease comprises Wiskott-Aldrich Syndrome and the gene is WAS. In some embodiments, the disease comprises von Hippel-Lindau disease and the gene is VHP. In some embodiments, the disease comprises Wilson disease and the gene is ATP7B. In some embodiments, the disease comprises Zellweger syndrome and the gene is selected from PEX1, PEX2, PEX3, PEX5, PEX6, PEX10, PEX12, PEX13, PEX14, PEX16, PEX19, and PEX26. In some embodiments, the disease comprises infantile myofibromatosis and the gene is CD34. In some embodiments, the disease comprises platelet glycoprotein IV deficiency and the gene is CD 36. In some embodiments, the disease comprises immunodeficiency with hyper-IgM type 3 and the gene is CD40. In some embodiments, the disease comprises hemolytic uremic syndrome and the gene is CD46.

[287] In some embodiments, the disease involves complement hyperactivation, angiopathic thrombosis, or protein-losing enteropathy and the gene is CD55. In some embodiments, the disease comprises hemolytic anemia and the gene is CD59. In some embodiments, the disease comprises calcification of joints and arteries and the gene is CD73. In some embodiments, the disease comprises immunoglobulin alpha deficiency and the gene is C D79 A. In some embodiments, the disease comprises C syndrome and the gene is CD96. In some embodiments, the disease comprises hairy cell leukemia and the gene is CD 123. In some embodiments, the disease comprises histiocytic sarcoma and the gene is CD163. In some embodiments, the disease comprises autosomal dominant deafness and the gene is CD 164. In some embodiments, the disease comprises immunodeficiency 25 and the gene is CD247. In some embodiments, the disease comprises methymalonic acidemia due to transcobalamin receptor defect and the gene is CD320.

[288] In some embodiments, the disease is pain and the gene is NAVI. 7. In some embodiments, the disease is glaucoma and the gene is selected from MYOC and ANGPTL7. In some embodiments, the disease is Tuberous Sclerosis or Subependymal Glioma and the gene is RPTOR. In some embodiments, the disease is Pitt-Hopkins Syndrome and the gene is TCF4. Cancer

[289] In some embodiments, the disease is cancer. In some embodiments, the cancer is a solid cancer (i.e., a tumor). In some embodiments, the cancer is selected from a blood cell cancer, a leukemia, and a lymphoma. The cancer can be a leukemia, such as, by way of non-limiting example, acute myeloid (or myelogenous) leukemia (AML), chronic myeloid (or myelogenous) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), and chronic lymphocytic leukemia (CLL). In some embodiments, the cancer is any one of colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, bladder cancer, cancer of the kidney or ureter, lung cancer, non-small cell lung cancer, cancer of the small intestine, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, brain cancer (e.g., glioblastoma), cancer of the head or neck, melanoma, uterine cancer, ovarian cancer, breast cancer, testicular cancer, cervical cancer, stomach cancer, Hodgkin's Disease, non-Hodgkin's lymphoma, and thyroid cancer.

[290] In some embodiments, mutations are associated with cancer or are causative of cancer. The target nucleic acid, in some embodiments, comprises a portion of a gene comprising a mutation associated with a disease, such as cancer, a gene whose overexpression is associated with cancer, a tumor suppressor gene, an oncogene, a checkpoint inhibitor gene, a gene associated with cellular growth, a gene associated with cellular metabolism, a gene associated with cell cycle, or a combination thereof. Non-limiting examples of genes comprising a mutation associated with a disease such as cancer are ABL, ACE, AF4/HRX, AKT-2, ALK, ALK/NPM, AML1, AML1/MTG8, APC, ATM, AXIN2, AXL, BAP1, BARD1, BCL-2, BCL-3, BCL- 6, BCR/ABL, BUM, BMPR1A, BRCA1, BRCA2, BRIP1, c- MYC, CASR, CCR5, CDC73, CDH1, CDK4, CDKN1B, CDKN1C, CDKN2A, CEBPA, CHEK2, CREBBP, CTNNA1, DBL, DEK/CAN, DICER1, DIS3L2, E2A/PBX1, EGER, ENL/HRX, EPCAM, ERG/TLS, ERBB, ERBB-2, ETS-1, EWS/FU-1, FH, FKRP, FLCN, FMS, FOS, FPS, GATA2, GCG, GLI, GPC3, GPGSP, GREM1, HER2/neu, HOX11, HOXB13, HRAS, HST, IL-3, INT-2, JAK1, JUN, KIT, KS3, K-SAM, LBC, LCK, LMO1, IMO2, L-MYC, LYL-1, LYT-10, LYT-10/Cal, MAS, MAX, MDM-2, MEN1, MET, MITF, MLH1, MLL, MOS, MSH1, MSH2, MSH3, MSH6, MTG8/AML1, MUTYH, MYB, MYH11/CBFB, NBN, NEU, NFI, NF2, N-MYC, NTHL1, OST, PALB2, PAX-5, PBX1/E2A, PCDC1, PDGFRA, PHOX2B, PIM-1, PMS2, POLDI, POLE, POTI, PPARG, PRAD-1, PRKAR1A, PTCHI, PTEN, RAD50, RAD51C, RAD51D, RAF, RAR/PML, RAS-H, RAS-K, RAS-N, RBI, RECQL4, REL/NRG, RET, RH0M1, RH0M2, ROS, RUNX1, SDHA, SDHAF, SDHAF2, SDHB, SDHC, SDHD, SET/CAN, SIS, SKI, SMAD4, SMARCA4, SMARCB1, SMARCE1, SRC, STK11, SUFU, TALI, TAL2, TAN-1, TIAM1, TERC, TERT, TIMP3, TMEM127, TNF, TP53, TRAC, TSC1, TSC2, TRK, VHL, WRN, and WT1. Non-limiting examples of oncogenes are KRAS, NRAS, BRAF, MYC, CTNNB1, and EGFR. In some embodiments, the oncogene is a gene that encodes a cyclin dependent kinase (CDK). Non-limiting examples of CDKs are Cdkl, Cdk4, Cdk5, Cdk7, Cdk8, Cdk9, Cdkl l and CDK20. Non-limiting examples of tumor suppressor genes are TP53, RBI, and PTEN. SEQUENCES AND TABLES

TABLE 1. Exemplary Effector Protein Amino Acid Sequences, Exemplary Guide Repeat Sequences, and Exemplary PAM Sequences

[291] TABLE 1 provides illustrative amino acid sequences of effector proteins, guide sequences, and PAM sequences that are useful in the compositions, systems and methods described herein. With regards to the PAM sequences: N is any nucleotide, V is A, C, or G; B is C, G, or T; and S is G or C.

[292] TABLE 1.1 provides exemplary amino acid alterations relative to SEQ ID NO: 1 useful in compositions, systems, and methods described herein.

TABLE 1.1 Exemplary Amino Acid Alterations Relative to SEQ ID NO: 1

TABLE 2: Exemplary RDDP Sequences

TABLE 3: Exemplary Engineered RDDPs

TABLE 4. Exemplary Target Genes

TABLE 5. Diseases and Syndromes

TABLE 6. Exemplary MMLV sequences

EXAMPLES

[293] The following examples are included for illustrative purposes only and are not intended to limit the scope of the disclosure.

Example 1. Cas Dimer Mediated Precision Editing

[294] A Cas protein that dimerizes is tested for its ability to precisely edit a target gene of interest. Briefly, HEK293T cells are transfected with plasmids encoding a precision editing system comprising: CasM.265466 (D220R), MMLV-RT, a guide RNA targeting a human gene, a template RNA designed to hybridize to the target strand of the gene, and/or a template RNA designed to hybridize to the nontarget strand of the gene. Such systems may be represented in FIG. 1A and FIG. IB. DNA cleavage and repair may take place as shown in FIG.2. CasM.265466 (D220R), which may form a homodimer with itself, may be fused to DNA repair proteins or DNA repair protein recruiting peptides as shown in FIG. 3 and FIG. 4

[295] The HEK293T cells are also transfected with a plasmid containing a region of the human gene containing a stop codon or frameshift insertion. This region is located between sequences in the plasmid encoding fluorescent proteins (eBFP and mRhubbarb). The precision editing system targets and edits the genomic region, removing the frameshift insertion or stop codon, leading to expression of mRhubbarb in addition to eBFP. The same system may edit corresponding genomic DNA.

[296] The intended edits tested are 4 or 5 nt insertions at the target locus or precise deletion with frame restoration of a fluorescent reporter. Cells are harvested 48 hours after transfections and editing analyzed by NGS and/or fluorescence-activated cell sorter (FACS).

Example 2 — Novel RNA-dependent DNA Polymerases for Precision Editing

[297] To identify novel RNA-dependent DNA polymerases (RDDPs) for precision editing, a metagenomic search was performed. 15 RDDP reference sequences (3 prokaryotic group II intron RDDPs and 12 viral RDDPs) were blasted against sequences available in the Integrated Microbial Genomes System and the UniProt Archive. Around 1.3 million sequences were identified through the metagenomic search. These preliminary sequences varied in lengths and % identity to the 15 reference sequences.

[298] Using guided sampling, the 1.3 million preliminary sequences were refined and filtered based on parameters such as (1) reference coverage over subject, (2) KO prediction, (3) host temperature, etc. Eventually, 46 RDDP candidates (23 prokaryotic group II intron RDDPs and 23 viral RDDPs) were identified.

[299] The 46 identified RDDP candidates were further tested in cultured cells using the PE3 system described in Anzalone et al., Nature. 2019 Dec 5; 576(7785): 149-157. Briefly, each of the RDDP candidates was fused to nCas9(H840A) on the N terminus or the C terminus, separated by an XTEN40 linker. Each fusion protein was tested against three targets in HEK293T cells for precision editing: (1) HEK site 3 for an A insertion at the +1 position relative to the non-target strand cut site, (2) FANCF for a G to T substitution at the +5 position, and (3) RNF2 for a T insertion at the +1 position. Two control RDDPs were included: the wild-type Moloney murine leukemia virus (M-MLV) RDDP (SEQ ID NO: 81) and the evolved M-MLV RDDP (SEQ ID NO: 82). Each PE3 composition comprising the polynucleotide encoding one of the 92 fusion proteins was transfected into HEK293T cells. 3 days post-transfection, the precision editing outcomes were assessed by next generation sequence (NGS). Each test had two independent replicates.

[300] Among the 46 tested RDDP candidates, five showed >1% editing efficiency. None of these five RDDP candidates (2691319, 2691323, 2691335, 2691339, 2691355) have been shown to exhibit reverse polymerase activity in human cells previously. The editing efficiency of the 5 RDDP candidates, as well as that of the wild-type M-MLV RDDP, is shown in FIG. 5. Three RDDP candidates show editing efficiency comparable to that of the wild-type M-MLV RDDP. Furthermore, the three group II intron RDDPs are substantially smaller in size than the wild-type M-MLV RDDP (-425 aa vs. 677 aa), making them suitable as a component of the precision editing system to be packaged into a single adeno-associated virus (AAV) vector.

Example 3 — Novel RNA-dependent DNA Polymerases for Precision Editing

[301] Additional RDDPs were identified according to the methods provided in Example 2 (SEQ ID: 48 - 70 in Table 2). On average, each of these newly identified RDDPs was approximately 30% smaller than the MMLV-RT (See Table 6). Each of the RDDP candidates was fused to nCas9(H840A) (SEQ ID NO: 109) on the N terminus or the C terminus, separated by an XTEN40 linker (SEQ ID NO: 110). Each fusion protein was tested against two targets in HEK293T cells for precision editing: (1) HEK site 3 for an A insertion at the +1 position relative to the non-target strand cut site and (2) FANCF for a G to T substitution at the +5 position. Two control RDDPs were included: the wild-type Moloney murine leukemia virus (M-MLV) RDDP (SEQ ID NO: 81) and the evolved M-MLV RDDP (SEQ ID NO: 82) Each composition comprising the polynucleotide encoding one of the fusion proteins was transfected into HEK293T cells. 3 days post-transfection, the precision editing outcomes were assessed by next generation sequence (NGS). Each test had two independent replicates.

[302] The results of this experiment are shown in FIG. 6A (HEK) and FIG. 6B (FANCF). As shown, up to 14% precise editing was achieved (See 21531).

Example 4 - Variant RNA-dependent DNA Polymerases for Precision Editing

[303] Variants ofthe 2691319 RDDP (SEQ ID NO: 13) and 2691323 RDDP (SEQ ID NO: 15) were generated and tested for their ability to perform precision editing. Combinations of arginine and lysine mutations were made at positions D12, N24, D72, N195, and N114 as follows: (a) 2691319 (D12R-D72R-N195R) - SEQ ID NO: 71

(b) 2691319 (D12R-N24R-D72R-N114K) - SEQ ID NO: 72

(c) 2691319 (D12R-N24R-D72R-N195R) - SEQ ID NO: 73

(d) 2691319 (D12R-D72R-N114K-N195R) - SEQ ID NO: 74

(e) 2691319 (D12R-N24R-D72R-N114K-N195R) - SEQ ID NO: 75

(!) 2691323 (D12R-N72R-N195R) - SEQ ID NO: 76

(g) 2691323 (D12R-N24R-N72R-N114K) - SEQ ID NO: 77

(h) 2691323 (D12R-N24R-N72R-N195R) - SEQ ID NO: 78

(i) 2691323 (D12R-N72R-N114K-N195R) - SEQ ID NO: 79

Q) 2691323 (D12R-N24R-N72R-N114K-N195R) - SEQ ID NO: 80.

[304] Each of the engineered RDDP candidates was fused to nCas9(H840A) (SEQ ID NO: 109) on the N terminus or the C terminus, separated by an XTEN40 linker (SEQ ID NO: 110). The results are shown in FIG. 7A and FIG. 7B. The above-described modifications increased editing by up to 4-fold compared to the editing observed with the parental RDDPs, increasing precision editing from -2.4% to -9.6%.

Example 5. Dual-cut dual-flap for precise deletions

[305] Various systems, referred to as dual-cut dual-flap systems, for precise deletions were tested in this experiment. A non-limiting representative illustration of a dual-cut, dual-flap system is provided in FIG. 8. These systems comprise two guide RNAs and two RT template RNAs (retRNA) to create precise deletions. The effector protein used in these systems was CasM.265466 with a D220R amino acid substitution, denoted in this example as 466(D220R). Various plasmids encoding the fusion proteins in TABLE 7 below were tested. Note however that the presence of a P2A peptide results in cleavage of the fusion protein at the site of the P2A peptide. Some of the protein fusions also have a Brex27 peptide fused that inhibits NHEJ. The amino acid sequence of the BREX27 peptide was set forth in SEQ ID NO: 112 and the amino acid sequence of the MCP (MS2 aptamer binding protein) was set forth in SEQ ID NO: 113. These systems were screened with and without a small peptide NHEJ inhibitor (UbvG08). UbvG08 was expressed separately and not fused to the effector protein or the RT. Guide RNA sequences and retRNA sequences used in this experiment are provided in TABLE 8. Results are provided in TABLE 9. Briefly, HEK293T cells were transfected with plasmids encoding the various proteins and guide RNAs. The retRNAs encoded a -38 precise deletion. Precise edits were quantified using amplicon NGS. Blasticidin selection was applied 24 hours after transfection. TABLE 7. Description of Fusion Proteins

TABLE 8. Description of gRNAs and retRNAs

TABLE 9. Precise Editing Achieved with Dual-Cut, Dual-Flap Systems

Example 6. CasM.265466 nuclease target strand (TS) precision editing

[306] This experiment demonstrates precision editing using a fully active nuclease (CasM.265466) that creates double stranded breaks. See FIG. 9 for a non-limiting exemplary illustration of such a system. In this instance, the strand being extended by the RT is the target strand. Precision editing with a nuclease and TS extension is different from standard RT editing done with a non-target strand nickase and non-target strand extension. The target strand extension with Type V Cas proteins, such as CasM.265466, allows one to edit the seed region or PAM of the target, preventing subsequent rounds of targeting, thereby reducing the amount of imprecise indels. The fusion proteins tested are described in TABLE 10 with their corresponding sequences described in Example 5. gRNAl, gRNA2, retRNA3 and retRNA4 are also described in Example 5. The sequence of retRNAl is UGCUCGCUUCGGCAGCACAUAUACUAGUCGACGGGCCGCACUCGCCGGUCCCAAGCCC GGAUAAAAUGGGAGGGGGCGGGAAACCGCCUAACCAUGCCGAGUGCGGCCGCAACU AAGCACAUGAGGAUCACCCAUGUGCACAGAGGCGUCCCCAGUACGAAGAACUCAU UACCGGUACCCUGCCCAAAAUUUGAAAAACAAGACCAGCAAUCAAGAGGCUAGAA CAAUCAUUACGGAUCGAAAAUUAACAGUGGCCGCGGUCGGCGUGGACUGUAGAACA CUGCCAAUGCCGGUCCCAAGCCCGGAUAAAAGUGGAGGGUACAGUCCACGCUCUAGA GCGGACUUCGGUCCGC (SEQ ID NO: 128), with the bolded sequence being the template sequence. Results are presented in TABLE 10. TABLE 10. Editing results

Example 7. CasM.265466 RT system generates precise insertions with gRNA fused to retRNA [307] Various fusions of CasM.265466 (D220R) to RTs were tested for their ability to generate precise edits with a gRNA fused to a retRNA. The fusion of the gRNA and retRNA may be referred to as a “gretRNA.” FIG. 10 provides a non-limiting exemplary illustration of a partial DNA plasmid encoding a gretRNA (below) in contrast to a plasmid encoding a split RNA system (above). Amino acid sequences of the CasM.265466 RT fusion proteins and nucleotide sequences of the gretRNAs tested are provided in TABLE 11. All systems generated precise edits. Without being bound by theory, these results were surprising because it was not previously known if a template RNA fused to the 5' end of a guide RNA would be tolerated, wherein the guide RNA comprises atracr sequence and a repeat sequence that together, generally, comprise complex secondary structure. Results are provided in TABLE 12.

TABLE 11. Plasmid Constructs for Precise Editing Systems

TABLE 12. Precise Editing with gretRNA

Example 8. CasM.265466 RD DP systems generate precise insertions

[308] Combinations of CasM.265466 D220R and various RDDPs, including those presented in TABLE 2, were tested fortheir ability to generate precise edits using a fluorescent reporter. The fluorescent reporter for editing was comprised of a CMV promoter directing expression of a GFP gene and a hPGK promoter directing expression of mCherry. A 789-base pair long intron is located between amino acids 95 and 96 of GFP. The first two base pairs of the intron, which are normally GT and constitute part of the splice acceptor site, were engineered to be CG. This defect in the splice acceptor which does not allow the intron to be properly spliced away and leads to a lack of expression of GFP. mCherry is present as a transfection control. When transfected into cells, the plasmid will express mCherry but not GFP. Employing RT editing to edit the CG into GT yields a functional splice acceptor and GFP expression. Transfecting the reporter plasmid into HEK293T cells together with plasmids encoding the effector and RDDP proteins, gRNA and retRNA, and using flow cytometry to measure the levels of GFP provided a readout for RT editing. Results are provided

FIG. 11.

Claims

1. A system for modifying a target strand (TS) and/or a non-target strand (NTS) of a double stranded DNA (dsDNA) molecule, the system comprising: a) an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein forms a dimer with itself in a cell; b) an RNA-directed DNA polymerase (RDDP) or a nucleic acid encoding the RDDP; c) a guide RNA or nucleic acid encoding the guide RNA, wherein the guide RNA comprises: i. a first region comprising a scaffold sequence, and ii. a second region comprising a spacer sequence that hybridizes to a target sequence of the TS, wherein the first region is located 5 ’ of the second region; d) a TS template RNA (retRNA) or nucleic acid encoding the TS retRNA, wherein the TS retRNA comprises: i. a TS primer binding sequence (PBS) that hybridizes to the TS, and ii. a TS template sequence; and e) a NTS retRNA or nucleic acid encoding the NTS retRNA, wherein the NTS retRNA comprises: i. a NTS PBS that hybridizes to the NTS, and ii. a NTS template sequence.

2. The system of claim 1, wherein the effector protein cuts the TS and the NTS to generate staggered ends, thereby generating single stranded DNA with a free 3 ’ end on the TS (TS ssDNA) and single stranded DNA with a free 3’ end on the NTS (NTS ssDNA).

3. The system of claim 2, wherein the TS PBS hybridizes to at least a portion of the TS ssDNA, and the NTS PBS hybridizes to at least a portion of the NTS ssDNA.

4. The system of claim 3, wherein the RDDP polymerizes a new strand of ssDNA on the TS that is complementary to the TS template sequence, and a new strand of ssDNA on the NTS that is complementary to the NTS template sequence.

5. The system of any one of claims 1-4, wherein the system comprises a DNA repair protein.

6. The system of claim 5, wherein the DNA repair protein is covalently linked to the effector protein.

7. The system of claim 5, wherein the DNA repair protein is covalently linked to the RDDP.

8. The system of any one of claims 1-7, wherein the RDDP is covalently linked to the effector protein.

9. The system of any one of claims 1-8, wherein the guide RNA is covalently linked to the retRNA.

10. The system of any one of claims 1-8, wherein the TS template sequence is located 5’ of the TS PBS and the NTS template is located 5’ of the NTS PBS, optionally wherein the 3’ end of the PBS is linked to the 5’ end of the template sequence.

11. The system of any one of claims 1-10, wherein the TS retRNA, NTS retRNA, or each of the TS retRNA and NTS retRNA comprise a protein localization sequence, and the RDDP comprises or is covalently linked to a peptide that binds the protein localization sequence, the protein localization sequence thereby localizing the RDDP to the retRNA.

12. The system of any one of claims 1-11, wherein the TS template sequence and/or NTS template sequence comprises a difference of at least one nucleotide relative to an otherwise identical sequence in the dsDNA molecule (providing the interchangeability of thymines and uracils).

13. The system of any one of claims 1-12, wherein the effector protein is a Type V Cas protein.

14. The system of any one of claims 1-13, wherein the length of the effector protein is 400 to 500, 400 to 600, 400 to 700, 400 to 800, or 400-900 linked amino acids.

15. The system of any one of claims 1-14, wherein the effector protein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1.

16. The system of any one of claims 1-14, wherein the amino acid sequence of the effector protein is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1.

17. The system of claim 15 or 16, wherein the effector protein comprises an amino acid substitution selected from TABLE 1.1 relative to SEQ ID NO: 1.

18. The system of any one of claims 15-17, wherein the scaffold sequence comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 83, 84, and 85.

19. The system of any one of claims 1-18, wherein the target sequence is a sequence present in a eukaryotic organism, optionally wherein the eukaryotic organism is a mammal, optionally wherein the mammal is a human.

20. The system of any one of claims 1-19, wherein the RDDP comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 2-82.

21. The system of any one of claims 1 -20, comprising an expression vector, wherein the expression vector comprises any combination of: the nucleic acid encoding the effector protein; the nucleic acid encoding the RDDP; the nucleic acid encoding the guide RNA; and the nucleic acids encoding the TS retRNA and NTS retRNA.

22. The system of claim 21, wherein the expression vector is an adeno-associated viral (AAV) vector, optionally wherein the AAV vector is an scAAV vector.

23. The system of any one of claims 1-21, comprising a lipid or lipid nanoparticle.

24. The system of claim 23, wherein the nucleic acid encoding the effector protein or the nucleic acid encoding the RDDP comprises a messenger RNA.

25. A composition for modifying a target strand (TS) and/or a non target strand (NTS) of a double stranded DNA (dsDNA) molecule, the composition comprising: a) an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein forms a dimer with itself in a cell; b) an RNA-directed DNA polymerase (RDDP) or a nucleic acid encoding the RDDP; c) a guide RNA or nucleic acid encoding the guide RNA, wherein the guide RNA comprises: i. a first region comprising a scaffold sequence, and ii. a second region comprising a spacer sequence that hybridizes to a target sequence of the TS, wherein the first region is located 5 ’ of the second region; d) a TS template RNA (retRNA) or nucleic acid encoding the TS retRNA, wherein the TS retRNA comprises: i. a TS primer binding sequence (PBS) that hybridizes to the TS, and ii. a TS template sequence; and e) a NTS retRNA or nucleic acid encoding the NTS retRNA, wherein the NTS retRNA comprises: i. a NTS PBS that hybridizes to the NTS, and ii . a NTS template sequence .

26. The composition of claim 25, comprising a lipid nanoparticle (LNP), wherein the nucleic acid encoding the effector protein comprises an mRNA, wherein the LNP contains the mRNA, optionally wherein the mRNA comprises the nucleic acid encoding the RDDP, optionally wherein the LNP comprises the guide RNA, the TS retRNA, the NTS retRNA, or a combination thereof.

27. The composition of claim 26, wherein at least one of the guide RNA, the TS retRNA, and the NTS retRNA comprises a chemical modification.

28. A pharmaceutical composition comprising the composition of any one of claims 25-27, and a pharmaceutically acceptable excipient.

29. An expression vector for modifying a target strand (TS) and/or a non target strand (NTS) of a double stranded DNA (dsDNA) molecule, the expression vector comprising: a) a first nucleotide sequence encoding an effector protein, wherein the effector protein forms a dimer with itself in a cell; b) a second nucleotide sequence encoding a RDDP; c) a third nucleotide sequence encoding a guide RNA, wherein the guide RNA comprises: i. a first region comprising a scaffold sequence, and ii. a second region comprising a spacer sequence that hybridizes to a target sequence of the TS, wherein the first region is located 5 ’ of the second region; d) a fourth nucleotide sequence encoding a TS retRNA, wherein the TS retRNA comprises: i. a TS primer binding sequence (PBS) that hybridizes to the TS, and ii. a TS template sequence; and e) a fifth nucleotide sequence encoding a NTS retRNA, wherein the NTS retRNA comprises: i. a NTS PBS that hybridizes to the NTS, and ii . a NTS template sequence .

30. The expression vector of claim 29, wherein the expression vector is an AAV vector.

31. A system for deleting a region of a double stranded DNA (dsDNA) molecule, the system comprising: a) an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein forms a dimer with itself in a cell; b) an RNA-directed DNA polymerase (RDDP) or a nucleic acid encoding the RDDP; c) a first guide RNA (gRNA) or a nucleic acid encoding the first gRNA, wherein the first gRNA comprises: i. a first scaffold sequence, and ii. a first spacer sequence that hybridizes to a first target sequence on a first strand of the dsDNA molecule, wherein the first scaffold sequence is located 5 ’ of the first spacer sequence, and wherein the effector protein and the first gRNA form a first RNP complex that cleaves the dsDNA molecule to form a first single stranded DNA (ssDNA) flap from the first strand of the dsDNA molecule; d) a second gRNA or a nucleic acid encoding the second gRNA, wherein the second gRNA comprises: i. a second scaffold sequence, and ii. a second spacer sequence that hybridizes to a second target sequence on a second strand of the dsDNA molecule, wherein the second scaffold sequence is located 5’ of the second spacer sequence, and wherein the effector protein and the second gRNA form a second RNP complex that cleaves the dsDNA molecule to form a second ssDNA flap from the second strand of the dsDNA molecule; e) a first template RNA (retRNA) or a nucleic acid encoding the first retRNA, wherein the first retRNA comprises: i. a first primer binding sequence (PBS) that hybridizes to at least a portion of the first ssDNA flap, and ii. a first template sequence; and f) a second retRNA or a nucleic acid encoding the second retRNA, wherein the second retRNA comprises: i. a second PBS that hybridizes to at least a portion of the second ssDNA flap, and ii. a second template sequence.

32. The system of claim 31, wherein the nucleic acid encoding the effector protein, the RDDP, the gRNAs, and the retRNAs are combined in a single AAV vector.

33. The system of claim 31 or 32, wherein the first spacer sequence and the second spacer sequence hybridize to the first target sequence and the second target sequence of the dsDNA molecule respectively, wherein the first target sequence and the second target sequence are 10 to 10,000 base pairs apart.

34. The system of any preceding claim, comprising a BREX27 peptide or a nucleic acid encoding the BREX27 peptide, optionally wherein the BREX27 peptide comprises an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 112.

35. A system for modifying a target strand (TS) of a double stranded DNA (dsDNA) molecule, the system comprising: a) an effector protein or a nucleic acid encoding the effector protein, wherein the effector protein forms a dimer with itself in a cell; b) an RNA-directed DNA polymerase (RDDP) or a nucleic acid encoding the RDDP; c) a guide RNA (gRNA) or a nucleic acid encoding the gRNA, wherein the gRNA comprises: i. a first region comprising a scaffold sequence, and ii. a second region comprising a spacer sequence that hybridizes to a target sequence of the TS of the dsDNA molecule, wherein the first region is located 5 ’ of the second region; and wherein the effector protein and the gRNA form a RNP complex that produces a double stranded break; and d) a TS template RNA (retRNA) or a nucleic acid encoding the TS retRNA, wherein the TS retRNA comprises: i. a TS primer binding sequence (PBS) that hybridizes to the TS of the dsDNA molecule, and ii. a TS template sequence.

36. The system of any one of claims 31-35, wherein the effector protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least

97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1.

37. The system of claim 36, wherein the effector protein comprises at least one amino acid substitution relative to SEQ ID NO: 1, wherein the amino acid substitution is D220R.

38. The system of any one of claims 31-36, wherein the effector protein is linked to RDDP to form a fusion protein.

39. The system of claim 38, wherein the fusion protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least

98%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 114-123.

40. The system of claim 38, wherein the fusion protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 115, 117, 119, 122, and 123, wherein the amino acid sequence comprises a P2A peptide that results in cleavage of the fusion protein at the site of the P2A.

41. The system of any one of claims 35-40, wherein the gRNA comprises a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 124 or 125.

42. The system of claim 35, wherein the nucleic acid sequences encoding the effector protein, the RDDP, the gRNA, and the TS retRNAs are combined in a single AAV vector.

43. The system of claim 42, wherein a nucleic acid sequence encoding the effector protein and a nucleic acid sequence encoding the RDDP is linked by a nucleic acid sequence encoding a P2A peptide.

44. A composition comprising an engineered guide nucleic acid or a nucleic acid encoding the same, wherein the guide nucleic acid comprises or consists of a nucleotide sequence according to SEQ ID NO: 129

45. A composition comprising an engineered guide nucleic acid or a nucleic acid encoding the same, wherein the guide nucleic acid comprises or consists of a nucleotide sequence selected from SEQ ID NO: 130, SEQ ID NO: 131, and a combination thereof.

46. The composition of claim 45, wherein SEQ ID NO: 131 is located 5’ of SEQ ID NO: 130.

47. The composition of any one of claims 44-46, wherein the nucleic acid encoding the engineered guide nucleic acid is located in a AAV vector.

48. The composition of any one of claims 44-47, wherein the composition comprises an effector protein or a nucleic acid encoding the same, wherein the effector protein comprises an amino acid sequence that is at least is 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1.

49. A cell modified by the system of any one of claims 1-24, and 31-43, the composition of any one of claims 25-28 and 44-48, or the expression vector of claim 29 or 30.

50. The cell of claim 49, wherein the cell is a eukaryotic cell, optionally wherein the eukaryotic cell is a mammalian cell, optionally wherein the mammalian cell is a human cell.

51. A method of modifying a dsDNA molecule, the method comprising contacting a target nucleic acid with the system of any one of claims 1-24 and 31-43, the composition of any one of claims 25-28 and 44-48, or the expression vector of claim 29 or 30.

52. The system, composition, cell or method of any preceding claim wherein at least one gRNA is covalently linked to at least one retRNA.

53. The system, composition, cell or method of any preceding claim wherein the gRNA(s) is not covalently linked to the retRNA(s).

54. A precision editing system comprising a fused RNA, wherein the fused RNA comprises: a) a guide RNA (gRNA) comprising from 5’ to 3’: a tracrRNA sequence, a repeat sequence, and a spacer sequence; and b) a template RNA (retRNA), wherein the retRNA is covalently linked to the 5’ end of the gRNA.

55. The precision editing system of claim 54, wherein the fused RNA comprises one or more linker nucleotides between: i) the gRNA and the template RNA; ii) the tracrRNA sequence and the repeat sequence; iii) the repeat sequence and the spacer sequence; or a combination thereof.

56. The precision editing system of claim 54 or 55, comprising an effector protein or nucleic acid encoding the same, and an RNA-directed DNA polymerase (RDDP) or a nucleic acid encoding the RDDP.

57. A composition comprising an engineered RDDP or a nucleic acid encoding the same, wherein the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 15 and comprises the amino acid substitutions of D12R, N24R, N72R, N114K, and N195R; wherein the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 56 and comprises the amino acid substitutions of N24R, N72R, N114K, and N195R; or wherein the engineered RDDP comprises an amino acid sequence that is at least 90%, at least 95%, or 100% identical to SEQ ID NO: 65 and comprises the amino acid substitutions of E12R, N24R, N72R, N114K, and N195R.