Small reverse transcriptases and gene editing systems comprising such

Abstract

Small-sized reverse transcriptase (RT) polypeptides derived from SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 and fusion polypeptides comprising such an RT polypeptide and an RNA-guided nucleases. Also provided herein are gene editing systems comprising the RT polypeptide, the RNA-guided nucleases, a guide RNA, and an RT donor RNA for use in gene editing.

Description

SMALL REVERSE TRANSCRIPTASES AND GENE EDITING SYSTEMS COMPRISING SUCHCROSS REFERENCE TO RELATED APPLICATIONSThis application claims the benefit of the filing dates of U.S. Provisional Application No. 63 / 788,498, filed April 14, 2025, U.S. Provisional Application No. 63 / 701,041, filed September 30, 2024, and U.S. Provisional Application No. 63 / 670,239, filed July 12, 2024, the entire contents of each of which is incorporated by reference herein.SEQUENCE LISTINGThe instant application contains a Sequence Listing which has been filed electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on July 10, 2025, is named 063586-54200 lWO_Seq-Listing_ST26.xml and is 178,286 bytes in size.BACKGROUNDClustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR- associated (Cas) genes, collectively known as CRISPR-Cas or CRISPR / Cas systems, are adaptive immune systems in archaea and bacteria that defend particular species against foreign genetic elements.Reverse transcriptases (RTs) are a family of enzymes that generate a strand of DNA that is complementary to an RNA template. The combination of reverse transcriptases and CRISPR / Cas systems has shown great potential in gene editing. CRISPR-guided reverse transcription allows for introduction of desired nucleotide substitutions at a genomic site.It is therefore of great interest to develop efficient and accurate reverse transcriptase- CRISPR gene editing systems for use in disease treatment.SUMMARY OF THE PRESENT DISCLOSUREThe present disclosure is based, at least in part, on the development of reverse transcriptase polypeptides and variants thereof having desired gene editing activities when coused with RNA-guided nucleases such as CRISPR nucleases. Small reverse transcriptase polypeptides are preferable in terms of delivery payload size and cost of synthesis. Accordingly, provided herein are reverse transcriptase (RT) polypeptides, fusion polypeptides comprising the RT polypeptides and RNA-guided nucleases, gene editing systems comprising the RT polypeptides, the RNA-guided nucleases, and RNA templates, and uses thereof in gene editing.In some aspects, the present disclosure features a reverse transcriptase (RT) polypeptide, which comprises a polymerase domain and optionally is free of an RNase domain (e.g., an RNase H domain). The polymerase domain comprises a fingers subdomain and a palm subdomain. In some instances, the polymerase domain is free of a thumb subdomain, a connection subdomain, or a combination thereof. The RT polypeptide may comprise a YXDD motif, in which X represents a naturally-occurring amino acid residue. Further, the RT polypeptide provided herein is small-sized, e.g., having about 250-350 amino acids in length.In some embodiments, the RT polypeptide provided herein may comprise an amino acid sequence at least 85% identical to SEQ ID NO: 2, for example, comprising at least one mutation relative to SEQ ID NO: 2. In some instances, the RT polypeptide comprises an amino acid sequence at least 90% identical to SEQ ID NO: 2 and one or more mutations relative to SEQ ID NO: 2. The one or more mutations comprise one or more amino acid residue substitutions, one or more deletions, or a combination thereof. In some examples, the one or more amino acid residue substitutions are arginine and / or lysine substitutions (e.g., arginine substitutions) at one or more of positions Vll, G12, S13, A24, D28, S35, N36, P55, E59, A60, P74, A83, H84, E117, S128, T153, T163, G166, S169, G175, Q182, A186, L199, E222, E225, E269, andA281 in SEQ ID NO: 2. Alternatively or in addition, the RT polypeptide may comprise or further comprises one or more non-arginine / lysine amino acid residue substitutions at one or more of positions 120, V92, A100, Q102, V104, Fl 16, SI 19, Y121, G122, Q142, H146, Y155, G157, 1159, T163, 1172, A188, D189, S190, A191, T192, V202, F220, F238, V240, L241, and L287 of SEQ ID NO: 2. Examples include I20V, V92I, A100V, Q102T, V104I, F116L, S119C, Y121F, G122A, Q142L, H146W, Y155F, G157D, I159V, T163L, I172V, A188G, D189V, S190W, A191D, T192G, V202I, F220L, F238L, V240L, L241M, and / or L287F.In one example, the RT polypeptide comprises, relative to SEQ ID NO: 2, (i) one or more arginine and / or lysine substitutions at one or more of positions A24, N36, H84, and El 17; (e.g., A24R, N36R, H84R, and / or E117R; or (ii) a non-arginine / lysine substitution at position QI 42 (e.g., Q142L).Alternatively, or in addition, the one or more deletions comprise deletions within residues 1-12 of SEQ ID NO: 2, within residues 295-316 of SEQ ID NO: 2, or a combination thereof. In some examples, the one or more deletions comprise a deletion of amino acid residues 1-12 of SEQ ID NO: 2, a deletion of amino acid residues 1-8 of SEQ ID NO: 2, a deletion of amino acid residues 298-316 of SEQ ID NO: 2, and / or a deletion of amino acid residues 295-316 of SEQ ID NO: 2. In specific examples, the RT polypeptide is a truncated version of SEQ ID NO: 2 having amino acid residues 13-297 of SEQ ID NO: 2. In other specific examples, the RT polypeptide isa truncated version of SEQ ID NO: 2 having amino acid residues 9-294 of SEQ ID NO: 2.In some examples, the RT polypeptide is a variant of SEQ ID NO: 2, the variant comprising (a) amino acid substitutions at positions A24, Y155, A188, V202, and L287 of SEQ ID NO: 2 (e.g., A24R, Y155F, A188G, V202I, and L287F); and (b) deletions of residues 1-12 and 298-316 of SEQ ID NO: 2.In some instances, the RT polypeptide is a variant of SEQ ID NO: 2, which may comprise the mutations listed in Table 18 relative to SEQ ID NO: 2. In some examples, the RT polypeptide comprises the amino acid sequence of any of the RT_A variants listed in Table 21.For example, the RT polypeptide may be a truncated version of SEQ ID NO: 2 comprising amino acid residues 13-297 of SEQ ID NO: 2 and contain an arginine or lysine substitutions position A24 (e.g., A24R). The RT polypeptide may comprise or farther comprise substitutions at Y155 (e.g., Y155F), Al 88 (e.g., A188G), and / or V202 (e.g., V202I).In other examples, the RT polypeptide may be a truncated version of SEQ ID NO: 2 comprising amino acid residues 13-297 of SEQ ID NO: 2 and comprises A24R and V202I substitutions relative to SEQ ID NO: 2. The RT polypeptide may comprise or farther comprise arginine and / or lysine substitutions at positions S35 (e.g., S35R), E117 (e.g., E117R), T153 (e.g., T153R), Al 86 (e.g., A186R), and / or E222 (e.g., E222R) in SEQ ID NO: 2.In one specific example, the RT polypeptide is a variant of SEQ ID NO: 2 comprising a substitution at position A24 (e.g., A24R). In another specific example, the RT polypeptide is a variant of SEQ ID NO: 2 comprising substitutions at positions A24, N36, H84, and Q142 (e.g., A24R, N36R, H84R, and Q142L). In yet another specific example, the RT polypeptide is a variant of SEQ ID NO: 2 comprising substitutions at positions A24, N36, H84, and Q142 (e.g., A24R, N36R, H84R, and Q142L), and the deletions of residues 1-12 and residues 298-316 of SEQ ID NO: 2. Alternatively, the RT polypeptide is a variant of SEQ ID NO: 2 comprising an amino acid substitution at position El 17 (e.g., E117R) and the deletions of residues 1-12 and residues 298-316 of SEQ ID NO: 2.In some embodiments, the RT polypeptide provided herein may comprise an amino acid sequence at least 85% identical to SEQ ID NO: 3, for example, comprising at least one mutation relative to SEQ ID NO: 3. For example, the RT polypeptide may comprise an amino acid sequence at least 90% identical to SEQ ID NO: 3 and one or more mutations relative to SEQ ID NO: 3. The one or more mutations comprise one or more amino acid residue substitution, one or more deletions, or a combination thereof. In some instances, the RT polypeptide comprises the one or more amino acid residue substitutions are arginine and / or lysine substitutions (e.g., arginine substitutions) at one or more of positions Q14, E26, Q30, Q32, Q71, N121, L122, N126,K130, C133, SI 93, E291, and A292 in SEQ ID NO: 3. Alternatively or in addition, the RT polypeptide comprises the one or more deletions, which may comprise deletions within residues 1-5 of SEQ ID NO: 3, residues 294-338 of SEQ ID NO: 2, or a combination thereof. In some examples, the RT polypeptide is a truncated version of SEQ ID NO: 3 having amino acid residues 6-293 of SEQ ID NO: 3 or a variant thereof comprising one or more of the mutations disclosed herein.In some examples, the RT polypeptide may comprise or further comprise one or more non-arginine / lysine substitutions, optionally at one or more of positions S63, A114, N121, and L257 of SEQ ID NO: 3, for example, S63G, A114C, N121G, and / or L257L. In some examples, the RT polypeptide is a variant of SEQ ID NO: 3, the variant comprising arginine or lysine substitutions at positions Q32 and Q71 of SEQ ID NO: 3, optionally arginine substitutions Q32R and Q71R; and deletions of residues 1-5 and 294-338 of SEQ ID NO: 3.In some examples, the RT polypeptide is a variant of SEQ ID NO: 3 and comprises the mutations listed in Table 19 relative to SEQ ID NO: 3. In specific examples, the RT polypeptide may comprise arginine and / or lysine substitutions at positions Q32 (e.g., Q32R), N121 (e.g., N121R), and / or E291 (e.g., E291R) of SEQ ID NO: 3. In other specific examples, the RT polypeptide is a truncated version of SEQ ID NO: 3 having amino acid residues 6-293 of SEQ ID NO: 3. The RT polypeptide may comprise or further comprise arginine and / or lysine substitutions at positions Q30 (e.g., Q30R), G39 (e.g., G39R), Q71 (e.g., Q71R), L122 (e.g., L122R), and / or N126 (e.g., N126R) of SEQ ID NO: 3.In one specific example, the RT polypeptide is a variant of SEQ ID NO: 3 comprising the E291R substitution. In another specific examples, the RT polypeptide is a variant of SEQ ID NO: 3 comprising an amino acid substitution at position E291 (e.g., E291R) and the deletions of residues 1-5 and residues 294-338 of SEQ ID NO: 3. In yet another specific example, the RT polypeptide is a variant of SEQ ID NO: 3 comprising substitutions at positions Q32, N121, and E291 (e.g., Q32R, N121R, and E291R). Alternatively, the RT polypeptide is a variant of SEQ ID NO: 3 comprising substitutions at positions Q32, N121, and E291 (e.g., Q32R, N121R, and E291R) and the deletions of residues 1-5 and residues 294-338 of SEQ ID NO: 3.In some instances, the RT polypeptide comprises the amino acid sequence of any one of the RT_B variants listed in Table 21.In some embodiments, the RT polypeptide provided herein may comprise an amino acid sequence at least 85% identical to SEQ ID NO: 4, for example, comprising at least one mutation relative to SEQ ID NO: 4. In some instances, the RT polypeptide may comprise an amino acid sequence at least 90% identical to SEQ ID NO: 4 and one or more mutations relative to SEQ IDNO: 4. The one or more mutations comprise one or more amino acid residue substitution, one or more deletions, or a combination thereof. In some examples, the RT polypeptide comprises one or more amino acid residue substitutions, which are arginine and / or lysine substitutions (e.g., arginine substitutions) at one or more of positions E24, A35, A28, G31, T32, P34, G36, V38, T51, T70, E56, G73, A85, E88, H102, N117, H145, S218, F275, and P296 in SEQ ID NO: 4. In some examples, the one or more arginine substitutions are at one or more of positions E24, A35, A28, G31, T32, P34, G36, V38, T70, G73, A85, E88, H102, N117, H145, S218, and F275, Alternatively, or in addition, the lysine substitution is at position P296.Alternatively, or in addition, the RT polypeptide may comprise one or more amino acid residue substitutions at one or more of positions G73 (e.g., G73P), R110 (e.g., R110H), and F275 (e.g., F275Y) in SEQ ID NO: 4.Alternatively, or in addition, the RT polypeptide may comprise one or more deletions, which may comprise deletions within residues 1-4 of SEQ ID NO: 4, residues 297-350 of SEQ ID NO: 4, or a combination thereof. In some examples, the RT polypeptide is a truncated version of SEQ ID NO: 4 having amino acid residues 5-296 of SEQ ID NO: 4. In other instances, the RT polypeptide may comprise one or more deletions within the fragment of residues 300-327 of SEQ ID NO: 4. In one example, the RT polypeptide is a deletion variant of SEQ ID NO: 4 having the 300-327 fragment deleted.In some examples, the RT polypeptide may comprise or further comprise one or more non-arginine / lysine amino acid residue substitutions at one or more of positions F25, E52, K48, G73, L83, A93, N100, N117, R110, L121, A127, G154, V170, M172, D180, K192, V195, A202, M213, M234, 1252, H266, T286, V287, S297, P298, and P299 in SEQ ID NO: 4. Examples include F25Y, E52P, K66P, G73N, L83V, A93V, N100L, N117E, R110H or R110N, L121 V, A127E, G154D, M172F, V170Y, D180P, K192I, V195I, A202S, M213L, M234F, I252L, H266Y, T286D, V287D, S297G, P298S, and / or P299S.In some examples, the RT polypeptide comprises the mutations listed in Table 20 relative to SEQ ID NO: 4. In some instances, the RT polypeptide may comprise an arginine or lysine substitution at T70 (e.g., T70R) of SEQ ID NO: 4, and optionally one or more additional amino acid residue substitutions. In some instances, the one or more additional amino acid residue substitutions can be at positions A25 (e.g., A25R), R110 (e.g., R110H), and / or F275 (e.g., F275Y) of SEQ ID NO: 4. In some examples, the RT polypeptide may comprise or farther comprise one or more arginine and / or lysine substitutions at positions T32 (e.g., T32R), G31 (e.g., G31R), V38 (e.g., V38R), E88 (e.g., E88R), and / or N117 (e.g., N117R) of SEQ ID NO: 4.In one specific example, the RT polypeptide is a variant of SEQ ID NO: 4 comprising anamino acid substitution at position T70 (e.g., T70R). In another specific example, the RT polypeptide is a variant of SEQ ID NO: 4 comprising an amino acid substitution at position T70 (e.g., T70R) and the deletions of residues 1-4 and residues 297-350 of SEQ ID NO: 4. In yet another specific example, the RT polypeptide is a variant of SEQ ID NO: comprising amino acid substitutions at positions T70 (e.g., T70R), S297 (e.g., S297G), P298 (e.g., P298S), and P299 (e.g., P299S), and the deletion of residues 300-327 of SEQ ID NO: 4. In still another specific example, the RT polypeptide is a variant of SEQ ID NO: 4 comprising an amino acid substitution at position T32 (e.g., T32R). Alternatively, the RT polypeptide is a variant of SEQ ID NO: 4 comprising an amino acid substitution at position T32 (e.g., T32R) and the deletions of residues 1-4 and residues 297-350 of SEQ ID NO: 4.In some specific examples, the RT polypeptide comprises an amino acid substitution at position T70 of SEQ ID NO: 4 (e.g., T70R). Alternatively, the RT polypeptide comprises amino acid substitutions at positions T32 and N117 of SEQ ID NO: 4 (e.g. , T32R and N117R or N117E). Such an RT polypeptide may further comprise amino acid substitutions at positions S297, P298, and P299 of SEQ ID NO: 4, for example, S297G, P298S, and P299S; and the deletion of residues 300-327 of SEQ ID NO: 4.Specific examples of the RT polypeptides may comprise the amino acid sequences of any of the RT_C variants provided in Table 21.In another aspect, the present disclosure features a fusion polypeptide, comprising any of the RT polypeptides disclosed herein and an RNA-guided nuclease. In some instances, the RT polypeptide in the fusion polypeptide may lack its native N-terminus Met residue. Alternatively or in addition, the RNA-guided nuclease in the fusion polypeptide may lack its native N- terminus Met residue. In some embodiments, the RNA-guided nuclease is a nickase. In some examples, the RNA-guided nuclease is a Type II CRISPR nuclease, for example, a Cas9 nuclease, which optionally can be a Cas9 nickase variant. In other examples, the RNA-guided nuclease is a Type V CRISPR nuclease, for example, a Casl2i2 nuclease. Any of the fusion polypeptide provided herein may further comprise one or more nuclear localization signals (NLSs), one or more peptide linkers, or a combination thereof.In yet other aspects, the present disclosure provides a nucleic acid, comprising a nucleotide sequence encoding any of the RT polypeptides disclosed herein or any of the fusion polypeptides comprising the RT polypeptide and the RNA-guided nuclease as also disclosed herein. In some embodiments, the nucleic acid is in a vector, for example, an expression vector. In some examples, the vector may be a viral vector.Further, the present disclosure provides a gene editing system comprising: (a) an RTpolypeptide as disclosed herein or a first nucleic acid encoding the RT polypeptide; (b) an RNA- guided nuclease or a second nucleic acid encoding the RNA-guided nuclease; (c) a guide RNA (gRNA) or a third nucleic acid encoding the gRNA; wherein the gRNA comprises a scaffold sequence recognizable by the RNA-guided nuclease and a spacer sequence specific to a target sequence within a genomic site of interest, the targeting sequence being adjacent to a cognate protospacer adjacent motif (PAM) sequence of the RNA-guided nuclease; and (d) a reverse transcription donor RNA (RT donor RNA) or a four nucleic acid encoding the RT donor RNA; wherein the RT donor RNA comprises a primer binding site (PBS) and a reverse transcription template (RTT) sequence.In some embodiments, the RT polypeptide and the RNA-guided nuclease form a fusion polypeptide (e.g., those provided herein). In some instances, the gene editing system comprises a nucleic acid encoding the fusion polypeptide. In one example, the nucleic acid is a DNA. In another example, the nucleic acid is a messenger RNA (mRNA).In some embodiments, any of the gene editing systems provided herein comprise an RNA molecule comprising the gRNA and the RT donor RNA. In some examples, the RNA molecule further comprises a protecting sequence, which optionally is located at the 5’ end of the RNA molecule.In some instances, the template sequence in the RT donor RNA is homologous to the genomic site of interest and comprises one or more nucleotide variations relative to the genomic site of interest. In some instances, at least one nucleotide variation is located within the target sequence. Alternatively or in addition, at least one nucleotide variation is located in the PAM sequence.Any of the gene editing systems provided herein may further comprise one or more delivery components or vehicles such as lipid excipients (which may form lipid nanoparticles), nanoparticles, liposomes, exosomes, microvesicles, and / or a gene-gun. The delivery vehicles may be associated with one or more of elements (a)-(d) in some instances. Alternatively or in addition, the gene editing systems may comprise one or more viral particles for producing one or more of the gene editing components disclosed herein.Also within the scope of the present disclosure are a pharmaceutical composition comprising any of the gene editing systems disclosed herein and a kit comprising the elements (a)-(d) of the gene editing system.Further, the present disclosure provides a gene editing method, comprising contacting the gene editing system as disclosed herein with host cells to allow for gene editing of a gene targeted by the gene editing system. In some embodiments, the host cells are cultured in vitro. Inother embodiments, the host cells are located in a subject who needs gene editing of the target gene.The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.DETAILED DESCRIPTION OF THE INVENTIONThe present disclosure is based, at least in part, on the development of small-sized reverse transcriptase (RT) polypeptides, including those with designed mutations (e.g., amino acid residue substitutions and / or deletions), which exhibited desirable activities in gene editing, for example, incorporating designed edits at a target genomic site. Accordingly, provided herein are RT polypeptides, fiision polypeptides combining both an RT polypeptide and an RNA-guided nuclease (e.g., nickase), and gene editing systems comprising such and RNA components for guiding gene editing, as well as gene editing methods involving such.The gene editing systems comprising the RT polypeptides provided herein have demonstrated successful incorporation of edits at target genomic sites. See Examples below. Such RT polypeptides and gene editing systems comprising such are expected to be effective in introducing desired nucleotide substitutions, insertions, and / or deletions at genetic sites of interest, thereby achieving desired therapeutic effects (e.g., correcting genetic defects). The gene editing system provided herein can also be used in other areas, for example, in breeding and genomic functional studies of animals and plants.I. Small Reverse Transcriptases and Fusion Polypeptides ThereofAs used herein, the terms “reverse transcriptase” or “RT” refer to a multi-functional enzyme that typically has two enzymatic activities including RNA- and DNA-dependent DNA polymerization activity. A reverse transcriptase can generate DNA from an RNA template.RT enzymes commonly used in gene editing, e.g., the Moloney Murine Leukemia Virus (MMLV) RT enzyme, typically comprise a polymerase domain and an RNase domain (e.g., an RNase H domain), the polymerase domain including subdomains such as the fingers subdomain, the palm subdomain, the thumb subdomain, and the connection subdomain.Differing from such common RT enzymes, the RT polypeptides provided herein are small in size (e.g., having 250-350 amino acids in length). The RT polypeptides comprise a polymerase domain. The polymerase domain comprises a fingers subdomain and a palm subdomain and may be free of a thumb subdomain and / or connection subdomain. The RTpolypeptides provided herein are also free of an RNase domain (e.g., an RNase H domain). In some embodiments, the RT polypeptide provided herein may comprise a YXDD domain. Such small-sized enzymes are preferred in terms of delivery and cost of synthesis.As used herein, the term “domain” refers to a distinct fimctional and / or structural unit of a polypeptide. In some instances, a functional domain may be linear. In other instances, a functional domain can be discontinuous and conformational. In some embodiments, a domain may comprise a conserved amino acid sequence.In some embodiments, provided herein are RT polypeptides derived from the RT polypeptide of SEQ ID NO: 2 (also referred to as RT_A), the RT polypeptide of SEQ ID NO: 3 (also referred to as RT_B), or the RT polypeptide of SEQ ID NO: 4 (also referred to as RT_C). The RT polypeptides of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 are also described herein as reference RTs. Such an RT polypeptide may comprise the reference RT or a variant thereof as disclosed herein, which may comprise at least one mutation relative to the reference RT. The variant RT polypeptides provided herein are expected to possess advantageous features relative to the reference RT, for example, higher gene editing efficiency as reported in Examples below.As used herein, the term “variant RT polypeptide” refers to an RT polypeptide comprising an alteration, e.g., a substitution, insertion, deletion and / or fusion, at one or more residue positions, compared to the reference RT enzyme provided herein (RT_A of SEQ ID NO: 2, RT_B of SEQ ID NO: 3), or RT_C of SEQ ID NO: 4). The variant RT polypeptides provided herein are expected to exhibit one or more modulated activities (e.g., enhanced) relative to the reference RT enzyme. As used herein, the term “activity” refers to a biological activity. In some embodiments, activity includes enzymatic activity, e.g., polymerase activity. In some examples, the variant RT polypeptides disclosed herein have an enhanced enzymatic activity relative to the reference RT enzyme, e.g., having an enzymatic activity at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 2-fold, 5-fold, 10-fold, or greater than that of the reference RT enzyme.In some embodiments, the variant RT polypeptide provided herein shares a high sequence homology relative to the reference RT enzyme. For example, the variant RT polypeptide may comprise an amino acid sequence at least 70% (e.g., at least 80%, 85%, 90%, 95%, or higher) identical to SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In some instances, the variant RT polypeptide may comprise an amino acid sequence at least 90% identical to SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In some instances, the variant RT polypeptide may comprise an amino acid sequence at least 95% identical to SEQ ID NO: 2, SEQ ID NO: 3, orSEQ ID NO: 4. In other instances, the variant RT polypeptide may comprise an amino acid sequence at least 97% (e.g., 98%, 99%, 99.5%, or greater) identical to SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.The “percent identity” (a.k.a., sequence identity) of two nucleic acids or of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873- 77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength- 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, word length=3 to obtain amino acid sequences homologous to the protein molecules of the invention. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389- 3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.In some instances, the variant RT polypeptide disclosed herein may comprise one or more arginine and / or lysine substitutions (e.g., arginine substitutions) relative to the reference RT polypeptides provided herein. “Arginine substitutions” and / or “lysine substitutions” refers to the replacement of a non-arginine or non-lysine residue in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 with an arginine or lysine residue.In some instances, one or more of the substituting arginine residues may be replaced by a conservative amino acid residue such as lysine or histidine. In some embodiments, the variant RT polypeptide provided herein may comprise one or more arginine substitutions, one or more lysine substitutions, or a combination thereof.Alternatively, or in addition, the variant RT polypeptide disclosed herein may comprise one or more amino acid residue substitutions, for example, conservative amino acid substitutions, either taken alone or in combination with the other types of mutations disclosed herein.As used herein, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York,1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.In some embodiments, the variant RT polypeptide may comprise one or more deletions, for example, at the N-terminus and / or the C-terminus, relative to the reference RT enzyme. In some instances, the deletion mutations can be in combination with the amino acid substitutions disclosed herein (e.g., arginine substitutions, non-arginine substitutions, and / or conservative substitutions). The deletion mutations may further reduce the size of the resultant variant RT polypeptide so as to facilitate delivery and other benefits associated with small size as provided herein.(A) RT_A Polypeptide of SEQ ID NO: 2 and Engineered Variants ThereofIn some embodiments, the RT polypeptide is derived from reference RT enzyme RT_A (SEQ ID NO: 2), including both the reference enzyme (SEQ ID NO: 2), or a variant thereof such as those disclosed herein. The variants of the RT polypeptide of SEQ ID NO: 2 may be produced via introducing one or more mutations (e.g., one or more amino acid substitutions and / or one or more deletions) to the reference enzyme of SEQ ID NO: 2 to modulate (e.g., enhance) one or more activities of the RT enzyme.The RT polypeptide of SEQ ID NO: 2 (see Table 1 below) is a small-sized RT enzyme with 316 amino acid residues and comprises the YADD (SEQ ID NO: 58) motif. This enzyme has a polymerase domain located at positions 14-294 of SEQ ID NO: 2. Within the polymerase domain, positions 14-93 and 161-205 are deemed as the finger subdomain and positions 94-160 and 206-294 are deemed as the palm subdomain. The RT active site is suggested to be at positions 234-237. The RT polypeptide of SEQ ID NO: 2 does not have a thumb subdomain or a connection subdomain in the polymerase domain. The RT polypeptide of SEQ ID NO: 2 also does not have an RNase domain (e.g., an RNase H domain), a PrimPol domain, or a protease domain. In some instances, the RT_A polypeptide in the fusion polypeptide may lack its native N-terminus Met residue. Alternatively, or in addition, the RNA-guided nuclease in the fusion polypeptide may lack its native N-terminus Met residue.Compared to the MMLV RT, which is commonly used in RT-mediated gene editing, the RT polypeptide of SEQ ID NO: 2 and variants thereof as disclosed herein are smaller, thereby benefiting delivery. Arginine and / or lysine substitutions (e.g., arginine substitutions), nonarginine substitutions (e.g., conservative substitutions) and / or deletions (e.g., at the N-terminusand / or the C-terminus) can be introduced into the RT polypeptide of SEQ ID NO: 2 to increase enzymatic activity and / or gene editing activity.Variants of RT_AVariants of the RT polypeptide of SEQ ID NO: 2 (RT_A) as provided herein can contain one or more alterations relative to SEQ ID NO: 2, e.g., one or more amino acid residue substitutions, one or more deletions, one or more insertions, one or more fiisions, or a combination thereof.(i) Arginine and / or Lysine SubstitutionsIn some embodiments, variants of the RT polypeptide of SEQ ID NO: 2 may comprise one or more arginine substitutions, one or more lysine substitutions, or a combination thereof relative to SEQ ID NO: 2. In some examples, the variant RT polypeptide may contain up to 20 arginine and / or lysine substitutions (e.g., up to 20 arginine substitutions, up to 20 lysine substitutions, or a combination thereof, e.g., up to 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 arginine substitutions, lysine substitutions, or a combination thereof. In specific examples, the variant RT polypeptide may contain 10, 9, 8, 7, 6, 5, 4, 3, or 2 arginine substitutions, lysine substitutions, or a combination thereof. In some examples, the variant RT polypeptide provided herein contains arginine substitutions.In some instances, the arginine and / or lysine substitutions may be located at one or more of positions: VI 1, G12, S13, A24, D28, S35, N36, P55, E59, A60, P74, A83, H84, El 17, S128, T153, T163, G166, S169, G175, Q182, A186, L199, E222, E225, E269, and A281 in SEQ ID NO: 2. In one example, a variant of the RT polypeptide of SEQ ID NO: 2 comprises an arginine or lysine substitution at position A24 (e.g., A24R) of SEQ ID NO: 2.(ii) Non-Arginine / Lysine SubstitutionsAlternatively, or in addition, a variant of the RT polypeptide of SEQ ID NO: 2 (RT_A) may comprise one or more amino acid residue substitutions (non-arginine / lysine substitutions) at one or more of positions: 120, V92, A100, Q102, V104, Fl 16, SI 19, Y121, G122, Q142, H146, Y155, G157, 1159, T163, 1172, A188, D189, S190, A191, T192, V202, F220, F238, V240, L241, and L287 of SEQ ID NO: 2. Exemplary amino acid substitutions at these positions include: I20V, V92I, A100V, Q102T, V104I, F116L, S119C, Y121F, G122A, Q142L, H146W, Y155F, G157D, I159V, T163L, I172V, A188G, D189V, S190W, A191D, T192G, V202I, F220L, F238L, V240L, L241M, and / or L287F.In one specific example, the non-arginine / lysine substitution can be at position QI 42 of SEQ ID NO: 2 (e.g., Q142L). In other examples, the non-arginine / lysine substitution can be at one or more positions of Y155, Al 88, V202, and L287 of SEQ ID NO: 2. Examples include Y155F, A188G, V202I, and L287F. In some instances, the replacement residue may be a conservative substitution of the current replacement residue.(iii) DeletionsAlternatively, or in addition, a variant of the RT polypeptide of SEQ ID NO: 2 (RT_A) may comprise deletions relative to SEQ ID NO: 2, for example, at the N-terminus of SEQ ID NO: 2, at the C-terminus of SEQ ID NO: 2, or a combination thereof. In some examples, the deletion may be within an N-terminal fragment of SEQ ID NO: 2, for example, within residues 1-14 (e.g., within residues 1-13 or 1-12) of SEQ ID NO: 2. In some examples, the deletion may be within a C-terminal fragment of SEQ ID NO: 2, for example, within residues 295-316 of SEQ ID NO: 2. In one example, a variant of the RT polypeptide of SEQ ID NO: 2 is an N-terminal truncated version of SEQ ID NO: 2, e.g., having a deletion of residues 1-12 or residues 1-8 of SEQ ID NO: 2. In another example, a variant of the RT polypeptide of SEQ ID NO: 2 is a C- terminal truncated version of SEQ ID NO: 2, e.g., having a deletion of residues 298-316 of SEQ ID NO: 2. In yet another example, a variant of the RT polypeptide of SEQ ID NO: 2 may have deletions at both the N-terminus and the C-terminus of SEQ ID NO: 2. Such a truncated version may have the amino acid residues 13-297 of SEQ ID NO: 2. Alternatively, the truncated version may have the amino acid residues 9-294 of SEQ ID NO: 2.(iv) Combination of MutationsIn some embodiments, a variant of the RT polypeptide of SEQ ID NO: 2 as disclosed herein may comprise a combination of arginine / lysine substitutions, non-arginine / lysine substitutions such as conversative substitutions, and deletions. Examples of such combined mutations are provided in Example 7 below, each of which is within the scope of the present disclosure.In some examples, the variant RT polypeptide is a truncated version of SEQ ID NO: 2 (e.g., with N-terminal deletions and / or C-terminal deletions as disclosed herein, such as the fragment of residues 13-297 of SEQ ID NO: 2, having the deletions of 1-12 and 298-316 residues in SEQ ID NO: 2) with further amino acid substitutions at position A24 (e.g., A24R), and optionally at position N36 and / or H84 (e.g., N36R and / or H84R). Such a variant RT polypeptide may fiirther comprise one or more substitutions at positions Y155, Al 88, and V202of SEQ ID NO: 2, for example, Y155F, Y155W, A188G, V202I, V202L, and V202M. Alternatively, the variant RT polypeptide of SEQ ID NO: 2 may comprise further comprise a substitution at position Q142, e.g., Q142L.In one specific example, the variant RT_A polypeptide comprises (e.g., consists of) a substitution at position A24 of SEQ ID NO: 2 (e.g., A24R). In another example, the variant RT polypeptide comprises (e.g., consists of) substitutions at positions A24, N36, and H84, Q142 in SEQ ID NO: 2, such as A24R / N36R / H84R / Q142L. In yet another example, the variant RT polypeptide may be the truncated 13-297 version of SEQ ID NO: 2 in combination with the substitutions described above.In another specific example, the variant RT_A polypeptide comprises (e.g., consists of) (a) amino acid substitutions at positions A24 (e.g., A24R), Y155 (e.g., Y155F), Al 88 (e.g., A188G), V202 (e.g., V202I), and L287 (e.g., L287F); and (b) a deletion of residues 1-12 and / or residues 298-316 of SEQ ID NO: 2.Additional variants of RT_A (SEQ ID NO: 2) can be found in U.S. Provisional Application Nos. 63 / 670,239 and 63 / 701,041, the relevant disclosures of each of which are incorporated by reference for the purpose and subject matter referenced herein.Any of the variant of the RT polypeptide of SEQ ID NO: 2 as provided herein may share a sequence identity at least 90% (e.g., 95%, 97%, 98%, 99%, 99.5%, or greater) with SEQ ID NO: 2 and carry any of the mutations or combination of mutations as disclosed herein. Specific examples can be found in Table 1 and 21 below. In some examples, the variant of RT_A comprises an amino acid sequence of any of the RT_A variants listed in Table 21. In some instances, the variant of RT_A may include an N-terminus Met residue relative to the sequences provided in Table 21 (which does not include the N-terminus Met residue). In other instances, the variant of RT_A may not include the N-terminus Met residue, for example, when the variant is fiised to a functional fragment (e.g., an NLS fragment) or when the variant is at the C-terminal part in the fiision polypeptide disclosed herein.(B) RT_B Polypeptide of SEQ ID NO: 3 and Engineered Variants ThereofIn some embodiments, the RT polypeptide is derived from reference RT enzyme RT_B (SEQ ID NO: 3), including both the reference RT_B enzyme (SEQ ID NO: 3), or a variant thereof such as those disclosed herein. A variant of the RT polypeptide of SEQ ID NO: 3 may be produced via introducing one or more mutations (e.g., one or more amino acid substitutions and / or one or more deletions) to the reference enzyme of SEQ ID NO: 3 to modulate (e.g., enhance) one or more activities of the RT enzyme.The RT polypeptide of SEQ ID NO: 3 (RT_B) (see Table 1 below) is a small-sized RT enzyme with 338 amino acid residues and comprises the YADD (SEQ ID NO: 58) motif. This enzyme has a polymerase domain located at positions 1-298 of SEQ ID NO: 3. Within the polymerase domain, positions 1-88 and 156-200 are deemed as the finger subdomain and positions 89-155 and 201-289 are deemed as the palm subdomain. The RT active site is suggested to be at positions 229-232. The RT polypeptide of SEQ ID NO: 3 does not have a thumb subdomain or a connection subdomain in the polymerase domain. The RT polypeptide of SEQ ID NO: 3 also does not have RNase domain (e.g., an RNase H domain), PrimPol domain, or protease domain. In some instances, the RT_B polypeptide in the fusion polypeptide may lack its native N-terminus Met residue. Alternatively, or in addition, the RNA-guided nuclease in the fusion polypeptide may lack its native N-terminus Met residue.Compared to the MMLV RT, the RT polypeptide of SEQ ID NO: 3 and variants thereof as disclosed herein are smaller, thereby benefiting delivery. Arginine and / or lysine substitutions (e.g., arginine substitutions), non-arginine substitutions (e.g., conservative substitutions), and / or deletions (e.g., at the N-terminus and / or the C-terminus) can be introduced into the RT polypeptide of SEQ ID NO: 3 to increase enzymatic activity and / or gene editing activity.Variants of RT_BA variant of the RT polypeptide of SEQ ID NO: 3 as provided herein can contain one or more alterations relative to SEQ ID NO: 3, e.g., one or more amino acid residue substitutions, one or more deletions, one or more insertions, one or more fusions, or a combination thereof.(i) Arginine and / or Lysine SubstitutionsIn some embodiments, a variant of the RT polypeptide of SEQ ID NO: 3 may comprise one or more arginine substitutions, one or more lysine substitutions, or a combination thereof relative to SEQ ID NO: 3. In some examples, the variant RT polypeptide may contain up to 20 arginine and / or lysine substitutions (e.g., up to 20 arginine substitutions, up to 20 lysine substitutions, or a combination thereof), e.g., up to 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 arginine substitutions, lysine substitutions, or a combination thereof. In specific examples, the variant RT polypeptide may contain 10, 9, 8, 7, 6, 5, 4, 3, or 2 arginine substitutions, lysine substitutions, or a combination thereof. In some examples, the variant RT polypeptide provided herein contains arginine substitutions.In some instances, the arginine and / or lysine substitutions may be located at one or more of positions: Q14, E26, Q30, Q32, Q71, N121, L122, N126, K130, C133, S193, E291, and A292in SEQ ID NO: 3. In some instances, a variant of the RT polypeptide of SEQ ID NO: 3 comprises one or more arginine substitutions at these positions, for example, at position Q32 and / or position Q71 of SEQ ID NO: 3.(ii) DeletionsAlternatively or in addition, a variant of the RT polypeptide of SEQ ID NO: 3 may comprise deletions relative to SEQ ID NO: 3, for example, at the N-terminus of SEQ ID NO: 3, at the C-terminus of SEQ ID NO: 3, or a combination thereof. In some examples, the deletion may be within an N-terminal fragment of SEQ ID NO: 3, for example, within residues 1 -5 of SEQ ID NO: 3. In some examples, the deletion may be within a C-terminal fragment of SEQ ID NO: 3, for example, within residues 294-338 of SEQ ID NO: 3. In one example, a variant of the RT polypeptide of SEQ ID NO: 3 is an N-terminal truncated version of SEQ ID NO: 3, e.g., having a deletion of residues 1 -5 of SEQ ID NO: 3. In another example, a variant of the RT polypeptide of SEQ ID NO: 3 is a C-terminal truncated version of SEQ ID NO: 3, e.g., having a deletion of residues 294-338 of SEQ ID NO: 3. In yet another example, a variant of the RT polypeptide of SEQ ID NO: 3 may have deletions at both the N-terminus and the C-terminus of SEQ ID NO: 3. Such a truncated version may have the amino acid residues 6-293 of SEQ ID NO: 3.(iii) Non-Arginine / Lysine SubstitutionsIn some instances, the variant of RT_B may comprise or further comprise one or more non-arginine / lysine amino acid substitutions. Such non-arginine / lysine substitutions may be at one or more of positions S63, Al 14, N121, and L257 of SEQ ID NO: 3. Examples include, but are not limited to, S63G, Al 14C, N121G, and L257L. In some instances, the replacement residue may be a conservative substitution of the current replacement residue.(iv) Combination of MutationsIn some embodiments, a variant of the RT polypeptide of SEQ ID NO: 3 as disclosed herein may comprise a combination of arginine / lysine substitutions, non-arginine / lysine substitutions such as conversative substitutions, and deletions. Examples of such combined mutations are provided in Example 7 below, each of which is within the scope of the present disclosure.In some examples, a variant of the RT polypeptide of SEQ ID NO: 3 may comprise amino acid substitutions such as arginine / lysine substitutions at one or more of positions Q32, N121, and E291 of SEQ ID NO: 3. In one example, the variant RT polypeptide comprises Q32R,N121R, and E291R substitutions. Such a variant RT polypeptide of RT_B may be a truncated version of SEQ ID NO: 3, for example, having the fragment 6-293 of SEQ ID NO: 3.Alternatively, or in addition, a variant of the RT polypeptide of SEQ ID NO: 3 as disclosed herein may comprise or farther comprise amino acid substitutions such as arginine / lysine substitutions at one or more of positions Q30, Q32, G39, Q71, LI 22, and / or N126 of SEQ ID NO: 3. In some examples, the variant RT polypeptide has arginine substitutions at one or more of these positions.In one specific example, the variant of RT_B may comprise (e.g., consists of) residue substitutions at positions Q32, N121, and E291 of SEQ ID NO: 3, for example, Q32R / N121R / E291R. Such a variant may fiirther comprise deletions of residues 1-5 and 294- 338 of SEQ ID NO: 3.In another specific example, the variant of RT_B may comprise (e.g., consists of) arginine and / or lysine substitutions at positions Q32 and Q71 of SEQ ID NO: 3 (e.g., arginine substitutions Q32R and Q71R) and deletions of residues 1 -5 and / or 204-338 of SEQ ID NO: 3.Any of the variants of the RT polypeptide of SEQ ID NO: 3 as provided herein may share a sequence identity at least 90% (e.g., 95%, 97%, 98%, 99%, 99.5%, or greater) with SEQ ID NO: 3 and carry any of the mutations or combination of mutations as disclosed herein. Specific examples can be found in Table 1 and 21 below. In some instances, the variant of RT_B may comprise an amino acid sequence of any of the RT_B variant provided in Table 21 below (e.g., SEQ ID NO: 108). In some instances, the variant of RT_B may include an N-terminus Met residue relative to the sequences provided in Table 21 (which does not include the N-terminus Met residue). In other instances, the variant of RT_B may not include the N-terminus Met residue, for example, when the variant is fased to a fimctional fragment (e.g., an NLS fragment) or when the variant is at the C-terminal part in the fusion polypeptide disclosed herein.(C) RT_C Polypeptide of SEQ ID NO: 4 and Engineered Variants ThereofIn some embodiments, the RT polypeptide is derived from the reference RT polypeptide RT_C (SEQ ID NO: 4), including both the reference enzyme (SEQ ID NO: 4), or a variant thereof such as those disclosed herein. A variant of the RT polypeptide of SEQ ID NO: 4 may be produced via introducing one or more mutations (e.g., one or more amino acid substitutions and / or one or more deletions) to the reference enzyme to modulate (e.g., enhance) one or more activities of the RT enzyme.The RT_C reference enzyme of SEQ ID NO: 4 (see Table 1 below) is a small-sized RT enzyme with 350 amino acid residues and comprises the YADD (SEQ ID NO: 58) motif. Thisenzyme has a polymerase domain located at positions 6-291 of SEQ ID NO: 4. Within the polymerase domain, positions 6-87 and 153-198 are deemed as the finger subdomain and positions 88-152 and 199-291 are deemed as the palm subdomain. The RT active site is suggested to be at positions 227-230. The RT polypeptide of SEQ ID NO: 4 does not have a thumb subdomain or a connection subdomain in the polymerase domain. The RT polypeptide of SEQ ID NO: 4 also does not have RNase domain (e.g., an RNase H domain), PrimPol domain, or protease domain. In some instances, the RT_C polypeptide in the fusion polypeptide may lack its native N-terminus Met residue. Alternatively, or in addition, the RNA-guided nuclease in the fusion polypeptide may lack its native N-terminus Met residue.Compared to the MMLV RT, the RT polypeptide of SEQ ID NO: 4 and variants thereof as disclosed herein are smaller, thereby benefiting delivery. Arginine and / or lysine substitutions (e.g., arginine substitutions), non-arginine substitutions (e.g., conservative substitutions) and / or deletions (e.g., at the N-terminus and / or the C-terminus) can be introduced into the RT polypeptide of SEQ ID NO: 4 to increase enzymatic activity and / or gene editing activity.Variants of RT_CA variant of the RT polypeptide of SEQ ID NO: 4 as provided herein can contain one or more alterations relative to SEQ ID NO: 4, e.g., one or more amino acid residue substitutions, one or more deletions, one or more insertions, one or more fusions, or a combination thereof.(i) Arginine and / or Lysine SubstitutionsIn some embodiments, a variant of the RT polypeptide of SEQ ID NO: 4 may comprise one or more arginine substitutions, one or more lysine substitutions, or a combination thereof relative to SEQ ID NO: 4. In some examples, the variant RT polypeptide may contain up to 20 arginine and / or lysine substitutions (e.g., up to 20 arginine substitutions, up to 20 lysine substitutions, or a combination thereof), e.g., up to 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 arginine substitutions, lysine substitutions, or a combination thereof. In specific examples, the variant RT polypeptide may contain 10, 9, 8, 7, 6, 5, 4, 3, or 2 arginine substitutions, lysine substitutions, or a combination thereof. In some examples, the variant RT polypeptide provided herein contains arginine substitutions.In some instances, the arginine and / or lysine substitutions may be located at one or more of positions: E24, A35, A28, G31, T32, P34, G36, V38, T51, T70, E56, G73, A85, E88, H102, N117, H145, S218, F275, P296 in SEQ ID NO: 4. For example, the RT_C variant may comprise one or more arginine substitutions at one or more of positions E24, A35, A28, G31, T32, P34,G36, V38, T70, G73, A85, E88, H102, N117, H145, S218, and F275 in SEQ ID NO: 4. Alternatively or in addition, the RT_C variant may comprise or further comprise a lysine substitution at position P296 of SEQ ID NO: 4.In one example, a variant of the RT polypeptide of SEQ ID NO: 4 comprises an arginine or lysine substitution at position T70 (e.g., T70R) of SEQ ID NO: 4. In another example, a variant of the RT polypeptide of SEQ ID NO: 4 comprises an arginine or lysine substitution at position T32 (e.g., T32R) of SEQ ID NO: 4. In yet another example, a variant of the RT polypeptide of SEQ ID NO: 4 comprises an arginine or lysine substitution at positions T32 and T70 (e.g., T32R / T70R) of SEQ ID NO: 4.(ii) Non-Arginine / Lysine SubstitutionsAlternatively, or in addition, a variant of the RT polypeptide of SEQ ID NO: 4 (RT_C) may comprise one or more amino acid residue substitutions (non-arginine / lysine substitutions) at one or more of positions: F25, E52, K48, G73, L83, A93, N100, N117, R110, L121, A127, G154, V170, M172, D180, K192, V195, A202, M213, M234, 1252, H266, T286, V287, S297, P298, and P299 of SEQ ID NO: 4. Exemplary amino acid substitutions at these positions include: F25Y, E52P, K48P, G73N, L83V, A93V, N100L, N117E, R110H or R1 ION, L121 V, A127E, G154D, M172F, V170Y, D180P, K192I, V195I, A202S, M213L, M234F, I252L, H266Y, T286D, V287D, S297G, P298S, and / or P299S.In some instances, the variant of RT_C may comprise a non-arginine / lysine substitution at position N117 of SEQ ID NO: 4, for example, N117E. Alternatively, or in addition, the variant of RT_C may comprise or farther comprise one or more non-arginine / lysine substitutions at one or more of positions S297, P298, and P299 of SEQ ID NO: 4, for example, S297G, P298S, and / or P299S. In some instances, the replacement residue may be a conservative substitution of the current replacement residue.(iii) DeletionsAlternatively, or in addition, a variant of the RT polypeptide of SEQ ID NO: 4 may comprise deletions relative to SEQ ID NO: 4, for example, at the N-terminus of SEQ ID NO: 4, at the C-terminus of SEQ ID NO: 4, or a combination thereof. In some examples, the deletion may be within an N-terminal fragment of SEQ ID NO: 4, for example, within residues 1-10, for example, within residues 1-7 or 1-5 (e.g., 1-4) of SEQ ID NO: 4. In some examples, the deletion may be within a C-terminal fragment of SEQ ID NO: 4, for example, within residues 297-350 of SEQ ID NO: 4 (e.g., residues 298-350 of SEQ ID NO: 4). In one example, a variant of the RTpolypeptide of SEQ ID NO: 4 is an N-terminal truncated version of SEQ ID NO: 4, e.g., having a deletion of residues 1-4 of SEQ ID NO: 4. In another example, a variant of the RT polypeptide of SEQ ID NO: 4 is a C-terminal truncated version of SEQ ID NO: 4, e.g., having a deletion of residues 297-350 of SEQ ID NO: 4. In some examples, the variant of the RT polypeptide of SEQ ID NO: 4 may have a deletion of residues 300-327 of SEQ ID NO: 4. In yet another example, a variant of the RT polypeptide of SEQ ID NO: 4 may have deletions at both the N-terminus and the C-terminus of SEQ ID NO: 4. Such a truncated version may have the amino acid residues 5- 296 of SEQ ID NO: 4. In some examples, the deletion may be within the polypeptide of SEQ ID NO: 4, for example, a deletion of residues 297-327 of SEQ ID NO: 4 (e.g., 300-327 of SEQ ID NO: 4). In some examples, the deletions may be within residues 297-318 and within residues 322-327 of SEQ ID NO: 4 (for example, deletions of the fragments of 297-318 and 322-327 of SEQ ID NO: 4).(iv) Combination of MutationsIn some embodiments, a variant of the RT polypeptide of SEQ ID NO: 4 (RT_C) as disclosed herein may comprise a combination of arginine / lysine substitutions, non- arginine / lysine substitutions such as conversative substitutions, and deletions. Examples of such combined mutations are provided in Example 7 below, each of which is within the scope of the present disclosure.In some examples, the variant RT polypeptide is a truncated version of SEQ ID NO: 4 (e.g., with N-terminal deletions and / or C-terminal deletions as disclosed herein, such as the fragment of residues 5-296 of SEQ ID NO: 4). Alternatively, or in addition, the variant RT polypeptide may comprise an amino acid substitution such as arginine / lysine substitution at position T70 (e.g., T70R). In some instances, the variant RT polypeptide may further comprise one or more substitutions at positions A25, R110, and / or F275 of SEQ ID NO: 4. Such additional substitutions may include A25R, A25K, A25H, R110H, R110K, F275Y, and F275W.A variant of the RT polypeptide of SEQ ID NO: 4 disclosed herein may comprise or further comprise arginine and / or lysine substitutions (e.g., arginine substitutions) at positions T32, G31, V38, E88, and / or N117 of SEQ ID NO: 4. In some instances, the variant RT polypeptide contains arginine substitutions at one or more of such positions.In one specific example, the variant of RT_C may comprise (e.g., consists of) the substitution of T70R and optionally deletions of residues 1-4 and 297-350 of SEQ ID NO: 4. In another specific example, the variant of RT_C may comprise (e.g., consists of) the substitution of T32R and optionally deletions of residues 1-4 and 297-350 of SEQ ID NO: 4. In yet anotherexample, the variant of RT_C comprises the T70R, S297G, P298S, and P299S substitutions and the deletion of 300-327 of SEQ ID NO: 4 (which is equivalent to a variant of RT_C comprising T70R and deletions of residues 297-318 and 322-327 of SEQ ID NO: 4).In another specific example, the variant of RT_C comprises amino acid substitutions at positions T32 and N117, for example, T32R and N117R or N117E. Such a variant of RT_C may further comprise amino acid substitutions at positions S297, P298, and P299, for example, S297G, P298S, and P299S. Further, the variant of RT_C may also include a deletion of residues 300-327 of SEQ ID NO: 4.In some instances, the variant of RT_C may comprise substitutions at positions T32, N117, S297, P298, and P299 (e.g., T32R, N117R, S297G, P298S, and P299S) of SEQ ID NO: 4 and deletion of residues 300-327 of SEQ ID NO: 4 (a.k.a., substitutions at positions T32 and N117 (e.g., T32R and N117R or N117E) of SEQ ID NO: 4 and deletions of 297-318 and 322- 327 of SEQ ID NO: 4). In some examples, the variant of RT_C may consist of the just-noted mutations. In other examples, the variant of RT_C may farther comprise one or more mutations as disclosed herein, for example, one or more substitutions at one or more of positions F25 (e.g., F25Y), K48 (e.g., K48P), T51 (e.g., T51R), E56 (e.g., E56K), A127 (e.g., A127E), K192 (e.g., KI 921), H266 (e.g., H266Y), and V287 (e.g., V287D). Examples of such RT_C variants include the KD3 variant and KD3 -containing variants provided in Table 21 below.Any of the variants of the RT polypeptide of SEQ ID NO: 4 as provided herein may share a sequence identity at least 90% (e.g., 95%, 97%, 98%, 99%, 99.5%, or greater) with SEQ ID NO: 4 and carry any of the mutations or combination of mutations as disclosed herein. Specific examples can be found in Table 1 and 21 below. In some instances, the variant of RT_C comprises an amino acid sequence of any one of the RT_C variants provided in Table 21, for example, SEQ ID NO: 66, 121, 147, or 151. In some instances, the variant of RT_C may include an N-terminus Met residue relative to the sequences provided in Table 21 (which does not include the N-terminus Met residue). In other instances, the variant of RT_C may not include the N-terminus Met residue, for example, when the variant is fused to a fanctional fragment (e.g., an NLS fragment) or when the variant is at the C-terminal part in the fusion polypeptide disclosed herein.(D) Fusion PolypeptidesIn some embodiments, the RT polypeptide derived from the RT_A polypeptide of SEQ ID NO: 2, the RT_B polypeptide of SEQ ID NO: 3, or the RT_C polypeptide of SEQ ID NO: 4 as provided herein may be a fusion polypeptide comprising an RT moiety as disclosed herein andone or more additional functional elements. In some instances, the one or more additional functional elements may be heterologous to the RT moiety.As used herein, the terms “fiision” and “fused” refer to the joining of at least two nucleotide or protein molecules. For example, “fusion” and “fused” can refer to the joining of at least two polypeptide domains that are encoded by separate genes in nature. The fiision can be an N-terminal fusion, a C-terminal fusion, or an intramolecular fusion. In some aspects, the domains are transcribed and translated to produce a single polypeptide.In some instances, the RT moiety in the fiision polypeptide may be the reference polypeptide of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. Alternatively, the RT portion in the fiision polypeptide may be a variant of any of the reference RT enzymes as disclosed herein. Exemplary additional functional moieties to include in the fiision polypeptide include a peptide tag, a fluorescent protein, a base-editing domain, a DNA methylation domain, a histone residue modification domain, a localization factor, a transcription modification factor, a lightgated control factor, a chemically inducible factor, a chromatin visualization factor, or a combination thereof.In some embodiments, the additional fimctional moiety may comprise a nuclear localization signal (NLS), a nuclear export signal (NES), or a combination thereof. In some examples, the fiision polypeptide may comprise an NLS, which may be located at either the N- terminus or the C-terminus. In specific examples, the fiision polypeptide may comprise a first NLS located at the N-terminus and a second NLS located at the C-terminus. The first and second NLS fragments may be identical. Alternatively, the two NLS fragments may be different. In some embodiments, the fiision polypeptide may comprise an NLS near the N-terminus and / or near the C-terminus (e.g., within about 1, 2, 3, 4, or 5 of the first amino acid or last amino acid of the RT polypeptide).In some embodiments, the additional fimctional moiety may be a flexible peptide linker, for example, an XTEN peptide linker, or a G / S rich peptide linker. Examples of such peptide linkers are provided in Example 2 and Example 3 below, which may be applicable to any of the RNA-guided nuclease polypeptides disclosed herein.In some embodiments, the fusion polypeptide provided herein may comprise any of the RT moieties disclosed herein and an RNA-guided nuclease, such as SpCas9 or Casl2i2. Such a fusion polypeptide can be used for gene editing. More details are provided in the Gene Editing System section below.Exemplary fusion polypeptides comprising an RT polypeptide, an RNA-guided nuclease, and optionally one or more NLSs and peptide linkers are provided in Tables 2, 7, and 11 below.(E) Preparation of RT Polypeptides and Fusion Polypeptides Comprising SuchThe RT polypeptides and fiision polypeptides comprising such as disclosed herein may be prepared by conventional methods or the methods disclosed herein. For example, the RT polypeptides and fusion polypeptides comprising such can be prepared by culturing host cells such as bacteria cells or mammalian cells, capable of producing the nuclease polypeptides, isolating the nuclease polypeptides thus produced, and optionally, purifying the nuclease polypeptides. A fusion polypeptide comprising the RT polypeptide and an RNA-guided nuclease may be complexed with a gRNA.The RT polypeptides and fiision polypeptides comprising such can be also prepared by an in vitro coupled transcription-translation system. Bacteria that can be used for preparation of the RT polypeptides and fiision polypeptides comprising such are not particularly limited as long as they can produce the RT polypeptides and fusion polypeptides comprising such. Some nonlimiting examples of the bacteria include E. coli cells described herein.Unless otherwise noted, all compositions and complexes and polypeptides provided herein are made in reference to the active level of that composition or complex or polypeptide, and are exclusive of impurities, for example, residual solvents or by-products, which may be present in commercially available sources. Enzymatic component weights are based on total active protein. All percentages and ratios are calculated by weight unless otherwise indicated. All percentages and ratios are calculated based on the total composition unless otherwise indicated. In the exemplified composition, the enzymatic levels are expressed by pure enzyme by weight of the total composition and unless otherwise specified, the ingredients are expressed by weight of the total compositions.(i) VectorsThe present disclosure provides vectors for expressing the RT polypeptides and fusion polypeptides comprising such. In some embodiments, a vector disclosed herein includes a nucleotide sequence encoding an RT polypeptide as provided herein or a fiision polypeptide thereof. In some embodiments, the vector comprises a Pol II promoter or a Pol III promoter.Expression of natural or synthetic polynucleotides is typically achieved by operably linking a polynucleotide encoding the RT polypeptides and fusion polypeptides comprising such to a promoter and incorporating the construct into an expression vector. The expression vector is not particularly limited as long as it includes a polynucleotide encoding the RT polypeptides and fusion polypeptides comprising such and can be suitable for replication and integration in eukaryotic cells.Typical expression vectors include transcription and translation terminators, initiation sequences, and promoters useful for expression of the desired polynucleotide. For example, plasmid vectors carrying a recognition sequence for RNA polymerase (pSP64, pBluescript, etc.), may be used. Vectors including those derived from retroviruses such as lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Examples of vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. The expression vector may be provided to a cell in the form of a viral vector.Viral vector technology is well known in the art and described in a variety of virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to phage viruses, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication fimctional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers.The kind of the vector is not particularly limited, and a vector that can be expressed in host cells can be appropriately selected. To be more specific, depending on the kind of the host cell, a promoter sequence to ensure the expression of the polypeptide(s) from the polynucleotide is appropriately selected, and this promoter sequence and the polynucleotide are inserted into any of various plasmids etc. for preparation of the expression vector.Additional promoter elements, e.g., enhancing sequences, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain fimctional elements downstream of the start site as well. Depending on the promoter, it appears that individual elements can fimction either cooperatively or independently to activate transcription.Further, the disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the disclosure. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence, which it is operatively linked when such expression is desired or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.The expression vector to be introduced can Also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, theselectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate transcriptional control sequences to enable expression in the host cells. Examples of such a marker include a dihydrofolate reductase gene and a neomycin resistance gene for eukaryotic cell culture; and a tetracycline resistance gene and an ampicillin resistance gene for culture of E. coll and other bacteria. By use of such a selection marker, it can be confirmed whether the polynucleotide encoding the polypeptide(s) of the present invention has been transferred into the host cells and then expressed without fail.The preparation method using recombinant expression vectors is not particularly limited, and examples thereof include methods using a plasmid, a phage or a cosmid.Any encoding nucleic acids for the RT polypeptides or fusion polypeptides comprising such, including vectors such as expression vectors, is also within the scope of the present disclosure.(ii) Methods of ExpressionThe present disclosure includes a method for protein expression, comprising translating the RT polypeptides and fusion polypeptides comprising such described herein.In some embodiments, a host cell described herein is used to express the RT polypeptides and fusion polypeptides comprising such. The host cell is not particularly limited, and various known cells can be preferably used. Specific examples of the host cell include bacteria such as E. coll, yeasts (budding yeast, Saccharomyces cerevisiae, and fission yeast, Schizosaccharomyces pombe), nematodes (Caenorhabditis elegans), Xenopus laevis oocytes, and animal cells (for example, CHO cells, COS cells and HEK293 cells). The method for transferring the expression vector described above into host cells, i.e., the transformation method, is not particularly limited, and known methods such as electroporation, the calcium phosphate method, the liposome method and the DEAE dextran method can be used.After a host is transformed with the expression vector, the host cells may be cultured, cultivated or bred, for production the RT polypeptides and fusion polypeptides comprising such. After expression, the host cells can be collected and the RT polypeptides and ftision polypeptides comprising such purified from the cultures etc. according to conventional methods (for example, filtration, centrifugation, cell disruption, gel filtration chromatography, ion exchange chromatography, etc.).A variety of methods can be used to determine the level of production of a mature RT polypeptide and ftision polypeptide comprising such in a host cell. Such methods include, but arenot limited to, for example, methods that utilize either polyclonal or monoclonal antibodies specific for the proteins or a labeling tag as described elsewhere herein. Exemplary methods include, but are not limited to, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (MA), fluorescent immunoassays (FIA), and fluorescent activated cell sorting (FACS). These and other assays are well known in the art (See, e.g., Maddox et al., J. Exp. Med. 158:1211

[1983] ).The present disclosure provides methods of in vivo expression of the RT polypeptides and fusion polypeptides comprising such. Such a method may comprise providing a polyribonucleotide encoding the RT polypeptide or the fusion polypeptide comprising such to a host cell in a subject (e.g., a human subject) wherein the polyribonucleotide encodes the RT polypeptide or the fusion polypeptide comprising such expressing the polypeptide from the cell.II. RT-Mediated Gene Editing SystemsIn some aspects, provided herein is an RT- mediated gene editing system, which involves at least two protein components, i.e., any of the RT polypeptides disclosed herein and an RNA- guided nuclease (e.g., a CRISPR nuclease polypeptide), and at least two RNA components, i.e., a guide RNA and an RT donor RNA. In specific embodiments, the two protein components can be located on a fusion polypeptide. Alternatively or in addition, the two RNA components may be located on a single RNA molecule. In some instances, the gene editing system may comprise the protein components and / or the RNA components. In other instances, the gene editing system may comprise nucleic acid(s) encoding the protein components, and / or nucleic acid(s) encoding the RNA components.In some instances, the RT-mediated gene editing system comprises an RNA-guided nuclease such as a CRISPR nuclease having nickase activity. Such a gene editing system is expected to achieve precise gene editing at a desired genomic target site.A. Protein ComponentsThe gene editing systems provided herein involve at least two enzymes, an RT polypeptide as disclosed herein and an RNA-guided nuclease such as a CRISPR nuclease. In some embodiments, the gene editing system comprises the two enzymes. In specific examples, the gene editing system may comprise a fusion polypeptide comprising the two enzyme components. Alternatively, the gene editing system may comprise one or more nucleic acids encoding the two enzyme components. For example, the gene editing system may comprise one or more expression vectors (e.g., viral vectors such as retroviral vectors, adenoviral vectors, oradeno-associated viral vectors) capable of expressing the RNA-guided nuclease, the RT, or the fusion polypeptide comprising such. In other examples, the gene editing system may comprise one or more mRNA molecules coding for the RNA-guided nuclease, the RT, or the fiision polypeptide comprising such.In some embodiments, the RNA-guided nuclease polypeptide and the RT polypeptide as disclosed herein may form a complex, which may be a heterodimer of the two protein components via a dimerization domain (e.g., a leucine zipper), an antibody, a nanobody, or an aptamer.(i) RNA-Guided Nuclease PolypeptidesRNA-guided nucleases refer to nucleases (e.g., nickases) that are complexed with guide RNAs to cleave at specific DNA sites determined by the guide RNAs. With a short guide RNA (gRNA), an RNA-guided nuclease can be guided to the target site for gene editing. The gRNA determines the efficacy and specificity of gene editing by the nuclease.In some embodiments, the RNA-guided nucleases are CRISPR-Cas RNA-guided nucleases (e.g., nickases), which are the primary tools used in gene editing methods. As used herein, the term “CRISPR nuclease” refers to an RNA-guided effector that is capable of binding a nucleic acid and introducing a single-stranded break or double-stranded break. As used herein, the term “CRISPR nickase” refers to an RNA-guided effector that is capable of binding a nucleic acid and cutting one strand of the nucleic acid (i.e., introducing a nick into one strand of a double-stranded nucleic acid.) Multiple types of CRISPR nucleases (e.g., Type I, Type II, Type III, Type IV, Type V, Type VI, and Type VII) have been identified.In some embodiments, the CRISPR nuclease is Cas9 (e.g., Cas9 and nCas9), Casl2a / Cpfl, Casl2b / C2cl, Casl2c / C2c3, Casl2d / CasY, Casl2e / CasX, Casl2g, Casl2h, Casl2i, and Casl2j / CasPhi). Non-limiting examples of Cas enzymes include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csnl or Csxl2), CaslO, CaslOd, Casl2a / Cpfl, Casl2b / C2cl, Casl2c / C2c3, Casl2d / CasY, Casl2e / CasX, Casl2g, Casl2h, Casl2i, Casl2j / Cas<b, Cpfl, Csyl, Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, Cse5e, Cscl, Csc2, Csa5, Csnl, Csn2, Csml, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csxl l, Csfl, Csf2, CsO, Csf4, Csdl, Csd2, Cstl, Cst2, Cshl, Csh2, Csal, Csa2, Csa3, Csa4, Csa5, a Type II CRISPR nuclease, a Type V CRISPR nuclease, a Type VI CRISPR nuclease, CARF, DinG, homologue thereof, or modified or engineered version thereof. Other CRISPR nucleases are also within the scope of this disclosure, although they may not bespecifically listed in this disclosure. See, e.g., Makarova et al. “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” CRISPRJ. l(5):325-36 (2018).In some embodiments, the CRISPR nuclease is a nuclease disclosed in WO2021055874, W02020206036, W02020191102, WO2020186213, W02020028555, W02020033601, WO2019126762, WO2019126774, W02019071048, WO2019018423, W02019005866, WO2018191388, WO2018170333, WO2018035388, WO2018035387, WO2017219027, WO2017189308, WO2017184768, WO2017106657, WO2016205749, W02017070605, WO2016205764, W02016205711, WO2016028682, WO2015089473, WO2014093595, WO2015089427, WO2014204725, WO2015070083, WO2014093655, WO2014093694, WO2014093712, WO2014093635, WO2021133829, W02021007177, WO2020197934, W02020181102, W02020181101, W02020041456, W02020023529, W02020005980, W02019104058, W02019089820, W02019089808, W02019089804, WO2019089796, WO2019036185, WO2018226855, WO2018213351, WO2018089664, WO2018064371, WO2018064352, WO2017106569, WO2017048969, WO2016196655, WO2016106239, WO2016036754, W02015103153, WO2015089277, WO2014150624, WO2013176772, WO2021119563, WO2021118626, WO2020247883, WO2020247882, WO2020223634, WO2020142754, W02020086475, W02020028729, WO2019241452, WO2019173248, WO2018236548, WO2018183403, WO2017027423, WO2018106727, WO2018071672, WO2017096328, W02017070598, W02016201155, WO2014150624, WO2013098244, WO2021113522, W02021050534, WO2021046442, WO2021041569, W02021007563, WO2020252378, W02020180699, W02020018142, WO2019222555, WO2019178428, WO2019178427, or W02019006471, which are incorporated by reference for the subject matter and purpose referenced herein.In some embodiments, the RNA-guided nuclease for use in the gene editing systems provided herein can be a Type II CRISPR nuclease. The Type II systems are farther subdivided into multiple subtypes, e.g., Cas9a, Cas9b, Cas9c, and Cas9d. Jinek et al., Science, 337(6096):816-821 (2012) showed that the CRISPR / Cas9 system is useful for Reprogrammable genome editing, and international patent application publication number WO2013 / 176772 provides numerous examples and applications of the CRISPR / Cas nuclease system for site-specific gene editing. In some examples, the Type II CRISPR nuclease may be a Cas9 enzyme, for example, Cas9 from S. pyogenes, supra. In some instances, the Cas9 enzyme can be a naturally-occurring enzyme, which comprises two nuclease domains, a HNH nuclease domain and a RuvC domain.In other instances, the Cas9 enzyme for use in the gene editing systems provided hereinmay be a variant Cas9 enzyme, for example, a nickase mutant of a naturally-occurring Cas9. In some embodiments, a nickase is a variant of a CRISPR nuclease that comprises a deactivated HNH domain. In some embodiments, a nickase is a variant of a CRISPR nuclease that comprises a deactivated RuvC domain. In one example, the Cas9 enzyme is a nickase mutant comprising a mutation at position H840 (e.g., H840A), e.g., an nSpCas9 comprising an amino acid sequence such as SEQ ID NO: 8. Alternatively, the CRISPR nuclease can be of a different species of a Cas9 CRISPR nuclease, for example, an SaCas9 CRISPR nuclease.In some embodiments, the RNA-guided nuclease for use in the gene editing systems provided herein can be a Type V CRISPR nuclease. Type V CRISPR nuclease is a subclass of CRISPR nucleases. See, e.g., Tong et al., Front. Cell Dev. Biol., 2021, Vol. 8, doi:10.3389 / fcell.2020.622103. In some embodiments, the Type V nuclease is a Casl2 CRISPR nuclease. In some embodiments, the Type V nuclease is a Casl2a (Cpfl), Casl2b (C2cl), Casl2c, Casl2d, Casl2e, Casl2f, Casl2h, Casl2i, or Casl2j (CasPhi) CRISPR nuclease. In some embodiments, the Type V nuclease is a variant (e.g., a functional variant) of a Casl2a (Cpfl), Casl2b (C2cl), Casl2c, Casl2d, Casl2e, Casl2f, Casl2h, Casl2i, or Casl2j (CasPhi) CRISPR nuclease. In some embodiments, the Type V nuclease comprises an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a wild-type Type V nuclease sequence (e.g., a wild-type amino acid sequence of Casl2a (Cpfl), Casl2b (C2cl), Casl2c, Casl2d, Casl2e, Casl2f, Casl2h, Casl2i, or Casl2j (CasPhi). In some embodiments, the Type V enzyme for use in the gene editing systems provided herein may be a variant Type V enzyme. For example, in some embodiments, the Type V enzyme is a nickase variant.In some embodiments, the Type V nuclease of the present invention is a Casl2i CRISPR nuclease. In some embodiments, the Casl2i CRISPR nuclease is a Casl2i2 CRISPR nuclease. In some embodiments, the CRISPR nuclease is a variant Casl2i2 polypeptide described in WO / 2021 / 202800, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein. In some embodiments, the variant Casl2i2 polypeptide comprises one or more of the amino acid substitutions listed in Table 2 of WO / 2021 / 202800. In some embodiments, the CRISPR nuclease is a variant Casl2i2 polypeptide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3 of PCT / US2021 / 025257. In some embodiments, the CRISPR nuclease is a variant Casl2i2 polypeptide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to theamino acid sequence of SEQ ID NO: 4 of PCT / US2021 / 025257. In some embodiments, the CRISPR nuclease is a variant Casl2i2 polypeptide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 5 of PCT / US2021 / 025257. In some embodiments, the CRISPR nuclease is a variant Casl2i2 polypeptide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 495 of PCT / US2021 / 025257. In some embodiments, the CRISPR nuclease is a variant Casl2i2 polypeptide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 496 of PCT / US2021 / 025257. In some embodiments, the CRISPR nuclease is a variant Casl2i2 polypeptide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 3-146 and 495-512 of WO / 2021 / 202800, which are incorporated by reference for the subject matter and purpose referenced herein.In one specific example, the RNA-guided nuclease is a Casl2i2 polypeptide comprising the amino acid sequenced of SEQ ID NO: 34.Additional CRISPR nucleases for use in the gene editing system disclosed herein can be found, e.g., in WO2021050534, WO2022192391, and W02024020567, the relevant disclosures of each of which are incorporated by reference herein for the subject matter and purpose referenced herein.(ii) RT PolypeptidesAny of the RT polypeptides, e.g., those derived from SEQ ID NO: 2 (RT_A), SEQ ID NO: 3 (RT_B), or SEQ ID NO: 4 (RT_C), as disclosed herein can be used in the gene editing systems provided herein. Examples are provided in Tables 1 and 14-20 (the RT portion). See relevant disclosures herein. Exemplary RT polypeptides are provided in Table 21.(iii) Fusion PolypeptidesIn some embodiments, the gene editing system provided herein comprise a fusion polypeptide that includes both the RT polypeptide as disclosed herein and the RNA-guided nuclease (e.g., nickase) such as a CRISPR nuclease (e.g., nickase) polypeptide as also disclosed herein. In some instances, the fusion polypeptide may further comprise one or more additionalfunctional elements, for example, NLSs and peptide linkers. Alternatively, the gene editing system may comprise a nucleic acid (e.g., a vector such as an expression vector) encoding the fusion polypeptide.In some embodiments, the fusion polypeptide may comprise the RT polypeptide at its N- terminus and the RNA-guided nuclease (e.g., nickase) polypeptide downstream to the RT polypeptide. In other embodiments, the fiision polypeptide may comprise the RNA-guided nuclease (e.g., nickase) polypeptide at its N-terminus and the RT polypeptide downstream to the RNA-guided nuclease (e.g., nickase) polypeptide. In some embodiments, the RT polypeptide may be fiised with the RNA-guided nuclease (e.g., nickase) polypeptide at an intramolecular position within the RT polypeptide, for example, the RNA-guided nuclease (e.g., nickase) polypeptide may be within a loop of the reverse transcriptase polypeptide.Any of the RT polypeptides disclosed herein and any of the RNA-guided nuclease (e.g., nickase) polypeptide such as those disclosed herein may be used for constructing the fusion polypeptides. In some instances, the RNA-guided nuclease (e.g., nickase) polypeptide may be a Cas9 nuclease (e.g., a nickase variant such as SEQ ID NO: 8).In some embodiments, any of the RT-nuclease (e.g., nickase) fusion polypeptides disclosed herein may comprise one or more additional functional elements, e.g., those provided herein. In some instances, the additional fimctional elements may be one or more NLS elements. In some examples, the fusion polypeptide may comprise an NLS at its N-terminus, at its C- terminus, or both. Alternatively or in addition, the additional functional elements may be a flexible peptide linker, which can be located between the RNA-guided nuclease (e.g., nickase) polypeptide and the RT polypeptide. Suitable peptide linkers include, but are not limited to, G / S rich peptide linkers and XTEN peptide linkers. Examples of NLS and peptide linkers are provided in Example 2 and Example 3 below.In some examples, the RT-nuclease (e.g., nickase) fusion polypeptide provided herein comprises a peptide linker (the first peptide linker) located between the RNA-guided nuclease (e.g., nickase) polypeptide and the RT polypeptide. In some instances, the RNA-guided nuclease (e.g., nickase) polypeptide is N-terminal to the RT polypeptide. In other instances, the RNA- guided nuclease (e.g., nickase) polypeptide is C-terminal to the RT polypeptide. In some instances, an additional peptide linker and / or one or more NLS signals may be located between the RNA-guided nuclease (e.g., nickase) polypeptide and the RT polypeptide. For example, an additional peptide linker and an NLS may be place between the RNA-guided nuclease (e.g., nickase) polypeptide and the RT polypeptide, in addition to the first peptide linker. In some specific examples, the peptide linker between the RNA-guided nuclease (e.g., nickase)polypeptide and the RT polypeptide is at least 20-aa in length, for example, ranging from about 20 amino acids to 100 amino acids.Alternatively or in addition, the RT-nuclease (e.g., nickase) fiision polypeptide provided herein may comprise at least two NLSs (the first NLS and the second NLS), at least one of which is located at the N-terminus or the C-terminus of the fiision polypeptide. In some examples, one of the two NLSs is located at the N-terminus and the other one is located at the C-terminus. In other examples, the two NLSs are located at the N-terminus. In yet other examples, the two NLSs are located at the C-terminus.In some instances, the RT-nuclease (e.g., nickase) fusion polypeptide provided herein may comprise one or more additional NLS(s) (e.g., a third NLS, and optionally a fourth NLS). Such additional NLS(s) may be located between the RNA-guided nuclease (e.g., nickase) polypeptide and the RT polypeptide. In other examples, the additional NLS(s) may be located between the RNA-guided nuclease (e.g., nickase) / RT polypeptide and a terminal NLS, optionally via a peptide linker.In other embodiments, the gene editing system provided herein may comprise a nucleic acid encoding the RT-nuclease (e.g., nickase) fusion polypeptide. In some examples, the nucleotide sequence encoding the fusion polypeptide described herein can be codon-optimized for use in a particular host cell or organism. In some examples, the nucleic acid encoding the fusion polypeptides as disclosed herein can be an mRNA molecule, which can be codon optimized. Exemplary codon-optimized nucleotide sequences encoding exemplary RT-nuclease (e.g., nickase) fusion polypeptides can be found in Tables 2, 7, 11 and 14-20 below, any of which is within the scope of the present disclosure.B. RNA ComponentsThe gene editing systems provided herein also involve at least two RNA components, a guide RNA (gRNA), which directs gene editing at a desired genetic site, and an RT donor RNA, which serves as the RNA template for the RT polypeptide in reverse transcription. The RT donor RNA comprises desired nucleotide substitutions to be inserted into the genetic site of interest. In some embodiments, the gene editing system comprises the two RNA molecules. In specific examples, the gene editing system may comprise a single RNA molecule comprising the gRNA and the RT donor RNA. Alternatively, the gene editing system may comprise one or more nucleic acids encoding the two RNA components. For example, the gene editing system may comprise one or more expression vectors (e.g., viral vectors such as retroviral vectors, adenoviral vectors, or adeno-associated viral vectors) capable of producing the gRNA, the RT donor RNA,or the single RNA molecule comprising such.In some embodiments, the gRNA and the RT donor RNA as disclosed herein may form a complex.Ci) Guide RNAsThe gene editing system disclosed herein further comprises one or more gRNAs or nucleic acid(s) encoding such. As used herein, the terms “RNA guide,” “RNA guide sequence,” or “guide RNA (gRNA)” refer to an RNA molecule or a modified RNA molecule that facilitates the targeting of an RNA-guided nuclease described herein to a genomic site of interest. For example, an RNA guide can be a molecule that comprises a spacer sequence and a scaffold sequence. The spacer sequence recognizes (e.g., binds to) a site in a non-PAM strand that is complementary to a target sequence in the PAM strand, e.g., designed to be complementary to a specific nucleic acid sequence. The scaffold sequence contains a nuclease binding sequence for binding to the RNA-guided nuclease. In some embodiments, the scaffold is an RNA sequence.In some instances, the gRNA disclosed herein may further comprise a linker sequence, a 5’ end and / or 3 ’ end protection fragment, or a combination thereof.Spacer SequencesAs used herein, the term “spacer” and “spacer sequence” (a.k.a., a DNA-binding sequence) is a portion in an RNA guide that is the RNA equivalent of the target sequence (a DNA sequence). The spacer contains a sequence capable of binding to the non-PAM strand via base-pairing at the site complementary to the target sequence (which is in the PAM strand). Such a spacer is also known as specific to the target sequence. In some instances, the spacer may be at least 75% identical to the target sequence (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%), except for the RNA-DNA sequence difference. In some instances, the spacer may be 100% identical to the target sequence except for the RNA-DNA sequence difference.The gene editing system disclosed herein comprises one or more gRNAs, each comprising a spacer for targeting a genomic site of interest and a scaffold, which is recognizable by the RNA-guided nuclease contained in the gene editing system. The target sequence can be adjacent to a protospacer adjacent motif (PAM) sequence recognizable by the RNA-guided nuclease. For example, if an SpCas9 polypeptide is used in the gene editing system, the PAM sequence is 5’-NGG-3’, in which N represents any of A, C, G, and T. When a Casl2i2 nuclease is used, the PAM sequence is 5’-TTN-3’, in which N presents any of A, C, G, and T.As used herein, the terms “protospacer adjacent motif’ or “PAM sequence” refer to a DNA sequence adjacent to a target sequence. In some embodiments, a PAM sequence is required for binding of the RNA-guided nuclease and / or indel activity. In a double-stranded DNA molecule, the strand containing the PAM sequence is called the “PAM-strand” and the complementary strand is called the “non-PAM strand.” The gRNA binds to a site in the non- PAM strand that is complementary to a target sequence disclosed herein, and the PAM sequence as described herein is present in the PAM-strand. The PAM sequence can be located upstream to the target sequence.As used herein, the term “adjacent to” refers to a nucleotide or amino acid sequence in close proximity to another nucleotide or amino acid sequence. In some embodiments, a nucleotide sequence is adjacent to another nucleotide sequence if no nucleotides separate the two sequences (i.e., immediately adjacent). In some embodiments, a nucleotide sequence is adjacent to another nucleotide sequence if a small number of nucleotides separate the two sequences (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides).A spacer sequence as disclosed herein may have a length of from about 15 nucleotides to about 30 nucleotides. For example, the spacer can have a length of from about 15 nucleotides to about 20 nucleotides, from about 15 nucleotides to about 25 nucleotides, from about 20 nucleotides to about 25 nucleotides, or from about 20 nucleotides to about 30 nucleotides. In some embodiments, the spacer in the gRNA may be generally designed to have a length of between 15 and 25 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25) and be complementary to a specific target sequence. In some embodiments, the spacer sequence may be designed to have a length of between 18-22 nucleotides (e.g., 20 nucleotides).In some embodiments, the spacer sequence may have at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to a target sequence as described herein and is capable of binding to the complementary region of the target sequence via base-pairing.In some embodiments, the spacer sequence comprises only RNA bases. In some embodiments, the spacer sequence comprises a DNA base (e.g., the spacer comprises at least one thymine). In some embodiments, the spacer sequence comprises RNA bases and DNA bases (e.g., the DNA-binding sequence comprises at least one thymine and at least one uracil).Scaffold SequenceThe scaffold sequence (also called direct repeat sequence) in the gRNA is recognizable by the RNA-guided nuclease polypeptide also in the gene editing system.For example, when the RNA-guided nuclease is SpCas9, the scaffold sequence may be: GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA AAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 13), or a variant thereof comprising a nucleotide sequence at least 80% (e.g., at least 85%, 90%, 95%, 98%, or greater) identical to SEQ ID NO: 13. The variant scaffold sequence may comprise deletions, nucleotide substitutions, or a combination thereof.Alternatively, when a Casl2i2 nuclease is used, the scaffold sequence can be AGAAAUCCGUCUUUCAUUGACGG (SEQ ID NO: 40), or a variant thereof comprising a nucleotide sequence at least 80% (e.g., at least 85%, 90%, 95%, 98%, or greater) identical to SEQ ID NO: 40. The variant scaffold sequence may comprise deletions, nucleotide substitutions, or a combination thereof.In a gRNA, the scaffold may be located at the 3’ end of the spacer or the 5’ end of the spacer. In some instances, the scaffold and spacer are connected directly. In other instances, the scaffold and spacer may be connected via a nucleotide linker.(ii) RT Donor RNAAs used herein, the terms “reverse transcription donor RNA” or “RT donor RNA” refer to an RNA molecule comprising a reverse transcription template sequence (RTT sequence) and a primer binding site (PBS). An RT donor RNA may be fused to an RNA guide at either the 5’ end or 3’ end of the RNA guide.Any of the RT donor RNAs disclosed herein comprises: (i) a primer binding site (PBS), and (ii) an RTT sequence. In some instances, the RT donor RNA may forther comprise: (iii) a nucleotide linker sequence, (iv) a 5’ end and / or 3’ end protection fragment (see disclosures herein), or a combination thereof. In some examples, the 5’ end or 3’ end protection fragment (e.g., 3’ extension) may comprise a pseudoknot motif to protect against 3’ exonuclease activity.In some embodiments, an RT donor RNA comprises an aptamer. In some embodiments, the aptamer recruits a reverse transcriptase polypeptide.Primer Binding Site (PBS)In some embodiments, the PBS in an RT donor RNA as disclosed herein is an RNA sequence capable of binding to a DNA strand via base-paring. The DNA strand has been or canbe nicked or cleaved by the RNA-guided nuclease polypeptide of the gene editing system disclosed herein. In some embodiments, the PBS comprises an RNA sequence capable of binding to a DNA strand (a PBS-targeting site) via base-pairing. The DNA strand may have a free 3’ end or a 3’ free end can be generated via cleavage by the RNA-guided nuclease polypeptide contained in the same gene editing system. In some examples, the PBS-targeting site may be located on the same DNA strand as the PAM sequence (the PAM strand).In some embodiments, the PBS may be about 5-50 nucleotides in length. For example, the PBS may be about 5-40, 5-30, or 5-20 nucleotides in length. In specific examples, the PBS may be about 5-20 (e.g., 7-17) nucleotides in length. In some examples, the PBS may contain 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, or 20 nucleotides.As used herein, the term “PBS-targeting site” refers to the region to which a PBS binds. The PBS-targeting site may be adjacent to (e.g., upstream to) the PAM sequence. In a gene editing system comprising an RNA-guided nuclease such as a CRISPR nuclease polypeptide that is a nickase variant (e.g., comprises a disrupted HNH nuclease domain as disclosed herein), the PBS in the RT donor RNA may bind to a region (the PBS-targeting site) on the PAM strand. In some embodiments, the PBS-targeting site may partially or completely overlap with the target sequence. In some instances, the PBS-targeting site may be located upstream to the PAM sequence. For example, the PBS-targeting site may be up to 100 nucleotides upstream to the PAM sequence, for example, up to 50 nucleotides, up to 30 nucleotides, up to 25 nucleotides, up to 20 nucleotides, up to 15 nucleotides, up to 10 nucleotides, or up to 5 nucleotides upstream to the PAM sequence. In specific examples, the PBS-targeting site may start about 3 nucleotides to about 10 nucleotides upstream of the PAM sequence (i.e., the 5 ’-most nucleotide of the PBS may bind about 3 nucleotides, 4, nucleotides, 5, nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides upstream of the PAM sequence.) In specific examples, the PBS-targeting site may start 1 nucleotide, 1-2 nucleotides, 1-3 nucleotides, 1-4 nucleotides, or 1-5 nucleotides, upstream of the PAM sequence. When a free 3’ end is generated by the RNA-guided nuclease polypeptide in the gene editing system within or nearby the target sequence, the PBS binding to the PAM strand at a site upstream to the PAM sequence could efficiently facilitate DNA synthesis by the RT polypeptide in the gene editing system, starting from the free 3’ end generated in the PAM strand.Reverse Transcription Template (RTT) SequenceThe reverse transcription template sequence (RTT sequence) serves as the template for the reverse transcription mediated by the RT polypeptide in the gene editing system disclosed herein. In some embodiments, the RTT sequence comprises a sequence with at least one encoded edit. In some embodiments, the RTT sequence comprises sequence homology to a target sequence or its complementary region with at least one encoded edit. In some embodiments, the RTT sequence is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length. In some embodiments, the RTT sequence is about 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 110 nucleotides, or 120 nucleotides in length or any length in between.In some embodiments, the RTT sequence is about 10 nucleotides. In some embodiments, the RTT sequence is about 11 nucleotides. In some embodiments, the RTT sequence is about 12 nucleotides. In some embodiments, the RTT sequence is about 13 nucleotides. In some embodiments, the RTT sequence is about 14 nucleotides. In some embodiments, the RTT sequence is about 15 nucleotides. In some embodiments, the RTT sequence is about 16 nucleotides. In some embodiments, the RTT sequence is about 17 nucleotides. In some embodiments, the RTT sequence is about 18 nucleotides. In some embodiments, the RTT sequence is about 19 nucleotides. In some embodiments, the RTT sequence is about 20 nucleotides. In some embodiments, the RTT sequence is about 21 nucleotides. In some embodiments, the RTT sequence is about 22 nucleotides. In some embodiments, the RTT sequence is about 23 nucleotides. In some embodiments, the RTT sequence is about 24 nucleotides. In some embodiments, the RTT sequence is about 25 nucleotides. In some embodiments, the RTT sequence is about 26 nucleotides. In some embodiments, the RTT sequence is about 27 nucleotides. In some embodiments, the RTT sequence is about 28 nucleotides. In some embodiments, the RTT sequence is about 29 nucleotides. In some embodiments, the RTT sequence is about 30 nucleotides.In some embodiments, the reverse transcription template sequence comprises at least oneencoded edit (e.g., at least two) relative to a target sequence. In some embodiments, the at least one encoded edit comprises at least one substitution, insertion, and / or deletion. In some embodiments, the edit in the target sequence comprises a substitution, an insertion, and / or a deletion relative to the sequence of a target sequence. In some embodiments, the reverse transcription template sequence comprises at least one LoxP site.In some embodiments, the edit can be a single or multi-nucleotide substitution, such as a G to T substitution, a G to A substitution, a G to C substitution, a T to G substitution, a T to A substitution, a T to C substitution, a C to G substitution, a C to T substitution, a C to A substitution, an A to T substitution, an A to G substitution, or an A to C substitution. In some embodiments, the change in sequence can convert a G:C base pair to a T:A base pair, a G:C base pair to an A:T base pair, a G:C base pair to C:G base pair, a T:A base pair to a G:C base pair, a T:A base pair to an A:T base pair, a T:A base pair to a C:G base pair, a C:G base pair to a G:C base pair, a C:G base pair to a T:A base pair, a C:G base pair to an A:T base pair, an A:T base pair to a T:A base pair, an A:T base pair to a G:C base pair, or an A:T base pair to a C:G base pair.In some embodiments, a template sequence described herein may farther introduce one or more silent mutations. As used herein, a silent mutation refers to a mutation that does not change the amino acid residue encoded by the codon comprising the mutation. The RTT sequence can be reverse transcribed into DNA by the reverse transcriptase of the gene editing system described herein. In some embodiments, the RTT sequence is reverse transcribed from 5’ to 3’ into DNA of the PAM strand.In some embodiments, two guides with overlapping RTT regions can be used for dual flap editing, by which a nucleotide fragment can be inserted into a target site. The nucleotide fragment for insertion may range from 20-800 base pairs, e.g., 20-500 base pairs.In some embodiments, the RTT sequence is 5’ of the PBS. In some embodiments, the RTT sequence is 3’ of the PBS. In some instances, the PBS and the RTT sequence in the RT donor RNA provided herein may be connected via a linker sequence to avoid steric hindrance between the two RNA components.(iii) Single RNA MoleculeIn some embodiments, the gene editing system provided herein comprises a single RNA molecule, which includes both the gRNA and the RT donor RNA, or a nucleic acid encoding the single RNA molecule. Such a single RNA molecule is capable of mediating cleavage at a target sequence within a genomic site of interest by the RNA-guided nuclease polypeptide andsynthesis of a DNA fragment from a free 3’ end of a free DNA strand generated by the RNA- guided nuclease polypeptide cleavage based on the RTT sequence in the single RNA molecule.In some embodiments, the single RNA molecule may comprise the RNA guide linked to the RT donor RNA, optionally via a linker. In some examples, the single RNA molecule, from 5’ to 3’ end, comprises a spacer sequence, a scaffold sequence recognizable by the RNA-guided nuclease polypeptide, an RTT sequence, and a PBS. See, e.g., Table 3 and Table 4. In specific examples, the single RNA molecule may comprise, from 5’ to 3’, a protecting fragment, an RTT sequence, a PBS, a scaffold sequence i.e., a direct repeat sequence), and a spacer sequence. See, e.g., Table 8 and Table 9.Any of the single RNA molecules provided herein may further comprise a linker, which may be located between a scaffold sequence and an RTT or following a PBS. In some examples, the linker may comprise a hairpin structure.In some examples, the 5’ end and / or the 3’ end of the single RNA molecule, or the gRNA and / or RT donor RNA, may contain a protection fragment, which may enhance resistance of the RNA molecule to exonuclease activity. In some instances, the end protection fragment may comprise a nucleotide sequence capable of forming a secondary structure, such as hairpin, a circularization, a pseudoknot, or a triplex structure. In other instances, the end protection fragment may comprise the sequence of an exoribonuclease-resistant RNA (xrRNA), a transfer RNA (tRNA), or a truncated tRNA. In some embodiments, the modification is a Zika-like pseudoknot, a murine leukemia virus pseudoknot (MLV-PK) sequence, a red clover necrotic mosaic virus (RCNMV) sequence, a sweet clover necrotic mosaic virus (SCNMV) sequence, a carnation ringspot virus (CRSV) sequence, preQ sequence, or an RNA bacteriophage MS2 sequence.In some examples, the 5’ end of the single RNA molecule (or the 5’ end of the gRNA and / or the RT donor RNA when separate RNA molecules are used) may contain a 5’ extension motif, which can be any of the protection fragments disclosed herein. In some examples, the 3’ end of the single RNA molecule (or the 3’ end of the gRNA and / or the RT donor RNA when separate RNA molecules are used) may contain a 3’ extension motif, which can be any of the protection fragments disclosed herein.(iv) Modifications of Nucleic AcidsAny of the RNA components in a gene editing system as disclosed herein, e.g., the single RNA molecule, the gRNA, and / or the RT donor RNA, may include one or more modifications.Exemplary modifications can include any modification to the sugar, the nucleobase, theintemucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage / to the phosphodiester backbone), and any combination thereof. Some of the exemplary modifications provided herein are described in detail below.Any of the RNA components disclosed herein may include any useful modification, such as to the sugar, the nucleobase, or the intemucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage / to the phosphodiester backbone). One or more atoms of a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). In certain embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and the intemucleoside linkage. Modifications may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additional modifications are described herein.In some embodiments, the modification may include a chemical or cellular induced modification. For example, some nonlimiting examples of intracellular RNA modifications are described by Lewis and Pan in “RNA modifications and structures cooperate to guide RNA- protein interactions” from Nat Reviews Mol Cell Biol, 2017, 18:202-210.Different sugar modifications, nucleotide modifications, and / or intemucleoside linkages (e.g., backbone structures) may exist at various positions in the sequence. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of the sequence, such that the function of the sequence is not substantially decreased. The sequence may include from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e. any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%>, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%).In some embodiments, sugar modifications (e.g., at the 2’ position or 4’ position) or replacement of the sugar at one or more ribonucleotides of the sequence may, as well asbackbone modifications, include modification or replacement of the phosphodiester linkages. Specific examples of a sequence include, but are not limited to, sequences including modified backbones or no natural intemucleoside linkages such as intemucleoside modifications, including modification or replacement of the phosphodiester linkages. Sequences having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this application, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their intemucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, a sequence will include ribonucleotides with a phosphorus atom in its intemucleoside backbone.Modified sequence backbones may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates such as 3 ’-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates such as 3 ’-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3 ’-5’ linkages, 2 ’-5’ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3’-5’ to 5’-3’ or 2’-5’ to 5’-2’. Various salts, mixed salts and free acid forms are also included. In some embodiments, the sequence may be negatively or positively charged.The modified nucleotides, which may be incorporated into the sequence, can be modified on the intemucleoside linkage (e.g., phosphate backbone). Herein, in the context of the polynucleotide backbone, the phrases “phosphate” and “phosphodiester” are used interchangeably. Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another intemucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).The a-thio substituted phosphate moiety is provided to confer stability to RNA and DNA polymers through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellularenvironment.In specific embodiments, a modified nucleoside includes an alpha-thio-nucleoside (e.g., 5’-O-(l-thiophosphate)-adenosine, 5’-O-(l-thiophosphate)-cytidine (a-thio-cytidine), 5’-O-(l- thiophosphate)-guanosine, 5’-O-(l-thiophosphate)-uridine, or 5’-O-(l-thiophosphate)- pseudouridine).Other intemucleoside linkages that may be employed according to the present invention, including intemucleoside linkages which do not contain a phosphorous atom, are described herein.In some embodiments, the sequence may include one or more cytotoxic nucleosides. For example, cytotoxic nucleosides may be incorporated into sequence, such as bifunctional modification. Cytotoxic nucleoside may include, but are not limited to, adenosine arabinoside, 5- azacytidine, 4’-thio-aracytidine, cyclopentenylcytosine, cladribine, clofarabine, cytarabine, cytosine arabinoside, 1 -(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine, decitabine, 5 -fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafiir ((RS)-5-fluoro-l-(tetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione), troxacitabine, tezacitabine, 2 ’-deoxy -2’ -methylidenecytidine (DMDC), and 6-mercaptopurine. Additional examples include fludarabine phosphate, N4-behenoyl-l-beta-D- arabinofuranosylcytosine, N4-octadecyl- 1 -beta-D-arabinofiiranosylcytosine, N4-palmitoyl- 1 -(2- C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl) cytosine, and P-4055 (cytarabine 5’-elaidic acid ester).In some embodiments, the sequence includes one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, poly- A sequence, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol groups and tyrosine residues, etc.). The one or more post-transcriptional modifications can be any post-transcriptional modification, such as any of the more than one hundred different nucleoside modifications that have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197) In some embodiments, the first isolated nucleic acid comprises messenger RNA (mRNA). In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of pyridin-4-one ribonucleoside, 5 -aza-uridine, 2-thio-5-aza-uridine, 2- thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5 -hydroxyuridine, 3 -methyluridine, 5- carboxymethyl-uridine, 1 -carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl- pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2 -thiouridine, l-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-l-methyl-pseudouridine, 2-thio-l-methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouridine, 2-thio-1 -methyl- 1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy- pseudouridine, and 4-methoxy-2-thio-pseudouridine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 5 -aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5 -formylcytidine, N4-methylcytidine,5 -hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo- pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-l- methyl-pseudoisocytidine, 4-thio- 1 -methyl- 1 -deaza-pseudoisocytidine, 1 -methyl- 1 -deaza- pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2- thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy- pseudoisocytidine, and 4-methoxy- 1-methyl-pseudoisocytidine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8- aza-2 -aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1- methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2- methoxy-adenine. In some embodiments, mRNA comprises at least one nucleoside selected from the group consisting of inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7- deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza- guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy- guanosine, 1 -methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo- guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, andN2,N2-dimethyl-6-thio-guanosine.The sequence may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotides (e.g., naturally -occurring nucleotides, purine or pyrimidine, or any one or more or all of A, G, U, C, I, pU) may or may not be uniformly modified in the sequence, or in a given predetermined sequence region thereof. In some embodiments, the sequence includes a pseudouridine. In some embodiments, the sequence includes an inosine, which may aid in the immune system characterizing the sequence as endogenous versus viral RNAs. The incorporation of inosine may also mediate improved RNA stability / reduced degradation. See for example, Yu, Z. et al. (2015) RNA editing by AD ARImarks dsRNA as “self’. Cell Res. 25, 1283-1284, which is incorporated by reference in its entirety.In some embodiments, any RNA sequence described herein, such as an editing template RNA, may comprise an end modification (e.g., a 5’ end modification or a 3’ end modification). In some embodiments, the end modification is a chemical modification. In some embodiments, the end modification is a structural modification. See disclosures herein.When a gene editing system disclosed herein comprises nucleic acids encoding the RNA- guided nuclease and / or the RT polypeptide, e.g., mRNA molecules, such nucleic acid molecules may contain any of the modifications disclosed herein, where applicable.III. Gene Editing MethodsAny of the gene editing systems can be used to genetically modify (edit) a target nucleic acid, which can be a genetic site of interest, e.g., a genetic site where gene editing is needed, for example, to fix a genetic mutation, to introduce a protective mutation, to introduce modifications for modulating expression of a gene, etc.A. Delivery of Gene Editing System to Target CellsComponents of any of the gene editing systems disclosed herein may be formulated, for example, including a carrier, such as a carrier and / or a polymeric carrier, e.g., a liposome, and delivered by known methods to a cell (e.g., a mammalian cell). Such methods include, but not limited to, transfection (e.g., lipid-mediated, cationic polymers, calcium phosphate, dendrimers); electroporation or other methods of membrane disruption (e.g., nucleofection), viral delivery (e.g., lentivirus, retrovirus, adenovirus, adeno-associated virus (AAV)), microinjection, microprojectile bombardment (“gene gun”), fugene, direct sonic loading, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, exosome-mediated transfer, lipid nanoparticle-mediated transfer, and any combination thereof. In some examples, the delivery method involves the use of lipid nanoparticles to mediate delivery of one or more components of the gene editing system disclosed herein.In some embodiments, the method comprises delivering one or more nucleic acids (e.g., nucleic acids encoding the RNA-guided nuclease polypeptide, the RT polypeptide, or the fusion polypeptide comprising both, the RNA guide, the RT donor RNA, or the single RNA molecule comprising both, etc.), one or more transcripts thereof, and / or a pre-formed RNA guide / RNA- guided nuclease polypeptide / RT polypeptide complex to a cell, where a ternary complex is formed. In some embodiments, an RNA guide and / or RT donor RNA, or a fusion thereof, and anRNA encoding an RNA-guided nuclease polypeptide or a RT polypeptide, or a fusion polypeptide comprising both, are delivered together in a single composition. In some embodiments, an RNA guide and an RNA encoding an RNA-guided nuclease polypeptide are delivered in separate compositions. In some embodiments, an RNA guide / RT donor RNA and an RNA encoding an RNA-guided nuclease polypeptide / RT polypeptide delivered in separate compositions are delivered using the same delivery technology. In some embodiments, an RNA guide / RT donor RNA and an RNA encoding an RNA-guided nuclease polypeptide / RT polypeptide delivered in separate compositions are delivered using different delivery technologies.In some embodiments, one or more of the protein components and one or more of the RNA components are delivered together. For example, the RNA-guided nuclease and / or RT polypeptide and the RNA guide and / or RT donor RNA are packaged together in a single AAV particle. In another example, the RNA-guided nuclease and / or RT polypeptide and the RNA guide and / or RT donor RNA are delivered together via lipid nanoparticles (LNPs). In some embodiments, the RNA-guided nuclease and / or RT polypeptides and the RNA guide and / or RT donor RNA are delivered separately. For example, the RNA-guided nuclease and / or RT polypeptides and the RNA guide and / or RT donor RNA are packaged into separate AAV particles. In another example, the RNA-guided nuclease and / or RT polypeptides is delivered by a first delivery mechanism and the RNA guide and / or RT donor RNA is delivered by a second delivery mechanism.Exemplary intracellular delivery methods, include, but are not limited to: viruses, such as AAV, or virus-like agents; chemical-based transfection methods, such as those using calcium phosphate, dendrimers, liposomes, or cationic polymers (e.g., DEAE-dextran or polyethylenimine); non-chemical methods, such as microinjection, electroporation, cell squeezing, sonoporation, optical transfection, impalefection, protoplast fusion, bacterial conjugation, delivery of plasmids or transposons; particle-based methods, such as using a gene gun, magnectofection or magnet assisted transfection, particle bombardment; and hybrid methods, such as nucleofection. In some embodiments, a lipid nanoparticle comprises an mRNA encoding a RT-nuclease fusion polypeptide, an editing template RNA, or an mRNA encoding such. In some embodiments, the present application further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells.B. Host Cells for Gene EditingAny of the gene editing systems disclosed herein can be delivered to a variety of cells e.g., to mammalian cells such as a mouse cell, a non-human primate cell, or a human cell). In some embodiments, the cell is in cell culture or a co-culture of two or more cell types. In some embodiments, the cell is ex vivo. In some embodiments, the cell is obtained from a living organism and maintained in a cell culture.In some embodiments, the cell is derived from a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, 293T, MF7, K562, HeLa, CHO, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, the cell is an immortal or immortalized cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is a stem cell such as a totipotent stem cell (e.g., omnipotent), a pluripotent stem cell, a multipotent stem cell, an oligopotent stem cell, or an unipotent stem cell. In some embodiments, the cell is an induced pluripotent stem cell (iPSC) or derived from an iPSC. In some embodiments, the cell is a differentiated cell. In some embodiments, the cell is a mammalian cell, e.g., a human cell or a murine cell. In some embodiments, the murine cell is derived from a wild-type mouse, an immunosuppressed mouse, or a disease-specific mouse model. In some embodiments, the cell is a cell within a living tissue, organ, or organism.Any of the genetically modified cells produced using any of the gene editing system disclosed herein is also within the scope of the present disclosure. Such modified cells may comprise a disrupted target gene.Any of the gene editing systems, compositions comprising such, vectors, nucleic acids, RNA guides and cells disclosed herein may be used in therapy. Gene editing systems, compositions, vectors, nucleic acids, RNA guides and cells disclosed herein may be used in methods of treating a disease or condition in a subject. Any suitable delivery or administration method known in the art may be used to deliver compositions, vectors, nucleic acids, RNA guides and cells disclosed herein. Such methods may involve contacting a target sequence with a composition, vector, nucleic acid, or RNA guide disclosed herein. Such methods may involve a method of editing a target sequence as disclosed herein. In some embodiments, a cell engineered using an RNA guide disclosed herein is used for ex vivo gene therapy.IV. Therapeutic ApplicationsAny of the gene editing systems or modified cells generated using such a gene editingsystem as disclosed herein may be used for treating a disease that is associated with the target gene, for example, a genetic defect in the target gene.In some embodiments, provided herein is a method for treating a target disease as disclosed herein comprising administering to a subject (e.g., a human patient) in need of the treatment any of the gene editing systems disclosed herein. The gene editing system may be delivered to a specific tissue or specific type of cells where the gene edit is needed. The gene editing system may comprise LNPs encompassing one or more of the components, one or more vectors (e.g., viral vectors) encoding one or more of the components, or a combination thereof. Components of the gene editing system may be formulated to form a pharmaceutical composition, which may further comprise one or more pharmaceutically acceptable carriers.In some embodiments, modified cells produced using any of the gene editing systems disclosed herein may be administered to a subject (e.g., a human patient) in need of the treatment. The modified cells may comprise a substitution, insertion, and / or deletion described herein. In some examples, the modified cells may include a cell line modified by the RNA- guided nuclease polypeptide, the RT polypeptide, or the RNA-guided nuclease-RT fusion polypeptide, and the RNA guide and RT donor RNA or the single RNA molecule comprising both. In some instances, the modified cells may be a heterogenous population comprising cells with different types of gene edits. Alternatively, the modified cells may comprise a substantially homogenous cell population (e.g., at least 80% of the cells in the whole population) comprising one particular gene edit in the target gene. In some examples, the cells can be suspended in a suitable media.In some embodiments, provided herein is a composition comprising the gene editing system or components thereof. Such a composition can be a pharmaceutical composition. A pharmaceutical composition that is useful may be prepared, packaged, or sold in a formulation suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, intra-lesional, buccal, ophthalmic, intravenous, intra-organ or another route of administration. A pharmaceutical composition of the disclosure may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition (e.g., the gene editing system or components thereof), which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one- half or one-third of such a dosage.A formulation of a pharmaceutical composition suitable for parenteral administration may comprise the active agent (e.g., the gene editing system or components thereof or the modified cells) combined with a pharmaceutically acceptable carrier, such as sterile water orsterile isotonic saline. Such a formulation may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Some injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Some formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Some formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.The pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the cells, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulation may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or saline. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which that are useful include those which may comprise the cells in a packaged form, in a liposomal preparation, or as a component of a biodegradable polymer system. Some compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.V. Kits and Uses ThereofThe present disclosure also provides kits that can be used, for example, to carry out a method described herein for genetical modification of a target gene. In some embodiments, the kits include an RNA guide and an RT donor RNA, or a single RNA molecule comprising both, an RNA-guided nuclease polypeptide, and an RT polypeptide, or a fusion polypeptide thereof. In some embodiments, the kits include the single RNA molecule and the RNA-guided nuclease-RT fusion polypeptide. In some embodiments, the kits include a polynucleotide that encodes the RNA-guided nuclease polypeptide, the RT polypeptide, or the RNA-guided nuclease-RT fusion polypeptide, and optionally the polynucleotide is comprised within a vector, e.g., as described herein. In some embodiments, the kits include a polynucleotide that encodes the RNA components disclosed herein. The RNA-guided nuclease polypeptide, the RT polypeptide, or a fusion polypeptide thereof (or polynucleotide encoding such) and the RNA components (e.g., as a ribonucleoprotein) can be packaged within the same or other vessel within a kit or can bepackaged in separate vials or other vessels, the contents of which can be mixed prior to use.The RNA-guided nuclease polypeptide, the RT polypeptide, and the RNA components can be packaged within the same or other vessel within a kit or can be packaged in separate vials or other vessels, the contents of which can be mixed prior to use. The kits can additionally include, optionally, a buffer and / or instructions for use of the RNA components, the RNA-guided nuclease polypeptide, and the RT polypeptide, or the fusion polypeptide thereof.General techniquesThe practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (L E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed. 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds.(1985»; Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984»; Animal Cell Culture (R.I. Freshney, ed. (1986»; Immobilized Cells and Enzymes (IRL Press, (1986»; and B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.).Without further elaboration, it is believed that one skilled in the art can, based on theabove description, utilize the present disclosure to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the present disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.EXAMPLESThe following examples are provided to fiirther illustrate some embodiments of the present disclosure but are not intended to limit the scope of the present disclosure; it will be understood by their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.Example 1 - Evaluation of Reverse Transcriptases with an In Vitro AssayIn this Example, reverse transcriptase (RT) polypeptides were tested in an in vitro assay to determine efficiency of transcription off ribonucleic acid (RNA) substrates. Each RT polypeptide was synthesized in an in vitro transcription and translation (IVTT) reaction. A synthesized RNA substrate was then provided, and generation of a complementary deoxyribonucleic acid (cDNA) was measured. The cDNA sequence was converted to doublestranded DNA, barcoded for multiplexing, and sequenced on an Illumina Next Generation Sequencer. The RNA templates contained a stretch of degenerate bases that served as unique molecular identifiers (UMIs); each unique recorded sequence represented a single reverse transcription event, thereby allowing the level of activity of each RT sequence to be assessed. Comparing the number of UMIs generated between different RTs allowed for quantification of the number of reverse transcription events in a particular time window.To generate the transcription template for the RNA used in the RT extension assay, overlapping primers were designed with a T7 promoter upstream of a 74bp sequence from six different RNAs of varying levels of secondary structure. The overlapping primers were filled in with DNA Polymerase I, Large (Klenow) Fragment (New England Biolabs®), and a T7 RNA synthesis reaction was performed using a HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs®). RNA products were purified using Zymo RNA Clean & Concentrator columns (Zymo Research) and eluted in nuclease-free water and pooled in equimolar fashion for use in the in vitro RT extension assay. gBlock HiFi gene fragments containing each RT polypeptide downstream of a T7 RNA polymerase promoter were ordered from Integrated DNA Technologies and used as templates for polymerase chain reactions (PCR) to generate linear amplicons encompassing the entire gBlock.Amplicons from this reaction were then used as templates for the PURExpress® In Vitro Protein Synthesis Kit (New England Biolabs®) to generate each RT polypeptide via IVTT.The IVTT reactions containing the RT polypeptides were combined with an RT primer at a final concentration of 1 pM, 500pM each dNTP, 5 pM RNA substrate (preparation described above), and 50nM ssDNA library prep control in a reaction buffer (50mM Tris-HCl, 75mM KC1, 3mM MgCh, and lOmM DTT). In each experiment, WT MMLV (amino acid sequence provided below; SEQ ID NO: 1), an active RT polypeptide, was included as a positive control for RNA extension activity.MLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEA RLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL TEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPWALNPATLLPLPEEG LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL SI IHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI (SEQ ID NO : 1 )The RT reaction was run at 37 °C for 30 minutes, and then heated to 80°C for 15 minutes to deactivate the RT polypeptide. RNase H (New England Biolabs®) and RNase cocktail (Thermo Fisher Scientific) were added, and the solution was incubated at 37°C for 20 minutes to remove the RNA template. Nucleic acids were purified using Agencourt AMPure XP (Beckman Coulter) and eluted in nuclease-free water.This solution containing first-strand cDNA was filled in using DNA Polymerase I, Large (Klenow) Fragment (New England Biolabs®), lOOpM each dNTP, 400nM primer, and NEBuffer2.1(New England Biolabs®) diluted to IX (50mM NaCl, lOmM Tris-HCl, lOmM MgC12, lOOpg / mL BSA) at 37°C for 20 minutes. Thermolabile Exonuclease I (New England Biolabs®) was added directly to the reaction and further incubated at 37°C for another 10 minutes to remove unused primer. Nucleic acids were purified using Agencourt AMPure XP (Beckman Coulter) and eluted in nuclease-free water.PCR was then performed to add on Illumina sequencing handles and barcodes, and samples were quantified with Qubit™ 3 Fluorometer (Thermo Fisher Scientific). Libraries were pooled in equimolar fashion, purified with agarose gel electrophoresis, and loaded onto the Illumina NextSeq 550 for paired-end sequencing with a Mid Output Kit (v2.5).For each RNA transcription sample, the UMI region of the transcript was counted to record the total number of transcription events within the reaction and normalized to the number of UMI counts measured from a ssDNA library preparation control doped into the transcriptionreaction. Most reverse transcriptases tested returned a normalized UMI count « 1. Three RTs, however, shown in Table 1, produced significantly more cDNA in the assay. Normalized UMI counts produced by an extension reaction with MMLV are shown as a comparison and were collected across multiple experiments.Table 1. Results from In Vitro RT AssayExample 2 - RNA-Templated Editing of Human Genes in HEK293T Cells Using nSpCas9- Reverse Transcriptase Fusion PolypeptidesIn this Example, the RT polypeptides identified in Example 1 were fused to the C- terminus of the H840A nickase variant of SpCas9 to introduce specific edits into the genome.A sequence encoding the nSpCas9 nickase-RT fusion polypeptide was cloned into a pcDNA3.1 vector (Invitrogen) transcribed by a CMV promoter. The fiision comprised the following components arranged from N- to C-termini: 1) SV40 NLS (MKRTADGSEFESPKKKRKV; SEQ ID NO: 5), 2) nSpCas9 coding sequence, 3) XTEN linker, (SGGSSGGSSGSETPGTSESATPESSGGSSGGSS; SEQ ID NO: 6), 4) an RT from Example 1 (human codon-optimized), and 5) nucleoplasmin NLS (KRPAATKKAGQAKKKK; SEQ ID NO: 7). The amino acid sequences of the nSpCas9-RT fusions are shown in Table 2 below. Table 2. nSpCas9-RT Fusion PolypeptidesEditing template RNAs were designed to be specific to the target sequences shown in Table 3 and cloned into a pUC19 plasmid comprising a U6 PolIII promoter and a 6x polyT terminator sequence. The editing templates were synthesized by GenScript and comprised the following four components, from 5’ to 3’: 1) spacer sequence, 2) scaffold motif, 3) reverse transcription template (RTT) encoding 1-4 nucleotide substitutions or insertions, and 4) primer binding site (PBS). Guides AAVS1-T2, EMX1-T6, VEGFA-T3, and RUNX1 were used. The sequences of each component and the frill-length editing template RNA sequences are shown in Tables 3 and 4. Nicking gRNAs were designed to create a nick within 100 bp of the target site to bias the DNA repair toward incorporating the edit introduced by the novel RT. The nicking gRNA sequences are shown in Table 4.Table 3. Sequences of Editing Template RNA ComponentsEncoded edits shown in bolded text Table 4. Full-Length Sequences Used for Editing with nSpCas9-RT Fusion PolypeptidesEncoded edits shown in bolded textFor each experiment, two or three plasmids were introduced into HEK293T cells. In all editing experiments, a plasmid expressing the nSpCas9-RT fusion polypeptide and a plasmid expressing the editing template RNA were included. Some experiments also included a plasmid expressing a nicking gRNA. Approximately 16 hours prior to transfection, 25,000 HEK293T cells in DMEM / 10%FBS+Pen / Strep (DIO media) were plated into each well of a 96-well plate. On the day of transfection, the cells were 50-70% confluent. For each well to be transfected, a mixture of Lipofectamine 2000™ (Thermo Fisher Scientific) and Opti-MEM™ (Thermo Fisher Scientific) was prepared and incubated at room temperature for 5 minutes (Solution 1). After incubation, the Lipofectamine 2000™: Opti-MEM™ mixture was added to a separate mixture containing the nSpCas9-RT fiision polypeptide, editing template RNA, and Opti-MEM™ (Solution 2) or nSpCas9-RT fiision polypeptide, editing template RNA, nicking gRNA, and Opti-MEM™ (Solution 2). Solutions 1 and 2 were mixed by pipetting up and down, then incubated at room temperature for 25 minutes. Following incubation, the Solution 1 and 2 mixture was added dropwise to each well of a 96-well plate containing the cells.Approximately 72 hours post transfection, cells were trypsinized by adding TrypLE™ (Thermo Fisher Scientific) to the center of each well and incubating at 37°C for approximately 5 minutes. D10 media was then added to each well and mixed to resuspend cells. The resuspended cells were centrifuged for 10 minutes to obtain a pellet, and the supernatant was discarded. The cell pellet was then resuspended in Quick Extract™ buffer (Lucigen®), and cells were incubated at 65°C for 15 minutes, 68°C for 15 minutes, and 98°C for 10 minutes.Samples were prepared for NGS by amplifying a region of approximately 200 bp centered at each editing target, then sequenced with an Illumina NovaSeq. For each target, the fraction of NGS reads containing indels were calculated for each sample. To determine thepercentage of edits installed in the target genes, NGS reads determined to contain indels were further analyzed for insertions or substitutions corresponding to the edits encoded by the editing template RNAs. For all three nSpCas9-RT fiision polypeptides, editing of the target loci was observed, both without a nicking gRNA (Table 5) and with a nicking gRNA (Table 6). About 24% of NGS reads at the VEGFA-T3 target site were determined to contain the edit encoded by the editing template RNA when using the nSpCas9-RT_C fusion polypeptide and when the nicking gRNA was included (see last row of Table 6).Table 5. Editing Efficiencies with nSpCas9-RT Fusion Polypeptide without a Nicking gRNATable 6. Editing Efficiencies with nSpCas9-RT Fusion Polypeptide with a Nicking gRNAOverall, this Example shows that the tested nSpCas9-RT fiision polypeptides incorporated substitutions and insertions encoded by editing template RNAs into human genes.Example 3 - RNA-Templated Editing of Human Genes in HEK293T Cells Using Casl2i2-Reverse Transcriptase Fusion PolypeptidesIn this Example, RT_A and RT_B, which were identified in Example 1, were fiised to the C-terminus of Casl2i2 to introduce specific edits into the genome using a modified version of the method described in US 20230023791, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein.A sequence encoding the Casl2i2-RT fusion polypeptide was cloned into a pcDNA3.1 vector (Invitrogen) transcribed by a CMV promoter. The fiision comprised the following components arranged from N- to C-termini: 1) His Tag (MKIEEGKGHHHHHH; SEQ ID NO: 33), 2) Casl2i2 coding sequence, nucleoplasmin NLS (KRPAATKKAGQAKKKK; SEQ ID NO: 7), 3) XTEN linker (SGGSSGGSSGSETPGTSESATPESSGGSSGGSS; SEQ ID NO: 6), 4) an RT from Example 1 (human codon-optimized), and 5) SV40 NLS (MKRTADGSEFESPKKKRKV; SEQ ID NO: 5). The amino acid sequences of the Casl2i2- RT fusion polypeptides are shown in Table 7.Table 7. Casl2i2-RT Fusion PolypeptidesEditing template RNAs were designed to be specific to target sequences in the EMX1 and AAVS1 genes downstream of a 5’-TTT-3’ PAM sequence. Each guide made two edits: 1) a transversion of the second T in the PAM sequence to an A, and 2) a 4bp transversion in the positions 13-16 bp downstream of the PAM sequence to their reverse complements. Each edit disrupted further targeting by Casl2i2 by interrupting the PAM sequence or spacer targeting sequence.Each template RNA comprised five components, as shown in Table 8: 1) protecting group containing a direct repeat and non-targeting spacer sequence (described in US 20230023791), 2) reverse transcriptase template (RTT) matching a non-target strand sequence(described in US 20230023791), 3) primer binding site (PBS), 4) Casl2i2 direct repeat sequence, and 5) spacer sequence. The full-length editing template RNA sequences are shown in Table 9. These guides were cloned into a pUC19 plasmid between a U6 PolIII promoter and 6x polyT terminator sequence.Table 8. Sequences of Editing Template RNA ComponentsEncoded edits shown in bolded text Table 9. Full-Length Sequences Used for Editing with Casl2i2-RT Fusion PolypeptidesEncoded edits shown in bolded textApproximately 16 hours prior to transfection, 25,000 HEK293T cells in DMEM / 10%FBS+Pen / Strep (DIO media) were plated into each well of a 96-well plate. On the day of transfection, the cells were 50-70% confluent. For each well to be transfected, a mixture of Lipofectamine 2000™ (Thermo Fisher Scientific) and Opti-MEM™ (Thermo Fisher Scientific) was prepared and incubated at room temperature for 5 minutes (Solution 1). After incubation, the Lipofectamine 2000™: Opti-MEM™ mixture was added to a separate mixture containing the Casl2i2-RT fiision polypeptide, editing template RNA, and Opti-MEM™ (Solution 2). Solutions 1 and 2 were mixed by pipetting up and down, then incubated at room temperature for 25 minutes. Following incubation, the Solution 1 and 2 mixture was added dropwise to each well of a 96-well plate containing the cells. Approximately 72 hours post transfection, cells were trypsinized by adding TrypLE™ (Thermo Fisher Scientific) to thecenter of each well and incubating at 37°C for approximately 5 minutes. DIO media was then added to each well and mixed to resuspend cells. The resuspended cells were centrifuged for 10 minutes to obtain a pellet, and the supernatant was discarded. The cell pellet was then resuspended in Quick Extract™ buffer (Lucigen®), and cells were incubated at 65°C for 15 minutes, 68°C for 15 minutes, and 98°C for 10 minutes.Samples were prepared for NGS as described in Example 2. For each of the Casl2i2- RT fusion polypeptides, incorporation of the edits encoded by the editing template RNAs was observed, as indicated in Table 10.Table 10. Editing Efficiencies with Casl2i2-RT Fusion PolypeptideOverall, this Example shows that the tested Casl2i2-RT fusion polypeptides incorporated substitutions and insertions encoded by editing template RNAs into human genes.Example 4 - RNA-Templated Editing of Human Genes in HEK293T Cells Using Casl2i2-Reverse Transcriptase Fusion PolypeptidesIn this Example, RT_C, which was identified in Example 1, is fused to the C- terminus of Casl2i2 to introduce specific edits into the genome using the method described in Example 3.A sequence encoding the Casl2i2- RT_C fusion polypeptide is cloned into a pcDNA3.1 vector (Invitrogen) as described in Example 3. The amino acid sequence of the Casl2i2-RT fiision is shown in Table 11.Table 11. Casl2i2-RT Fusion PolypeptidesExperiments are conducted to determine editing efficiency using the methods and editing template RNAs described in Example 3. Incorporation of the edits encoded by the editing template RNAs is expected at the target loci.Example 5 - Effectiveness of Variant RT Polypeptides for Targeting of Exemplary Human GenesThis Example describes editing assessment of exemplary human genes using variant RTs fiised to nSpCas9 and transfected into HEK293T cells.Single substitutions were designed based on the primary sequence of the RT, the predicted tertiary structure of the RT, or through arginine mutagenesis of residues identified with 10 A2of solvent-exposed surface area. The substitutions were individually cloned into the nSpCas9-RT fiision plasmids described in Example 2. The plasmids of Example 2 are herein referred to as the reference plasmids.Each of the mutated sequences was tested for its ability to introduce edits encoded by editing template RNAs at one or more of four potential targets - the three targets described in Table 3 and Table 4 for AAVS1-T2, EMX1-T6, and VEGFA-T3, as well as an additional target in AAVS1 where two different edits off each exposed ‘flap’ from the individual nicking reactions overlap to introduce the complete 50 bp sequence. The sequences of the two guides used to create the 50 bp insertion (AGGTTTGTACCGTACACCACTGAGACCGCGGTGG TTGACCAGACAAACCT; SEQ ID NO: 49) are shown in Table 12 and Table 13. Each mutation was tested in an arrayed screening layout, using the method described in Example 2. For edits introducing the 50 bp insertion with two template RNA guides, the nicking guide described in Example 2 was replaced by the second templating guide.Table 12. Sequences of Editing Template RNA Components for Dual Flap EditingTable 13. Full-Length Sequences Used for Editing with nSpCas9-variant RT Fusion PolypeptidesFor nSpCas9-RT_A, 178 individual mutations were tested. Of those, 140 exhibited lower editing activity than that of the reference plasmid, while 38 yielded at least equivalent levels of editing at one or more targets compared to the reference plasmid. 26 variants yielded a >1.5x increase in editing level at one or more of the tested targets; this data is summarized in Table 14.Table 14. Fold-Increase in Editing for nSpCas9-variant RT_A Polypeptides Relative to the nSpCas9-RT_A Polypeptide ReferenceFor nSpCas9-RT_B, 198 individual mutations were tested. Of those, 144 exhibited lower editing activity than that of the reference plasmid, while 54 yielded at least equivalent levels of editing activity at one or more targets compared to the reference plasmid. 9 variants yielded a >1.5x increase in editing at one or more targets compared to the reference plasmid; this data is summarized in Table 15.Table 15. Fold-Increase in Editing for nSpCas9-variant RT_B Polypeptides Relative to the nSpCas9-RT_B Polypeptide ReferenceFor nSpCas9- RT_C, 238 individual mutations were tested. Of those, 182 exhibited lower editing activity than that of the reference plasmid, while 56 yielded at least equivalent levels of editing activity at one or more targets compared to the reference plasmid. Of those, 15 yielded a >1.5x increase in editing at one or more targets compared to the reference plasmid; this data is summarized in Table 16.Table 16. Fold-Increase in Editing for nSpCas9-variant RT_C Polypeptides Relative to the nSpCas9-RT_C Polypeptide ReferenceThis Example thus shows that fusions of the engineered RT variants and nSpCas9 introduced edits encoded by editing template RNAs into human genes.6 - Effectiveness of Truncated RTHumanGenesThis Example describes editing assessment of exemplary human genes using truncated RTs fiised to nSpCas9 and transfected into HEK293T cells.To create truncations, the tertiary structure of each RT was first predicted using AlphaFold2 (Tunyasuvunakool et al., Nature 596 (2021)). Then, each predicted structure was examined for domain boundaries, specifically looking for sequence on the N- or C-terminus that appeared unstructured or not contributing to the core reverse transcriptase domain / fold. After removing the sequence from both termini, the shortened reverse transcriptase sequences were cloned as nSpCas9 fusions and tested as described in Example 5. Editing data is shown in Table 17 (numbers denote the amino acid positions included from the polypeptide sequences in Table 1). Data shown was calculated as the percentage of NGS reads comprising the edit encoded by the editing template RNA used.Table 17. Editing Efficiencies Using nSpCas9-Truncated RTs at Exemplary Targets, as Measured by NGSFor RT_A, truncation to 9-297 retained activity close to the reference (untruncated) polypeptide. Additional truncations 13-297 and 9-294 were taken forward to combine with RT variants in Example 7. For RT_B, truncation to 6-293 exhibited activity higher or comparable to the reference (untruncated) polypeptide and was taken forward to combine with RT variants in Example 7. For RT_C , truncation to 5-296 exhibited activity comparable to the reference (untruncated) polypeptide at two of three targets but lower activity at one target relative to the reference polypeptide.This Example thus shows that fusions of the engineered truncated RT polypeptides and nSpCas9 introduced edits encoded by editing template RNAs into human genes.Example 7 - Effectiveness RT Variant Polypeptides with Multiple Substitutions or Truncations for Targeting of Exemplary Human GenesThis Example describes combining the beneficial substitutions identified in Example 5 and truncations identified in Example 6 and assessing the ability of the resulting variant polypeptides to introduce edits into the human genome at exemplary targets.To prepare fusion polypeptides with multiple substitutions, up to four of the most active individual substitutions from Example 5 were combined. In some cases, additional variants were included based on the expectation of an increase in solubility, further interaction with the RNA / DNA template, or increasing RT activity. In some cases, beneficial substitutions were combined with the truncations from Example 6 or additional truncations designed using theAlphaFold2 -predicted structures.Each variant RT polypeptide sequence was cloned into an expression plasmid as a fusion to the C-terminus of nSpCas9 as described in Example 2. The nSpCas9-variant RT fusion polypeptides were tested using the method described in Example 5. Results are shown in Table 18, Table 19, and Table 20 (numbers denote the amino acids positions included from the polypeptide sequences in Table 1). Data shown was calculated as the percentage of NGS reads comprising the edit encoded by the editing template RNA used or the 50 bp insertion (for the dual flap insertion condition). Table 18. Editing Efficiencies Using nSpCas9-variant RT_A Polypeptides at Exemplary Targets, as Measured by NGSFor RT_A, when fused to nSpCas9, the 13-297 truncation demonstrated one of the largest increases in editing when combined with A24R. Adding Y155F, A188G, or V202I to the 13-297 A24R polypeptide further increased activity 2-fold or more at the VEGFA-T3 target. In particular, the A24R V2021 13-297 polypeptide demonstrated the highest activity. Combining additional mutations from Table 15 (e.g., S35R, E117R, T153R, A186R, and E222R) with the A24R V2021 13-297 polypeptide is anticipated to further increase editing.Table 19. Editing Efficiencies Using nSpCas9-variant RT_B Polypeptides at Exemplary Targets, as Measured by NGSFor RT_B, when fused to nSpCas9, the variant containing Q32R, N121R, and E291R showed the highest level of editing. Truncating the RT_B polypeptide to 6-293 with the Q32R, N121R, and E291R substitutions showed similar or improved editing at the two targets tested.The 6-293 Q32R N121R E291R variant showed the highest editing overall. Combiningadditional mutations from Table 14 (e.g., Q71R, Q30R, L122R, N126R, and G39R) with the 6-293 Q32R N121R E291R variant is anticipated to further increase editing.Table 20. Editing Efficiencies Using nSpCas9-variant RT_C Polypeptides at Exemplary Targets, as Measured by NGSFor RT_C, when fused to nSpCas9, many of the combined substitutions showed increased editing over the single substitution T70R. The highest activity on VEGFA-T3 was achieved using the A25R T70R F275Y R110H variant, while the highest level of the 50 bp insertion at the AAVS1 locus was achieved without A25R. Combining additional substitutions from Table 16 (e.g., T32R, N117R, G31R, V38R, and E88R) with the T70R F275Y R110H variant is anticipated to further increase editing. Table 21. Exemplary Reverse Transcriptase (RT) VariantsAny of the RT variants provided in the above Table is within the scope of the present disclosure.OTHER EMBODIMENTSAll of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.EQUIVALENTSWhile several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims andequivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”“Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

Claims

WHAT IS CLAIMED IS:

1. A reverse transcriptase (RT) polypeptide, which comprises a polymerase domain, wherein the polymerase domain comprises a fingers subdomain and a palm subdomain; wherein the RT polypeptide comprises a YXDD motif, in which X represents a naturally-occurring amino acid residue, optionally wherein the YXDD motif is YADD (SEQ ID NO: 58); and wherein the RT polypeptide has a length of about 250-350 amino acids; optionally wherein the polymerase domain is free of a thumb subdomain, a connection subdomain, or a combination thereof; and optionally wherein the RT polypeptide is free of an RNase domain.

2. The RT polypeptide of claim 1, which comprises an amino acid sequence at least 85% identical to SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4; optionally wherein the RT polypeptide comprises at least one mutation relative to SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

3. The RT polypeptide of claim 2, comprising an amino acid sequence at least 90% identical to SEQ ID NO: 2 and one or more mutations relative to SEQ ID NO: 2; wherein the one or more mutations comprise one or more amino acid residue substitutions, one or more deletions, or a combination thereof.

4. The RT polypeptide of claim 3, wherein the one or more amino acid residue substitutions are arginine and / or lysine substitutions, optionally arginine substitutions, at one or more of positions Vll, G12, S13, A24, D28, S35, N36, P55, E59, A60, P74, A83, H84, E117, S128, T153, T163, G166, S169, G175, Q182, A186, L199, E222, E225, E269, and A281 in SEQ ID NO: 2.

5. The RT polypeptide of claim 3 or claim 4, wherein RT polypeptide comprises or fiirther comprises one or more non-arginine or lysine amino acid residue substitutions at one or more of positions 120, V92, A100, Q102, V104, Fl 16, SI 19, Y121, G122, Q142, H146, Y155, G157, 1159, T163, 1172, A188, D189, S190, A191, T192, V202, F220, F238, V240, L241, and L287 of SEQ ID NO: 2; optionally wherein the substitutions are I20V, V92I, A100V, Q102T, V104I, F116L, S119C, Y121F, G122A, Q142L, H146W, Y155F, G157D, I159V, T163L, I172V, A188G, D189V, S190W, A191D, T192G, V202I, F220L, F238L, V240L, L241M, and / or L287F.

6. The RT polypeptide of claim 3, wherein the RT polypeptide comprises, relative to SEQ ID NO: 2,(i) one or more arginine and / or lysine substitutions at one or more of positions A24, N36, H84, and El 17; optionally A24R, N36R, H84R, and / or E117R; or(ii) a non-arginine / lysine substitution at position QI 42, optionally Q142L.

7. The RT polypeptide of any one of claims 3-6, wherein the one or more deletions comprise deletions within residues 1-12 of SEQ ID NO: 2, within residues 295-316 of SEQ ID NO: 2, or a combination thereof.

8. The RT polypeptide of claim 7, wherein the one or more deletions comprise a deletion of amino acid residues 1-12 of SEQ ID NO: 2, a deletion of amino acid residues 1-8 of SEQ ID NO: 2, a deletion of amino acid residues 298-316 of SEQ ID NO: 2, and / or a deletion of amino acid residues 295-316 of SEQ ID NO: 2; optionally wherein the RT polypeptide is a truncated version of SEQ ID NO: 2 having amino acid residues 13-297 of SEQ ID NO: 2 or amino acid residues 9-294 of SEQ ID NO: 2.

9. The RT polypeptide of claim 3, wherein the RT polypeptide is a truncated version of SEQ ID NO: 2 comprising amino acid residues 13-297 of SEQ ID NO: 2 and an arginine or lysine substitution as position A24, optionally A24R.

10. The RT polypeptide of claim 9, which further comprises substitutions at Y155, Al 88, and / or V202, optionally Y155F, Al 88G, and / or V202I.

11. The RT polypeptide of claim 3, wherein the RT polypeptide is a truncated version of SEQ ID NO: 2 comprising amino acid residues 13-297 of SEQ ID NO: 2 and comprises A24R and V202I substitutions relative to SEQ ID NO: 2.

12. The RT polypeptide of any one of claims 9-11, which farther comprises arginine and / or lysine substitutions at positions S35, E117, T153, A186, and / or E222 in SEQ ID NO: 2, optionally S35R, E117R, T153R, A186R, and / or E222R.

13. The RT polypeptide of claim 3, wherein the RT polypeptide comprises the following features:(i) the substitution of A24R;(ii) the substitutions of A24R, N36R, H84R, and Q142L;(iii) the substitutions of A24R, N36R, H84R, and Q142L, and the deletions of residues 1-12 and residues 298-316 of SEQ ID NO: 2; or(iv) the substitution of El 17R and the deletions of residues 1-12 and residues 298-316 of SEQ ID NO: 2.

14. The RT polypeptide of claim 3, wherein the RT polypeptide is the variant of SEQ ID NO: 2, the variant comprising (a) amino acid substitutions at positions A24, Y155, Al 88, V202, and L287 of SEQ ID NO: 2, optionally A24R, Y155F, A188G, V202I, and L287F; and (b) deletions of residues 1-12 and 298-316 of SEQ ID NO: 2.

15. The RT polypeptide of claim 3, wherein the RT polypeptide comprises the mutations listed in Table 18 relative to SEQ ID NO: 2; optionally wherein the RT polypeptide comprises the amino acid sequence of any of the RT_A variants listed in Table 21.

16. The RT polypeptide of claim 2, comprising an amino acid sequence at least 90% identical to SEQ ID NO: 3 and one or more mutations relative to SEQ ID NO: 3; wherein the one or more mutations comprise one or more amino acid residue substitution, one or more deletions, or a combination thereof.

17. The RT polypeptide of claim 16, wherein the one or more amino acid residue substitutions are arginine and / or lysine substitutions, optionally arginine substitutions, at one or more of positions Q14, E26, Q30, Q32, Q71, N121, L122, N126, K130, C133, S193, E291, and A292 in SEQ ID NO: 3.

18. The RT polypeptide of claim 16 or claim 17, wherein the one or more deletions comprise deletions within residues 1-5 of SEQ ID NO: 3, residues 294-338 of SEQ ID NO: 2, or a combination thereof; optionally wherein the RT polypeptide is a truncated version of SEQ ID NO: 3 having amino acid residues 6-293 of SEQ ID NO: 3 or a variant thereof comprising one or more mutations.

19. The RT polypeptide of claim 16, wherein the RT polypeptide comprises arginine and / or lysine substitutions at positions Q32, N121, and / or E291 of SEQ ID NO: 3;optionally Q32R, N121R and E291R.

20. The RT polypeptide of claim 16 or claim 19, which is a truncated version of SEQ ID NO: 3 comprising the deletions of residues 1-5 and residues 294-338 of SEQ ID NO: 3.

21. The RT polypeptide of claim 19 or claim 20, which further comprises arginine and / or lysine substitutions at positions Q30, G39, Q71, L122, and / or N126 of SEQ ID NO: 3; optionally Q30R, G39R, Q71R, L122R, and / or N126R.

22. The RT polypeptide of claim 16, wherein the RT polypeptide comprises the following features:(i) the substitution of E291R;(ii) the substitution of E291R and the deletions of residues 1-5 and residues 294-338 of SEQ ID NO: 3;(iii) the substitutions of Q32R, N121R, and E291R; or(iv) the substitutions of Q32R, N121R, and E291R and the deletions of residues 1-5 and residues 294-338 of SEQ ID NO: 3.

23. The RT polypeptide of any one of claims 16-22, wherein the one or more amino acid substitutions comprise or further comprise one or more non-arginine / lysine substitutions, optionally at one or more of positions S63, A114, N121, and L257 of SEQ ID NO: 3, optionally S63G, A114C, N121G, and / or L257L.

24. The RT polypeptide of claim 16, wherein the RT polypeptide is a variant of SEQ ID NO: 3, the variant comprising arginine or lysine substitutions at positions Q32 and Q71 of SEQ ID NO: 3, optionally arginine substitutions Q32R and Q71R; and deletions of residues 1-5 and 294-338 of SEQ ID NO: 3.

25. The RT polypeptide of claim 16, wherein the RT polypeptide comprises the mutations listed in Table 19 relative to SEQ ID NO: 3; optionally wherein the RT polypeptide comprises the amino acid sequence of any one of the RT_B variants listed in Table 21.

26. The RT polypeptide of claim 2, comprising an amino acid sequence at least 90% identical to SEQ ID NO: 4 and one or more mutations relative to SEQ ID NO: 4; wherein the one or more mutations comprise one or more amino acid residue substitution, one or more deletions, or a combination thereof.

27. The RT polypeptide of claim 26, wherein the one or more amino acid residue substitutions are arginine and / or lysine substitutions, optionally arginine substitutions, at one or more of positions E24, A35, A28, G31, T32, P34, G36, V38, T51, T70, E56, G73, A85, E88, H102, N117, H145, S218, F275, P296 in SEQ ID NO: 4; optionally wherein the one or more arginine substitutions are at one or more of positions E24, A35, A28, G31, T32, P34, G36, V38, T70, G73, A85, E88, H102, N117, H145, S218, and F275, and optionally wherein the lysine substitution is at position P296.

28. The RT polypeptide of any one of claims 26-27, wherein RT polypeptide comprises or farther comprises one or more non-arginine / lysine amino acid residue substitutions at one or more of positions F25, E52, K48, G73, L83, A93, N100, N117, R110, L121, A127, G154, V170, M172, D180, K192, V195, A202, M213, M234, 1252, H266, T286, V287, S297, P298, and P299 in SEQ ID NO: 4; optionally wherein the substitutions are F25Y, E52P, K66P, G73N, L83V, A93V, N100L, N117E, R110H or R1 ION, L121 V, A127E, G154D, M172F, V170Y, D180P, K192I, V195I, A202S, M213L, M234F, I252L, H266Y, T286D, V287D, S297G, P298S, and / or P299S.

29. The RT polypeptide of any one of claims 26-28, wherein the one or more deletions comprise deletions within residues 1-4 of SEQ ID NO: 4, within residues 297-350 of SEQ ID NO: 4, optionally within residues 300-327 of SEQ ID NO: 4, or a combination thereof; optionally wherein the RT polypeptide is a truncated version of SEQ ID NO: 4 having amino acid residues 5-296 of SEQ ID NO: 4 or a deletion variant having the fragment of residues 300-327 of SEQ ID NO: 4 deleted.

30. The RT polypeptide of claim 26, which comprises an arginine or lysine substitution at T70 of SEQ ID NO: 4, optionally T70R, and one or more additional amino acid residue substitutions.

31. The RT polypeptide of claim 30, wherein the one or more additional amino acid residue substitutions are at positions A25, R110, and / or F275 of SEQ ID NO: 4, optionally wherein the substitutions comprise A25R, R110H, and F275Y.

32. The RT polypeptide of claim 30 or claim 31 , which further comprises one or more arginine and / or lysine substitutions at positions T32, G31, V38, E88, and / or Nil 7 of SEQ ID NO: 4; optionally T32R, G31R, V38R, E88R, and / or N117R.

33. The RT polypeptide of claim 26, wherein the RT polypeptide comprises amino acid substitutions at positions S297, P298, and P299 of SEQ ID NO: 4, optionally S297G, P298S, and P299S; and the deletion of residues 300-327 of SEQ ID NO: 4.

34. The RT polypeptide of claim 26, wherein the RT polypeptide comprises the following features:(i) the substitution of T70R;(ii) the substitution of T70R and the deletions of residues 1-4 and residues 297-350 of SEQ ID NO: 4;(iii) the substitutions of T70R, S297G, P298S, P299S, and the deletion of residues 300-327 of SEQ ID NO: 4;(iv) amino acid substitutions at positions T32 and N117 of SEQ ID NO: 4, optionally T32R and N117R or N117E, amino acid substitutions at positions S297, P298, and P299 of SEQ ID NO: 4, optionally S297G, P298S, and P299S; and the deletion of residues 300-327 of SEQ ID NO: 4(v) the substitution of T32R; or(vi)the substitution of T32R and the deletions of residues 1-4 and residues 297-350 of SEQ ID NO: 4.

35. The RT polypeptide of claim 26, wherein the RT polypeptide comprises the mutations listed in Table 20 relative to SEQ ID NO: 4; optionally wherein the RT polypeptide comprises the amino acid sequence of any one of the RT_C variants listed in Table 21.

36. A fiision polypeptide, comprising an RT polypeptide set forth in any one of claims 1-35 and an RNA-guided nuclease; optionally wherein the RT polypeptide lacks its N-terminus methionine residue in the fusion polypeptide.

37. The fasion polypeptide of claim 36, wherein the RNA-guided nuclease is a nickase.

38. The fasion polypeptide of claim 36 or claim 37, wherein the RNA-guided nuclease is a Type II CRISPR nuclease.

39. The fasion polypeptide of claim 38, wherein the Type II CRISPR nuclease is a Cas9 nuclease, which optionally is a Cas9 nickase variant.

40. The fasion polypeptide of claim 36 or claim 37, wherein the RNA-guided nuclease is a Type V CRISPR nuclease.

41. The fasion polypeptide of claim 40, wherein the Type V CRISPR nuclease is Casl2i2.

42. The fasion polypeptide of any one of claims 36-41, which farther comprises one or more nuclear localization signals (NLS), one or more peptide linkers, or a combination thereof.

43. A nucleic acid, comprising a nucleotide sequence encoding an RT polypeptide set forth in any one of claims 1 -36 or encoding a fusion polypeptide set forth in any one of claims 36-41.

44. The nucleic acid of claim 43, which is in a vector, which optionally is a viral vector.

45. A gene editing system comprising:(a) an RT polypeptide set forth in any one of claims 1-35 or a first nucleic acid encoding the RT polypeptide;(b) an RNA-guided nuclease or a second nucleic acid encoding the RNA- guided nuclease;(c) a guide RNA (gRNA) or a third nucleic acid encoding the gRNA;wherein the gRNA comprises a scaffold sequence recognizable by the RNA-guided nuclease and a spacer sequence specific to a target sequence within a genomic site of interest, the targeting sequence being adjacent to a cognate protospacer adjacent motif (PAM) sequence of the RNA-guided nuclease; and(d) a reverse transcription donor RNA (RT donor RNA) or a four nucleic acid encoding the RT donor RNA; wherein the RT donor RNA comprises a primer binding site (PBS) and a reverse transcription template (RTT) sequence.

46. The gene editing system of claim 45, wherein the RT polypeptide and the RNA-guided nuclease form a fusion polypeptide.

47. The gene editing system of claim 46, wherein the fusion polypeptide is set forth in any one of claims 37-42.

48. The gene editing system of claim 46 or claim 47, wherein the gene editing system comprises a nucleic acid encoding the fusion polypeptide.

49. The gene editing system of claim 48, wherein the nucleic acid is a DNA.

50. The gene editing system of claim 48, wherein the nucleic acid is a messenger RNA (mRNA).

51. The gene editing system of any one of claims 45-50, which comprises an RNA molecule comprising the gRNA and the RT donor RNA.

52. The gene editing system of any one of claims 45-51, wherein the template sequence in the RT donor RNA is homologous to the genomic site of interest and comprises one or more nucleotide variations relative to the genomic site of interest.

53. The gene editing system of claim 52, wherein at least one nucleotide variation is located within the target sequence; and / or wherein at least one nucleotide variation is located in the PAM sequence.

54. The gene editing system of any one of claims 51-53, wherein the RNA molecule further comprises a protecting sequence, which optionally is located at the 5’ end of the RNA molecule.

55. The gene editing system of any one of claims 45-54, wherein the gene editing system comprises one or more lipid excipients associated with one or more of elements (a)- (d); optionally wherein the lipid excipients form lipid nanoparticles; and / or wherein the gene editing system comprises one or more viral particles for producing one or more of elements (a)-(d).

56. A pharmaceutical composition comprising the gene editing system of any one of claims 45-55.

57. A kit comprising the elements (a)-(d) of the gene editing system set forth in any one of claims 45-55.

58. A gene editing method, comprising contacting the gene editing system of any one of claims 45-55 with host cells to allow for gene editing of a gene targeted by the gene editing system.

59. The gene editing method of claim 58, wherein the host cells are cultured in vitro.

60. The gene editing method of claim 59, wherein the host cells are located in a subject who needs gene editing of the target gene.