Compact gene editors for delivery by adeno-associated viral vectors

Abstract

A fusion protein comprising (a) a nuclease polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and (b) a reverse transcriptase (RT) polypeptide comprising a polymerase domain, which 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. Also provided herein are gene editing systems comprising the nuclease polypeptide and the RT polypeptide for use in nuclease-guided reverse transcription-mediated gene editing.

Description

Attorney Docket No.: 063586-545001 WOCOMPACT GENE EDITORS FOR DELIVERY BY ADENO-ASSOCIATED VIRAL VECTORSCROSS REFERENCE TO RELATED APPLICATIONSThis application claims the benefit of the filing dates of U.S. Provisional Application No. 63 / 878,613, filed September 9, 2025, U.S. Provisional Application No. 63 / 788,492, filed April 14, 2025, and U.S. Provisional Application No. 63 / 701,014, filed September 30, 2024, the entire contents of each of which are 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 September 29, 2025, is named 063586-545001WO_SeqListing_ST26.xml and is 663,946 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 enzymes that generate a strand of DNA that is complementary to an RNA or DNA template. The combination of reverse transcriptases and CRISPR / Cas systems has shown great potential in genetic editing. Nuclease-guided reverse transcription allows for introduction of desired nucleotide substitutions at a genomic site. However, delivering the gene editing nucleases and RT transcriptases together to particular host cells for gene editing is challenging due to the large sizes of the enzymes and the size constraints of some delivery technologies (e.g. AAV-mediated delivery).It is therefore of great interest to develop efficient, accurate, and easy-to-deliver reverse transcriptase-nuclease 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 nucleases (e.g., RNA-guided nucleases such as CRISPR nucleases) and RT enzymes, the fusion polypeptides of which exhibited superior gene editing efficiencies and accuracies. Further, the coding sequences of such fusion polypeptides are small enough to fit into the adeno-associated viralAttorney Docket No.: 063586-545001 WO vectors along with the editing template RNA for delivering a gene editing system into target host cells to implement the designed gene editing.Accordingly, some aspects of the present disclosure feature a fusion protein comprising (a) a nuclease polypeptide, and (b) a reverse transcriptase (RT) polypeptide. The nuclease polypeptide comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. Each of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 comprises an HNH nuclease domain and an RuvC nuclease domain. The RT polypeptide comprises a polymerase domain, which comprises a fingers subdomain and a palm subdomain. The RT polypeptide comprises a YXDD motif, in which X represents a naturally-occurring amino acid residue, for example, YADD (SEQ ID NO: 70). In some embodiments, the RT polypeptide has a length of about 250-350 amino acids.In some embodiments, the nuclease polypeptide is a variant of SEQ ID NO: 1 (Nuclease A), comprising the following feature relative to SEQ ID NO: 1 : (i) one or more mutations in the HNH nuclease domain or in the RuvC nuclease domain; (ii) one or more arginine and / or lysine substitutions (non-arginine / lysine); (iii) one or more deletions and / or insertions, optionally an N-terminal truncation; (iv) one or more non-arginine and / or lysine amino acid substitutions; or (v) a combination of (i), (ii), (iii); and / or (iv).In some examples, the variant of SEQ ID NO: 1 comprises the one or more mutations in the HNH nuclease domain or in the RuvC nuclease domain, e.g., at positions D396, H397, N420, D24, E337, and / or D524 of SEQ ID NO: 1. In some instances, the variant of SEQ ID NO: 1 comprises a mutation at position H397 of SEQ ID NO: 1 (e.g., H397A, H397R, or H397K, H397W, or H397Y).Alternatively, or in addition, the variant of SEQ ID NO: 1 comprises or further comprises one or more arginine or lysine substitutions at one or more of positions D56, E59, G60, E63, 167, G71, T98, E102, E206, V210, R258, R259, R261, E490, E491, E495, W508, 1519, D533, D536, S564, D568, W593, T605, E655, E694, E706, G713, R720 in SEQ ID NO: 1. In some examples, the one or more arginine substitutions are at one or more of positions D56, E59, G60, E63, 167, G71, E206, E491, S564, D568, W593, T605, E655, E694, and E706 in SEQ ID NO: 1. In other examples, the one or more lysine substitutions are at one or more of positions T98, E102, V210, R258, R259, R261, E490, E491, E495, W508, 1519, D533, D536, G713, and R720 in SEQ ID NO: 1. In one specific example, the variant of SEQ ID NO: 1 comprises or further comprises arginine and / or lysine substitutions at positions 167, D568, and E706 of SEQ ID NO: 1 (e.g, arginine substitutions I67R, D568R, and E706R). In another specific example, the variant of SEQ ID NO: 1 comprises or further comprises arginine and / orAttorney Docket No.: 063586-545001 WO lysine substitutions at positions 167, D568, W593, and E706 of SEQ ID NO: 1 (e.g., I67R, D568R, W593R, and E706R).Alternatively, or in addition, the variant of SEQ ID NO: 1 comprises or further comprises the N-terminal truncation, which is a deletion within residues 1-15 of SEQ ID NO: 1. In some examples the N-terminal truncation is the deletion of residues 1-14 or 1-15 of SEQ ID NO: 1.In one example, the variant of SEQ ID NO: 1 comprises, relative to SEQ ID NO: 1 : (a) a mutation at position H397 of SEQ ID NO: 1 (e.g., H397A, H397K, H397W, or H397Y); (b) arginine and / or lysine substitutions at positions 167, D568, and E706 of SEQ ID NO: 1 (e.g., I67R, D568R, and E706R); and (c) a deletion of residues 1-15 of SEQ ID NO: 1. Alternatively, the variant of SEQ ID NO: 1 comprises, relatively to SEQ ID NO: 1, (a) a mutation at position H397 (e.g., H397A); (b) arginine and / or lysine substitutions at positions 167, D568, W593, and E706 (e.g., I67R, D568R, W593R, and E706R); (c) and a deletion of residues 1-15 of SEQ ID NO: 1.Alternatively, or in addition, the variant of SEQ ID NO: 1 comprises or further comprises, relative to SEQ ID NO: 1 : (i) one or more non-arginine and / or lysine amino acid substitutions at one or more of positions Q72, K76, T83, N87, 196, T98, E102, Cl 11, S134, E137, E144, A174, D225, L232, E241, T255, F265, F275, K288, E300, A307, 1406, V321, 1335, K348, E367, K366, E367, T391, 1406, E418, L434, H478, M479, E495, Q497, 1500, W508, D533, S548, T562, R567, R568, S570, N571, T574, P575, Y577, S587, L588, T596, E603, 1607, S608, Y611, K613, W622, N667, T673, L684, R699, S729, V733, R755, R757, and T766 of SEQ ID NO: 1; (ii) one or more deletions, (iii) one or more insertions, optionally an insertion between position 538 and 539 of SEQ ID NO: 1; or a combination of (i), (ii), and / or (iii).In some examples, the non-arginine and / or lysine amino acid substitution(s) is Q72S, K76M or K76V, T83A, T83N, T83S, or N87S, I96T, T98N, Cl 11 A, S134D, E137D, E144A, A174L, D225N, L232I, E241L, T255E, F265E, F275D or F275G, K288E, E300Q, A307N, I406V, V321I, I335V, K348M, E367D, K366N, E367D, T391G or T391S, I406V, E418G, L434V, H478W, M479L, Q497V, I500T, S548N, T562S, R567M, R568I, R568M, S570G, N571S, T574S, P575A, Y577H, S587G, L588H, T596V, E603D, I607F, S608T, Y611D, K613E, W622F, N667D, T673S, L684P, R699S, S729G, R755S, R757S, V733I, T766V, and / or T766I. In specific examples, the non-arginine / lysine substitutions may be at one or more of the following positions in SEQ ID NO: 1 : K76, F265, F275, K288, E300, 1406, E367, E418, L434, H478, 1500, W508, T562, or R699. Exemplary amino acid substitutions include: K76V,Attorney Docket No.: 063586-545001 WOF265E, F275G, K288E, E300Q, I406V, E367D, E418G, L434V, H478W, I500T, W508K, T562S, and / or R699S.In some examples, the deletion can be Q277 and / or positions E206-D207 of SEQ ID NO: 1. In some examples, the insertion is L between position 538 and 539 of SEQ ID NO: 1.In some specific examples, the variant of SEQ ID NO: 1 comprises: (a) a mutation at position H397 of SEQ ID NO: 1 (e.g., H397A); (b) arginine and / or lysine substitutions at positions 167, D568, W593, and E706 of SEQ ID NO: 1 (e.g., I67R, D568R, W593R, and E706R); (c) a deletion of residues 1-15 of SEQ ID NO: 1; and (d) a non-arginine / lysine substitutions at the following positions relative to SEQ ID NO: 1 : T83, N87, D225, R261, P575, W622, and S729 (e.g, T83N, N87S, D225N, R261K, P575A, W622F, and / or S729G). Such a variant of SEQ ID NO: 1 may further comprise the non-arginine / lysine substitutions at the following positions relative to SEQ ID NO: 1 : W508, T562, or R699 (e.g, W508K, T562S, or R699S).In one example, the variant of SEQ ID NO: 1 in the fusion protein disclosed herein may comprise: (a) a mutation in the RuvC nuclease domain, which is at position H397 of SEQ ID NO: 1 (e.g., H397A, H397K, H397R, H397W, or H397Y); (b) arginine or lysine substitutions at positions 167, D568, W593, and E706 of SEQ ID NO: 1 (e.g., I67R, D568R, W593R, and E706R); (c) amino acid substitutions at positions T83, N87, D225, R261, P575, W622, and S729 of SEQ ID NO: 1 (e.g., T83N, N87S, D225N, R261K, P575A, W622F, and S729G); and (d) deletion of residues 1-15 of SEQ ID NO: 1.In some instances, such a variant of SEQ ID NO: 1 may further comprise (a) an arginine / lysine substitution at one or more of the following positions relative to SEQ ID NO: 1 : E206, E490, E491, W508, 1519, and G713 (e.g., E206R, E490K, E491K or E491R, W508K, I519K, and / or G713K); (b) one or more non-arginine / lysine substitutions at one or more of the following positions relative to SEQ ID NO: 1 : Q72, K76, T255, F265, F275, K288, E300, E367, 1406, E418, L434, H478, 1500, W508, T562, L588, E603, 1607, S608, Y611, N667, L684, and R699 (e.g., Q72S, K76V, T255E, F265E, F275G, K288E, E300Q, E367D, I406V, E418G, L434V, H478W, I500T, W508K, T562S, L588H, E603D, I607F, S608T, Y611D, N667D, L684P, and / or R699S); or (c) a combination of (a) and (b).In specific examples, the variant of SEQ ID NO: 1 disclosed herein may comprise: (a) a mutation in the RuvC nuclease domain, which is at position H397 of SEQ ID NO: 1 (e.g., H397A, H397K, H387R, H397W, or H397Y); (b) arginine or lysine substitutions at positions 167, D568, W593, and E706 of SEQ ID NO: 1 (e.g., I67R, D568R, W593R, and E706R); (c) amino acid substitutions at positions T83, N87, D225, R261, P575, W622, and S729 of SEQAttorney Docket No.: 063586-545001 WOID NO: 1, optionally T83N, N87S, D225N, R261K, P575A, W622F, and S729G; and (d) deletion of residues 1-15 of SEQ ID NO: 1. Such a variant may further comprise one or more amino acid substitutions at one or more of positions E206, R258, R259, F275, K288, L434, E490, E491, L588, 1607, S608, Y611, N667, L684, and R699 of SEQ ID NO: 1 (e.g., E206R, R258K, R259K, F275G, K288E, L434V, E490K, E491R, L588H, I607F, S608T, Y611D, N667D, L684P, and R699S). In one specific example, the one or more further amino acid substitutions are at positions E206, E490, E491, and R699 (e.g., E206R, E490K, E491R, and / or R699S).Any of the variant of Nuclease A (SEQ ID NO: 1) may comprise an amino acid sequence at least 95%, optionally at least 98%, identical to SEQ ID NO: 1. In specific examples, the Nuclease A variant comprises (e.g., consists of) an amino acid sequence of any one of SEQ ID NOs: 90, 134, 148, 153, 163, 428-443, and 469-480. Any of the Nuclease A variants disclosed herein is also with the scope of the present disclosure.In some embodiments, the nuclease polypeptide in any of the fusion polypeptides disclosed herein is a variant of SEQ ID NO: 2 (Nuclease K), the variant comprising the following feature relative to SEQ ID NO: 2: (i) one or more mutations in the HNH nuclease domain or in the RuvC nuclease domain of SEQ ID NO: 2; (ii) one or more arginine and / or lysine substitutions; (iii) one or more deletions, optionally an N-terminal truncation; or (iv) a combination of any one of (i)-(iii).In some examples, the variant of SEQ ID NO: 2 comprises one or more mutations in the HNH nuclease domain or in the RuvC nuclease domain at positions D374, H375, and / or N398 in SEQ ID NO: 2. In some instances, the mutation is at position H375 (e.g., H375A).Alternatively, or in addition, the variant of SEQ ID NO: 2 comprises or further comprises one or more arginine and / or lysine substitutions, optionally one or more arginine substitutions, at one or more of positions G42, D46, 153, F83, E128, E541, F570, E576, D582, C630, 1631, E683, S719, and T734 in SEQ ID NO: 2. In some examples, the variant of SEQ ID NO: 2 comprises or further comprises arginine and / or lysine substitutions at the following positions relative to SEQ ID NO: 2: G42 and D582, e.g., G42R and D582R. In some examples, the variant of SEQ ID NO: 2 comprises mutations H375A, G42R, and D582R relative to SEQ ID NO: 2.Any of the Nuclease K variants disclosed herein may comprise an amino acid sequence at least 95%, optionally 98%, identical to SEQ ID NO: 2. In some examples, the Nuclease K variant comprises (e.g., consists of) the amino acid sequence of SEQ ID NO: 12.In some embodiments, the nuclease polypeptide in the fusion polypeptide disclosedAttorney Docket No.: 063586-545001 WO herein is a variant of SEQ ID NO: 3 (Nuclease BT), comprising the following feature relative to SEQ ID NO: 3 : (i) one or more mutations in the HNH nuclease domain or in the RuvC nuclease of SEQ ID NO: 3; (ii) one or more arginine and / or lysine substitutions, optionally one or more arginine substitutions; (iii) one or more non-arginine / lysine amino acid substitutions; (iv) one or more deletions, optionally an N-terminal truncation; or (v) a combination of any one of (i)-(iv).In some examples, the variant of SEQ ID NO: 3 comprises one or more mutations in the HNH or RuvC nuclease domain, which are at positions H231, H232, H255, D67, E176, and / or D329 of SEQ ID NO: 3. In one example, the variant of SEQ ID NO: 3 comprises a mutation at position H232 of SEQ ID NO: 3 (e.g., H232A).Alternatively, or in addition, the variant of SEQ ID NO: 3 comprises or further comprises one or more arginine and / or lysine substitutions, optionally one or more arginine substitutions, at one or more of positions R31, N34, R39, E99, Q102, E105, T106, T113, K123, R135, R151, M186, E191, Q194, 1206, N239, V256, 1272, A277, K241, L292, K318, E401, E432, and V433 in SEQ ID NO: 3. In some instances, the one or more arginine substitutions are at one or more of positions E99, Q102, E105, T106, T113, K123, E191, 1206, N239, V256, 1272, A277, K241, K318, E401, E432, and V433 in SEQ ID NO: 3. Examples include R31, N34, R39, R135, R151, M186, Q194, and L292Q in SEQ ID NO: 3.In some specific examples, the variant of SEQ ID NO: 3 comprises or further comprises arginine and / or lysine substitutions at the following positions relative to SEQ ID NO: 3: Q102, 1206, and V433, for example, the Q102R, I206R, and V433R substitutions. In one specific example, the variant of SEQ ID NO: 3 comprises mutations H232A, Q102R, I206R, and V433R relative to SEQ ID NO: 3.Alternatively, or in addition, the variant of SEQ ID NO: 3 comprises or further comprises one or more non-arginine / lysine substitutions at one or more of positions R2, N14, H18, N34, D70, C75, R84, V85, L88, H94, 1100, R102, T106, M109, Y116, 1128, H131, P133, E166, V168, 1182, 1190, R206, R207, A208, A222, P227, F228, Q229, Q238, N239, K241, K257, T260, K263, Q269, 1272, S290, L292, L296, G301, V305, S313, D323, N336, V339, T343, V350, K353, V379, E401, K428, K429, E437, N445, D448, S454, C481. Examples include, but are not limited to, R2M, N14G, H18N, N34L, D70N, C75S, R84C, V85G, L88A, L88F, L88I, L88M, or L88V, H94G, HOOF, R102I or R102H, T106G, M109G, Y116N, H28E, H131Y, P133G, E166A, V168L, H82V, H90V, R207H, R207A, A208E, A222G, P227I, F228L, Q229E, Q238C, N239G, K241E, K241I, K241H, or K241Q, K257A, T260N, K263A, Q269L, I272N, S290P, L292N or L292Q, L296A, L296G, L296P, or L296V, G301P, V305I,Attorney Docket No.: 063586-545001 WOS313T, D323P, N336C, V339I, T343V, V350F, K353E or K353N, V379I, E401A, K428N or K428S, K429G, E437P, N445G, D448G, S454T, C481V.In some examples, the variant of SEQ ID NO: 3 comprises (a) arginine and / or lysine substitutions at positions Q102, 1206, and V433 of SEQ ID NO: 3 (e.g., Q102R, I206R, and V433R); (b) a non-arginine / lysine substitution at position L292 of SEQ ID NO: 3 (e.g., L292Q); and optionally (c) a substitution at position H232 of SEQ ID NO: 3 (e.g., H232A).Any of the Nuclease BT variants disclosed herein may comprise an amino acid sequence at least 95%, optionally at least 98%, identical to SEQ ID NO: 3. In one example, the Nuclease BT variant comprises (e.g., consisting of) the amino acid sequence of SEQ ID NO: 166. In another example, the Nuclease BT variant is a nickase variant of SEQ ID NO: 166, e.g., comprising the H232A substitution.Any of the nuclease polypeptides in the fusion protein may be free of its native N- terminus methionine.The RT polypeptide in the fusion polypeptide disclosed herein may comprise an amino acid sequence at least 90%, optionally at least 95% or at least 98%, identical to SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.In some embodiments, the RT polypeptide is a variant of SEQ ID NO: 4 (RT_C) comprising:(i) one or more arginine and / or lysine substitutions, optionally one or more arginine substitutions, at 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: 4;(ii) one or more 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: 4; optionally wherein the substitutions are I20V, V92I, A100V, Q102T, VI 041, Fl 16L, SI 19C, Y121F, G122A, Q142L, H146W, Y155F, G157D, I159V, T163L, I172V, A188G, D189V, S190W, A191D, T192G, V202I, F220L, F238L, V240L, L241M, and / or L287F;(iii) one or more deletions comprise deletions within residues 1-14, optionally 1-12, of SEQ ID NO: 4, within residues 295-316 of SEQ ID NO: 4, or a combination thereof; or(iv) a combination of any one of (i)-(iii).In some examples, the variant of SEQ ID NO: 4 comprises, relative to SEQ ID NO: 4: one or more arginine and / or lysine substitutions at one or more of positions A24, N36, H84, and El 17 of SEQ ID NO: 4 (e.g, A24R, N36R, H84R, and / or El 17R). In other examples, theAttorney Docket No.: 063586-545001 WO variant of SEQ ID NO: 4 comprises, relative to SEQ ID NO: 4: a non-arginine / lysine substitution at position Q142 of SEQ ID NO: 4 (e.g., Q142L).Alternatively, or in addition, the variant of SEQ ID NO: 4 further comprises the deletions of residues 1-12 and / or residues 298-316 of SEQ ID NO: 4.In some specific examples, the variant of SEQ ID NO: 4 comprises the following features: (i) the substitution of A24R; (ii) the substitutions of A24R, N36R, H84R, and Q142L,; or (iii) the substitutions of A24R, N36R, H84R, and Q142L, and the deletions of residues 1-12 and residues 298-316 of SEQ ID NO: 4. The nuclease polypeptide fusion partner for such a variant of SEQ ID NO: 4 may Nuclease A or a variant thereof, or Nuclease K or a variant thereof. In other specific examples, the variant of SEQ ID NO: 4 comprises mutation El 17R and the deletions of residues 1-12 and residues 298-316 of SEQ ID NO: 4. The nuclease polypeptide fusion partner for such a variant of SEQ ID NO: 4 may Nuclease BT or a variant thereof.In some specific examples, the variant of SEQ ID NO: 4 comprises (a) amino acid substitutions at positions A24, Y155, A188, V202, and L287 of SEQ ID NO: 4 (e.g., A24R, Y155F, A188G, V202I, and L287F); and (b) deletions of residues 1-12 and 298-316 of SEQ ID NO: 4. In one example, the variant of SEQ ID NO: 4 comprises (e.g., consisting of) the amino acid sequence of SEQ ID NO: 272.In some embodiments, the RT polypeptide is a variant of SEQ ID NO: 5 (RT_B) comprising: (i) one or more arginine and / or lysine substitutions, optionally one or more arginine substitutions, at positions Q14, E26, Q30, Q32, Q71, N121, L122, N126, K130, C133, SI 93, E291, and A292 in SEQ ID NO: 5; (ii) one or more deletions comprise deletions within residues 1-5 of SEQ ID NO: 5, within residues 294-338 of SEQ ID NO: 5, or a combination thereof; (iii) one or more non-arginine / lysine amino acid residue substitutions; or (iv) a combination of (i), (ii), and / or (iii). In some examples, the variant of SEQ ID NO: 5 comprises, relative to SEQ ID NO: 5: one or more arginine and / or lysine substitutions at one or more of positions Q32, N121, and E291 (e.g., Q32R, N121R, and / or E291R). Such a variant of SEQ ID NO: 5 may further comprise the deletions of residues 1-5 and residues 294-338 of SEQ ID NO: 5.In some examples, the variant of SEQ ID NO: 5 comprises the following features: (i) the substitution of E291R; or (ii) the substitution of E291R and the deletions of residues 1-5 and residues 294-338 of SEQ ID NO: 5. The nuclease polypeptide fusion partner of such a variant of SEQ ID NO: 5 can be Nuclease A or a variant thereof, or Nuclease K or a variant thereof. In other examples, the variant of SEQ ID NO: 5 comprises the following features: (i)Attorney Docket No.: 063586-545001 WO the substitutions of Q32R, N121R, and E291R; or (ii) the substitutions of Q32R, N121R, and E291R and the deletions of residues 1-5 and residues 294-338 of SEQ ID NO: 5. The nuclease polypeptide fusion partner of such a variant of SEQ ID NO: 5 can be Nuclease BT or a variant thereof.Alternatively, or in addition, the variant of SEQ ID NO: 5 comprises or further comprises one or more non-arginine / lysine amino acid residue substitutions at one or more of positions S63, Al 14, N121, and L257, for example, S63G, Al 14C, N121G, and / or L257L.In some specific examples, the variant of SEQ ID NO: 5 comprises arginine or lysine substitutions at positions Q32 and Q71 of SEQ ID NO: 5 (e.g., Q32R and Q71R); and deletions of residues 1-5 and 294-338 of SEQ ID NO: 5. In one example, the variant of SEQ ID NO: 5 comprises (e.g., consisting of) the amino acid sequence of SEQ ID NO: 290.In some embodiments, the RT polypeptide is a variant of SEQ ID NO: 6 (RT_C), comprising: (i) one or more arginine and / or lysine substitutions, optionally one or more arginine substitutions, at one or more of positions E24, T32, A35, A28, K29, G31, T32, P34, G36, V38, T51, T70, E56, K68, G73, A85, E88, N100, H102, N117, R122, H145, S218, F275, and P296 in SEQ ID NO: 6; optionally wherein the one or more arginine substitutions are at one or more of positions E24, T32, A35, A28, K29, G31, T32, P34, G36, V38, K68, T70, G73, A85, E88, H102, N117, H145, S218, and F275, and optionally wherein the lysine substitution is at position N100, N117, R122, and / or P296; (ii) one or more deletions comprise deletions within residues 1-10, optionally 1-7 or 1-4, of SEQ ID NO: 6, within residues 297-350 of SEQ ID NO: 6, deletion of position 50 in SEQ ID NO: 6, or a combination thereof; (iii) one or more non-arginine / lysine amino acid substitutions; or (iv) a combination of (i), (ii), and / or (iii).In some instances, the variant of SEQ ID NO: 6 comprises, relative to SEQ ID NO: 6, one or more arginine and / or lysine substitutions at one or more of positions K29, T32, K68, T70, N100, H102, N117, and R122 (e.g., K29R, T32R, K68R, T70R, N100K, H102R, N117R, and / or R122K). Alternatively, or in addition, the variant of SEQ ID NO: 6 comprises or further comprises (a) the deletions of residues 1-4 and residues 297-350 of SEQ ID NO: 6; (b) the deletion of residues 297-325 of SEQ ID NO: 6, or (c) deletion of residue 50 in SEQ ID NO: 6.In some examples, the variant of SEQ ID NO: 6 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: 6; or (iii) the substitution of T70R and the deletion of residues 297-325 of SEQ ID NO: 6. The nuclease polypeptide fusion partner of such an RT variant of SEQ ID NO: 6 can be Nuclease A or a variant thereof, or Nuclease K or a variant thereof. In other examples, the variant of SEQ ID NO: 6 comprises the following features: (i)Attorney Docket No.: 063586-545001 WO the substitution of T32R; or (ii) the substitution of T32R and the deletions of residues 1-4 and residues 297-350 of SEQ ID NO: 6. The nuclease polypeptide fusion partner of such a variant of SEQ ID NO: 6 can be Nuclease BT or a variant thereof.Alternatively, or in addition, the variant of SEQ ID NO: 6 comprises or further comprises the one or more non-arginine / lysine amino acid substitutions at one or more of positions H8, F25, R43, E52, K48, G73, L83, A93, N100, M101, F103, N117, R110, L121, A127, H145, D146, G154, V170, M172, D180, K192, V195, 1199, V201, A202, M213, M234, L249, 1252, H266, V268, T286, V287, S297, P298, and P299 of SEQ ID NO: 6. Examples include, but are not limited to, H8N, F25Y, R43N or R43S, E52P, K66P, G73N, L83V, A93V, N100L, M101 A, F103I, N117E, N117M or N117P, R110H or R110N, L121 V, A127E, H145E or H145S, D146A or D146M, G154D, M172F, V170Y, D180P, K192I, V195I, I199F. V201N, A202S, M213L, M234F,L249A, I252L, H266Y, V268M, T286D, V287D, S297G, P298S, and P299S.In some instances, the variant of the SEQ ID NO: 6 comprises or further comprises, relative to SEQ ID NO: 6, one or more non-arginine / lysine substitutions at the following positions: H8, R43, M101, F103, N117, H145, D146, 1199, V201, A202, L249, V268, S297, P298, and P299; optionally wherein the non-arginine / lysine substitution(s) is H8N, R43N or R43S, M101A, F103I, N117E, N117M, or N117P, H145S or H145E, D146A or D146M. I199F, V201N, A202S, L249A, V268M, S297G, P298S, and / or P299S.In some specific examples, the RT variant of SEQ ID NO: 6 comprises, relative to SEQ ID NO: 6: an amino acid substitution at position T70 of SEQ ID NO: 6 (e.g, T70R). In other specific examples, the RT variant of SEQ ID NO: 6 comprises amino acid substitutions at positions T32 and N117 of SEQ ID NO: 6 (e.g., T32R and N117R or N117E). Such an RT variant of SEQ ID NO: 6 may further comprise amino acid substitutions at positions S297, P298, and P299 of SEQ ID NO: 6 (e.g, S297G, P298S, and P299S); and / or the deletion of residues 300-327 of SEQ ID NO: 6.In some examples, the variant of SEQ ID NO: 6 comprises the following features: (a) arginine or lysine substitutions at positions T32 and N117 of SEQ ID NO: 6, optionally T32R and N117R or N117K; (b) non-arginine / lysine substitutions at positions S297, P298, and P299 of SEQ ID NO: 6, optionally S297G, P298S, and P299S; and (c) deletion of residues 300-327 of SEQ ID NO: 6. Such a variant may further comprise: (d) one or more arginine or lysine substitutions at one or more of positions W7, K29, N100, and H102, of SEQ ID NO: 6 (e.g., W7K, K29R, N100K, and / or H102R); (e) one or more non-arginine / lysine substitutions at one or more of positions F25, R43, L83, M101, F103, H145, D146, V153, 1199, V201, L249, andAttorney Docket No.: 063586-545001 WOT286 of SEQ ID NO: 6 (e.g., F25Y, R43E, L83I, M101 A, F103I, N117E, N117M, or N117P, H145S or H145E, D146A or D146M, V153T, I199F, V201N, L249A, and / or T286D); or (f) a combination of (d) and (e).In other examples, the variant of SEQ ID NO: 6 may comprise the following features: (a) arginine or lysine substitutions at positions T32 of SEQ ID NO: 6 (e.g., T32R), (b) non- arginine / lysine substitutions at positions S297, P298, and P299 of SEQ ID NO: 6, optionally S297G, P298S, and P299S; and (c) deletion of residues 300-327 of SEQ ID NO: 6. The variant of SEQ ID NO: 6 may further comprise a non-arginine or lysine substitution at position N117 of SEQ ID NO: 6, for example, N117E, N117M, or N117P. In some instances, the variant of SEQ ID NO: 6 may further comprise (d) one or more arginine or lysine substitutions at one or more of positions W7, K29, N100, and H102, of SEQ ID NO: 6 (e.g., W7K, K29R, N100K, and / or H102R); (e) one or more non-arginine / lysine substitutions at one or more of positions F25, R43, L83, M101, F103, H145, D146, V153, 1199, V201, L249, and T286 of SEQ ID NO: 6 (e.g, F25Y, R43E, L83I, M101A, F103I, , H145S or H145E, D146A or D146M, V153T, I199F, V201N, L249A, and / or T286D); or (f) a combination of (d) and (e).In some examples, the variant of SEQ ID NO: 6 further comprises a non-arginine / lysine substitution at position V201 (e.g, V201N), H145 (e.g., H145E), D146 (e.g., D146M, or N117 (e.g., N117M or N117P) of SEQ ID NO: 6. In some examples, the variant of SEQ ID NO: 6 further comprises arginine / lysine substitutions at positions N100 and Hl 02 (e.g., N100K and H102R) and non-arginine / lysine substitutions at positions M101 and F103 (e.g., M101A and F103I). In other examples, the variant of SEQ ID NO: 6 further comprises arginine / lysine substitutions at positions N117 (e.g., N117K) and non-arginine / lysine substitutions at positions H145 and D146 (e.g., H145S and D146A). In yet other examples, the non-arginine / lysine substitutions at positions H145 and D146 (e.g., H145E and D146M).In one example, the variant of SEQ ID NO: 6 comprises (e.g., consisting of) the amino acid sequence of SEQ ID NO: 26, 303, 329, 333, 444-466, and 481-491. Any of the SEQ ID NO: 6 variants (RT_C variants) disclosed herein is also within the scope of the present disclosure.Exemplary nuclease polypeptide for use in the fusion polypeptides disclosed herein may comprises any one of the amino acid sequences listed in Sequence Table 1. Alternatively, or in addition, exemplary RT polypeptides for use in the fusion polypeptides disclosed herein may comprise any one of the amino acid sequences listed in Sequence Table 2.In some instances, the fusion protein disclosed herein may comprise, from N-terminus to C-terminus: (i) the nuclease polypeptide comprising SEQ ID NO: 9 and the RT polypeptideAttorney Docket No.: 063586-545001 WO comprising SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 26, or SEQ ID NO: 27; (ii) the nuclease polypeptide comprising SEQ ID NO: 12 and the RT polypeptide comprising SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 26, or SEQ ID NO: 27; or (iii) the nuclease polypeptide comprising SEQ ID NO: 15 and the RT polypeptide comprising SEQ ID NO: 20, SEQ ID NO: 24, or SEQ ID NO: 28.In other instances, the fusion protein comprises, from N-terminus to C-terminus: (i) the RT polypeptide comprising SEQ ID NO: 17, SEQ ID NO: 21, or SEQ ID NO: 25 and the nuclease polypeptide comprising SEQ ID NO: 9; (ii) the RT polypeptide comprising SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 29 and the nuclease polypeptide comprising SEQ ID NO: 10; (iii) the RT polypeptide comprising SEQ ID NO: 17, SEQ ID NO: 21, or SEQ ID NO: 25 and the nuclease polypeptide comprising SEQ ID NO: 12; or (iv) the RT polypeptide comprising SEQ ID NO: 20, SEQ ID NO: 24, or SEQ ID NO: 28 and the nuclease polypeptide comprising SEQ ID NO: 15.In specific examples, the nuclease polypeptide and the RT polypeptide in the fusion polypeptide as disclosed herein comprises the following pair of amino acid sequences, respectively:(1) SEQ ID NO: 90 and SEQ ID NO: 303;(2) SEQ ID NO: 134 and SEQ ID NO: 329;(3) SEQ ID NO: 134 and SEQ ID NO: 333;(4) SEQ ID NO: 148 and SEQ ID NO: 329;(5) SEQ ID NO: 153 and SEQ ID NO: 329;(6) SEQ ID NO: 163 and SEQ ID NO: 329;(7) SEQ ID NO: 90 and SEQ ID NO: 290;(8) SEQ ID NO: 90 and SEQ ID NO: 272;(9) SEQ ID NO: 12 and SEQ ID NO: 26;(10) SEQ ID NO: 166 and SEQ ID NO: 303;(11) SEQ ID NO: 430 and SEQ ID NO: 329;(12) SEQ ID NO: 431 and SEQ ID NO: 444, or(13) SEQ ID NO: 431 and SEQ ID NO: 329.In some examples, the nuclease polypeptide in the fusion protein may be any one of SEQ ID NOs: 134, 148, 143, 163, 428-443, and 469-480 and the RT polypeptide in the fusion protein may be any one of SEQ ID NOs: 329, 333, 444-466, and 481-491.Any of the fusion proteins disclosed herein may further comprise one or more nuclear localization signals (NLSs); and / or one or more peptide linkers. In one example, the fusionAttorney Docket No.: 063586-545001 WO protein may comprise, from N-terminus to C-terminus, a first NLS, the nuclease polypeptide, a peptide linker, the RT polypeptide, and a second NLS. In another example, the fusion protein may comprise, from N-terminus to C-terminus, first NLS, the RT polypeptide, a peptide linker, the nuclease polypeptide, and a second NLS.Examples of fusion protein as disclosed herein are provided in Sequence Table 3. In these examples, the NLS fragments, peptide linkers, and protein tags are for illustration and / or research purposes only. The NLS fragment and peptide linkers can be replaced with other suitable ones as known to those skilled in the art. The protein tags can be removed.In other aspects, provided herein is a nucleotide sequence encoding a fusion protein as disclosed herein. The nucleic acid may be a vector, such as an expression vector.In yet other aspects, the present disclosure features an adeno-associated viral (AAV) vector, comprising a transgene, which comprises a first nucleotide sequence encoding a fusion protein as disclosed herein, the encoding nucleotide sequence being in operable linkage to a promoter; wherein the transgene is flanked by a 5’ - inverted terminal repeat (ITR) sequence and a 3 ’-ITR sequence. In some embodiments, the transgene further comprises a second nucleotide sequence encoding an editing template RNA.Also provided herein is an adeno-associated viral (AAV) particle, comprising an AAV genome, which comprises a transgene flanked by a 5’ - inverted terminal repeat (ITR) sequence and a 3 ’-ITR sequence; wherein the transgene comprises a first nucleotide sequence encoding a fusion protein disclosed herein, the encoding nucleotide sequence being in operable linkage to a promoter; and optionally a second nucleotide sequence encoding an editing template RNA.Further, the present disclosure provides a gene editing system comprising:(a) a nuclease polypeptide or a nucleic acid encoding such, the nuclease polypeptide comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; wherein each of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 comprises an HNH nuclease domain and an RuvC nuclease domain;(b) a reverse transcriptase (RT) polypeptide or a nucleic acid encoding such, wherein the RT polypeptide comprises a polymerase domain, which 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: 70); and wherein the RT polypeptide has a length of about 250- 350 amino acids;(c) a guide RNA (gRNA) or a nucleic acid encoding such; and(d) a reverse transcription donor RNA (RT donor RNA) or a nucleic acid encodingAttorney Docket No.: 063586-545001 WO such.In some instances, the gRNA comprises (i) a scaffold sequence recognizable by the nuclease polypeptide and (ii) a spacer sequence specific to a target sequence within a genomic site of interest. The target sequence is adjacent to a protospacer adjacent motif (PAM) specific to the nuclease polypeptide. In some instances, the RT donor RNA comprises a primer binding site (PBS) and a template sequence.In some examples, the gene editing system may comprise a fusion protein comprising the nuclease polypeptide of (a) and the RT polypeptide of (b), or a nucleic acid encoding the fusion protein. For example, the gene editing system may comprise any of the fusion proteins disclosed herein or a nucleic acid encoding such.In some instances, the gene editing system comprises a single editing template RNA comprising the gRNA of (c) and the RT donor RNA of (d), or a nucleic acid encoding the single editing template RNA. The single editing template RNA further comprises a linker and / or a 3’ extension. In some instances, the single editing template RNA comprises, from 5’ to 3’, the spacer sequence, the scaffold sequence, the template sequence, the PBS, the linker, and the 3’ extension.In some examples, the gene editing system may comprise an AAV vector as disclosed herein or an AAV particle as also disclosed herein.In some examples, the nuclease polypeptide involved in the gene editing system disclosed herein may be Nuclease A or a variant thereof. The corresponding PAM for such a nuclease polypeptide is 5’-NGG-3’, in which N represents any nucleotide, and the corresponding scaffold sequence comprises a nucleotide sequence at least 70% identical to SEQ ID NO: 63 or a fragment thereof. In some instances, the scaffold sequence comprises the nucleotide sequence of any one of SEQ ID NOs: 63-66.In some examples, the nuclease polypeptide involved in the gene editing system is Nuclease K or a variant thereof. The corresponding PAM for such a nuclease polypeptide is 5 ’ - NGG-3’, in which N represents any nucleotide, and the scaffold sequence can be a variant of SEQ ID NO: 66, comprising one or more deletions, one or more nucleotide substitutions, or a combination thereof, as compared with SEQ ID NO: 66. In some instances, the scaffold sequence has a length of about 100-120 nucleotides. In some examples, the scaffold sequence may comprise one or more of the following features relative to SEQ ID NO: 66, the scaffold sequence comprises: (i) a 3’ end deletion, optionally within nucleotides 126-179 of SEQ ID NO: 66; (ii) a deletion within nucleotides 8-31 of SEQ ID NO: 66; and (iii) one or more nucleotide substitutions.Attorney Docket No.: 063586-545001 WOIn some examples, the scaffold sequence for Nuclease K or a variant thereof (as well as for Nuclease A or a variant thereof) is at least 90% identical to SEQ ID NO: 65 and comprises one or more mutations relative to SEQ ID NO: 65. Specific examples include: Nuclease K Scaffold Variant 2, Nuclease K Scaffold Variant 7, Nuclease K Scaffold Variant 16, Nuclease K Scaffold Variant 28, Nuclease K Scaffold Variant 32, Nuclease K Scaffold Variant 33, Nuclease K Scaffold Variant 34, Nuclease K Scaffold Variant 36, Nuclease K Scaffold Variant 41, or Nuclease K Scaffold Variant 42. The nucleotide sequences of these exemplary scaffold sequences are provided in Sequence Table 4 below.Any of the Nuclease K scaffold variants as disclosed herein is also within the scope of the present disclosure.In other embodiments, the nuclease polypeptide involved in the gene editing system disclosed herein is Nuclease BT or a variant thereof. The PAM for such a nuclease polypeptide is 5’-RRT-3’, or 5’-NRT-3’, in which R represents A, and N represents any nucleotide; and the scaffold sequence comprises a nucleotide sequence at least 70% identical to SEQ ID NO: 67. In some examples, the scaffold sequence comprises the nucleotide sequence of any one of SEQ ID NOs: 67-69 and 78.Also provided herein are a pharmaceutical composition comprising the gene editing system disclosed herein and a kit comprising the elements of the gene editing system.Moreover, the present disclosure features a gene editing method, comprising contacting the gene editing system as disclosed herein with host cells to allow for editing of a gene targeted by the gene editing system. In some embodiments, the host cells are cultured in vitro. In other embodiments, the host cells are located in a subject who needs gene editing of the target gene. In some examples, the gene editing system for use in the method comprises the AAV particle as also disclosed herein.Also with the scope of the present disclosure is a gene editing system as disclosed herein for use in modifying a target gene in host cells, as well as use of such a gene editing system for manufacturing a medicament for genetically modifying a target gene in a subject so as to treat diseases associated with 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.Attorney Docket No.: 063586-545001 WOBRIEF DESCRIPTION OF THE DRAWINGSThe following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawing in combination with the detailed description of specific embodiments presented herein.FIGS. 1A-1O are schematic illustrations depicting predicted secondary structures of exemplary scaffold sequences. FIG. 1A: Nuclease A Reference Scaffold (SEQ ID NO: 63). FIG. IB: Nuclease A Truncated Reference Scaffold (SEQ ID NO: 64). FIG. 1C: Nuclease K Scaffold Variant 1 (SEQ ID NO: 65). FIG. ID: Nuclease Reference Scaffold (SEQ ID NO: 66). FIG. IE: Nuclease K Scaffold Variant 2 (SEQ ID NO: 348). FIG. IF: Nuclease K Scaffold Variant 7 (SEQ ID NO: 353). FIG. 1G: Nuclease K Scaffold Variant 16 (SEQ ID NO: 362). FIG. 1H: Nuclease K Scaffold Variant 28 (SEQ ID NO: 374). FIG. II: Nuclease K Scaffold Variant 32 (SEQ ID NO: 378). FIG.1J: Nuclease K Scaffold Variant 33 (SEQ ID NO: 379). FIG. IK: Nuclease K Scaffold Variant 34 (SEQ ID NO: 380). FIG. IL: Nuclease K Scaffold Variant 36 (SEQ ID NO: 382). FIG. IM: Nuclease K Scaffold Variant 41 (SEQ ID NO: 387). FIG. IN: Nuclease K Scaffold Variant 42 (SEQ ID NO: 388). FIG. IO: Nuclease K Scaffold Variant 43 (SEQ ID NO: 467).FIG. 2 is a diagram illustrating four exemplary AAV constructs. The vectors are depicted in the 5’ to 3’ orientation and illustrate the relative transcriptional orientation of the expression cassettes. Orientation 1 : Fusion Polypeptide Expression Cassette - Editing Template RNA Expression Cassette. Orientation 2: Fusion Polypeptide - Editing Template RNA Expression Cassette in the reverse transcriptional orientation. Orientation 3 : Editing Template RNA Expression Cassette - Fusion Polypeptide Expression Cassette. Orientation 4: Editing Template RNA Expression Cassette in the reverse transcriptional orientation - Fusion Polypeptide Expression Cassette.DETAILED DESCRIPTION OF THE INVENTIONThe present disclosure provides RNA-guided nucleases such as CRISPR nucleases and reverse transcriptases (RTs) for use in gene editing mediated by nuclease-guided reverse transcription, which allows for introduction of desired nucleotide substitutions and / or insertions at a genomic site of interest. The present disclosure also provides gene editing systems comprising the nuclease and the RT enzymes provided herein or a nucleic acid(s) encoding such, as well as necessary RNA components such as a guide RNA and a RT donor RNA or encoding nucleic acids thereof for use in genetic editing target genes, for example,Attorney Docket No.: 063586-545001 WO introducing nucleotide substitutions and / or inserting a nucleotide fragment at designed genomic sites. In some instances, the nuclease and the RT enzymes can be in fusion polypeptide format. Alternatively or in addition, the guide RNA and the RT donor RNA can form a single RNA molecule (z.e., editing template RNA). In some examples, the gene editing system provided herein may comprise AAV vectors or particles comprising coding sequences for the fusion polypeptide and optionally the editing template RNA.The nucleases reported herein have shown high gene editing efficiencies. When used as RT-fusion polypeptides in the gene editing systems provided herein, they have shown successful substitution of nucleotides at target sites. See Examples below. Such gene editing systems are expected to be effective in introducing desired nucleotide substitutions at genetic sites of interests, thereby achieving desired genetic modifications and / or therapeutic effects (e.g., correcting genetic defects or inserting desirable nucleotide sequences at target genomic site which may have a desired therapeutic outcome). The gene editing system provided herein can also be used in other areas, for example, in the generation of animal and cell models with specific genetic modifications.I. Enzymes for Nuclease-Guided Reverse Transcription-Mediated Gene EditingThe nuclease-guided reverse transcription-mediated gene editing approach disclosed herein involves at least two enzyme polypeptides, an RNA-guided nuclease polypeptide (e.g., a CRISPR nuclease) and an RT polypeptide. In some embodiments, the two enzyme components may form a fusion polypeptide, which may further comprise additional functional motifs, such as nuclear localization signals (NLS), peptide linkers, or a combination thereof.In some embodiments, the CRISPR nuclease, the RT polypeptide, or both, lack the native N-terminus methionine residue, for example, when the polypeptide is in the C-terminal part of a fusion protein and / or when a functional fragment (e.g., an NLS) is fused to its N- terminus.A. Nuclease PolypeptidesAs used herein, the term “RNA-guided 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. An RNA-guided nuclease may be a CRISPR nuclease, which typically comprises multiple functional domains, e.g., nuclease domains (e.g., RuvC and / or HNH), bridge helix (BH) domain, nucleic acid recognition (REC) domain, phosphate lock loop (PLL), wedge domain (WED), PAM-interacting domain (PID), or a combination thereof. As used herein, theAttorney Docket No.: 063586-545001 WO term “domain” refers to a distinct functional 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, the nuclease polypeptide for use in the gene editing system disclosed herein can be derived from the reference Nuclease A (SEQ ID NO: 1), Nuclease K (SEQ ID NO: 2), or Nuclease BT (SEQ ID NO: 3). Such a nuclease polypeptide may comprise the reference nuclease or a variant thereof as disclosed herein. In some instances, the nuclease polypeptides provided herein may be a variant nuclease of the reference nuclease comprising at least one mutation relative to the reference nuclease.As used herein, the term “variant nuclease polypeptide” refers to a nuclease polypeptide comprising an alteration, e.g., a substitution, insertion, deletion and / or fusion, at one or more residue positions, compared to the reference nuclease (Nuclease A, Nuclease K, or Nuclease BT).The variant nuclease polypeptides provided herein are expected to exhibit one or more modulated activities (e.g., enhanced or reduced) relative to the reference nuclease. As used herein, the term “activity” refers to a biological activity. In some embodiments, activity includes enzymatic activity, e.g., catalytic ability of an effector. For example, activity can include nuclease activity. In some embodiments, activity includes binding activity, e.g., binding of an effector (e.g., a nuclease) to an RNA guide and / or target nucleic acid. In some examples, the variant nuclease polypeptides disclosed herein have an enhanced binding to a cognate guide RNA (gRNA) as compared with the reference nuclease, e.g., having a binding 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 nuclease. A cognate gRNA refers to a gRNA having a scaffold recognizable by the nuclease.In some examples, the variant nuclease polypeptides disclosed herein have an enhanced enzymatic activity relative to the reference nuclease, 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 nuclease. In other examples, the variant nuclease polypeptides disclosed herein have a decreased enzymatic activity relative to the reference nuclease, e.g., having an enzymatic activity at least 20%, 30%, 40%, 50%, 60%, or 70% lower than that of the reference nuclease. In some instances, the decreased enzymatic activity is achieved by reducing or diminishing the nuclease activity of the RuvC domain. In some instances, the decreased enzymatic activity is achieved by reducing or diminishing the nuclease activity ofAttorney Docket No.: 063586-545001 WO the HNH domain.In some instances, the variant nuclease polypeptides disclosed herein have enhanced indel activity relative to the reference nuclease. As used herein, the term “indel activity” refers to the ability of a nuclease to introduce an indel (insertion / deletion) into a sequence (e.g., a genomic target).In some embodiments, the variant nuclease polypeptide provided herein share a high sequence homology relative to the reference nuclease. For example, the variant nuclease 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: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some instances, the variant nuclease polypeptide may comprise an amino acid sequence at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some instances, the variant nuclease polypeptide may comprise an amino acid sequence at least 95% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In other instances, the variant nuclease polypeptide may comprise an amino acid sequence at least 97% (e.g., 98%, 99%, 99.5%, or greater) identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.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 embodiments, the variant nuclease polypeptides may comprise one or more mutations (e.g., arginine substitutions, lysine substitutions, or a combination thereof) relative to the reference Nuclease A (SEQ ID NO: 1), Nuclease K (SEQ ID NO: 2), or Nuclease BT (SEQ ID NO: 3) provided herein. “Arginine substitutions” and / or “lysine substitutions” refers to the replacement of a non-arginine or non-lysine residue in SEQ ID NO: 1 (Nuclease A), SEQ ID NO: 2 (Nuclease K), or SEQ ID NO: 3 (Nuclease BT) with an arginine or lysine residue.Attorney Docket No.: 063586-545001 WOAlternatively, or in addition, the variant nuclease polypeptide may comprise one or more mutations in either the RuvC nuclease domain or the HNH nuclease domain. Such mutations (e.g., nickase mutations) may reduce or eliminate the nuclease activity of either the RuvC or the HNH nuclease domain, leading to a variant exhibiting nickase activity. As used herein, the term “nickase” refers to an enzyme that cuts one strand of a double-stranded DNA at a specific recognition nucleotide sequence (e.g., the target sequence disclosed herein). A nickase may interact with one strand of the DNA duplex to produce DNA molecules that are cut at one strand (a.k.a.. nicked). In some embodiments, a nickase is a variant of a nuclease that comprises a deactivated HNH domain. In some embodiments, a nickase is a variant of a nuclease that comprises a deactivated RuvC domain.Alternatively, or in addition, the variant nuclease polypeptide may comprise one or more non-arginine / lysine substitutions at one or more positions relative to the reference nuclease. A non-arginine / lysine substitution refers to an amino acid residue substitution where the native amino acid residue is replaced with an amino acid residue that is not arginine or lysine.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 nuclease polypeptide provided herein may comprise one or more arginine substitutions, one or more lysine substitutions, or a combination thereof.In some instances, a variant nuclease polypeptide provided herein may contain one or more conservative amino acid residue 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 instances, the variant nuclease polypeptide may be a truncated version of the reference nuclease. A truncated version may comprise one or more deletions at certain regionsAttorney Docket No.: 063586-545001 WO within the reference nuclease, for example, at the N-terminus, at the C-terminus, or both.The variant nuclease polypeptides provided herein are expected to possess advantageous features relative to the reference nuclease, for example, increased binding to a cognate guide RNA, higher nuclease activity, etc. As such, the variant nuclease polypeptides disclosed herein would be expected to exhibit better activities in gene editing relative to the reference nuclease, e.g., nickase activity, higher efficiency and accuracy in gene editing involving strand replacement.(a) Nuclease A and Variants ThereofIn some embodiments, the nuclease polypeptide is derived from Nuclease A, including both the wild-type Nuclease A (SEQ ID NO: 1), a variant thereof such as those disclosed herein (e.g. , a Nickase A variant), or a fusion polypeptide comprising such. The variant nuclease polypeptides of Nuclease A may be produced via introducing one or more mutations to the reference nuclease to modulate (e.g., enhance or reduce) one or more activities of the nuclease.The reference nuclease of Nuclease A (SEQ ID NO: 1) (see Sequence Table 1 below) is a nuclease that comprises both a RuvC nuclease domain (located at residues 15-53, 308-338, and 472-566 of SEQ ID NO: 1) and a HNH domain (located at residues 339-471 of SEQ ID NO: 1). The RuvC nuclease domain and the HNH nuclease domain coordinate cleavage of the DNA strand adjacent to the 5’-NGG-3’ PAM motif, in which N represents any nucleotide. Positions D24, E337 and D524 are deemed the active sites in the RuvC domain, and positions D396, H397, and N420 are deemed the active sites in the HNH domain. R506 and H521 may also be important for the nuclease activity of the RuvC domain. In addition to the nuclease domains, the reference nuclease of SEQ ID NO: 1 also includes a BH domain (residues 54-89 of SEQ ID NO: 1), a REC domain (residues 90-307 of SEQ ID NO: 1), a PLL domain (residues 567-580 of SEQ ID NO: 1), a WED domain (residues 581-672 of SEQ ID NO: 1), and a PID domain (residues 673-776 of SEQ ID NO: 1).Compared to the CRISPR-Cas9 nuclease from Streptococcus pyogenes, the reference CRISPR nuclease of SEQ ID NO: 1 disclosed herein is smaller. The scaffold utilized by the reference CRISPR nuclease of SEQ ID NO: 1 and variants thereof can be miniaturized relative to the reference scaffold of SEQ ID NO: 63. This scaffold comprises a distinctive structure compared to the SpCas9 nuclease scaffold. The distinctive scaffold is expected to allow for decreased size of some domains (e.g., the REC domain) and thus contribute to the smaller nuclease size. These features would be beneficial for delivery. Arginine and / or lysine substitutions (e.g., arginine substitutions) can be introduced into the nuclease of SEQ ID NO: 1Attorney Docket No.: 063586-545001 WO to increase indel activity.Further, the RuvC domain of Nuclease A (SEQ ID NO: 1) cleaves the non-target strand of a target nucleic acid within 4 to 8 nucleotides upstream of the PAM, and the HNH domain cleaves the target strand between 3 and 4 nucleotides upstream of the PAM, each leading to a cut site with an overhang of 0-5 nucleotides (most commonly a 3-5 nucleotide overhang). In contrast, the RuvC domain of SpCas9 cleaves the non-target strand of a target nucleic acid within 3 to 5 nucleotides upstream of the cognate PAM (5’-NGG-3’, in which N is any nucleotide), and the HNH domain cleaves the target strand between 3 and 4 nucleotides upstream of the PAM, each leading to an overhang of 0-3 nucleotides (mostly commonly a blunt cut).Additionally, use of gene editing systems comprising Nuclease A (SEQ ID NO: 1) or variants thereof can result in the introduction of indels into a target nucleic acid that are larger than those capable of being introduced by SpCas9. For example, insertions induced by use of Nuclease A or variants thereof can range from about 1 -nucleotide to about 7-nucleotides (most commonly about 4-nucleotides). Deletions induced by use of Nuclease A or variants thereof can range from about 1 -nucleotide to about 25-nucleotides or larger (most commonly about 11- nucleotides). Further, since the reference nuclease of SEQ ID NO: 1 comprises a RuvC domain and an HNH domain, nickase variants can be engineered, e.g., via disrupting nuclease activity of either the RuvC domain or the HNH domain.Variants of Nuclease A PolypeptideThe variant nuclease polypeptide of Nuclease A as provided herein can contain one or more alterations relative to Nuclease A (SEQ ID NO: 1), 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. In some instances, the alterations may be introduced into the BH domain, the PLL domain, the WED domain, the PID domain, or a combination thereof. In some instances, no alterations are introduced into the RuvC and / or the HNH nuclease domains, or at the active sites and / or sites involved in activity in these domains as provided herein. Alternatively, or in addition, conservative amino acid substitutions may be introduced into SEQ ID NO: 1, including in the RuvC and / or the HNH nuclease domains.(i) Arginine and / or Lysine SubstitutionsIn some embodiments, the variant nuclease polypeptide of Nuclease A may comprise one or more arginine substitutions, one or more lysine substitutions, or a combination thereofAttorney Docket No.: 063586-545001 WO relative to SEQ ID NO: 1. In some examples, the variant CRISPR nuclease 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 nuclease polypeptide may contain 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 arginine substitutions, lysine substitutions, or a combination thereof. In some examples, the variant CRISPR nuclease polypeptide provided herein contains arginine substitutions.In some instances, the arginine and / or lysine substitutions may be located in the BH domain, in the PLL domain, in the WED domain, in the PID domain, or in any of the combination thereof of Nuclease A. In some examples, the variant CRISPR nuclease polypeptide derived from Nuclease A may contain one or more of the arginine and / or lysine substitutions at one or more of the following positions in SEQ ID NO: 1 : D56, E59, G60, E63, 167, G71, T98, E102, E206, V210, R258, R259, R261, E490, E491, E495, W508, 1519, D533, D536, S564, D568, W593, T605, E655, E694, E706, G713, and R720. In some examples, the variant CRISPR nuclease polypeptide derived from Nuclease A comprises one or more of the arginine substitutions at one or more of positions D56, E59, G60, E63, 167, G71, E206, E491, S564, D568, W593, T605, E655, E694, and E706 in SEQ ID NO: 1. In other examples, the variant CRISPR nuclease polypeptide derived from Nuclease A comprises one or more lysine substitutions at one or more of positions of SEQ ID NO: 1 : T98, E102, V210, R258, R259, R261, E490, E491, E495, W508, 1519, D533, D536,G713, and R720.In some instances, the arginine and / or lysine substitution may not be at the active sites and / or sites involved in activity in the RuvC and / or the HNH nuclease domains of Nuclease A (e.g., not at positions D24, E337, R506, H521, and / or D524 in the RuvC domain and / or at positions D396, H397, and / or N420 in the HNH domain of SEQ ID NO: 1). In some examples, the arginine and / or lysine substitution may not be in the RuvC and / or HNH domains of Nuclease A.Additional structural information for Nuclease A can be found in WO2024206758, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein.In some embodiments, a variant of Nuclease A provided herein exhibits nuclease activity and may not have mutations at the above positions. Alternatively, a variant nuclease may be a dead nuclease (e.g, for use in base editing) having mutations at one or more of these positions.In some instances, a variant nuclease comprising arginine and / or lysine substitutions atAttorney Docket No.: 063586-545001 WO a combination of 2 or more (e.g., 3 or 4) positions, for example, of D56, 167, E206, E491, 1519, D568, T605, E694, E706, and / or G713 of SEQ ID NO: 1. Examples include, but are not limited to, (a) 167, D568, and E706; (b) D56, 167, and D568; (c) 167, D568, and T605; (d) D56; 167, and E706; (e) 167, T605, and E706; (f) 167, D568, and E694; (g) D56, 167, E694, and E706; (h) D56, 167, T605, and E706; (i) 167, D568, W593, and E706, (j) ) 167, W508, D568, W593, E706, (k) 167, E490, E491, D568, W593, E706, (1) 167, W508, D568, W593, E706, (k) 167, E206, E490, E491, D568, W593, E706, (m) 167, 1519, D568, W593, E706, and (n) 167, D568, W593, E706, and G713. In some specific examples, the engineered CRISPR nuclease polypeptide comprises the arginine substitutions of 167, D568, and E706. In other specific examples, the engineered CRISPR nuclease polypeptide comprises the arginine substitutions of 167, D568, W593, and E706. In some examples, the variant CRISPR nuclease comprise arginine substitutions at the combined positions listed herein.Addition exemplary combinations of arginine and / or lysine substitutions e.g., arginine substitutions) include (relative to SEQ ID NO: 1): (i) at least two positions of D56, E59, G60, E63, 167, G71, S564, D568, T605, E655, E694, and E706; (ii) at least two positions D56, 167, T605, E694, and E706; (iii) at least two positions of D56, 167, D568, T605, E694, and E706; (iv) at least two positions of D56, E63, G71, D568, T605, E655, E694, and E706; and (v) at least two positions of D56, 167, G71, D568, T605, E655, E694, and E706.In some specific examples, the variant nuclease polypeptide of Nuclease A provided herein may comprise arginine substitutions of I67R, D568R and E706R relative to SEQ ID NO: 1. In other specific examples, the variant nuclease polypeptide of Nuclease A provided herein may comprise arginine substitutions of I67R, D568R, W593R, and E706R to SEQ ID NO: 1. In some instances, the variant nuclease polypeptide of Nuclease A may further comprise arginine / lysine substitutions at one or more of positions E206, R258, R259, E490, E491, W508, 1519, and G713 in SEQ ID NO: 1, for example, E206R, R258K, R259K, E490K or E490R, E491R or E491R, I519K, or G713K.(ii) Deletions and / or InsertionsAlternatively, or in addition, the engineered variant nuclease polypeptide of Nuclease A disclosed herein may have an N-terminal truncation relative to Nuclease A of SEQ ID NO: 1. Such an engineered variant CRISPR nuclease polypeptide has a fragment at the N-terminus of Nuclease A deleted. In some instances, the deleted N-terminal fragment has up to 80 amino acid residues, for example, up to 70 amino acid residues, up to 60 amino acid residues, up to 50 amino acid residues, up to 40 amino acid residues, up to 30 amino acid residues, or up to 20Attorney Docket No.: 063586-545001 WO amino acid residues. In some examples, the N-terminal truncation is a deletion within the residues 1-15 of SEQ ID NO: 1. In one specific example, the N-terminal truncation is a deletion of residues 1-14 of SEQ ID NO: 1. In another specific example, the N-terminal truncation is a deletion of residues 1-15 of SEQ ID NO: 1.Alternatively, or in addition, the engineered nuclease polypeptide disclosed herein may have a C-terminal truncation relative to the reference nuclease A of SEQ ID NO: 1. Such an engineered CRISPR nuclease polypeptide has a fragment at the C-terminus of the reference nuclease deleted. In some instances, the deleted C-terminal fragment has up to 10 amino acid residues, for example, up to 5 amino acid residues, up to 4 amino acid residues, up to 3 amino acid residues, up to 2 amino acid residues, or up to 1 amino acid residues. In some examples, the C-terminal truncation is a deletion of the final residue, the final two residues, the final three residues, the final four residues, or the final five residues of SEQ ID NO: 1.In specific example, a truncated variant of SEQ ID NO: 1 may have the deletion of amino acid residues 1-15 of SEQ ID NO: 1 (z.e., the 16-775 truncation version).In some instances, the variant of SEQ ID NO: 1 does not include the N-terminus methionine residue. Such a variant of SEQ ID NO: 1 may have a functional fragment (e.g., an NLS) fused to its N-terminus or is at the C-terminal part of the fusion polypeptides disclosed herein.Alternatively or in addition, the variant of Nuclease A provided herein may comprise or further comprise a deletion of Q277, a deletion of E206 and / or D207, or a combination thereof. In other examples, the variant of Nuclease A provided herein may comprise or further comprise an insertion between positions 538 and 539 (described herein as the 539L insertion). The amino acid position numberings refer to SEQ ID NO: 1.(iii) Nickase MutationsAlternatively, or in addition, any of the variant nuclease polypeptides of Nuclease A provided herein may comprise one or more nickase mutations within either the RuvC or the HNH nuclease domain to reduce or eliminate the nuclease activity of the target domain, thereby producing a variant with nickase activity. Such mutations may be deletions, insertions, amino acid substitutions, or a combination thereof. In some embodiments, the mutations within either the RuvC or the HNH nuclease domain are amino acid substitutions, of which the substituting amino acid residue is not a conservative substitution of the native amino acid residue at the position of the mutation. For example, if the native amino acid residue is R, the substituting residue can be any amino acid residue except for K. Similarly, if the native aminoAttorney Docket No.: 063586-545001 WO acid residue is K, the substituting residue can be any amino acid residue except for R. Groups of conservative amino acid residue substitutions are provided herein.Positions D24, E337, and D524 are identified as putative catalytic residues in the RuvC domain of Nuclease A, and positions D396, H397, and N420 are identified as putative catalytic residues in the HNH domain of Nuclease A of SEQ ID NO: 1. In some examples, the one or more mutations may be within the RuvC nuclease domain to reduce or inactivate the RuvC domain (e.g., at positions D24, E337, and / or D524 in the RuvC domain). In some specific examples, the variant CRISPR nuclease polypeptide derived from Nuclease A may contain substitution(s) of D24A, E337A, and / or D524A. In other examples, any of D24, E337, and D524 may be substituted by an amino acid residue similar to A, for example, G, S, or L.In some examples, the one or more mutations may be within the HNH domain to reduce or inactivate the HNH domain e.g., at positions D396, H397, and / or N420 in the HNH domain). In some specific examples, the variant nuclease polypeptide may contain substitutions of H397A, H397K, H397W, or H397Y, D396A, and / or N420A or N420R. Alternatively, any of H397, D396, and N420 may be replaced with an amino acid residue similar to A, K, W, or Y, for example, G, L, S, R, or F. In one specific example, the variant CRISPR nuclease polypeptide may contain substitution of H397A or H397L. In other examples, the variant CRISPR nuclease polypeptide may contain substitution of H397R, H397K, H397W, H397Y, or N420R.In yet other examples, the one or more mutations may be within both the RuvC domain and the HNH domain of Nuclease A to reduce or diminish the nuclease enzymatic activity.(iv) Non-Arginine and / or Lysine Amino Acid SubstitutionsIn some instances, the variant of Nuclease A may comprise or further comprise one or more non-arginine / lysine amino acid substitutions. As used herein, a non-arginine / lysine amino acid substitution refers to an amino acid substitution in which the native amino acid residue is replaced by an amino acid that is neither arginine nor lysine. Such non-arginine / lysine substitutions may be at one or more of positions K76, T83, N87, 196, T98, E102, Cl 11, S134, E137, E144, A174, D225, L232, E241, E265, F275, K288, E300, A307, V321, 1335, K348, E367, K366, T391, 1406, E418, L434, M479, H478, E495, Q497, 1500, W508, D533, S548, T562, R567, R568, S570, N571, T574, P575, Y577, S587, L588, T596, E603, 1607, S608, Y611, K613, W622, T673, L684, W622, R699, S729, V733, R755, R757, and T766 of SEQ ID NO: 1. Examples include, but are not limited to, K76M or K76V, T83A, T83N, T83S, or N87S, I96T, T98N, C111A, S134D, E137D, E144A, A174L, D225N, L232I, E241L, E265E, F275DAttorney Docket No.: 063586-545001 WO or F275G, K288E, E300Q,A307N, I406V, V321I, I335V, K348M, E367D, K366N, T391G or T391S, E418G, L434V, M479L, H478W, Q497V, I500T, S548N, T562S, R567M, R568I, R568M, S570G, N571S, T574S, P575A, Y577H, S587G, L588H, T596V, E603D, I607E, S608T, Y611D, K613E, W622F, T673S, L684P, R699S, S729G, R755S, R757S, V733I, T766V, T766I.In some examples, the variant of Nuclease A may comprise non-arginine / lysine substitutions at one or more of positions T83, N87, D225, R261, P575, W622, and S729 of SEQ ID NO: 1. Examples include T83N, N87S, D225N, R261K, P575A, W622F, and / or S729G. In some instances, the variant of Nuclease A may further comprise one or more non- arginine / lysine substitutions at positions Q72 (e.g., Q72S), K76 (e.g., K76V), T255 (e.g., T255E), F265 (e.g, F265E), F275 (e.g, F275G), K288 (e.g., K288E), E300 (e.g., E300Q), 1406 (e.g., I406V), E367 (e.g., E367D), E418 (e.g., E418G), L434 (e.g., L434V), H478 (e.g., H478W), 1500 (e.g., I500T), L588 (e.g., L588H), E603 (e.g., E603D), 1607 (e.g., I607F), S608 (e.g., S608T), Y611 (e.g., Y611D), N667 (e.g., N667D), L684 (e.g., L684P), and R699 (e.g., R699S) in SEQ ID NO: 1. In some instances, any of the replacement residue may be a conservative substitution of the current replacement residue.(v) Combination of MutationsIn some embodiments, the variant nuclease polypeptide derived from Nuclease A can comprise a combination of the mutations disclosed herein, e.g., arginine and / or lysine substitutions, deletions and / or insertions, nickase mutations, and / or non-arginine / lysine amino acid substitutions.In some examples, the variant nuclease polypeptide provided herein may comprise both arginine / lysine substitutions (e.g., arginine substitutions), e.g., at one or more positions provided herein, and nickase mutations. For example, the variant nuclease polypeptide may comprise arginine / lysine substitutions (e.g., arginine substitutions) at positions 167, D568, and / or E706, and further comprise an amino acid substitution at H397 (e.g., H397A or H397L) in SEQ ID NO: 1. In some instances, such a variant may further comprise an N-terminal truncation as disclosed herein, e.g., a deletion within residues 1-15 of SEQ ID NO: 1 such as the deletion of 1-14 aa or 1-15 aa of SEQ ID NO: 1. In some instances, such a variant may further comprise a C-terminal truncation as disclosed herein, e.g., a deletion within the final five residues of SEQ ID NO: 1.In some instances, the variant of Nuclease A (SEQ ID NO: 1) may comprise a combination of (a) one or more arginine / lysine substitution; (b) one or more mutations in eitherAttorney Docket No.: 063586-545001 WO the RuvC nuclease domain or the HNH nuclease domain, and / or (c) truncation. In some examples, an N-terminal or C-terminal truncated variant may further comprise one or more of the mutations disclosed herein, for example, one or more arginine / lysine substitutions, one or more mutations resulting in nickase activity, or a combination thereof. In some specific examples, the N-terminal truncated variant (e.g., having residues 1-15 deleted) may further comprises arginine substitutions of I67R, D568R, E706R relative to SEQ ID NO: 1. In other specific examples, the N-terminal truncated variant (e.g., having residues 1-15 deleted) further comprises arginine substitutions of I67R, D568R, W593R, and E706R relative to SEQ ID NO: 1. Such a variant may further comprise a nickase mutation such as a mutation at position H397 (e.g., H397A, H397K, H397W, or H397Y).In some examples, the variant of Nuclease A includes the 16-775 region of SEQ ID NO: 1 and comprises amino acid substitutions I67R, D568R, W593R, and E706R. In some instances, such a Nuclease A variant may further comprise one or more amino acid substitutions (e.g., arginine substitutions, lysine substitutions, non-arginine / lysine substitutions, or a combination thereof). Examples of additional arginine / lysine substitutions may be at one or more of positions E206, R258, R259, E490, E491, W508, 1519, and G713 in SEQ ID NO: 1, for example, E206R, R258K, R259K, E490K or E490R, E491R or E491R, I519K, or G713K. Examples of non-arginine / lysine substitutions may be at one or more of positions Q72 (e.g., Q72S), K76 (e.g., K76V), T255 (e.g., T255E), F265 (e.g., F265E), F275 (e.g., F275G), K288 (e.g., K288E), E300 (e.g., E300Q), 1406 (e.g., I406V), E367 (e.g., E367D), E418 (e.g., E418G), L434 (e.g., L434V), H478 (e.g., H478W), 1500 (e.g., I500T), L588 (e.g., L588H), E603 (e.g., E603D), 1607 (e.g., I607F), S608 (e.g., S608T), Y611 (e.g., Y611D), N667 (e.g., N667D), L684 (e.g., L684P), and R699 (e.g., R699S) in SEQ ID NO: 1. in SEQ ID NO: 1.In specific examples, the variant of Nuclease A is a Nickase A variant (e.g. , comprising the H397A, H397K, H397W, or H397Y substitution) and may further comprise deletions of 1- 15 of SEQ ID NO: 1 (known as the 16-775 truncation), and amino acid substitutions I67R, D568R, W593R, and E706R. In some instances, such a Nickase A variant may further comprise one or more amino acid substitutions (e.g., arginine substitutions, lysine substitutions, non-arginine / lysine substitutions, or a combination thereof). Such a Nuclease A variant may further comprise one or more non-arginine / lysine amino acid substitutions, for example, at one or more of the positions T83, N87, D225, R261, P575, W622, and S729 relative to SEQ ID NO: 1. Examples include T83N, N87S, D225N, R261K, P575A, W622F, and S729G. The Nuclease A variant may further comprise non-arginine / lysine substitutions at the position of W508, T562, or R699 of SEQ ID NO: 1, for example, W508K, T562S, orAttorney Docket No.: 063586-545001 WOR699S.In some instances, the Nickase A variant may further comprise (a) arginine / lysine substitution(s) at one or more of positions E206 (e.g., E206R), R258 (e.g., R258K), R259 e.g., R259K), E490 (e.g, E490K), and / or E491 (e.g, E491R) in SEQ ID NO: 1, (b) non- arginine / lysine substation(s) at one or more of positions F265 (e.g., F265E), F275 (e.g., F275G), K288 (e.g., K288E), L434 (e.g., L434V), L588 (e.g., L588H), 1607 (e.g., I607F), S608 (e.g., S608T), L684 (e.g., L684P), Y611 (e.g., Y611D), N667 (e.g., N667D), and / or R699 (e.g., R699S) in SEQ ID NO: 1, or (c) a combination of (a) and (b).In some instances, the Nuclease A variant may comprise the combination of substitutions at positions R699 and N117 of SEQ ID NO: 1, for example, R699S + N117E or R699S + N117M. In other instances, the Nuclease A variant may comprise the combination of substitutions at positions K76 and N117 of SEQ ID NO: 1, for example, K76V + N117P. Such variants may comprise further mutations as those disclosed herein.In some embodiments, a Nuclease A variant disclosed herein may be free of one or more of the substitutions R51C, R158G, V182D, C286R, F399S, and N457S relative to SEQ ID NO: 1. In some examples, the Nuclease A variant may not have mutations at one or more of the involved positions relative to SEQ ID NO: 1.Any of the Nuclease A variants provided herein may share a sequence identity at least 90% (e.g., 95%, 97%, 98%, 99%, 99.5%, or greater) with SEQ ID NO: 1. Exemplary engineered variant CRISPR nuclease polypeptide of Nuclease A is listed in Sequence Table 1, which is within the scope of the present disclosure. In specific examples, the variant CRISPR nuclease polypeptide may comprise (e.g., consist of) an amino acid sequence of any one of SEQ ID NOs: 90, 134, 148, 153, 163, 428-443, and 469-480.(b) Nuclease K and Variants ThereofIn some embodiments, the nuclease polypeptide is derived from Nuclease K, including both the wild-type Nuclease K (SEQ ID NO: 2), a variant thereof such as those disclosed herein, or a fusion polypeptide comprising such. The variant CRISPR nuclease polypeptides of Nuclease K may be produced via introducing one or more mutations to the reference CRISPR nuclease to modulate (e.g., enhance or reduce) one or more activities of the nuclease.The reference Nuclease K (SEQ ID NO: 2; see Sequence Table 1 below) is a CRISPR nuclease that comprises both a RuvC nuclease domain (located at residues 1-39, 286-320, and 448-543 of SEQ ID NO: 1) and a HNH domain (located at residues 321-447 of SEQ ID NO: 2). The RuvC nuclease domain and the HNH nuclease domain coordinate cleavage of the DNAAttorney Docket No.: 063586-545001 WO strand adjacent to the 5’-NGG-3’ PAM motif, in which N represents any nucleotide. Positions D10, E315 and D500 are deemed the active sites in the RuvC domain and positions D374, H375, and N398 are deemed the active sites in the HNH domain. R482 and H497 may also be important for the nuclease activity of the RuvC domain. In addition to the nuclease domains, the reference CRISPR nuclease of SEQ ID NO: 2 also includes a BH domain (residues 40-77 of SEQ ID NO: 2), a REC domain (residues 78-285 of SEQ ID NO: 2), a PLL domain (residues 544-556 of SEQ ID NO: 2), a WED domain (residues 557-648 of SEQ ID NO: 2), and a PID domain (residues 649-747 of SEQ ID NO: 2).Compared to the CRISPR-Cas9 nuclease from Streptococcus pyogenes, the reference CRISPR Nuclease K of SEQ ID NO: 2 disclosed herein is smaller. The scaffold utilized by the reference CRISPR nuclease of SEQ ID NO: 2 and variants thereof may be miniaturized relative to the cognate scaffold of SEQ ID NO: 66. This scaffold comprises a distinctive structure compared to the SpCas9 nuclease scaffold. The distinctive scaffold is expected to allow for decreased size of some domains (e.g., the REC domain) and thus contribute to the smaller CRISPR nuclease size. These features would be beneficial for delivery. Arginine and / or lysine substitutions can be introduced into the nuclease of SEQ ID NO: 2 to increase indel activity.Further, the RuvC domain of Nuclease K cleaves the non-target strand within 4 to 6 nucleotides upstream of the PAM and the HNH domain cleaves the target strand between 3 and 4 nucleotides upstream of the PAM, each leading to a cut site with an overhang of 0-3 nucleotides (most commonly a 3-nucleotide overhang). This cutting pattern is different from that of SpCas9 as described above. Additionally, use of gene editing systems comprising Nuclease K or variants thereof can result in the introduction of indels into a target nucleic acid that are larger than those capable of being introduced by SpCas9. For example, insertions induced by use of Nuclease K or variants thereof can range from about 1 -nucleotide to about 10-nucleotides (most commonly about 4-nucleotides). Deletions induced by use of Nuclease A or variants thereof can range from about 1 -nucleotide to about 25 -nucleotides or larger (most commonly about 11 -nucleotides). Further, since Nuclease K comprises a RuvC domain and an HNH domain, nickase variants can be engineered, e.g., via disrupting nuclease activity of either the RuvC domain or the HNH domain.Variants of Nuclease K PolypeptidesThe variant of Nuclease K polypeptide provided herein can contain one or more mutations relative to Nuclease K (SEQ ID NO: 2), e.g., one or more amino acid residueAttorney Docket No.: 063586-545001 WO substitutions, one or more deletions, one or more insertions, one or more fusions, or a combination thereof. In some instances, the alterations may be introduced into the BH domain, the WED, the PID, or a combination thereof of Nuclease K. In some instances, no alterations are introduced into the RuvC and / or the HNH nuclease domains of Nuclease K, or at the active sites and / or sites involved in activity in these domains as provided herein. Alternatively, conservative amino acid substitutions may be introduced into SEQ ID NO: 2, including in the RuvC and / or the HNH nuclease domains of Nuclease K. In some instances, the variant of Nuclease K polypeptide does not include an N-terminus methionine residue, for example, when the N-terminus of the Nuclease K variant is fused to a functional fragment (e.g., an NLS) or when the Nuclease K variant is located at the C-terminal part in the fusion polypeptide as disclosed herein.(i) Arginine and / or Lysine SubstitutionsIn some embodiments, the variant nuclease polypeptide derived from Nuclease K provided herein 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 CRISPR nuclease polypeptide derived from Nuclease K 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 l9, 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 CRISPR nuclease polypeptide derived from Nuclease K may contain 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 arginine substitutions, lysine substitutions, or a combination thereof. In some examples, the variant CRISPR nuclease polypeptide derived from Nuclease K provided herein contains arginine substitutions.In some instances, the arginine and / or lysine substitutions may be located in the BH domain, in the WED domain, in the PID domain, or in any of the combinations thereof of Nuclease K. For example, the arginine and / or lysine substitution may be introduced into one or more of the following positions: G42, D46, 153, F83, E128, E541, F570, E576, D582, C630, 1631, E683, S719, and T734 of SEQ ID NO: 2. In some instances, the arginine and / or lysine substitutions may be at position G42 and / or D582 of SEQ ID NO: 2. In other instances, the arginine and / or lysine substitutions may be at position 153, D582, and / or T734 of SEQ ID NO: 2.In some examples, the variant nuclease polypeptide derived from Nuclease K (SEQ ID NO: 2) may contain one or more of the following arginine substitutions: G42R, D46R, I53R,Attorney Docket No.: 063586-545001 WOE128R, D582R, F570R, E576R, C630R, 1631R, E683R, T734R, and S719R. In other examples, the variant CRISPR nuclease polypeptide derived from Nuclease K may contain one or more of the following arginine substitutions: G42R, D46R, I53R, F83R, E541R, F570R, D582R, E683R, and S719R. In some specific examples, the variant CRISPR nuclease polypeptide derived from Nuclease K may contain arginine substitutions at position G42 and / or position D582. In other specific examples, the variant CRISPR nuclease polypeptide may contain arginine substitutions at position 153, D582, and / or T734.In some instances, the arginine and / or lysine substitution may not be at the active sites and / or sites involved in the activity of the RuvC and / or the HNH nuclease domains of Nuclease K (e.g., not at positions D10, E315, R482, H497, and / or D500 in the RuvC domain and / or at positions D374, H375, and / or N398 in HNH domain). In some examples, the arginine and / or lysine substitution may not be in the RuvC and / or HNH domains of Nuclease K.(ii) Nickase MutationsAlternatively or in addition, the variant nuclease polypeptide derived from Nuclease K provided herein may comprise one or more mutations (z.e., nickase mutations) within either the RuvC or the HNH nuclease domain to reduce or eliminate the nuclease activity of the target domain, thereby producing a variant with nickase activity. Such mutations may be deletions, insertions, amino acid substitutions, or a combination thereof. In some embodiments, the mutations within either the RuvC or the HNH nuclease domain of Nuclease K are amino acid substitutions, of which the substituting amino acid residue is not a conservative substitution of the native amino acid residue at the position of the mutation. For example, if the native amino acid residue is R, the substituting residue can be any amino acid residue except for K. Similarly, if the native amino acid residue is K, the substituting residue can be any amino acid residue except for R. Groups of conservative amino acid residue substitutions are provided herein.Positions D10, E315, and D500 are deemed the active sites in the RuvC domain of Nuclease K (SEQ ID NO: 2), and positions D374, H375, and N398 are deemed the active sites in the HNH domain. R482 and H497 may also be important for nuclease activity of the RuvC domain of Nuclease K. In some examples, the one or more mutations may be within the RuvC nuclease domain to reduce or inactivate the RuvC domain e.g., at positions D10, E315, R482, H497, and / or D500 in the RuvC domain). In some specific examples, the variant CRISPR nuclease polypeptide may contain substitution(s) of D10A, E315A, and / or D500A. In other examples, any of D10, E315, and D500 may be substituted by an amino acid residue similar toAttorney Docket No.: 063586-545001 WOA, for example, G, S, or L.In some examples, the one or more mutations may be within the HNH domain (e.g., at positions H375, D374, and / or N398 in the HNH domain of SEQ ID NO: 2). In some specific examples, the variant CRISPR nuclease polypeptide derived from Nuclease K may contain substitutions ofH375A, D374A, and / or N398A. Alternatively, any ofH375, D374, and N398 may be replaced with an amino acid residue similar to A, for example, G, L, or S. In one specific example, the variant CRISPR nuclease polypeptide derived from Nuclease K may contain substitution ofH375A or H375L.(iii) Combination of MutationsIn some embodiments, the variant nuclease polypeptide derived from Nuclease K can comprise a combination of the mutations disclosed herein, e.g., arginine and / or lysine substitutions, deletions and / or insertions, nickase mutations, and / or non arginine / lysine amino acid substitutions.In some embodiments, the variant CRISPR nuclease polypeptide derived from Nuclease K provided herein may comprise both arginine / lysine substitutions (e.g., arginine substitutions), e.g., at one or more positions provided herein, and nickase mutations, e.g., at one or more of the positions also provided herein. In some instances, the variant nuclease polypeptide of Nuclease K may comprise arginine / lysine substitutions (e.g., arginine substitutions) listed in Sequence Table 1 below. Such variant CRISPR nuclease polypeptide may further comprise one or more mutations that reduce or inactivate the RuvC and / or HNH nuclease domain (e.g., one or more mutations within the RuvC and / or HNH nuclease domain). For example, the variant CRISPR nuclease polypeptide derived from Nuclease K may comprise arginine / lysine substitutions (e.g., arginine substitutions) at positions D582, 153, and T734 (e.g., I53R / D582R / T734R), and an amino acid substitution at H375 (e.g., H375A) in SEQ ID NO: 2.In another example, the variant CRISPR nuclease polypeptide derived from Nuclease K may comprise arginine / lysine substitutions (e.g., arginine substitutions) at positions G42 and D582 (e.g., G42R / D582R), and an amino acid substitution at H375 (e.g., H375A).Any of the variant CRISPR nuclease polypeptide derived from Nuclease K 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. Exemplary engineered variant CRISPR nuclease polypeptide of Nuclease A is listed in Sequence Table 1, which is within the scope of the present disclosure. In specific examples, the variant CRISPR nuclease polypeptide may comprise (e.g., consist of)Attorney Docket No.: 063586-545001 WO the amino acid sequence of SEQ ID NOs: 12.(c) Nuclease BT and Variants ThereofIn some embodiments, the nuclease polypeptide is derived from Nuclease BT, including both the wild-type Nuclease BT (SEQ ID NO: 3), a variant thereof such as those disclosed herein, or a fusion polypeptide comprising such. The variant nuclease polypeptides of Nuclease BT may be produced via introducing one or more mutations to the reference nuclease to modulate (e.g., enhance or reduce) one or more activities of the nuclease.Nuclease BT of SEQ ID NO: 3 (see Sequence Table 1 below) is an RNA-guided nuclease that comprises both a RuvC nuclease domain (located at residues 60-96, 148-181, and 272-341 of SEQ ID NO: 3) and a HNH domain (located at residues 182-271 of SEQ ID NO: 3). The RuvC nuclease domain and the HNH nuclease domain coordinate cleavage of the DNA strand adjacent to the 5’-RRT-3’ PAM or 5’-NRT-3’ motif, in which R represents A or G and N represents any nucleotide. Positions D67, El 76, and D329 are deemed the active sites in the RuvC domain and positions H231, H232, and H255 are deemed the active sites in the HNH domain. In addition to the nuclease domains, the reference Nuclease BT of SEQ ID NO: 3 also includes a PLMP domain (residues 1-59 of SEQ ID NO: 3), a BH domain (residues 97-125 of SEQ ID NO: 3), a REC domain (residues 126-147 of SEQ ID NO: 3), a PLL domain (residues 342-355 of SEQ ID NO: 3), a WED domain (residues 356-426 of SEQ ID NO: 3), and a PID domain (residues 427-484 of SEQ ID NO: 3).Compared to the CRISPR Cas9 nuclease, Nuclease BT of SEQ ID NO: 3 disclosed herein is smaller. Its cognate scaffold comprises a distinctive structure compared to the Cas9 nuclease scaffold. The distinctive scaffold is expected to allow for decreased size of some domains (e.g., the REC domain) and thus contribute to the smaller size of Nuclease BT. Additionally, the cognate scaffold can be miniaturized relative to the reference scaffold of SEQ ID NO: 3. These features would be beneficial for delivery. Arginine and / or lysine substitutions (e.g., arginine substitutions) can be introduced into Nuclease BT of SEQ ID NO: 3 to increase indel activity. Additionally, since Nuclease BT of SEQ ID NO: 3 comprises a RuvC domain and an HNH domain, nickase variants can be engineered via disruption of the nuclease activity of one of the two domains. The cutting pattern of and PAMs capable of being recognized by Nuclease BT of SEQ ID NO: 3 are different than those of Cas9, allowing for additional gene targets to be edited. Also, unlike Cas9, Nuclease BT of SEQ ID NO: 3 comprises a PLMP domain. The PLMP domain is expected to bind a helix on the 3’ end of the scaffold and provide an increased binding affinity of Nuclease BT to its cognate scaffold.Attorney Docket No.: 063586-545001 WOVariants of Nuclease BTThe variant of Nuclease BT provided herein are derived from Nuclease BT of SEQ ID NO: 3, e.g., via introducing one or more mutations to the reference Nuclease BT to modulate (e.g., enhance or reduce) one or more activities of the nuclease.The variant of Nuclease BT provided herein can contain one or more alterations relative to Nuclease BT of SEQ ID NO: 3, e.g., one or more amino acid residue substitutions, one or more deletions, one or more insertions, fusion, or a combination thereof. In some instances, the alterations may be introduced into the BH domain, the PLL domain, the WED domain, the PID domain, or a combination thereof. In some instances, no alterations are introduced into the RuvC and / or the HNH nuclease domains, or at the active sites and / or sites involved in activity in these domains as provided herein. Alternatively, conservative amino acid substitutions may be introduced into SEQ ID NO: 3, including in the RuvC and / or the HNH nuclease domains.In some instances, the variant of Nuclease BT polypeptide does not include an N- terminus methionine residue, for example, when the N-terminus of the Nuclease BT variant is fused to a functional fragment (e.g., an NLS) or when the Nuclease BT variant is located at the C-terminal part in the fusion polypeptide as disclosed herein.(i) Arginine and / or Lysine SubstitutionsIn some embodiments, the variant of Nuclease BT provided herein may comprise one or more arginine substitutions, one or more lysine substitutions, or a combination thereof relative to SEQ ID NO: 3. In some instances, the arginine and / or lysine substitutions may be located in the BH domain, in the PLL domain, in the WED domain, in the PID domain, or in any of the combination thereof. In some instances, the one or more arginine and / or lysine substitutions (e.g., arginine substitutions) may be located at one or more of positions R31, N34, R39, E99, Q102, E105, T106, T113, K123, R135, R151, M186, E191, Q194, 1206, N239, V256, 1272, A277, K241, L292, K318, E401, E432, and V433 in SEQ ID NO: 1. In some instances, the Nuclease BT variant may comprise one or more arginine substitutions at one or more of positions E99, Q102, E105, T106, T113, K123, E191, 1206, N239, V256, 1272, A277, K241, K318, E401, E432, and V433. Alternatively, or in addition, the Nuclease BT variant may comprise one or more lysine substitutions at one or more of positions R31, N34, R39, R135, R151, M186, Q194, and L292.In some specific examples, the variant of Nuclease BT may comprise arginine and / or lysine substitutions (e.g., arginine substitutions) at the following positions relative to SEQ IDAttorney Docket No.: 063586-545001 WONO: 1 : a) QI 02, 1206, and V433; b) QI 02, V256, and V433; c) QI 02, E191, 1272, and V433; d) Q102, V256, and V433; e) Q102, E191, 1206, and V433; f) Q102, 1272, and V433; g) E105, V256, and V433; h) E105, E191, V256, and V433; i) E105, E191, 1272, E401, and 433; and j) E105, E191, N239, 1272, and V433. In one specific example, the variant of Nuclease BT comprises arginine and / or lysine substitutions at Q102, 1206, and V433 (e.g., Q102R / I206R / V433R).In some examples, the variant of Nuclease BT may contain one or more of the following arginine substitutions relative to SEQ ID NO: 3: V433R, E105R, Q102R, E432R, T113R, I272R, I206R, V256R, E99R, E191R, E401R, N239R, T106R, and A277R. By way of non-limiting examples, the one or more arginine substitutions may include a) Q102R, I206R, and V433R; b) Q102R, V256R, and V433R; c) Q102R, E191R, I272R, and V433R; d) Q102R, V256R, and V433R; e) Q102R, E191R, I206R, and V433R; f) Q102R, I272R, and V433R; g) E105R, V256R, and V433R; h) E105R, E191R, V256R, and V433R; i) E105R, E191R, I272R, E401R, and 433R; or j) E105R, E191R, N239R, I272R, and V433R. In some specific examples, the variant of Nuclease BT comprises the arginine substitutions of Q102R, I206R, and V433R.In some instances, the arginine and / or lysine substitution may not be at the active sites and / or sites involved in activity in the RuvC and / or the HNH nuclease domains (e.g., not at positions D67, El 76, and / or D329 in the RuvC domain and / or at positions H231, H232, and / or H255 in the HNH domain). In some examples, the arginine and / or lysine substitution may not be in the RuvC and / or HNH domains.Additional structural information for Nuclease BT can be found in International Patent Application No. PCT / US25 / 21457, filed on March 26, 2025, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein.(ii) Nickase MutationsAlternatively, or in addition, the variant of Nuclease BT provided herein may comprise one or more mutations within either the RuvC or the HNH nuclease domain to reduce or eliminate the nuclease activity of the target domain, thereby producing a variant with nickase activity. As used herein, the term “nickase” refers to an enzyme that cuts one strand of a double-stranded DNA at a specific recognition nucleotide sequence (e.g., the target sequence disclosed herein). A nickase may interact with one strand of the DNA duplex to produce DNA molecules that are cut at one strand (a.k.a., nicked). In some embodiments, the variant of Nuclease BT disclosed herein is a nickase comprising one or more nickase mutations in theAttorney Docket No.: 063586-545001 WOHNH domain to deactivate the HNH domain. In other embodiments, a variant of Nuclease BT can be a nickase comprising one or more nickase mutations in the RuvC domain to deactivate the RuvC domain. The variant of Nuclease BT may share a high sequence homology relative to the reference Nuclease BT (e.g., at least 85% sequence identity).The one or more mutations may be deletions, insertions, amino acid substitutions, or a combination thereof. In some embodiments, the mutations within either the RuvC or the HNH nuclease domain are amino acid substitutions, of which the substituting amino acid residue is not a conservative substitution of the native amino acid residue at the position of the mutation. For example, if the native amino acid residue is R, the substituting residue can be any amino acid residue except for K. Similarly, if the native amino acid residue is K, the substituting residue can be any amino acid residue except for R. Groups of conservative amino acid residue substitutions are provided herein.Positions D67, El 76, and D329 are identified as putative catalytic residues in the RuvC domain, and positions H231, H232, and H255 are identified as putative catalytic residues in the HNH domain. In some examples, the one or more mutations may be within the RuvC nuclease domain to reduce or inactivate the RuvC domain (e.g., at positions D67, El 76, and / or D329 in the RuvC domain). In some specific examples, the variant of Nuclease BT may contain substitution(s) of D67A, E176A, and / or D329A. In other examples, any of D67, E176, and D329 may be substituted by an amino acid residue similar to A, for example, G, S, or L.In some examples, the one or more mutations may be within the HNH domain to reduce or inactivate the HNH domain (e.g., at positions H231, H232, and / or H255 in the HNH domain). In some specific examples, the variant of Nuclease BT may contain substitutions of H231A, H232A, and / or H255A. Alternatively, any of H231, H232, and H255 may be replaced with an amino acid residue similar to A, for example, G, L, or S. In one specific example, the variant of Nuclease BT may contain substitution of H232A or H232L.In some embodiments, the variant of Nuclease BT provided herein may comprise both arginine / lysine substitutions, e.g., at one or more positions provided herein, and nickase mutations, which are mutations that lead to nickase activity. For example, the variant of Nuclease BT may comprise arginine / lysine substitutions (e.g., arginine substitutions) at positions Q102, 1206, and / or V433, and further comprise an amino acid substitution at H232 (e.g., H232A or H232L) in SEQ ID NO: 3. In one specific example, the variant of Nuclease BT comprises (e.g., consists of) the amino acid substitutions Q102R, I206R, and V433R.In one specific example, the variant of Nuclease BT comprises (e.g., consists of) the amino acid substitutions H232A, Q102R, I206R, and V433R.Attorney Docket No.: 063586-545001 WO(iii) Non-Arginine / Lysine SubstitutionsIn some instances, the variant of Nuclease BT 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 R2, N14, H18, N34, D70, C75, R84, V85, L88, H94, 1100, R102, T106, M109, Y116, 1128, H131, P133, E166, V168, 1182, 1190, R206, R207, A208, A222, P227, F228, Q229, Q238, N239, K241, K257, T260, K263, Q269, 1272, S290, L292, L296, G301, V305, S313, D323, N336, V339, T343, V350, K353, V379, E401, K428, K429, E437, N445, D448, S454, C481 of SEQ ID NO: 3. Examples include, but are not limited to, R2M, N14G, H18N, N34L, D70N, C75S, R84C, V85G, L88A, L88F, L88I, L88M, or L88V, H94G, HOOF, R102I or R102H, T106G, M109G, Y116N, I128E, H131Y, P133G, E166A, V168L, Il 82V, Il 90V, R207H, R207A, A208E, A222G, P227I, F228L, Q229E, Q238C, N239G, K241E, K241I, K241H, or K241Q, K257A, T260N, K263A, Q269L, I272N, S290P, L292N or L292Q, L296A, L296G, L296P, or L296V, G301P, V305I, S313T, D323P, N336C, V339I, T343V, V350F, K353E or K353N, V379I, E401 A, K428N or K428S, K429G, E437P, N445G, D448G, S454T, C481V.In some specific examples, the variant of Nuclease BT may comprise an amino acid substitution at position L292 of SEQ ID NO: 3, for example, L292Q. Residues similar to Q (e.g., conservative substitutions) may also be used as the replacement residue at position L292.(iv) Combination of MutationsIn some embodiments, the variant nuclease polypeptide derived from Nuclease BT can comprise a combination of the mutations disclosed herein, e.g., arginine and / or lysine substitutions, deletions and / or insertions, nickase mutations, and / or non arginine / lysine amino acid substitutions.In some examples, the variant of Nuclease BT comprises arginine and / or lysine substitutions at positions Q102, 1206, and V433 of SEQ ID NO: 3, for example, arginine substitutions Q102R, I206R, and V433R. In some instances, such a Nuclease BT variant may further comprise one or more amino acid substitutions (e.g., arginine substitutions, lysine substitutions, non-arginine / lysine substitutions, or a combination thereof). For example, such a Nuclease BT variant may further comprise a non-arginine / lysine substitution at position L292 of SEQ ID NO: 3, for example, L292Q. In some instances, the variant Nuclease BT may further comprise a nickase mutation, for example at position H232 (e.g., H232A).In some examples, the variant nuclease polypeptide provided herein may share a sequence identity at least 90% (e.g., 95%, 97%, 98%, 99%, 99.5%, or greater) with SEQ IDAttorney Docket No.: 063586-545001 WONO: 3. Exemplary engineered variant nuclease polypeptide of Nuclease BT is listed in Sequence Table 1, which is within the scope of the present disclosure. In specific examples, the variant nuclease polypeptide of Nuclease BT may comprise (e.g., consist of) an amino acid sequence of any one of SEQ ID NO: 166. Alternatively, the variant nuclease polypeptide derived from Nuclease BT can be a variant of SEQ ID NO: 166 comprising a nickase mutation such as H232A.B. Reverse Transcriptase PolypeptidesAs 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 RT polypeptides 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 functional 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: 4 (also referred to as RT_A), the RT polypeptide of SEQ ID NO: 5 (also referred to as RT_B), or the RT polypeptide of SEQ ID NO: 6 (also referred to as RT_C). The RT polypeptides of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 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 inAttorney Docket No.: 063586-545001 WOExamples 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: 4, RT B of SEQ ID NO: 5, or RT C of SEQ ID NO: 6). See, e.g., Sequence Table 2 below. 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 share 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: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In some instances, the variant RT polypeptide may comprise an amino acid sequence at least 90% identical to SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In some instances, the variant RT polypeptide may comprise an amino acid sequence at least 95% identical to SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. 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: 4, SEQ ID NO: 5, or SEQ ID NO: 6.Any of the RT polypeptides disclosed herein may lack its native N-terminus methionine residue, for example, when fused with a nuclease polypeptide or a functional fragment (e.g., NLS) at its N-terminus.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: 4, SEQ ID NO: 5, or SEQ ID NO: 6 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.Attorney Docket No.: 063586-545001 WOIn 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 and Variants ThereofIn some embodiments, the RT polypeptide is derived from reference RT enzyme RT_A (SEQ ID NO: 4), including both the reference enzyme (SEQ ID NO: 4), or a variant thereof such as those disclosed herein. The variants 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 of SEQ ID NO: 4 to modulate (e.g., enhance) one or more activities of the RT enzyme.The RT polypeptide of SEQ ID NO: 4 (see Sequence Table 2 below) is a small-sized RT enzyme with 316 amino acid residues and comprises the YADD (SEQ ID NO: 70) motif. This enzyme has a polymerase domain located at positions 14-294 of SEQ ID NO: 4. 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: 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 an RNase domain (e.g., an RNase H domain), a PrimPol domain, or a protease domain.Compared to the MMLV RT, which is commonly used in RT-mediated gene editing, 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.RT A VariantsVariants of the RT polypeptide of SEQ ID NO: 4 (RT_A) as provided herein can contain one or more alterations relative to SEQ ID NO: 4, e.g., one or more amino acid residueAttorney Docket No.: 063586-545001 WO 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, variants 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: 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: 4. In one example, a variant of the RT polypeptide of SEQ ID NO: 4 comprises an arginine or lysine substitution at position A24 (e.g, A24R) 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_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, F116, S119, 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: 4. 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 Q142 of SEQ ID NO: 4 (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: 4. Examples include Y155F, A188G, V202I, and L287F. In some instances, the replacement residue may be a conservative substitution of the current replacement residue.Attorney Docket No.: 063586-545001 WO(iii) DeletionsAlternatively, or in addition, a variant of the RT polypeptide of SEQ ID NO: 4 (RT_A) 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 1-14 (e.g., within residues 1-13 or 1-12) 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 295-316 of SEQ ID NO: 4. In one example, a variant of the RT polypeptide of SEQ ID NO: 4 is an N- terminal truncated version of SEQ ID NO: 4, e.g., having a deletion of residues 1-12 or residues 1-8 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 298-316 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 13-297 of SEQ ID NO: 4. Alternatively, the truncated version may have the amino acid residues 9-294 of SEQ ID NO: 4.In some examples, the variant of the RT polypeptide of SEQ ID NO: 4 may lack the N- terminus methionine residue, for example, when the variant is fused to a functional fragment (e.g., an NLS fragment) or when the variant is at the C-terminal part in the fusion polypeptide disclosed herein.(iv) Combination of MutationsIn some embodiments, a variant of the RT polypeptide of SEQ ID NO: 4 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 Examples 1, 3, and 9 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 13-297 of SEQ ID NO: 4, having the deletions of 1-12 and 298-316 residues in SEQ ID NO: 4) 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 further comprise one or more substitutions at positions Y155, Al 88, and V202 of SEQ ID NO: 4, for example, Y155F, Y155W, A188G, V202I, V202L, and V202M. Alternatively, the variant RT polypeptide of SEQ ID NO: 4 may comprise further comprise aAttorney Docket No.: 063586-545001 WO 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: 4 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: 4, 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: 4 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), A188 (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: 4.Additional variants of RT_A (SEQ ID NO: 4) 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: 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 a combination of any of the mutations as disclosed herein. Specific examples can be found in Sequence Table 2 below. In some examples, the variant of RT_A comprises the amino acid sequence of SEQ ID NO: 272.(b) RT_B and Variants ThereofIn some embodiments, the RT polypeptide is derived from reference RT enzyme RT_B (SEQ ID NO: 5), including both the reference RT_B enzyme (SEQ ID NO: 5), or a variant thereof such as those disclosed herein. A variant of the RT polypeptide of SEQ ID NO: 5 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: 5 to modulate (e.g., enhance) one or more activities of the RT enzyme.The RT polypeptide of SEQ ID NO: 5 (RT_B) (see Sequence Table 2 below) is a small-sized RT enzyme with 338 amino acid residues and comprises the YADD (SEQ ID NO: 70) motif. This enzyme has a polymerase domain located at positions 1-298 of SEQ ID NO: 5. 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: 5 does not have a thumb subdomain or a connection subdomain in the polymerase domain. The RTAttorney Docket No.: 063586-545001 WO polypeptide of SEQ ID NO: 5 also does not have RNase domain (e.g., an RNase H domain), PrimPol domain, or protease domain.Compared to the MMLV RT, the RT polypeptide of SEQ ID NO: 5 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: 5 to increase enzymatic activity and / or gene editing activity.Variants ofRT BA variant of the RT polypeptide of SEQ ID NO: 5 as provided herein can contain one or more alterations relative to SEQ ID NO: 5, 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: 5 may comprise one or more arginine substitutions, one or more lysine substitutions, or a combination thereof relative to SEQ ID NO: 5. 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 A292 in SEQ ID NO: 3. In some instances, a variant of the RT polypeptide of SEQ ID NO: 5 comprises one or more arginine substitutions at these positions, for example, at position Q32 and / or position Q71 of SEQ ID NO: 5.(ii) DeletionsAlternatively or in addition, a variant of the RT polypeptide of SEQ ID NO: 5 may comprise deletions relative to SEQ ID NO: 5, for example, at the N-terminus of SEQ ID NO: 5, at the C-terminus of SEQ ID NO: 5, or a combination thereof. In some examples, the deletion may be within an N-terminal fragment of SEQ ID NO: 5, for example, within residuesAttorney Docket No.: 063586-545001 WO1-5 of SEQ ID NO: 5. 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: 5. In one example, a variant of the RT polypeptide of SEQ ID NO: 5 is an N-terminal truncated version of SEQ ID NO: 5, e.g., having a deletion of residues 1-5 SEQ ID NO: 5. In another example, a variant of the RT polypeptide of SEQ ID NO: 5 is a C-terminal truncated version of SEQ ID NO: 5, e.g., having a deletion of residues 294-338 of SEQ ID NO: 5. In yet another example, a variant of the RT polypeptide of SEQ ID NO: 5 may have deletions at both the N-terminus and the C- terminus of SEQ ID NO: 5. Such a truncated version may have the amino acid residues 6-293 of SEQ ID NO: 5.In some examples, the variant of the RT polypeptide of SEQ ID NO: 5 may lack the N- terminus methionine residue, for example, when the variant is fused to a functional fragment (e.g., an NLS fragment) or when the variant is at the C-terminal part in the fusion polypeptide disclosed herein.(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, A114, N121, and L257 of SEQ ID NO: 5. Examples include, but are not limited to, S63G, A114C, 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: 5 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 Examples 1, 3, and 9 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: 5 may comprise amino acid substitutions such as arginine / lysine substitutions at one or more of positions Q32, N121, and E291 of SEQ ID NO: 5. 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: 5, for example, having the fragment 6-293 of SEQ ID NO: 5.Alternatively, or in addition, a variant of the RT polypeptide of SEQ ID NO: 5 as disclosed herein may comprise or further comprise amino acid substitutions such asAttorney Docket No.: 063586-545001 WO arginine / lysine substitutions at one or more of positions Q30, Q32, G39, Q71, L122, and / or N126 of SEQ ID NO: 5. 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: 5, for example, Q32R / N121R / E291R. Such a variant may further comprise deletions of residues 1-5 and 294- 338 of SEQ ID NO: 5.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: 5 (e.g., arginine substitutions Q32R and Q71R) and deletions of residues 1-5 and / or 204-338 of SEQ ID NO: 5.Any of the variants of the RT polypeptide of SEQ ID NO: 5 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: 5 and carry any of the mutations or a combination of any of the mutations as disclosed herein. Specific examples can be found in Sequence Table 2 below, e.g., SEQ ID NO: 290.(c) RT_C and Variants ThereofIn some embodiments, the RT polypeptide is derived from the reference RT polypeptide RT_C (SEQ ID NO: 6), including both the reference enzyme (SEQ ID NO: 6), or a variant thereof such as those disclosed herein. A variant of the RT polypeptide of SEQ ID NO: 6 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: 6 (see Sequence Table 2 below) is a small-sized RT enzyme with 350 amino acid residues and comprises the YADD (SEQ ID NO: 70) motif. This enzyme has a polymerase domain located at positions 6-291 of SEQ ID NO: 6. 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: 6 does not have a thumb subdomain or a connection subdomain in the polymerase domain. The RT polypeptide of SEQ ID NO: 6 also does not have RNase domain (e.g., an RNase H domain), PrimPol domain, or protease domain.Compared to the MMLV RT, the RT polypeptide of SEQ ID NO: 6 and variants thereof as disclosed herein are smaller, thereby benefiting delivery. Arginine and / or lysine substitutionsAttorney Docket No.: 063586-545001 WO(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: 6 to increase enzymatic activity and / or gene editing activity.Variant ofRT CA variant of the RT polypeptide of SEQ ID NO: 6 as provided herein can contain one or more alterations relative to SEQ ID NO: 6, 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: 6 may comprise one or more arginine substitutions, one or more lysine substitutions, or a combination thereof relative to SEQ ID NO: 6. 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: K29, T32, E24, A35, A28, G31, T32, P34, G36, V38, T51, K68, T70, E56, G73, A85, E88, N100, H102, N117, R122, H145, S218, F275, and P296 in SEQ ID NO: 6. For example, the RT_C variant may comprise one or more arginine substitutions at one or more of positions K29, T32, E24, A35, A28, G31, T32, P34, G36, V38, K68, T70, G73, A85, E88, H102, N117, H145, S218, and F275 in SEQ ID NO: 6. Alternatively or in addition, the RT C variant may comprise or further comprise a lysine substitution at position N100, N117, R122, and / or P296 of SEQ ID NO: 6.In one example, a variant of the RT polypeptide of SEQ ID NO: 6 comprises an arginine or lysine substitution at position T70 (e.g., T70R) of SEQ ID NO: 6. In another example, a variant of the RT polypeptide of SEQ ID NO: 6 comprises an arginine or lysine substitution at position T32 (e.g., T32R) of SEQ ID NO: 6. In yet another example, a variant of the RT polypeptide of SEQ ID NO: 6 comprises an arginine or lysine substitution at positions T32 and T70 (e.g, T32R / T70R) of SEQ ID NO: 6. In some instances, the variant of SEQ ID NO: 6 may comprise arginine and / or lysine substitutions at one or more of positions K29 (e.g.,Attorney Docket No.: 063586-545001 WOK29R), K68 (e.g., K68R), N100 (e.g., N100K), Hl 02 (e.g., H102R), N117 (e.g., N117K or N117R), and R122 (e.g., R122K) of SEQ ID NO: 6.(ii) Non-Arginine / Lysine SubstitutionsAlternatively, or in addition, a variant of the RT polypeptide of SEQ ID NO: 6 (RT_C) may comprise one or more amino acid residue substitutions (non-arginine / lysine substitutions) at one or more of positions: H8, F25, R43, E52, K48, G73, L83, A93, N100, M101, F103, N117, R110, L121, A127, H145, D146, G154, V170, M172, D180, K192, V195, 1199, V201, A202, M213, M234, L249, 1252, H266, V268, T286, V287, S297, P298, and P299 of SEQ ID NO: 6. Exemplary amino acid substitutions at these positions include: H8N, F25Y, R43N or R43S, E52P, K48P, G73N, L83V or L83I, A93V, N100L, M101A, F103I, N117E, N117M or N119P, R110H or RHON, L121V, A127E, H145E or H145S, D146A or D146M, G154D, M172F, V170Y, D180P, K192I, V195I, A202S, M213L, M234F, L249A, I252L, H266Y, V268M, T286D, V287D, S297G, P298S, and / or P299S.In some instances, the variant of RT_C may comprise a non-arginine / lysine substitution at position Nil 7 of SEQ ID NO: 6, for example, N117, N117M, or N117P. Alternatively, or in addition, the variant of RT_C may comprise or further comprise one or more non- arginine / lysine substitutions at one or more of positions S297, P298, and P299 of SEQ ID NO: 6, for example, S297G, P298S, and / or P299S. In some instances, the replacement residue may be a conservative substitution of the current replacement residue. In some instances, the variant of RT_C (SEQ ID NO: 6) may comprise one or more non-arginine / lysine substitutions at the following positions: H8, R43, M101, F103, N117, H145, D146, 1199, V201, A202, L249, V268, S297, P298, and P299. In specific examples, the non-arginine / lysine substitution(s) is H8N, R43N or R43S, M101A, F103I, N117E, N117M, or N117P, H145S or H145E, D146A or D146M. I199F, V201N, A202S, L249A, V268M, S297G, P298S, and / or P299S.(iii) DeletionsAlternatively, or in addition, a variant of the RT polypeptide of SEQ ID NO: 6 may comprise deletions relative to SEQ ID NO: 6, for example, at the N-terminus of SEQ ID NO: 6, at the C-terminus of SEQ ID NO: 6, or a combination thereof. In some examples, the deletion may be within an N-terminal fragment of SEQ ID NO: 6, for example, within residues 1-10, for example, within residues 1-7 or 1-5 (e.g, 1-4) of SEQ ID NO: 6. In some examples, the deletion may be within a C-terminal fragment of SEQ ID NO: 6, for example, within residues 297-350 of SEQ ID NO: 6. In one example, a variant of the RT polypeptide of SEQ IDAttorney Docket No.: 063586-545001 WONO: 6 is an N-terminal truncated version of SEQ ID NO: 6, e.g., having a deletion of residues 1-4 of SEQ ID NO: 6. In another example, a variant of the RT polypeptide of SEQ ID NO: 6 is a C-terminal truncated version of SEQ ID NO: 6, e.g., having a deletion of residues 297-350 of SEQ ID NO: 6. In some examples, the variant of the RT polypeptide of SEQ ID NO: 6 may have a deletion of residues 300-327 of SEQ ID NO: 6. In yet another example, a variant of the RT polypeptide of SEQ ID NO: 6 may have deletions at both the N-terminus and the C- terminus of SEQ ID NO: 6. Such a truncated version may have the amino acid residues 5-296 of SEQ ID NO: 6. In some examples, the deletion may be within the polypeptide of SEQ ID NO: 6, for example, a deletion of residues 297-325 of SEQ ID NO: 6.Alternatively, or in addition, the variant of SEQ ID NO: 6 may comprise or further comprise a deletion at position 50 of SEQ ID NO: 6.(iv) Combination of MutationsIn some embodiments, a variant of the RT polypeptide of SEQ ID NO: 6 (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 Examples 1, 3, and 9-12 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: 6 (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: 6). 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: 6. Such additional substitutions may include A25R, A25K, A25H, R110H, R110K, F275Y, and F275W.A variant of the RT polypeptide of SEQ ID NO: 6 disclosed herein may comprise or further comprise arginine and / or lysine substitutions (e.g., arginine substitutions) at positions T32, G31, V38, E88, and / or Nil 7 of SEQ ID NO: 6. 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: 6. 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: 6. In yet another example, the variant of RT_C comprises the T70R substitution and the deletion of 297-325Attorney Docket No.: 063586-545001 WO relative to SEQ ID NO: 6.In another specific example, the variant of RT_C comprises amino acid substitutions at positions T32 and N117, for example, T32R and N117E, N117K, N117M, N117P, or N117R. 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: 6. The variant of RT_C comprising the T70R, S297G, P298S, and P299S substitutions and the deletion of 300-327 of SEQ ID NO: 6 is equivalent to a variant of RT_C comprising T70R and deletions of residues 297-318 and 322-327 of SEQ ID NO: 6.In specific examples, the variant of SEQ ID NO: 6 comprises the following features: (i) arginine or lysine substitutions at positions T32 and N117 of SEQ ID NO: 6, optionally T32R and Nil 7R or N117K; (ii) non-arginine / lysine substitutions at positions S297, P298, and P299 of SEQ ID NO: 6, optionally S297G, P298S, and P299S; and (iii) deletion of residues 300-327 of SEQ ID NO: 6. In other specific examples, the variant of SEQ ID NO: 6 comprises the following features: (i) arginine or lysine substitutions at positions T32 SEQ ID NO: 6, optionally T32R; (ii) non-arginine / lysine substitutions at positions N117, S297, P298, and P299 of SEQ ID NO: 6, optionally N117E, N117M, or N117P, S297G, P298S, and P299S; and (iii) deletion of residues 300-327 of SEQ ID NO: 6.Such a variant may further comprise: (iv) arginine or lysine substitution(s) at position W7, K29, N100, and / or H102, of SEQ ID NO: 6 (e.g., W7K, K29R, N100K, and / or H102R), (v) non-arginine / lysine substitution(s) at position F25, R43, L83, M101, F103, N117, H145, D146, V153, 1199, V201, L249, and / or T286 of SEQ ID NO: 6 (e.g., F25Y, R43E, L83I, M101A, F103I, N117E, N117M, or N117P, H145S or H145E, D146A or D146M, V153T, I199F, V201N, L249A, and / or T286D), (vi) deletion of residue 50 of SEQ ID NO: 6; or (vii) a combination of any one of (iv)-(vi).In one example, such a variant, comprising the features of (i)-(iii), may further comprise a combination of substitutions at positions N100, M101, Hl 02, and Fl 03 of SEQ ID NO: 6 (e.g., N100K, M101A, H102R, and F103I). In another example, the variant may further comprise a combination of substitutions at positions H145 and D146 of SEQ ID NO: 6 (e.g., H145S and D146A or H145E, and D146M). In yet another example, the variant may further comprise a combination of substitutions at positions F25, R43, and L83 of SEQ ID NO: 6 (e.g., F25Y, R43E, and L83I).Any of the variants of the RT polypeptide of SEQ ID NO: 6 as provided herein may share a sequence identity at least 90% (e.g., 95%, 97%, 98%, 99%, 99.5%, or greater) withAttorney Docket No.: 063586-545001 WOSEQ ID NO: 6 and carry any of the mutations or a combination of mutations as disclosed herein. Specific examples can be found in Sequence Table 2 below, for example, any one of SEQ ID NO: 26, 303, 329, 333, 444-466, or 481-491.C. Fusion PolypeptidesIn some embodiments, the nuclease polypeptide in the gene editing systems disclosed herein (e.g., the reference nuclease of SEQ ID NO: 1 or any of the variants thereof as disclosed herein, the reference nuclease of SEQ ID NO: 2 or any of the variants thereof as disclosed herein, or the reference nuclease of SEQ ID NO: 3 or any of the variants thereof as disclosed herein) may be fused with any of the RT polypeptides disclosed herein (e.g., RT_A (SEQ ID NO: 4) or a variant thereof as disclosed herein, RT_B (SEQ ID NO: 5) or a variant thereof as disclosed herein, or RT_C (SEQ ID NO: 6) or a variant thereof as disclosed herein) to form a fusion polypeptide.As used herein, the terms “fusion” 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 fusion 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 embodiments, the fusion polypeptide may comprise the reverse transcriptase polypeptide at its N-terminus and the RNA-guided nuclease polypeptide downstream to the RT polypeptide. In other embodiments, the fusion polypeptide may comprise the RNA-guided nuclease polypeptide at its N-terminus and the RT polypeptide downstream to the RNA-guided nuclease polypeptide. In some embodiments, the RT polypeptide may be fused with the RNA- guided nuclease polypeptide at an intramolecular position within the RT polypeptide, for example, the RNA-guided nuclease polypeptide may be within a loop of the reverse transcriptase polypeptide.In some instances, the fusion polypeptide may further comprise one or more additional functional moieties, such as nuclear localization signals and / or peptide linkers. Exemplary additional functional moieties to include in the fusion 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 light-gated control factor, a chemically inducible factor, a chromatin visualization factor, or a combination thereof.In some embodiments, the additional functional moiety may comprise an NLS, aAttorney Docket No.: 063586-545001 WO nuclear export signal (NES), or a combination thereof. In some examples, the fusion polypeptide may comprise an NLS, which may be located at either the N-terminus or the C- terminus. In specific examples, the fusion 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 fusion 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 nuclease). In some embodiments, the fusion polypeptide may comprise an NLS within a flexible loop of the nuclease. Exemplary NLS fragments are provided in Sequence Table 3 below.Alternatively, or in addition, the additional functional elements may be a flexible peptide linker, which can be located between the RNA-guided nuclease 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 Sequence Table 1 below. See also Examples 1-3 and 9-12.In some examples, the nuclease-RT fusion polypeptide provided herein may comprise a peptide linker located between the nuclease polypeptide and the RT polypeptide. In some instances, the nuclease polypeptide is N-terminal to the RT polypeptide. In some instances, the nuclease 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 nuclease polypeptide and the RT polypeptide. For example, an additional peptide linker and an NLS may be placed between the nuclease polypeptide and the RT polypeptide, in addition to the first peptide linker. In some specific examples, the peptide linker between the RNA-guided nuclease 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 nuclease-RT fusion polypeptide provided herein may comprise two NLSs. 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 some embodiments, the nuclease-RT fusion polypeptide provided herein may comprise Nuclease A or a variant thereof as disclosed herein and one of the RT_A, RT_B, RT_C, or a variant thereof as also provided herein. Specific Nuclease A variants and the RT variants for use in constructing the nuclease-RT fusion polypeptides are provided in Examples 1 and 9-12 below. See also Sequence Table 3. The Nuclease A or a variant thereof may be N- terminal to the RT polypeptide. Alternatively, the RT polypeptide may be at the N-terminal toAttorney Docket No.: 063586-545001 WO the Nuclease A or the variant thereof. In some instances, the nuclease-RT fusion polypeptide may contain an NLS at both the N-terminus and the C-terminus and a peptide linker such as an XTEN linker provided herein between the nuclease and the RT polypeptides.In other embodiments, the nuclease-RT fusion polypeptide provided herein may comprise Nuclease K or a variant thereof as disclosed herein and one of the RT_A, RT_B, RT_C, or a variant thereof as also provided herein. Specific Nuclease A variants and the RT variants for use in constructing the nuclease-RT fusion polypeptides are provided in Examples2 and 9-12 below. See also Sequence Table 3. The Nuclease A or a variant thereof may be N- terminal to the RT polypeptide. Alternatively, the RT polypeptide may be at the N-terminal to the Nuclease A or the variant thereof. In some instances, the nuclease-RT fusion polypeptide may contain an NLS at both the N-terminus and the C-terminus and a peptide linker such as an XTEN linker provided herein between the nuclease and the RT polypeptides.In yet other embodiments, the nuclease-RT fusion polypeptide provided herein may comprise Nuclease BT or a variant thereof as disclosed herein and one of the RT_A, RT_B, RT_C, or a variant thereof as also provided herein. Specific Nuclease A variants and the RT variants for use in constructing the nuclease-RT fusion polypeptides are provided in Examples3 and 9-12 below. See also Sequence Table 3. The Nuclease A or a variant thereof may be N- terminal to the RT polypeptide. Alternatively, the RT polypeptide may be at the N-terminal to the Nuclease A or the variant thereof. In some instances, the nuclease-RT fusion polypeptide may contain an NLS at both the N-terminus and the C-terminus and a peptide linker such as an XTEN linker provided herein between the nuclease and the RT polypeptides.In some examples, the fusion polypeptide disclosed herein may comprise a variant of Nuclease A variant in fusion with a variant of RT_A, RT_B, or RT_C.The Nuclease A variant may be a Nickase A variant (e.g., comprising the H397A substitution) and may further comprise deletions of 1-15 of SEQ ID NO: 1 (known as the 16- 775 truncation), and amino acid substitutions I67R, D568R, W593R, and E706R. Such a Nickase A variant may comprise (e.g., consisting of) SEQ ID NO: 90. In some instances, such a Nickase A variant may further comprise one or more amino acid substitutions (e.g., arginine substitutions, lysine substitutions, non-arginine / lysine substitutions, or a combination thereof). Such a Nuclease A variant may further comprise one or more non-arginine / lysine amino acid substitutions, for example, at one or more of the positions T83, N87, D225, R261, P575, W622, and S729 relative to SEQ ID NO: 1. Examples include T83N, N87S, D225N, R261K, P575A, W622F, and S729G. In one example, the Nickase A variant may comprise (e.g., consisting of) SEQ ID NO: 134. The Nuclease A variant may further comprise non-Attorney Docket No.: 063586-545001 WO arginine / lysine substitutions at the position of W508, T562, or R699 of SEQ ID NO: 1, for example, W508K, T562S, or R699S. An exemplary Nuclease A variant may comprise any of the amino acid sequence of SEQ ID NO: 134, 143, 148, 163, 428-443 and 469-480 (e.g., SEQ ID NO: 430, 431, or 480).The RT_A variant in the fusion polypeptide may comprises (e.g., consists of) (a) amino acid substitutions at positions A24 (e.g., A24R), Y155 (e.g., Y155F), A188 (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: 4. In some examples, the variant of RT_A comprises (e.g., consists of) the amino acid sequence of SEQ ID NO: 272.The RT_B variant in the fusion polypeptide may comprise arginine and / or lysine substitutions at positions Q32 and Q71 of SEQ ID NO: 5 (e.g., arginine substitutions Q32R and Q71R) and deletions of residues 1-5 and / or 204-338 of SEQ ID NO: 5. In one example, the RT_B variant comprises (e.g., consisting of) SEQ ID NO: 290.The RT_C variant in the fusion polypeptide may comprise amino acid substitutions at positions T32 and N117, for example, T32R and N117R, N117E, N117M, N117Q, or N117P 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: 6. for example, any one of SEQ ID NO: 26, 303, 329, 333, 444-466, and 481-491 (e.g., SEQ ID NO: 329, or 444).Fusion polypeptides comprising a Nuclease A variant and any of an RT_A, RT_B, and RT_C variants as disclosed herein may further comprise one or more NLS fragments, one or more peptide linkers as disclosed herein, or a combination thereof. Examples of such fusion polypeptides are provided in Sequence Table 3, each of which is within the scope of the instant disclosure.In some examples, the fusion polypeptide disclosed herein may comprise a variant of Nuclease K variant in fusion with a variant of RT_A, RT_B, or RT_C. In some instances, the variant of Nuclease K may comprise arginine / lysine substitutions (e.g., arginine substitutions) at positions G42 and D582 (e.g., G42R / D582R), and an amino acid substitution at H375 (e.g., H375A). In specific examples, the variant CRISPR nuclease polypeptide may comprise (e.g., consist of) the amino acid sequence of SEQ ID NOs: 12. In some instances, the RT polypeptide in a fusion polypeptide comprising the Nuclease K variant may be a variant of RT_C. Such an RT_C variant may comprise a T70R substation relative to SEQ ID NO: 6. In one example, the RT_C variant may comprise (e.g., consists of) SEQ ID NO: 26.Fusion polypeptides comprising a Nuclease K variant and any of an RT_A, RT_B, andAttorney Docket No.: 063586-545001 WORT_C variants as disclosed herein may further comprise one or more NLS fragments, one or more peptide linkers as disclosed herein, or a combination thereof. Examples of such fusion polypeptides are provided in Sequence Table 3, each of which is within the scope of the instant disclosure.In some examples, the fusion polypeptide disclosed herein may comprise a variant of Nuclease BT variant in fusion with a variant of RT_A, RT_B, or RT_C. In some instances, the variant of Nuclease BT comprises arginine and / or lysine substitutions at positions Q102, 1206, and V433 of SEQ ID NO: 3, for example, arginine substitutions Q102R, I206R, and V433R. In some instances, such a Nuclease BT variant may further comprise one or more amino acid substitutions (e.g., arginine substitutions, lysine substitutions, non-arginine / lysine substitutions, or a combination thereof). For example, such a Nuclease BT variant may further comprise a non-arginine / lysine substitution at position L292 of SEQ ID NO: 3, for example, L292Q. In some instances, the variant Nuclease BT may further comprise a nickase mutation, for example at position H232 (e.g., H232A). In specific examples, the variant nuclease polypeptide of Nuclease BT may comprise (e.g., consist of) an amino acid sequence of any one of SEQ ID NO: 166. Alternatively, the variant nuclease polypeptide derived from Nuclease BT can be a variant of SEQ ID NO: 166 comprising a nickase mutation such as H232A.The RT fusion partner in the Nuclease BT variant-containing fusion polypeptide may be a RT_C variant. In some instances, the RT_C variant may comprise the T32R and N117R substitutions relative to SEQ ID NO: 6. In one example, the RT_C variant may comprise (e.g., consisting of) SEQ ID NO: 303.Fusion polypeptides comprising a Nuclease BT variant and any of an RT_A, RT_B, and RT_C variant as disclosed herein may further comprise one or more NLS fragments, one or more peptide linkers as disclosed herein, or a combination thereof. Examples of such fusion polypeptides are provided in Sequence Table 3, each of which is within the scope of the instant disclosure.Of note, the NLS and peptide linker sequences in any of the exemplary fusion polypeptides provided in Sequence Table 3 are for illustration purposes only and can be replaced with other suitable NLS and / or peptide linkers as known to those skilled in the art.II. Nuclease-Guided Reverse Transcription-Mediated Gene Editing SystemsThe present disclosure also provides nuclease-guided reverse transcription-mediated gene editing systems comprising (a) any of the nuclease polypeptides provided herein or a nucleic acid encoding such, (b) any of the RT polypeptides provided herein or a nucleic acidAttorney Docket No.: 063586-545001 WO encoding such, (c) a guide RNA (gRNA) or a nucleic acid encoding such, the gRNA comprising a spacer sequence and a scaffold sequence recognizable by the nuclease polypeptide, and (d) a reverse transcription donor RNA or a nucleic acid encoding such, the donor RNA comprising a primer binding site (PBS) and a template sequence (z.e., RTT). In some instances, the nuclease polypeptide and the RT polypeptide form a fusion polypeptide (e.g., any of the fusion proteins provided herein). Alternatively, or in addition, the gRNA and the donor RNA form a single RNA molecule (i.e., an editing template RNA).In some instances, the gene editing system provided herein comprises the enzymes or the fusion protein comprising such. Alternatively, the gene editing system comprises the encoding nucleic acids for the enzymes or the fusion protein comprising such. Alternatively, or in addition, the gene editing system comprises the RNA components. Alternatively, the gene editing system comprises the encoding nucleic acid(s) for the RNA components.A. Protein Components and Encoding Nucleic AcidsThe gene editing systems provided herein involve at least two enzymes, a nuclease and an RT such as those provided herein. 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. Examples are provided herein. In some embodiments, the 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.In some embodiments, the fusion polypeptide may comprise the reverse transcriptase polypeptide at its N-terminus and the nuclease polypeptide downstream to the RT polypeptide. In other embodiments, the fusion polypeptide may comprise the nuclease polypeptide at its N- terminus and the RT polypeptide downstream to the nuclease polypeptide. In some embodiments, the RT polypeptide may be fused with the nuclease polypeptide at an intramolecular position within the RT polypeptide, for example, the nuclease polypeptide may be within a loop of the reverse transcriptase polypeptide.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, or adeno-associated viral vectors) capable of expressing the 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 nuclease, the RT, or the fusion polypeptideAttorney Docket No.: 063586-545001 WO comprising such.In specific examples, the gene editing system may comprise an adeno-associated (AAV) viral vector encoding the nuclease-RT fusion protein or an AAV particle comprising an AAV genome encoding the fusion protein. In some instances, the AAV vector or genome may further encode the RNA components of the gene editing system disclosed herein. In some instances, the total size of the nuclease and the RT polypeptide, taken together, can be less than less than 1150 amino acids, for example, e.g., less than 1145 amino acids, less than 1120 amino acids, less than 1110 amino acids, less than 1100 amino acids, less than 1090 amino acids, or less than 1085 amino acids. In some examples, the total size of the nuclease and the RT polypeptides can be less than 1143 or 1142 amino acids. One advantageous feature of the nuclease / RT pair disclosed herein (e.g., those described in Examples 1-3 and 9-12 below. See also Sequence Table 3 below) is that their sizes are small enough to fit into an AAV vector with an Editing Template RNA and other regulatory components to facilitate delivery to particular host cells of interest.Any of the nuclease polypeptide disclosed herein and any of the RT polypeptides disclosed herein, as well as fusion polypeptides comprising such, can be used for the gene editing systems provided herein. See, e.g., descriptions of the enzymes and the fusion polypeptides elsewhere herein and also in Examples 1-3 and 9-12 and Sequence Tables 1-3, as well as variants thereof with protein tags removed.In other embodiments, the gene editing system provided herein may comprise a nucleic acid encoding the nuclease polypeptide. In some examples, the nucleotide sequence encoding the nuclease polypeptide described herein can be codon-optimized for use in a particular host cell or organism. For example, the nucleic acid can be codon-optimized for any non-human eukaryote including mice, rats, rabbits, dogs, livestock, or non-human primates. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at the world wide web site of kazusa.orjp / codon / and these tables can be adapted in a number of ways. See Nakamura et al. Nucl. Acids Res. 28:292 (2000), which is incorporated herein by reference in its entirety. Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA). In some examples, the nucleic acid encoding the nuclease polypeptides as disclosed herein can be an mRNA molecule, which can be codon optimized.Preparation of Protein ComponentsThe nuclease polypeptide, the RT polypeptide, or the nuclease-RT fusion polypeptideAttorney Docket No.: 063586-545001 WO disclosed herein may be prepared by conventional methods or the methods disclosed herein. For example, the nuclease polypeptide, the RT polypeptide, or the nuclease-RT fusion polypeptide 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. The nuclease polypeptide, the RT polypeptide, or the nuclease-RT fusion polypeptide can be also prepared by an in vitro coupled transcription-translation system.Host cells that can be used for preparation of the nuclease polypeptide, the RT polypeptide, or the nuclease-RT fusion polypeptide are not particularly limited as long as they can produce the nuclease polypeptide, the RT polypeptide, or the nuclease-RT fusion polypeptide. Some nonlimiting examples of the host cells include bacteria cells (e.g., E. coli cells), yeast cells, insect cells, or mammalian cells.(i) VectorsThe present disclosure provides vectors for expressing the nuclease polypeptide, the RT polypeptide, or the nuclease-RT fusion polypeptide. In some embodiments, a vector disclosed herein includes a nucleotide sequence encoding nuclease polypeptide, the RT polypeptide, or the nuclease-RT fusion polypeptide. 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 nuclease polypeptide, the RT polypeptide, or the nuclease-RT fusion polypeptide 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 nuclease polypeptide, the RT polypeptide, or the nuclease-RT fusion polypeptide 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 virologyAttorney Docket No.: 063586-545001 WO 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 functional 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 functional elements downstream of the start site as well. Depending on the promoter, it appears that individual elements can function 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 may be 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, the selectable 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. coli 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 particularlyAttorney Docket No.: 063586-545001 WO limited, and examples thereof include methods using a plasmid, a phage or a cosmid.(ii) Methods of ExpressionThe present disclosure includes a method for protein expression, comprising translating the nuclease polypeptide, the RT polypeptide, or the nuclease-RT fusion polypeptide described herein.In some embodiments, a host cell described herein is used to express the nuclease polypeptide, the RT polypeptide, or the nuclease-RT fusion polypeptide. 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. coh. 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, z.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 nuclease polypeptide, the RT polypeptide, or the nuclease-RT fusion polypeptide. After expression, the host cells can be collected and the nuclease polypeptide, the RT polypeptide, or the nuclease-RT fusion polypeptide 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 nuclease polypeptide, a mature RT polypeptide, or a mature nuclease-RT fusion polypeptide in a host cell. Such methods include, but are not 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 (RIA), 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 nuclease polypeptide, the RT polypeptide, or the nuclease-RT fusion polypeptide (and optionally the gRNA and / or the RT donor RNA in the gene editing system disclosed herein). Such a method may comprise providing a polyribonucleotide encoding the nuclease polypeptide, the RTAttorney Docket No.: 063586-545001 WO polypeptide, or the nuclease-RT fusion polypeptide to a host cell in a subject e.g., a human subject) wherein the polyribonucleotide encodes the nuclease polypeptide, the RT polypeptide, or the nuclease-RT fusion polypeptide and expressing the nuclease polypeptide, the RT polypeptide, or the nuclease-RT fusion polypeptide from the cell.B. RNA Components and Encoding Nucleic AcidsThe 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.In some specific examples, the gene editing system may comprise an AAV vector or AAV particle that comprises nucleic acids encoding both the nuclease and RT enzymes (e.g, a fusion polypeptide comprising such) and the gRNA and RT donor RNA (e.g., a single editing RNA template comprising both, z.e., an editing template RNA).(a) Guide RNAThe 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 a 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 nuclease. In some embodiments, the scaffold is an RNA sequence.In some instances, the gRNA disclosed herein may further comprise a linker sequence,Attorney Docket No.: 063586-545001 WO a 5’ end and / or 3’ end protection fragment, or a combination thereof. i. Spacer SequenceAs 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 variant nuclease polypeptide contained in the gene editing system.In association with Nuclease A or a variant thereof as disclosed herein, the target sequence can be adjacent to (e.g., upstream to or 5’ to) a PAM of 5’-NGG-3’, in which N represents any nucleotide.In association with Nuclease K or a variant thereof as disclosed herein, the target sequence can be adjacent to (e.g., upstream to or 5’ to) a PAM of 5’-NGG-3’, in which N represents any nucleotide.In association with Nuclease BT or a variant thereof as disclosed herein, the target sequence can be adjacent to (e.g., upstream to or 5’ to) a PAM of 5’-RRT-3’ or 5’-NRT-3’, in which R represents A or G and N represents any nucleotide.As used herein, the term “protospacer adjacent motif’ or “PAM sequence” refers to a DNA sequence adjacent to a target sequence. In some embodiments, a PAM sequence is required for binding of the nuclease and / or indel activity. In a double-stranded DNA molecule, the strand containing the PAM motif 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 motif 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, aAttorney Docket No.: 063586-545001 WO nucleotide sequence is adjacent to another nucleotide sequence if no nucleotides separate the two sequences (z.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). In some embodiments, a first sequence is adjacent to a second sequence if the two sequences are separated by about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments, a first sequence is adjacent to a second sequence if the two sequences are separated by up to 2 nucleotides, up to 5 nucleotides, up to 8 nucleotides, up to 10 nucleotides, up to 12 nucleotides, or up to 15 nucleotides. In some embodiments, a first sequence is adjacent to a second sequence if the two sequences are separated by 2-5 nucleotides, 4-6 nucleotides, 4-8 nucleotides, 4-10 nucleotides, 6-8 nucleotides, 6-10 nucleotides, 6-12 nucleotides, 8-10 nucleotides, 8-12 nucleotides, 10-12 nucleotides, 10-15 nucleotides, or 12-15 nucleotides.In specific examples, the spacer targets a nucleotide sequence (target sequence) that is immediately adjacent to the PAM motif. In other specific examples, the target sequence and the PAM motif have a small gap of less than 5 (e.g., 1, 2, 3, 4, or 5) 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).Attorney Docket No.: 063586-545001 WO ii. Scaffold SequenceThe scaffold sequence in the gRNA is recognizable by the nuclease polypeptide also in the gene editing system. In a gRNA, the scaffold may be located at the 3’ 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.Scaffold Sequence for Nuclease AIn some instances, the scaffold sequence is recognizable by Nuclease A (SEQ ID NO: 1) or any engineered variants thereof as disclosed herein. Such a scaffold sequence may comprise SEQ ID NO: 63 provided in Sequence Table 4 below.The predicted secondary structure of the reference scaffold sequence of SEQ ID NO: 63 is provided in FIG. 1A. In other instances, the scaffold sequence recognizable by Nuclease A (SEQ ID NO: 1) or any engineered variants thereof may be a variant derived from SEQ ID NO: 63. Such a variant scaffold sequence may comprise a nucleotide sequence at least 70% (e.g., at least 73%, at least 75%, at least 80%, 85%, 90%, 95%, 98%, or greater) identical to SEQ ID NO: 63. Alternatively or in addition, the variant scaffold sequence may comprise deletions, nucleotide substitutions, or a combination thereof. The variant CRISPR nuclease polypeptide may have increased binding to the variant scaffold sequence as compared with the scaffold of SEQ ID NO: 63. In some examples, the variant scaffold may be a fragment of SEQ ID NO: 63 or a variant thereof as disclosed herein. For example, the variant scaffold for use in the gRNAs provided herein may have a length ranging from 100-150.In some embodiments, the scaffold sequence recognizable by Nuclease A (SEQ ID NO: 1) or any engineered variants thereof can be a truncated variant of SEQ ID NO: 63. Any of the truncation variants provided herein may be about 110-140-nucleotide in length.In some examples, the truncated variant scaffold sequence recognizable by Nuclease A (SEQ ID NO: 1) or any engineered variants thereof may have a 3’ truncation relative to SEQ ID NO: 2. Such a 3’ truncation may remove one or more of the stem structures P6a, P6b, and P6c depicted in FIG. 1A. In specific examples, the 3’ truncation may comprise a deletion within residues 143-202 of SEQ ID NO: 63, e.g., deletion of the 143-202 fragment of SEQ ID NO: 63. See, FIG. IB.Alternatively, or in addition, the variant scaffold sequence can be a truncated variant of SEQ ID NO: 63 having an internal truncation relative to SEQ ID NO: 63. For example, such a truncation variant may comprise a deletion of whole or part of the stem structure Pl depicted in FIG. 1A. In some examples, the truncation variant may comprise a deletion within residues 14-Attorney Docket No.: 063586-545001 WO20 of SEQ ID NO: 63, within residues 25-32 of SEQ ID NO: 63, or a combination thereof. In specific examples, the truncation variant may comprise deletions of the 14-20 fragment and 25- 32 fragment of SEQ ID NO: 63. See, e.g., FIGs. IB and 1C. In some instances, the truncation variant may comprise a deletion in the loop consisting of residues 77-86 of SEQ ID NO: 63, which may result in shortening of the stem structure P5 depicted in FIG. 1A. In some examples, the truncation variant may comprise a deletion within residues 81-85 (e.g., deletion of the 81-85 fragment) of SEQ ID NO: 63.In some examples, the variant scaffold sequence recognizable by Nuclease A (SEQ ID NO: 1) or any engineered variants thereof may comprise a combination of the 3’ truncation and one or more of the internal truncations as disclosed herein. Alternatively, or in addition, the variant scaffold sequence may comprise one or more nucleotide variations relative to the corresponding residues in SEQ ID NO: 63.In one specific examples, the scaffold sequence recognizable by Nuclease A (SEQ ID NO: 1) or any engineered variants thereof comprises (e.g., consists of) the nucleotide sequence of SEQ ID NO: 64 shown in Sequence Table 4 below. For example, the variant scaffold sequence for use in the gRNAs provided herein may have a length ranging from 100-150 nt. Alternatively, the variant scaffold sequence may be about 115-130-nt in length.The cognate scaffold sequence for Nuclease A is highly homology to the scaffold sequence for Nuclease K. As such, the scaffold sequences for Nuclease A and Nuclease K may be used interchangeably. For example, the cognate scaffold sequence of Nuclease K (SEQ ID NO: 66) and variants thereof as disclosed herein (e.g., SEQ ID NO: 65) can also be used as the scaffold sequence for Nuclease A.Scaffold Sequence for Nuclease KIn some instances, the scaffold sequence is recognizable by Nuclease K (SEQ ID NO: 2) or any engineered variants thereof as disclosed herein. Such a scaffold sequence may comprise SEQ ID NO: 66 shown in Sequence Table 4 below.The projected secondary structure of the Nuclease K reference scaffold sequence (SEQ ID NO: 66) is depicted in FIG. ID. This scaffold sequence includes multiple stem-loop structures, including stem -loop Pl, stem-loop P2 (including P2a and P2b), stem-loop P3 (including P3a and P3b), stem-loop P4, stem -loop P6, stem-loop P7, and stem-loop P8. Further, the Nuclease K reference scaffold sequence includes stem P5, which is formed via base-pairing between the loop sequence in P2b and the segment connecting stem-loop P2a and stem-loop P6.Attorney Docket No.: 063586-545001 WOIn some instances, the scaffold sequence recognizable by Nuclease K (SEQ ID NO: 2) or any engineered variants thereof may be a variant derived from SEQ ID NO: 66. Such a variant scaffold sequence may comprise a nucleotide sequence at least 70% (e.g., at least 73%, at least 75%, at least 80%, at least 85%, 90%, 95%, 98%, or greater) identical to SEQ ID NO: 66. Alternatively or in addition, the variant scaffold sequence may comprise deletions, nucleotide substitutions, or a combination thereof. The variant CRISPR nuclease polypeptide may have increased binding to the variant scaffold sequence as compared with the scaffold of SEQ ID NO: 66.In some examples, the variant scaffold may be a truncated version of SEQ ID NO: 66, for example, with the 3’ end stem -loops P6-P8 removed, with a shortened Pl stem-loop, or a combination thereof. For example, the truncated version may have a 3’ end deletion within nucleotides 126-179 of SEQ ID NO: 66. Alternatively or in addition, the truncated version may have a deletion within nucleotides 8-31 of SEQ ID NO: 66. The variant scaffold sequence for use in the gRNAs provided herein may have a length ranging from 100-150 nt. Alternatively, the variant scaffold sequence may be about 115-130-nt in length. One exemplary truncated version of Nuclease K scaffold is SEQ ID NO: 65, the projected secondary structure of which is shown in FIG. 1C.Alternatively or in addition, one or more nucleotide substitutions can be introduced into the Nuclease K reference scaffold or the variant of SEQ ID NO: 65 to produce additional Nuclease K scaffold variants. In some instances, the nucleotide substitutions may change one or more U-A base pairs (e.g., in stem Pl and / or stem P2) in a stem structure to a C-G pair. In other instances, the nucleotide substitution may correct a mismatched base pair (e.g., A-C in P2b to G-C) in a stem structure.In some examples, the scaffold sequence for Nuclease K (and Nuclease A as well) to be used in the gene editing systems disclosed herein may comprise a nucleotide sequence at least 90% (e.g., at least 93%, at least 95%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 65. Examples are provided in Sequence Table 4 below, each of which is within the scope of the present disclosure. Specific examples include SEQ ID NO: 348, 353, 362, 374, 378, 379, 380, 382, 387, 388, or 467.The cognate scaffold sequence for Nuclease K is highly homology to the scaffold sequence for Nuclease A. As such, the scaffold sequences for Nuclease A and Nuclease K may be used interchangeably. For example, the cognate scaffold sequence of Nuclease A (SEQ ID NO: 63) and variants thereof as disclosed herein (e.g., SEQ ID NO: 64) can also be used as the scaffold sequence for Nuclease K. Similarly, the Nuclease K scaffold variants provided inAttorney Docket No.: 063586-545001 WOSequence Table 4 can also be used for Nuclease A and variants thereof.In some instances, the scaffold sequence is recognizable by Nuclease BT (SEQ ID NO: 3) or any engineered variants thereof as disclosed herein. Such a scaffold sequence may comprise SEQ ID NO: 67 shown in Sequence Table 4 below.In other instances, the scaffold sequence may be a variant derived from SEQ ID NO: 67. Such a variant scaffold sequence may comprise a nucleotide sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, or greater) identical to SEQ ID NO: 67. Alternatively or in addition, the variant scaffold sequence may comprise deletions, nucleotide substitutions, or a combination thereof. The variant RNA-guided nuclease polypeptide may have increased binding to the variant scaffold sequence as compared with the scaffold of SEQ ID NO: 67. In some examples, the variant scaffold may be a variant of SEQ ID NO: 67 as disclosed herein. For example, the variant scaffold for use in the gRNAs provided herein may have a length ranging from 100-150 nucleotides.In one specific example, the scaffold sequence comprises (e.g., consists of) the nucleotide sequence of SEQ ID NO: 68. In another specific example, the scaffold sequence comprises (e.g., consists of) SEQ ID NO: 69. In yet another specific example, the scaffold sequence comprises (e.g., consists of) SEQ ID NO: 78.(b) 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 (i.e., an 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 further 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. i. 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 canAttorney Docket No.: 063586-545001 WO be nicked or cleaved by the 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 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. In a gene editing system comprising a 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 (z.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.) 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 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.Attorney Docket No.: 063586-545001 WO ii. Reverse Transcription Template (RTT) Sequence (Template RNA)The 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.Attorney Docket No.: 063586-545001 WOIn some embodiments, the reverse transcription template sequence comprises at least one encoded 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 GG 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 further 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 transcribed into DNA by the reverse transcriptase of the gene editing system described herein. In some embodiments, the RTT sequence is transcribed from 5’ to 3’ into DNA of the PAM strand.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. In some embodiments, the RTT and an end protection fragment (e.g., a 3’ end protection fragment) may be connected via a linker sequence to avoid steric hindrance between the two RNA components.(c) Single Editing Template RNAIn some embodiments, the gene editing system provided herein comprises a single RNA molecule (z.e., a single editing template RNA), 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 nuclease polypeptide and synthesis of a DNA fragment from a free 3’ end of a freeAttorney Docket No.: 063586-545001 WODNA strand generated by the 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 nuclease polypeptide, a RTT sequence, and a PBS. In specific examples, the single RNA molecule may comprise, from 5’ to 3’, a spacer sequence, a scaffold sequence, an RTT sequence, a PBS, and a protection fragment.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 linker may comprise an aptamer domain.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, a preQi aptamer sequence, a truncated preQi aptamer sequence, a boxB RNA sequence, or an RNA bacteriophage MS2 sequence.In some specific 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. One specific example of the 3’ extension motif is provided in Examples below.(d) Modification 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, theAttorney Docket No.: 063586-545001 WO internucleoside 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 in a gene editing system as disclosed herein, e.g., the editing template RNA or any of the nucleic acid sequences encoding the components, may include one or more modifications, such as to the sugar, the nucleobase, or the internucleoside 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). One or more atoms of a purine 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 internucleoside 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, any of the RNA components in a gene editing system as disclosed herein comprises an abasic site (i.e., a location that does not have a purine or a pyrimidine). In some embodiments, the abasic site (also referred to as an apurinic / apyrimidinic site) is present in an editing template RNA. For example, an abasic site can be present in the RTT of an editing template RNA. In some embodiments, activity of a reverse transcriptase is halted at or near an abasic site.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 internucleoside 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%Attorney Docket No.: 063586-545001 WO 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 as backbone 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 internucleoside 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 internucleoside 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 theAttorney Docket No.: 063586-545001 WO oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside 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 cellular environment.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 internucleoside linkages that may be employed according to the present invention, including internucleoside 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 the 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, l-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)- cytosine, decitabine, 5-fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafur ((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-arabinofuranosylcytosine, 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,Attorney Docket No.: 063586-545001 WO 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-taurinom ethylpseudouridine, 5-taurinomethyl-2 -thio-uridine, l-taurinomethyl-4-thio-uridine, 5-methyl- uridine, 1-methyl-pseudouridine, 4-thio-l-methyl-pseudouridine, 2-thio-l -methylpseudouridine, 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 -thiopseudouridine. 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-l- methyl-l-deaza-pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocyti dine, 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-l- 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-m ethyladenine, 2-methylthio-adenine, and 2-m ethoxy-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,Attorney Docket No.: 063586-545001 WO6-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, and N2,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 ARI marks dsRNA as “self’. Cell Res. 25, 1283-1284, which is incorporated by reference in its entirety.In some embodiments, any RNA sequence described herein 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 nuclease, such nucleic acid molecules may contain any of the modifications disclosed herein, where applicable.III. Methods for Nuclease-Guided Reverse Transcription-Mediated Gene EditingAny 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 genetic 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 Systems to 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,Attorney Docket No.: 063586-545001 WO 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 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 / 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 an RNA encoding a 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 a nuclease polypeptide are delivered in separate compositions. In some embodiments, an RNA guide / RT donor RNA and an RNA encoding a 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 a 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 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 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 nuclease and / or RT polypeptides and the RNA guide and / or RT donor RNA are delivered separately. For example, the nuclease and / or RT polypeptides and the RNA guide and / or RT donor RNA are packaged into separate AAV particles. In another example, the nuclease and / or RT polypeptides can be 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 orAttorney Docket No.: 063586-545001 WO 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 nuclease-RT 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.In specific examples, the gene editing system provided herein can be delivered via the AAV gene delivery system as known in the art or provided herein, taking advantage of the small sizes of the nuclease and RT polypeptides used herein.B. Genetically Modified CellsAny 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 systemAttorney Docket No.: 063586-545001 WO 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 editing system 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. Alternatively, the gene editing system may comprise viral vectors such as AAV vectors or viral particles such as AAV particles to mediate delivery of the gene editing components into host cells.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 nuclease polypeptide, the RT polypeptide, or the 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 geneAttorney Docket No.: 063586-545001 WO 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 or sterile 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 releaseAttorney Docket No.: 063586-545001 WO 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 for Nuclease-Guided Reverse Transcription-Mediated Gene EditingThe 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, a nuclease polypeptide, and an RT polypeptide, or a fusion polypeptide thereof. In some embodiments, the kits include the single RNA molecule and the nuclease-RT fusion polypeptide. In some embodiments, the kits include a polynucleotide that encodes the nuclease polypeptide, the RT polypeptide, or the 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 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 be packaged in separate vials or other vessels, the contents of which can be mixed prior to use.The 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 nuclease polypeptide, and the RT polypeptide, or the fusion polypeptide thereof.Sequence Table 1. Nuclease and Nickase Variant SequencesAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOSequence Table 2. Reverse Transcriptase (RT) and Variant SequencesAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOSequence Table 3. Fusion Polypeptides and Components ThereofAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOSequence Table 4. Scaffold SequencesAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOGeneral 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 (J. 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.Attorney Docket No.: 063586-545001 WO(1986»; Immobilized Cells and Enzymes (1RL 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 the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.EXAMPLESWhile the present disclosure has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the disclosure. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit, and scope of the present disclosure. All such modifications are intended to be within the scope of the disclosure.Example 1: RNA-Templated Editing of Human Genes in HEK293T Cells Using CRISPR Nickase A-Reverse Transcriptase PolypeptidesThis Example shows genetic modification of human genes utilizing CRISPR nickasereverse transcriptase fusion polypeptides comprising i) a CRISPR Nickase A or variants thereof, and ii) a reverse transcriptase polypeptide selected from: RT_A, RT_B, RT_C, or variants thereof. These fusion polypeptides were used along with an editing template RNA to install sequence substitutions in target genes.CRISPR Nickase A (SEQ ID NO: 8) comprised an H397A substitution relative to the CRISPR nuclease A sequence of SEQ ID NO: 1, which conferred nickase activity. All substitutions described herein are relative to the reference Nuclease A (SEQ ID NO: 1). Variants of CRISPR Nickase A further comprised the following substitutions / modifications: i) I67R, D568R, and E706R) (SEQ ID NO: 9) or ii) I67R, D568R, W593R, E706R, and a truncation of the first 15 residues of CRISPR nuclease A (referred to as the 16-775 truncation) (SEQ ID NO: 90).The RT polypeptides used in this Example includes variants of RT_A, RT_B, and RT_C. The amino acid numbering of the variant sequences is relative to the reference RT_A (SEQ ID NO: 1), RT_B (SEQ ID NO: 2), or RT C (SEQ ID NO: 3), which contain the N-Attorney Docket No.: 063586-545001 WO terminus Methionine residue. This Met residue can be removed from the RT portion in the fusion polypeptides when additional N-terminal fragments are present or added (e.g. : NLS, proteins tags, etc.).Variants of RT_A comprised the following substitutions / modifications: i) A24R, ii) A24R, Q142L, N36R, and H84R, or iii) A24R, Q142L, N36R, H84R, and a truncation of the first 12 residues and last 19 residues of RT_A (referred to as the 13-297 truncation). See Sequence Table 2 above.Variants of RT_B comprised the following substitutions / modifications: i) E291R or ii) E291R and a truncation of the first 5 residues and last 45 residues of RT_B (referred to as the 6-293 truncation). Sequence Table 2 above.Variants of RT_C comprised the following substitutions / modifications: i) T70R or ii) T70R and truncation of the first four residues and the last 54 residues of RT_C (referred to as the 5-296 truncation). Sequence Table 2 above.Fusion polypeptides were generated with the RT polypeptide positioned N terminal or C terminal to the Nickase polypeptide, for example: C-Terminal RT-RT-fusion polypeptides included NLS- Nickase-Linker-RT-NLS, and N-Terminal RT-fusion polypeptides included NLS-RT-Linker- Nickase-NLS. The order of the enzymes in the fusion polypeptides is reflected in the descriptions in Sequence Table 3 above, where the first mentioned enzyme is located at the N-terminus.Specifically, this example used the following NLS and Linker sequences: the Bipartite SV40 NLS (SEQ ID NO: 30), the XTEN linker (SEQ ID NO: 31), and the nucleoplasmin NLS (SEQ ID NO: 33). The fusion configurations tested comprised the following sequence configurations, the C-terminal RT fusion: Bipartite SV40 NLS-CRISPR Nickase A-XTEN Linker-RT -Nucleoplasmin NLS, and the N-terminal RT fusion: Bipartite SV40 NLS-RT- XTEN Linker-CRISPR Nickase A-Nucleoplasmin NLS.Editing template RNAs were designed to be specific to an AAVS1 or EMX1 target sequence 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 five components, from 5’ to 3’ : 1) spacer sequence, 2) scaffold sequence, 3) reverse transcription template (RTT) encoding 6-nucleotide substitutions, 4) primer binding site (PBS), and 5) a 3’ extension motif. The editing template sequences encoded by the plasmid are shown in Table 1 below. The U6 PolIII promoter uses a +1 G at the start of the transcript (z.e., the 5’ end of the editing template RNA) for more efficient transcription that is excluded from the sequences in Table 1.Attorney Docket No.: 063586-545001 WOTable 1. Editing Template RNA Sequences*Spacer sequences are underlined, scaffold sequence is shown in bold, and the 6-nucleotide substitutions are shown in lowercaseApproximately 16 hours prior to transfection, 25,000 HEK293T cells in DMEM / 10%FBS+Pen / Strep (D10 media) were plated into each well of a 96-well plate. On the day of transfection, the cells were 50-90% 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 CRISPR nickase-RT fusion 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 approximately 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 either at room temperature or 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 150 bp centered near each editing target, then sequenced with an Illumina sequencer (e.g., NovaSeq 6000 sequencer). For each target and sample, the fraction of NGS reads containing substitutions corresponding to the edits encoded by the editing template RNAs were calculated.Attorney Docket No.: 063586-545001 WOResults are shown in Table 2 and Table 3.Table 2. Editing Efficiencies of CRISPR Nickase A-Reverse Transcriptase Polypeptides.Table 3. Editing Efficiencies of CRISPR Nickase A-Reverse Transcriptase Polypeptides.Attorney Docket No.: 063586-545001 WOAs shown in Table 2, each of the variant CRISPR Nickase A (H397A, I67R, D568R, E706R)-reverse transcriptase fusion polypeptides tested installed the substitutions encoded by editing template RNAs 1 and 2. These edits were above the background rates of 0.06 and <0.01 for editing template RNAs 1 and 2, respectively. Fusions to RT_C variants displayed the highest activity in both fusion configurations at both targets, compared to RT_A and RT_B. Use of RT_C in the N-terminal RT-fusion configuration with editing template RNA 1 resulted in the highest average percentage of NGS reads comprising the encoded substitution. As shown in Table 3, each of the variant CRISPR Nickase A (H397A, I67R, D568R, W593R, E706R, 16-775 truncation)-reverse transcriptase fusion polypeptides tested installed the substitutions encoded by editing template RNAs 1 and 2. These edits were above the background rate of <0.01 for both editing template RNAs. Use of RT_C (T70R) with editing template RNA 1 resulted in the highest average percentage of NGS reads comprising the encoded substitution at 8.57% of NGS reads.This Example thus shows that fusions between CRISPR Nickase A variants and variants of RT_A, RT_B, and RT_C install substitutions into human genes. This Example also shows Nickase A is able to use the same editing template RNA sequences as Nickase K.Example 2: RNA-Templated Editing of Human Genes in HEK293T Cells Using CRISPR Nickase K-Reverse Transcriptase PolypeptidesThis Example shows genetic modification of human genes utilizing CRISPR nickasereverse transcriptase fusion polypeptides comprising i) a CRISPR Nickase K or variants thereof and ii) a reverse transcriptase polypeptide selected from: RT_A, RT_B, RT_C, or variants thereof. These fusion polypeptides were used along with an editing template RNA to install sequence substitutions in target genes.CRISPR Nickase K comprised an H375A substitution relative to the CRISPR nuclease K sequence of SEQ ID NO: 2 which conferred nickase activity. All substitutions described herein are relative to CRISPR Nuclease K (SEQ ID NO: 2). Variants of CRISPR Nickase K further comprised the following substitutions: G42R and D582R. Variants of RT_A, RT_B, and RT_C, fusion polypeptide configurations, and editing template sequences were asAttorney Docket No.: 063586-545001 WO described in Example 1. See also Sequence Tables 1 and 2 for nuclease and RT variant sequences.Amino acid numbering of the variant sequences is relative to the Nuclease K (SEQ ID NO: 2), RT_A (SEQ ID NO: 4), RT_B (SEQ ID NO: 5), or RT C (SEQ ID NO: 6), which contain the +1 Methionine. The +1M is removed from the variant or fusion polypeptides when additional N-terminal polypeptides are present or added (e.g. NLS, proteins tags, etc.).Also, HEK293T cells were transfected, and edits were analyzed by NGS as described in Example 1, using the editing template RNA sequences in Table 1. Results are shown in Table 4Table 4. Editing Efficiencies of CRISPR Nickase K-Reverse Transcriptase Polypeptides.*One technical replicate was excluded because fewer than 10,000 NGS reads were generated * The sequences of the RT variants are provided in Sequence Table 2 above. The N-terminal M residue was removed from RT sequence when part of the fusion protein.As shown in Table 4, each of the variant CRISPR Nickase K (H375A, G42R, D582R)- reverse transcriptase fusion polypeptides tested installed the substitutions encoded by editingAttorney Docket No.: 063586-545001 WO template RNAs 1 and 2. These edits were above the background rates of 0.06 and <0.01 for editing template RNAs 1 and 2, respectively. Use of RT_C variants resulted in the highest activity in both fusion configurations at both targets, compared to RT_A and RT_B. Fusion to RT_C in N-terminal RT-fusion configuration when paired with editing template RNA 1 resulted in the highest average percentage of NGS reads comprising the encoded substitution. The C-terminally fused RT_C T70R variant (SEQ ID NO: 346) is a close second.This Example thus shows that fusions between a variant CRISPR Nickase K and variants of RT_A, RT_B and RT_C installed substitutions into human target genes.Example 3: RNA-Templated Editing of Human Genes in HEK293T Cells Using RNA- Guided Nickase BT-Reverse Transcriptase PolypeptidesThis Example shows genetic modification of human genes utilizing RNA-guided nickase-reverse transcriptase fusion polypeptides comprising i) an RNA-guided Nickase BT or variants thereof and ii) a reverse transcriptase polypeptide selected from: RT_A, RT_B, RT_C, or variants thereof. Specifically, these fusion polypeptides were used to install sequence substitutions in target genes.RNA-guided Nickase BT comprised an H232A substitution relative to the RNA-guided nuclease BT sequence of SEQ ID NO: 3, which conferred nickase activity. Variants of RNA- guided Nickase BT further comprised the following substitutions: V433R, Q102R, and I206R (SEQ ID NO: 15). Variants of RT_A comprised the following substitution / modification: El 17R as well as a truncation of the first 12 residues and last 19 residues of RT_A (referred to as the 13-297 truncation). Variants of RT_B comprised the following substitutions / modifications: Q32R, N121R, E291R as well as a truncation of the first 5 residues and last 45 residues of RT_B (referred to as the 6-293 truncation). Variants of RT_C comprised the following substitution: T32R. Configurations of the fusion polypeptides and editing template RNAs were as described in Example 1.Amino acid numbering of the variant sequences is relative to the Nuclease BT (SEQ ID NO: 3), RT_A (SEQ ID NO: 4), RT_B (SEQ ID NO: 5), or RT C (SEQ ID NO: 6), which contain the N-terminus Methionine. This Met residue can be removed from the variant or fusion polypeptides when additional N-terminal polypeptides are added (e.g. : NLS, proteins tags, etc.).The editing template sequences encoded by the plasmids are shown in Table 5 below. The U6 PolIII promoter uses a +1 G at the start of the transcript (z.e., the 5’ end of the ending template RNA) for more efficient transcription that is excluded from the sequences describedAttorney Docket No.: 063586-545001 WO in Table 5. HEK293T cells were transfected, and edits were analyzed by NGS as described in Example 1. Results are shown in Table 6.Table 5. Editing Template RNA SequencesTable 6. Editing Efficiencies of Nickase BT-Reverse Transcriptase Polypeptides.Attorney Docket No.: 063586-545001 WO*One technical replicate was excluded because fewer than 10,000 NGS reads were generated * The sequences of the RT variants are provided in Sequence Table 2. The N-terminal M residue was removed from RT sequence when part of the fusion protein.As shown in Table 6, each of the variant CRISPR Nickase BT (H232A, V433R, Q102R, I206R)-reverse transcriptase fusion polypeptides installed substitutions at levels above background for particular targets. The background rates for these editing template RNAs were determined to be <0.001, <0.001, and 0.002 for editing template RNAs 1, 2, and 3, respectively. For all editing template RNAs, use of RT_C (T32R) resulted in the highest activity in both fusion configurations tested. Use of RT_C (T32R) in the N-terminal RT -fusion configuration when paired with editing template RNA 2 resulted in the highest average percentage of NGS reads comprising the encoded substitution at 0.038% of NGS reads.This Example thus shows that fusions between variant CRISPR Nickase BT and variants of RT_A, RT_B, and RT_C installed substitutions into human target genes.Example 4: Engineering of AAV Vectors Encoding CRISPR Nickase-RT Fusion Polypeptides and Editing Template RNAIn this Example, AAV plasmid vectors comprising expression cassettes encoding a fusion polypeptide and an editing template RNA were produced. The AAV plasmids comprised nucleic acid sequences encoding a Fusion Polypeptide with 5’ and 3’ NLS sequences and a non-coding RNA serving as an editing template RNA flanked by 5’ and 3’ inverted terminal repeats (ITRs). 5’ and 3’ ITRs facilitate viral genome replication and packaging.The editing template RNA encoded by the AAV vector comprised, in the 5' to 3' direction, a spacer sequence complementary to the target, a scaffold sequence, an RT template (RTT) sequence, a PBS sequence, a linker, and a 3' extension sequence. Exemplary editing template RNA coding sequences are shown in Table 1 and Table 7. Exemplary fusion polypeptides are described in Examples 5, 6 and 7.Attorney Docket No.: 063586-545001 WOTable 7. Full-length Sequences of Editing Template RNA Components* Spacer sequences are underlined, scaffold sequence is shown in bold, and the gene edit is shown in underlined italics (e.g.: insertion or 6-nucleotide substitution)Four different AAV constructs were designed to test the expression of the fusion polypeptide and editing template RNA when the expression cassettes are placed in the same transcriptional orientation or in reverse transcriptional orientation as shown in FIG. 2. The expression cassettes can also include promoters, transcriptional initiation, and / or polyadenylation sequences to further regulate expression of the fusion polypeptide and / or editing template RNA.DNA vectors were designed for use in in Example 5 and Example 6. The maximum packaging capacity for adeno-associated virus (AAV) is generally around 4.8 kilobases (kb) of genetic material including the ITR sequences on 5’ and 3’ end. Exceeding this limit can lead to inefficient packaging and reduced viral vector production.Example 5: RNA-Templated Editing of AAVS1 and DNMT1 using AAV Vector Constructs via Plasmid DeliveryThis Example shows genetic modification of human AA VS1 and mouse DNMT1 genes in human and mouse cell lines, respectively, utilizing the vectors produced in Example 4. In this example the vector constructs were delivered as plasmidApproximately 16 hours prior to transfection, 25,000 HEK293T cells or 20,000 N2A cells in DMEM / 10%FBS+Pen / Strep (D10 media) were plated into each well of a 96-well plate. On the day of transfection, the cells were 50-90% confluent. For each well to be transfected, a mixture of Lipofectamine 2000™ (ThermoFisher Scientific) and Opti-MEM™ (ThermoFisher 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 AAV vectors and Opti-MEM™ (Solution 2). Solutions 1 and 2 were mixed by pipetting up and down, then incubated at room temperature for approximately 25 minutes.Attorney Docket No.: 063586-545001 WOFollowing 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 either at room temperature or 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 and analyzed. For each target, the fraction of NGS reads containing indels were calculated for each sample and its cognate no protein control. To determine the percentage of encoded edits installed, sequencing reads comprising the edits encoded by the editing template RNAs were analyzed and quantified. The percentage of NGS reads comprising indels and the encoded edits for the DNMT1 (dna methytransferase 1) target in Mouse N2A cells is shown in Table 8 and Table 9. The percentage of NGS reads comprising indels and the encoded edits for the AAVS1 target in HEK293T cells is shown in Table 10. AAV vector orientations 1-4 refer to the AAV vector orientations described in Example 4 (e.g., FIG. 2), and the data is the average of three technical replicates.Table 8. DNMT1 Editing Efficiencies in Mouse N2A cells with the N-Terminal RT Fusion Polypeptide: RT_B (E291R, 6-293 truncation) + [CRISPR Nickase A (H397A, I67R, D568R, W593R, E706R, 16-775 truncation)]As shown in Table 8 and Table 9, AAV vectors comprising CRISPR Nickase A variant fused to RT_B or RT_C variants incorporated encoded edits into human and mouse genes. Each of the vector orientations tested resulted in edit incorporation. For both targets, edit incorporation was higher with the N-Terminal RT_C fusion polypeptides than the N- Terminal RT_B fusion polypeptides in all construct orientations tested. On average across the targets tested, orientations 2 and 4 showed the higher editing efficiencies.Attorney Docket No.: 063586-545001 WOTable 9. DNMT1 Editing Efficiencies in Mouse N2A cells with the N-Terminal RT Fusion Polypeptide: RT C (T70R) + [CRISPR Nickase A (I67R, H397A, D568R, W593R, E706R, 16-775 truncation)]Table 10. AAVS1 Editing Efficiencies in human HEK293T cells with the N-Terminal Fusion Polypeptide: RT C (T70R) + [CRISPR Nickase A (I67R, H397A, D568R, W593R, E706R, 16-775 truncation)]This Example thus shows that a single AAV plasmid comprising a CRISPR nickase (e.g., a variant CRISPR Nickase A), an RT (e.g., RT_B or RT_C), and an editing template RNA introduced edits into human and mouse gene targets.Example 6: RNA-Templated Editing of AAVS1 and DNMT1 using AAV Vector Constructs via Viral DeliveryThis Example shows genetic modification of human and mouse genes using fusion polypeptides and editing template RNAs that were packaged into AAV. AAV plasmid vectors from Example 4 were packaged into AAV and evaluated for editing of human AAVS1 and mouse DNMT1 genes in human and mouse cell lines, respectively.AAVS1 editins in human SHSY-5Y cellsApproximately 16 hours prior to transduction, 10,000 SHSY-5Y cells (ATCC) in DMEM / 10%FBS+Pen / Strep (D10+P / S) were plated into each well of a 96-well plate. On the day of transduction, the media was removed and replaced with D10+P / S 10,000 viral particles of Human Adenovirus Type 5 (Ad5, ATCC) and incubated for 1 hour. At the end of the 1-hour incubation, the Ad5-containing media was removed and replaced with media containing le7Attorney Docket No.: 063586-545001 WO viral particles per pL. No transduction controls were prepared by replacing the media of a notransfection control with D10+P / S only. The SHSY-5Y cells were collected at day 3 and day 7 post transduction and analyzed by NGS as described in Example 5. Edit incorporation is shown in Table 11.Table 11. AAVS1 Editing Efficiencies in Human SHSY-5Y Cells with the Fusion Polypeptide: RT C (T70R) + [CRISPR Nickase A (I67R, H397A, D568R, W593R, E706R, 16-775 truncation)] and AAVS1 editing guide RNA 1 (Table 1)As shown in Table 11, edits were installed at the human AAVS1 locus in SHSY-5Y cells treated with AAV virus. AAV virus constructed in orientation 4 showed a higher level of edit incorporation in the dividing SHSY-5Y cells compared to orientation 2. There was a time dependent increase in encoded edits from Day 3 to Day 7 when le6 AAV viral particles per pL were added, which was also observed for other doses tested.DNMT1 editing in primary mouse hepatocytesNext, 25,000 Primary Mouse Hepatocytes were plated in William’s E medium, which was replaced after approximately 4 to 5 hours with media containing 1 e5 viral particles per pL. No-transduction negative controls were prepared replacing media with William’s media only. Approximately 7 days post transduction, cells were resuspended in Quick Extract™ buffer (Lucigen®) and analyzed by NGS as described in Example 5. Edit incorporation is shown in Table 12Table 12. DNMT1 Editing Efficiencies in Mouse Primary HepatocytesAttorney Docket No.: 063586-545001 WON-terminal fusion polypeptides used with DNMT1 editing guide RNA 6 (Table 7)This example demonstrates that CRISPR nickase RT -fusion polypeptides can install intended edits at the DNMT1 locus in Primary Mouse Hepatocytes.AAVS1 editing in human iP SC-derived motor neurons (iMNs)Human motor neurons derived from induced pluripotent stem (iPS) cells were purchased from BrainXCell and cultured in the laboratory using manufacturer’s protocols. Briefly, motor neurons were thawed from liquid nitrogen and 75,000 cells were seeded on 96-well microplates coated with poly-D-lysine in motor neuron seeding media. Motor neuron seeding media was prepared using 1 : 1 DMEM / F12: Neurobasal medium, supplemented with lx B27, lx N2, 0.5 mM GlutaMAX, 10 ng / ml BDNF, 10 ng / ml GDNF, 1 ng / ml TGF-pi, 15 pg / ml Geltrex, and lx Motor Neuron Seeding Supplement. On day 5, motor neurons were also treated with the same vector SHSY5Y cells were treated with. Cells were treated with 1E4, 3E4 and 9E4 vector genomes per cell and harvested at day 10 or day 60 post transduction and analyzed by NGS as described in Example 5. Average % edit incorporation is shown in Table 13.Table 13. AAVS1 Editing Efficiencies in iPSC-derived human motor neurons (iMNs)Attorney Docket No.: 063586-545001 WOThe results summarized in Table 13 demonstrate that CRISPR Nickase A RT-fusion polypeptides can install intended edits in iPSC-derived human motor neurons.This Example shows that an AAV virus delivering a fusion polypeptide comprising a CRISPR nickase e.g, a variant CRISPR Nickase A) and an RT (e.g., RT_B or RT_C variant), along with an editing template RNA can introduce desired edits into human and mouse gene targets in both dividing (SHSY-5Y) and non-dividing (primary mouse hepatocytes and iPSC- derived human motor neuron) cells.Example 7: Optimization of Fusion Polypeptide: CRISPR Nickase A (I67R, H397A, D568R, W593R, E706R, 16-775 truncation) and RT C (T70R) for RNA- Templated EditingThe Fusion Polypeptides described in Example 4 were further engineered to design and test smaller fusions, which would be beneficial to AAV delivery. The AAV constructs were designed according to AAV vector orientation 2 in Example 4 and utilized an N-terminal polypeptide comprising a variant of CRISPR Nickase A and a variant of RT_C, as well as the DNMT1 editing template RNA (Table 7)Earlier fusion polypeptides were tested with the XTEN linker, this example further examines the function of fusion polypeptides designed with linkers ranging from 15-99 nucleotides to determine if very short linkers could maintain or increase editing activity. N- Terminal Fusion polypeptides designed with the following configuration: NLS-(protein tag)- RT-linker-Nickase-(protein tag)-NLS were tested using the linkers in Table 14 and the results are shown in Table 14. The constructs were tested in N2A cells as described in Example 5.The N-terminal Fusion Polypeptide comprising the RT variant: RT_C (T70R), and the Nickase variant: Nickase A (I67R, H397A, D568R, W593R, E706R, 16-775 truncation) wereAttorney Docket No.: 063586-545001 WO evaluated with the linkers of varying sizes.Table 14. Editing Efficiencies Associated with Various LinkersOverall, the linkers tested yielded similar editing efficiencies with some linkers slightly performing better than others. Fusion Polypeptides containing the shortest linker, L7, produced the shortest fusion polypeptide with a length of 3342 nt (1114 amino acids) and performed slightly better than other linkers. Such size reductions allow for further miniaturizing the fusion polypeptide. To this end, additional modifications were made to further miniaturize the fusion polypeptide by truncating the RT_C variant protein (RT_C variant (T70R), A297-325; SEQ ID NO: 29). The results in Table 15 below show that the RT_C variant (T70R), A297-325 performs similarly to the RT_C variant (T70R), when tested at the DNMT1 locus. The new RT variant further reduced the size of fusion polypeptide to 3258 nt (1086 amino acids) allowing for the addition of other regulatory elements to boost edit installation.Table 15. N-Terminal Fusion Polypeptides with a PAPAP LinkerAttorney Docket No.: 063586-545001 WOThis Example thus shows that CRISPR-RT fusions Polypeptides comprising, which are capable of being delivered via AAV virus, can further be miniaturized. Reducing the size of fusion protein domains while maintaining their activity enhances flexibility for incorporating additional sequences, such as larger promoters, enhancers, single-stranded binding proteins, or microRNA binding sites, thereby enabling further regulation and optimization of activity.Example 8: Optimization of RNA Scaffolds Recognized by CRISPR Nuclease A and Fusion Polypeptide Variants for RNA-Templated EditingThis Example describes the genomic editing of exemplary target genes (AAVS1, EMX1, and / or DNMT1) by the fusion polypeptide comprising a CRISPR Nickase A variant and a RT_C variant when combined with editing template RNAs comprising alternative scaffold sequences. Editing template RNAs were designed by replacing the RNA scaffold sequences from Editing template RNAs 1, 2, and / or 6 (See Tables 1 and 7) with the RNA scaffold sequences of Sequence Table 4. Mutations relative to the parent scaffold are capitalized. Cloning was performed as described in Example 1. The structures of exemplary RNA scaffold sequences utilized here are illustrated in FIGS. 1D-1N. Exemplary editing template RNA sequences are shown in Table 16.Table 16: Exemplary Editing Template RNA SequencesAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WO*Spacer sequences are underlined, scaffold sequence is shown in bold (bold capital letters indicate mutations relative to Nuclease K Scaffold Variant 1), and the gene edit is shown in lowercase italics (e.g., insertions or 6-nucleotide substitutions)The compact RT editor of SEQ ID NO: 44 (RT_C (T70R) + Nickase A (I67R, H397A, D568R, W593R, E706R, and 16-775 truncation)) with N- and C-terminal NLS tags as described in Example 1 was combined with the editing template RNAs comprising alternate RNA scaffolds described above and was introduced into either HEK293T cells (for RNAs targeting AAVS1-T3 and EMX1-T7) or Neuro-2A cells (for RNAs targeting DNMT1) by lipid-based transient transfection as described in Example 1. A water-only negative control transfection was also performed where both the RT editor and editing template RNA components were replaced by water and transfected. Genomic DNA was recovered approximately 72 hours post-transfection, and samples were prepared for NGS and analyzed as described in Example 1. The percentage of NGS reads containing substitutions shown in Tables 17 and 18 were calculated as the average of three technical replicates, unless indicated otherwise.The lipid-based transient transfections were performed to assess the impact of the alternate scaffolds and were carried out across two experiments, representing distinct engineering rounds. The first engineering round focused on assessing Nuclease K Scaffold Variants 1-31, which comprise mutations that were focused on improving one general region of the RNA. Variant 2 contains a single base deletion from the 3’ end of Variant 1. Variants 3-Attorney Docket No.: 063586-545001 WO31 use Variant 2 as the parent construct and apply mutations and / or truncations to this sequence. Variants 3-7 contain mutations and / or truncations within the Pl helix. Variants 8-14 contain mutations within the P2 helix. Variants 15-21 contain mutations within or near to the P3a helix. Variants 22-24 contain mutations within the P3b helix. Variants 25-31 contain mutations within or near to the P5 helix, which is a long-range pseudoknot motif. Results for samples transfected with editing template RNAs (Table 17) comprising these scaffolds (Sequence Table 4) are shown in Table 18.Table 17. Editing activity of the RT_C Variant (T70R) + Nickase A Variant (I67R, H397A, D568R, W593R, E706R, and 16-775 truncation) Fusion PolypeptidePaired with Editing Template RNAs Comprising Alternate RNA ScaffoldsAttorney Docket No.: 063586-545001 WOAs shown in Table 17, all of the screened editing template RNAs comprising alternate Nuclease K scaffold variants resulted in editing levels that were above the background levels observed for the water-only negative control both of the targets tested (AAVS1-T3 and DNMT1). Additionally, nearly all of the screened editing template RNAs resulted in editing levels that was similar to or higher than the parent constructs (Nuclease K Scaffold Variants 1 and 2) at one or both of the targets tested. Nuclease K Scaffold Variant 2 resulted in editing levels that were roughly equivalent to or slightly higher than those obtained with Nuclease K Variant Scaffold 1 at AAVS1, indicating that the terminal base of Scaffold 1 is dispensable.Two scaffold variants, Nuclease K Scaffold Variants 16 and 28, resulted in elevated editing levels at both AAVS1-T3 and DNMT1 relative to Nuclease K Scaffold Variant 2. Scaffold Variant 16 contains a mutated base pair within the P3a helix and a single base change at the top of the P2 helix and resulted in 17% and 30% improvements in activity at AAVS1-T3 and DNMT1, respectively (relative to Nuclease K Scaffold Variant 2). Scaffold Variant 28 contains a mutated base pair within the P5 helix and resulted in 10% and 50% improvements in activity at AAVS1-T3 and DNMT1, respectively (relative to Nuclease K Scaffold Variant 2). Scaffold Variant 7 was also notable as it utilized a truncated Pl helix (decreased in size by 2 base pairs) with higher GC content and resulted in editing levels that were equivalent or slightly improved relative to the parent construct (Nuclease K Variant Scaffold 2) at both targets.Following these results, editing template RNAs were designed comprising Nuclease K Variant Scaffolds 32-42. Scaffold Variant 32 combined the mutations contained within Scaffold Variants 16 and 28 (the top two scaffold variants from the first engineering round). Scaffold Variant 33 combined mutations present in Scaffold Variants 7, 16, and 28 (the top 3 scaffold variants identified in the first engineering round). Scaffold Variants 34-42 use Scaffold Variant 33 as the parent sequence and apply additional mutations and / or truncations to this sequence. Scaffold Variant 34 includes an alternate P5 helix sequence. Scaffold Variant 35 includes an additional mutation in the P2 helix. Scaffold Variant 36 includes an additional mutation in the P3b helix. Scaffold Variants 37-40 include alternate Pl sequences with varying helix lengths and / or alternate mutations. Scaffold Variants 41-42 remove the P4 helix and replace it with either a 4-nucleotide GAAA linker sequence or no linker sequence. Results forAttorney Docket No.: 063586-545001 WO samples transfected with editing template RNAs comprising these scaffolds are shown inTable 18Table 18. Editing activity of the RT_C Variant (T70R) + Nickase A Variant (I67R, H397A, D568R, W593R, E706R, and 16-775 truncation) fusion polypeptide paired with Editing Template RNAs Comprising Alternate RNA Scaffolds” = Not tested*One technical replicate was excluded because fewer than 10,000 NGS reads were generatedAs shown in Table 19, all the editing template RNAs comprising alternate Nuclease K Scaffold Variants that were screened displayed editing levels that were above background levels observed for the water-only negative control at all three targets (AAVS1-T3, EMX1-T7, and DNMT1). Additionally, all the Scaffold Variants resulted in editing that matched the activity observed with Scaffold Variant 2 at one or more of the three targets.Three of the scaffold variants stood out for providing the largest improvement in activity across the three targets tested. These were Nuclease K Scaffold Variants 33, 34, and 36. Scaffold Variant 33 combines mutations within or near to the Pl, P3a, and P5 helices and resulted in 1.1-fold, 2.1-fold, and 1.6-fold improvements in activity at AAVS1-T3, EMX1-T7 and DNMT1, respectively (relative to Nuclease K Scaffold Variant 2). Scaffold Variant 34 also combines mutations within or near to the Pl, P3a, and P5 helices and resulted in 1.0-fold, 3.1- fold, and 1.7-fold improvements in activity at AAVS1-T3, EMX1-T7 and DNMT1,Attorney Docket No.: 063586-545001 WO respectively (relative to Nuclease K Scaffold Variant 2). Scaffold Variant 36 is identical to Scaffold Variant 33, except that it contains an additional mutation within the P3b helix. Scaffold Variant 36 resulted in 1.2-fold, 2.3-fold, and 1.6-fold improvements in activity at AAVS1-T3, EMX1-T7 and DNMT1, respectively (relative to Nuclease K Scaffold Variant 2).Nuclease K Scaffold Variants 39-42 are also notable as these each comprise truncations within the Pl or P4 helices and retain significant activity relative to their parent scaffold (Scaffold Variant 33). Scaffold Variants 39 and 40 have an additional 1 and 2 base pairs truncated from their Pl helices, respectively, relative to Scaffold Variant 33. Both displayed editing levels that were either equivalent or slightly lower than Scaffold Variant 33 at all three targets tested. Scaffold Variant 41 replaced the P4 helix with a 4 nucleotide GAAA linker sequence. Scaffold Variant 42 removed the P4 helix completely. Both variants showed reduced activity compared to Scaffold Variant 33, with Variant 41 showing slightly higher editing levels. However, Scaffold Variant 41 displayed levels near to the levels observed with Scaffold Variant 2.This Example thus shows that editing template RNAs comprising the scaffold variants described in Table 16 are functional editing template RNAs and these scaffolds, in many cases, provide significantly improved activity compared to Nuclease K Scaffold Variants 1 and 2. These scaffolds are therefore capable of being recognized by Nuclease / Nickase A. Mutations that preserve the predicted base pairing of the Pl, P2, P3a, P3b, and P5 helices were well tolerated. Truncations within the upper region of the Pl helix of the RNA scaffold were also well tolerated. In many cases, variants that improved editing efficiencies comprised mutations predicted to increase the stability of the predicted fold of the RNA scaffold (e.g. by increasing the number of GC base pairs) and / or removed predicted misfolding pathways (i.e. alternate nonproductive folds). Furthermore, the P4 helix is dispensable and not required for activity. However, it is preferrable to replace the P4 helix with a short linker (e.g. a GAAA linker) rather than removing it in its entirety. Editing template RNAs comprising Nuclease K Scaffold Variants 33, 34, and 36 led to the highest increase in activity over Nuclease K Scaffold Variants 1 and 2.Following these results, additional editing template RNAs were designed and tested comprising additional variant scaffolds. Variant scaffolds were designed that used Scaffold Variant 33 as the parent sequence and applied additional base pair mutations within the P2 helix to increase the GC content of this helix. Variant 43 (SEQ ID NO: 467) is provided as an example of a scaffold comprising additional P2 modifications. Variant 43 improved editing efficiencies compared to Variant 33 when tested by transient transfection into HEK239T cells.Attorney Docket No.: 063586-545001 WOSee Figure FIG. IO for the putative secondary structure of Variant 43.Example 9: Engineering of RT Enzymes Fused to CRISPR Nickase A (I67R, D568R, W593R, E706R, 16-775 truncation) for Increased RNA-Templated Editing in Human or Mouse CellsThis example describes engineering performed on RT_A, RT_B, and RT_C as N- terminal peptide fusions to a CRISPR Nickase A variant. Variants of RT_A, RT_B, and RT_C were selected and fused to Nickase A (I67R, H397A, D568R, W593R, E706R, 16-775 truncation) (SEQ ID NO: 90) with other fusion polypeptides (Sequence Table 3), in the following order: SV40 NLS - RT variant peptide - XTEN linker - Nickase A Variant (I67R, H397A, D568R, W593R, E706R, 16-775 truncation) (SEQ ID NO: 90) - Nucleoplasmin NLS. This example used the following NLS and Linker sequences: the Bipartite SV40 NLS (SEQ ID NO: 30), the XTEN linker (SEQ ID NO: 31), and the nucleoplasmin NLS (SEQ ID NO: 33). Each these peptide fusions included variants of RT_A (SEQ ID NO: 16), RT_B (SEQ ID NO: 21), or RT_C (SEQ ID NO: 25) and the variant substitutions are listed in Table 19. The peptide fusions were cloned and then tested for precise editing at the AAVS1 and EMX1 targets in HEK293T cells using Editing Template RNAs 1 and 2 (Table 1), respectively, and the DNMT1 target in N2A cells with Editing Template RNA 6 (Table 7), as described in Example 1. The amino acid sequences of the RT variants are provided in Sequence Table 2 below. The results are tabulated in Table 19 below, with average fold change calculated as the relative increase in editing across all three targets compared to the unmutated reference RT (RT_A, RT_B, or RT_C).Table 19. Editing activity of RT_A, RT_B, and RT_C Variants in Human or Mouse Cells Fused to Nickase A (I67R, H397A, D568R, W593R, E706R, 16-775 Truncation)Attorney Docket No.: 063586-545001 WO“The sequences of the RT variants are provided in Sequence Table 2 below. The N-terminal M residue was removed from RT sequence when part of the fusion protein.As shown in in Table 19, all the fusions containing an RT_A variant show an increased level of precise editing in at least one of the three targets examined relative to the original RT_A sequence. The N117R (13-297 truncation) variant (SEQ ID NO: 20) and A24R, V202I (13-297 truncation) (SEQ ID NO: 255) were particularly improved, showing over 2-fold increase across the three targets on average. Similarly, the RT_B variants showed an increase in editing activity, except for the 6-293 truncation that results in an approximate 40% drop in activity at the AAVS1 target, but does not impact the editing levels of the DNMT1 and EMX1 targets. The highest performing mutant is Q32R, N121R, E291R (6-293 truncation) (SEQ ID NO: 24), which showed clear improvement across all three targets and further reduced the size of the peptide fusion. Last, all the RT_C variants tested demonstrated an increase editing activity, with the N117R mutant demonstrating the highest level of activity (SEQ ID NO: 20). Each RT peptide was then subjected to further mutagenesis and truncation. First, an additional set of variants of RT_A listed in Table 20 with amino acid sequences provided in Sequence Table 2 below were generated using combinations of the mutations from Table 19 and cloned into the same RT editing fusion polypeptide with Nickase A as described above inAttorney Docket No.: 063586-545001 WO this Example. These variants were assayed for precise editing activity at AAVS1, DNMT1, and EMX1 for precise genome editing, and the results of the variants tested are tabulated as described above in this Example in Table 20.Table 20. Editing activity of Additional RT_A Variants in Human or Mouse Cells Fused to Nickase A (I67R, H397A, D568R, W593R, E706R, 16-775 Truncation)“The sequences of the RT A variants are provided in Sequence Table 2 below. The N- terminal M residue was removed from RT sequence when part of the fusion protein.Across the three targets, RT_A variants N117R, S35R (13-297 truncation) (SEQ ID NO: 258) and N117R, Y155F (13-297 truncation) (SEQ ID NO: 262) gave the highest levels of genome editing, although all the combinations tested generated editing at similar levels, over 10-fold higher than the 13-297 truncation reference.An additional set of variants for RT_A was also constructed using a peptide fusion comprising, in order: SV40 NLS (SEQ ID NO: 30), RT_A Variant, XTEN linker (SEQ ID NO: 31), Nickase A (I67R, H397A, D568R, W593R, E706R, 16-775 truncation) (SEQ ID NO: 90), Nucleoplasmin NLS (SEQ ID NO: 33). Additional mutations were added to the RT_A A24R, V202I (13-297 truncation) (SEQ ID NO: 255) portion of the RT editing fusion. The RT_A substitutions are listed in Table 21 with amino acid sequences provided in Sequence Table 2 below. The new RT_A-Nickase A fusion variants were assayed for precise genome editing at DNMT1, EMX1, and AAVS1 as described earlier in this Example. The results and specific mutants are summarized in Table 21.Attorney Docket No.: 063586-545001 WOTable 21. Editing Activity of Additional RT_A Variants in Human or Mouse Cells Fused to Nickase A (I67R, H397A, D568R, W593R, E706R, 16-775 truncation)As shown in in Table 21, many of the additional mutations in RT_A improve editing activity for at least one of the targets, or has similar levels of editing to the parent sequence. The addition of mutations G46T (SEQ ID NO: 265), L287F (SEQ ID NO: 266), or SI 19C (SEQ ID NO: 267) stand out as particularly high across all three targets as individual mutantsAttorney Docket No.: 063586-545001 WO added to the RT_A variant A24R, V202I, (13-297 truncation). RT_A variant A24R, V202I, Y155F, A188G, L287F (13-297 truncation) (SEQ ID NO: 272) performed best overall.Next, RT_B was subjected to additional mutagenesis as part of the RT editor comprising, in order, SV40 NLS (SEQ ID: 30), RT_B Variant, XTEN linker (SEQ ID: 31), Nickase A (I67R, H397A, D568R, W593R, E706R, 16-775 truncation) (SEQ ID: 77), Nucleoplasmin NLS (SEQ ID: 33). The new variants listed in Table 22 with amino acid sequences shown in Sequence Table 2 below combined some of the top performing mutants described in Table 6 above with some new mutations. Each of the new RT_B variants were tested in the context of the RT_B-Nickase A RT editing peptide fusion for genome editing activity at the AAVS1, DNMT1, and EMX1 targets as described above in this Example. Results and specific combinations of mutations are tabulated in Table 22.Table 22. Editing Activity of Additional RT_B Variants in Human or Mouse Cells Fused to Nickase A (I67R, H397A, D568R, W593R, E706R, 16-775 Truncation)Attorney Docket No.: 063586-545001 WOAll the RT_B mutation data collected in Table 22 showed increases in genome precise editing levels, with a few variants exhibiting improvements across all three targets. Namely, the combination of a 5 amino acid deletion and E291R with N121G (SEQ ID NO: 285), S63G (SEQ ID NO: 286), or Al 14C (SEQ ID NO: 287) were top performing mutants. Additionally, combinations of mutations from Table 21, showed significant improvement in editing activity, with the Q32R, Q71R (6-293 truncation) (SEQ ID NO: 290), Q32R, E26R, N121R, E291R (6- 293 truncation) (SEQ ID NO: 297), and Q32R, Q30R (6-293 truncation) (SEQ ID NO: 289) variants as particularly high performing.RT_C was also subjected to additional mutagenesis as part of an RT editor comprising, in order: SV40 NLS (SEQ ID NO: 30), RT_C Variant, XTEN linker (SEQ ID NO: 31), Nickase A (I67R, H397A, D568R, W593R, E706R, 16-775 truncation) (SEQ ID NO: 90), Nucleoplasmin NLS (SEQ ID NO: 33). The new variants combined some of the top performing mutants described in Table 23 with amino acid sequences provided in Sequence Table 2 below. Each of the new variants were tested for genome editing activity at the AAVS1, DNMT1, and EMX1 targets as described above in this Example. Results and specific combinations of mutations are tabulated in Table 23.Table 23. Editing Activity of Additional RT_C Variants in Human or Mouse Cells Fused to Nickase A (I67R, H397A, D568R, W593R, E706R, 16-775 Truncation)Attorney Docket No.: 063586-545001 WOFrom the data in Table 23, two mutants showed higher levels of genome editing: T70R, N117R (SEQ ID NO: 302), and T32R, N117R (SEQ ID NO: 303). Additionally, mutations V195I (SEQ ID NO: 311), A93V (SEQ ID NO: 307), and T286D (SEQ ID NO: 315) added on top of the T70R (5-296 truncation) variant (SEQ ID NO: 27) all led to increased editing levels relative to the T70R variant (SEQ ID NO: 26).This Example demonstrates additional mutations in the reverse transcriptase domain of the RT editing peptide fusion that increase the amount of precise genome editing relative to the unmutated versions of the RT_A (SEQ ID NO: 4), RT_B (SEQ ID NO: 5), or RT C (SEQ ID NO: 6) fused to a Nickase A variant (SEQ ID NO: 90). The top performing RT variant fusions to the Nickase A variant for each RT are: RT_A A24R V202I Y155F A188G L287F (13-297 truncation) (SEQ ID NO: 272), RT_B Q32R Q71R (6-293 truncation) (SEQ ID NO: 290), and RT_C T32RN117R (SEQ ID NO: 303).Example 10: Engineering of Nickase A-RT C Peptide Fusions for Increased RNA- Templated Editing in Human or Mouse CellsThis Example describes mutagenesis performed on the Nickase A-RT C peptide fusion to identify new variants with higher precise editing activity.In the first examination of new variants of the Nickase A-RT C fusions, a set ofAttorney Docket No.: 063586-545001 WO mutants were added in combination of the following peptide fusion, in order: SV40 NLS (SEQ ID NO: 30), an RT C T70R (SEQ ID NO: 26), XTEN linker (SEQ ID NO: 31), Nickase A Variant, HiBiT Tag (SEQ ID NO: 337), Nucleoplasmin NLS (SEQ ID NO: 33). All of the mutations were within the Nickase A peptide and converted certain residues to the positively charged residues arginine (R) or lysine (K). All new variants (listed in Table 24 with amino acid sequences provided in Sequence Table 2 below) were tested for precise editing in HEK293T at the EMX1 and AAVS1 targets with Editing Template RNA 1 and 2 (Table 1) or N2A at the DNMT1 target with Editing Template RNA 6 (Table 7) cells, as described in Example 9. The results are tabulated in Table 24 below.Table 24. Fold Change of Editing Activity with Additional Mutations within Nickase A (I67R, H397A, D568R, W593R, E706R, 16-775 truncation) in Human or Mouse Cells Fused to the RT_C T70R VariantThe addition of positive charge at the positions outlined in Table 24 led to increased precise editing activity across all three targets in most cases. Of particular interest are the E490K and E491K mutations that in combination (SEQ ID NO: 91 (containing E490K), SEQ ID NO: 92 (containing E491K), and SEQ ID NO: 93 (containing the combination)) led to an 85% increase in precise editing activity across the three targets tested.In a second examination of new variants for the Nickase A-RT C fusion, a new set of mutants was generated on top of the following peptide fusion, in order: SV40 NLS (SEQ ID NO: 30), an RT C T70R (5-296 truncation) (SEQ ID NO: 27), XTEN linker (SEQ ID NO: 31), Nickase A Variant, Nucleoplasmin NLS (SEQ ID NO: 33). In this set of mutations, onlyAttorney Docket No.: 063586-545001 WO residues in Nickase A Variant peptide sequence were modified compared to the parent Nickase A (I67R, H397A, D568R, W593R, E706R, 16-775 Truncation) (SEQ ID NO: 90), which is summarized in Table 25 with amino acid sequences provided in Sequence Table 1 below. All of the variants were tested for precise editing in HEK293T (at EMX1 and AAVS1) or N2A (at DNMT1) cells, as described earlier in this Example. The results are tabulated in Table 25 below.Table 25. Editing Activity of Fusion Peptides with Additional Mutations within Nickase A (I67R, H397A, D568R, W593R, E706R, 16-775 Truncation) in Human or Mouse Cells Fused to the RT_C T70R (5-296 Truncation) VariantAttorney Docket No.: 063586-545001 WOWhen added to Nickase A (I67R, H397A, D568R, W593R, E706R, 16-775 truncation) (SEQ ID NO: 90), mutations D225N, S729G, N87S, T83N, R261K, V733I, Cl 11 A, T391S, W622F, and P575A were particularly effective at increasing the precision editing levels across the average of the three targets without significantly lowering any of them. In a third examination of new variants for the Nickase A-RT C fusion, a new set of mutants was generated on top of the following peptide fusion, in order: SV40 NLS (SEQ ID NO: 30), an RT_C Variant, XTEN linker (SEQ ID NO: 31), Nickase A Variant, Nucleoplasmin NLS (SEQ ID NO: 33). In this set of mutations, residues in both the Nickase A (I67R, D568R, W593R, E706R, 16-775 Truncation) variant and RT_C T32R N117R variant peptides were modified, as summarized in Table 25 with corresponding amino acid sequences provided in Sequence Tables 1 and 2. All of the variants were tested for precise editing in HEK293T (at EMX1 and AAVS1) or N2A (at DNMT1) cells, as described earlier in this Example. The results are summarized in Table 26 below. Table 26. Editing Activity of Fusion Peptides with Additional Mutations within Nickase A (I67R, H397A, D568R, W593R, E706R, 16-775 Truncation) or RT C T32R N117R in Human or Mouse cells.Attorney Docket No.: 063586-545001 WOThe additional mutations captured in Table 35 broadly increase editing activity across all the targets. Of particular note is switching back H residue at position 397 in the Nickase A variant (SEQ ID NO: 90), which reverts the Nickase A variant back to a nuclease and increases editing activity. Further, N117K (SEQ ID NO: 327) shows increased activity above the original N117R mutation (SEQ ID NO: 303) that was identified in Table 25 above. The addition H145E to RT_C T32R N117R (SEQ ID NO: 326) also shows clear improvement in editing activity for DNMT1 and EMX1, but is neutral for AAVS1.In a fourth examination of new variants for the Nickase A-RT C fusion, combinations of mutations from Table 23 and Table 25 were added on top of the following peptide fusion, in order: SV40 NLS (SEQ ID NO: 30), an RT C T32R, N117R (SEQ ID NO: 303), XTEN linker (SEQ ID NO: 31), Nickase A (I67R, H397A, D568R, W593R, E706R, 16-775 truncation) (SEQ ID NO: 90), Nucleoplasmin NLS (SEQ ID NO: 33). Additionally, a variant of RT_C was generated by replacing residues 296-327 of RT_C T32R N117R (SEQ ID NO: 303) with a GSS tripeptide to create the KD3 variant (SEQ ID NO: 329; RT_C Variant KD3). Additional mutations were added on top of the RT_C Variant KD3 as part of the following peptide fusion, in order: SV40 NLS (SEQ ID NO: 30), GSG, RT_C Variant, the Short Linker (SEQ ID: 32), Nickase A Variant, Nucleoplasmin NLS (SEQ ID NO: 33). Mutations tested in either the Nickase A and / or RT_C peptide component are summarized in Table 27. The amino acid sequences of the additional Nickase A mutants are provided in Sequence Table 1 above.All of the variants were tested for precise editing in HEK293T (at EMX1 and AAVS1) or N2A (at DNMT1) cells, as described earlier in this Example. The results are summarized in Table 27 below.Table 27. Editing Activity of Fusion Peptides with Additional Mutations within Nickase A (I67R, H397A, D568R, W593R, E706R, 16-775 Truncation) or RT C in Human or Mouse Cells.Attorney Docket No.: 063586-545001 WOThe variants captured in Table 26 all show increased editing activity for at least one of the three targets tested without negatively impacting the others. The Nickase A variant containing the additional T83N, N87S, D225N, R261K, P575A, W622F, S729G mutations (SEQ ID NO: 134), named as the ‘4R+7C’ Nickase A mutant, showed the highest increase at the DNMT1 and EMX1 targets, and a neutral impact at AAVS1. The peptide fusions containing RT_C KD3 (SEQ ID NO: 329) demonstrated increased editing at AAVS1 (the highest of all of the variants tested in this set) and EMX1, and was neutral at DNMT1.In a fifth examination of new variants for the Nickase A-RT C fusion, a new peptide fusion was created to combine the best features of the variants tested in Table 26. In order, the new peptide fusion comprised: SV40 NLS (SEQ ID NO: 30), GSG, RT_C KD3 Variants, the Short Linker (SEQ ID NO: 32), Nickase A 4R+7C Variant, HiBiT Tag (SEQ ID NO: 337), Nucleoplasmin NLS (SEQ ID NO: 33). Additional single mutations were added to this fusionAttorney Docket No.: 063586-545001 WO peptide, either in the Nickase A peptide or the RT_C peptide shown in Table 28 with amino acid sequences provided in Sequence Tables 1 and 2 above, and the resulting sequences were tested for precise editing in HEK293T cells at AAVS1 and EMX1 with Editing Template RNAs 19 and 20 (Table 17), respectively, or N2A cells at DNMTl, using Editing Template RNA 21 (Table 17), using the transfection conditions and analysis method described in Example 9. The results are summarized in Table 28 below.Table 28. Editing Activity of Fusion Peptides with Additional Mutations within Nickase A 4R+7C or RT_C KD3 in Human or Mouse cellsAttorney Docket No.: 063586-545001 WOAll of the mutations in Table 27 exhibited increased precise genome editing for at least one target, with an average minimum of 10% increase in activity without negatively impacting any given taken by more than 5%. The mutants that generated the highest levels of precise editing improvement are: N117E, A127E, and V287D in RT_C KD3 (SEQ ID NO: 333, 335, and 336, respectively) and W508K, R699S, T83S, T766V, T562S, R757S in Nickase A 4R+7C (SEQ ID NO: 163, 148, 152, 160, 153, and 145, respectively). Of particular interest are N117E and T83S, which mutate previously identified mutations (N117R in RT_C and T83N in Nickase A) to new, higher performing mutations in the context of the fusion peptide containing Nickase A 4R+7C and RT_C KD3.This Example demonstrates that Nickase A RT_C peptide fusions can be engineered to increase precise genome editing rates at human targets. Additionally, this Example describes how the content of the peptide fusion outside of the Nickase A and RT_C components can impact and increase the amount of editing in the genome. Combining the highest performing mutants and fusion architecture has led to multiple high-performing peptide fusions of a Nickase A variant (SEQ ID NO: 90, 134, 148, 153, or 163) and RT C variant (SEQ ID NO: 26, 303, 329, or 333) (Sequence Table 3).Example 11: Improving RNA-Templated Editing of Human Genes in HEK293T Cells Using RNA-Guided Nuclease BT-Reverse Transcriptase PolypeptidesThis example describes a set of mutations that increase the level of a compact editing editor comprised of RT_C and Nuclease BT.An RT editor comprised of RT_C and Nuclease BT was constructed by fusing theAttorney Docket No.: 063586-545001 WO following peptides, in order: SV40 NLS (SEQ ID NO: 30), an RT C T32R, N117R (SEQ ID NO: 203), XTEN linker (SEQ ID NO: 31), Nuclease BT variant, Nucleoplasmin NLS (SEQ ID NO: 33). The Nuclease BT variant Q102R, I206R, and V433R was further modified with a set of individual single mutations, summarized in Table 30 with amino acid sequences provided in Sequence Table 2 above. These new variants were tested for precise editing in HEK293T (at an EMX1 target) or N2A (at a DNMT1 target) cells using the templating guides described in Table 29, with the transfection and data analysis methods as described in Example 1. The results are summarized in Table 30 below. Table 29. Full-length Sequences of Nuclease BT Editing Template RNA Components*Spacer sequences are underlined, scaffold sequence is shown in bold, and the gene edit is shown in lowercase italics (e.g.: insertion or 6-nucleotide substitution)Table 30. Fold-Change in precise editing activity of Nuclease BT Variant -RT C T32R, N 117R fusion mutantsAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOAttorney Docket No.: 063586-545001 WOThe mutations listed in Table 30 represent all the changes made to the fusion peptide described above that increased precise editing by at least 10%, without reducing one of the targets by more than 5%. The four highest performing mutations L292Q, K241I, L292K, R102I increased the average editing rate of the two targets by 4-fold or greater.This Example thus shows that fusions between variants of Nuclease BT and variants of RT_C can be engineered to increase the frequency of installed substitutions into human target genes. Converting the top performing RT_C -Nuclease BT peptide fusion to a nickase variant results in SEQ ID NO 347, which is expected to be the highest performing nickase peptide fusion containing Nickase BT.Example 12: RNA-Templated Editing of Human and Mouse Genes Using RT C- CRISPR Nickase A Fusion PolypeptidesThis Example describes the genomic editing of an exemplary target gene at the DNMT1, mACTb (mouse beta-actin), hPAH (human phenylalanine hydroxylase), and hHEK3 (human - human embryonic kidney) loci by RT C-CRISPR Nickase A variant fusion polypeptides and editing template RNAs.Editing template RNAs were designed as described in Example 1 and cloned into a pUC19 plasmid following the U6 PolIII promoter and terminated with a 6x polyT sequence. Editing template RNAs were designed to be specific to the targets within the DNMT1,Attorney Docket No.: 063586-545001 WO mACTb, hPAH, and hHEK3 loci and utilized a 20-nucleotide spacer sequence. The U6 PolIII promoter uses a +1 G at the start of the transcript (z.e., the 5’ end of the RNA) for more efficient transcription that is excluded from the sequences described here. The RNA guide sequences are shown in Table 31.Table 31. Full-Length Sequences of Nickase K Editing Template RNA ComponentAttorney Docket No.: 063586-545001 WO*Spacer sequences are underlined, scaffold sequence is shown in bold, and the gene edit is shown in lowercase italics (e.g.: insertion or 6-nucleotide substitution)Two RT C-CRISPR Nickase A RT fusion peptides were test separately with the editing template RNAs of Table 31 and introduced into HEK293T cells or Neuro-2A cells by lipid-based transient transfection as described in Example 9. Specifically, the RT and Nickase combinations tested were: 1) RT_C KD3 (SEQ ID NO: 329) - Nickase A I67R, H397A, D568R, W593R, E706R, T83N, N87S, D225N, R261K, P575A, W622F, S729G (16-775 truncation)(SEQ ID NO: 134), and 2) RT C T32R N117R (SEQ ID NO: 303) - Nickase A I67R, H397A, D568R, W593R, E706R, (16-775 truncation)(SEQ ID NO: 90). Genomic DNA was recovered approximately 72 hours post-transfection, and samples were prepared for NGS and analyzed as described in Example 1. The percentage of NGS reads containing substitutions shown in Table 32 were calculated as the average of three technical replicates, unless indicated otherwise.Table 32. Editing Activity of RT C-CRISPR Nickase A Fusion Polypeptides at Multiple TargetsAttorney Docket No.: 063586-545001 WO*One technical replicate was excluded because fewer than 10,000 NGS reads were generatedAs shown in Table 32, all of the editing template RNAs displayed editing levels above 1% for both RT C-CRISPR Nickase A variants tested. The RT_C -Nickase A RT editor peptide fusion containing the Nickase A 4R+7C variant (Nickase A I67R, H397A, D568R, W593R, E706R, T83N, N87S, D225N, R261K, P575A, W622F, S729G (16-775 truncation); SEQ ID NO: 134) displayed higher editing levels for 7 out of 8 of the editing template RNAs tested.This Example thus shows that the RT_C -Nickase A RT editor peptide fusion containing the Nickase A 4R+7C variant (SEQ ID NO: 134) has increased activity compared to the fusion containing the Nickase A 4R variant (Nickase A I67R, D568R, W593R, E706R, (16-775 truncation); SEQ ID NO: 90) across a broad range of targets.Example 13: Nickase A-RT C Fusions Packaged into AAVs to Create Precise Genomic Edits in vitroThis example demonstrates the ability of exemplary compact editors to precisely edit the genome of mammalian cells when delivered by AAV and packaged along with the requisite template guide RNA.A nucleic acid encoding a fusion protein containing a Nickase A variant (I67R, H397A, D568R, W593R, E706R, T83N, N87S, D225N, R261K, P575A, W622F, S729G, 16- 775 truncation; SEQ ID NO: 134) and an RT C variant (RT_C Variant T32R, N117R, S297G, P298S, P299S, deletion (300-327); SEQ ID NO: 329) was packaged into an AAV vector under a Pol II promoter. The fusion protein comprises, from N-terminus to C-terminus, an NLS, linker, RT_C variant, Linker, Nickase A variant and a second NLS. The AAV also included the Editing Template RNA 1 (Table 1) expressed with a U6 Pol III promoter.Two non-dividing cell types were transduced to test the ability of the compact RT editor to make precise genome edits: primary human hepatocytes and human motor neurons derived from iPSCs.To test editing in human motor neurons, iPSCs were differentiated into iMNs via an 18-Attorney Docket No.: 063586-545001 WO day protocol that used similar differentiation protocol that was pervious described in: Lepine S, et al. iScience 2024, Du Z, et al. Nat. Comm. 2015, and Castellanos-Montiel M, et al. Cells 2023. Differentiated iMNs were plated 4 days prior to transduction, then the AAVs were added to the culture at MOIs ranging from 103-105. Cells were harvested after 10, 20, or 30 days, and the DNA was extracted, sequenced, and analyzed for precise editing levels according to the methods described in Example 1. Indels were also measured after 10 or 20 days. The results are summarized in Table 33.Table 33. Precise Editing Activity in Induced Motor Neurons by AAV Delivery of Nickase_A-RT_C Fusion and Template Guide RNAThe fusion protein demonstrated increased levels of precise editing with increasing dose and time. Maximum precise editing for the RT fusion was achieved with the maximum dose of 105 MOI after 30 days, 1.4% editing, and would likely continue to rise over time.To test for editing in primary human hepatocytes, donor cells purchased from ThermoFisher Scientific were thawed and plated according to the manufacturer’s protocol. The transduction protocol was performed similarly to the induced motor neuron experiment described above, with cells being treated with the AAVs at two doses and harvested for analyses after 10 or days after transduction. Analysis was performed to measure precise editing levels as described in Example 1.Attorney Docket No.: 063586-545001 WOTable 34. Precise Editing Activity in Primary Human Hepatocytes by AAV Delivery of Nickase A- RT_C Fusion and Template Guide RNALike the induced motor neuron experiment described above, precise editing in human primary hepatocytes increased with both dose and time. A maximum precise editing level of 4.13% was reached after 20 days using the higher dose.Results across both cell types demonstrated that the compact RT editor disclosed herein can be packaged into an AAV vector with the template editing guide RNA to produce precise editing in vitro. This is a significant advantage over state of the art, which requires two different viral particles to deliver all of the components necessary for RT editing.Example 14: Precise Genome Editing Activity of Compact RT Editing Systems by RNA DeliveryThis example demonstrates the ability of exemplary compact editors to precisely edit the genome when delivered as an mRNA expressing the compact editor fusions protein along with the request guide RNA. In this example, fusion compact editors were in vitro transcribed as mRNA, which delivered to human cells via electroporation alongside chemically synthesized editing template RNAs.To test the compact RT editor fusions in an mRNA format, fusion proteins were cloned into an in vitro transcription compatible DNA vector, such that each clone comprised, from N- terminus to C-terminus: NLS (SEQ ID NO: 30), GSG Linker, RT variant, Linker (SEQ ID NO: 32), Nickase / Nuclease, Tag (SEQ ID NO: 337, optional), NLS (SEQ ID NO: 33).The plasmids encoding the fusion protein sequence were linearized with a restriction enzyme and used to generate mRNA with an in vitro mRNA transcription kit (New England BioLabs). Editing Template RNA 1 (SEQ ID NO: 71) was chemically synthesized, with three 2'-0Me and phosphorothioate modifications at each of the 5' and 3' ends. Approximately 1.5 ug of mRNA and 500 pmol of gRNA were co-nucleofected (Lonza) into ~300k HEK239T cells and grown for 3 days in DMEM+10% FBS before DNA extraction and measuring precise editing as described in Example 1. The results are summarized in Tables 35A and 35B.Attorney Docket No.: 063586-545001 WOA. Precision Editing o f Compact Editors Delivered by mRNANuclease alone or a compact editor fusion proteins comprising the nuclease and an RT polypeptide for use in this study are provided Table 35A below.Table 35A: Precise Editing Activity of Compact Editors in HEK293T Cells Delivered by Simultaneous Nucleofection of mRNA and gRNACo-delivering a synthesized editing template RNA noted above and mRNA encoding only the nuclease resulted in high levels of genome editing activity (>90% as shown in Table 35A above), while co-delivering the synthesized editing template RNA and mRNA encoding the nuclease-RT fusion editor exhibited -80% editing activity (see Table 35A). Fusion proteins containing an RT domain also exhibited high levels of precise editing, which is not detected when mRNA encoding only the nuclease was used.B. Variants ofRT C Increase Precise Genome Editins Activity of Nickase A-RT C Fusions in Human Cells via mRNA ElectroporationTo test the precision editing of compact RT editor fusions in an mRNA format, two Nickase A variants (SEQ ID NO: 90 and SEQ ID NO: 134) were cloned as fusions to RT_C in an in vitro transcription compatible DNA vector, such that each clone comprised: SV40 NLS (SEQ ID NO: 30), GSG linker, RT C Variant, Short Linker (SEQ ID NO: 32), Nickase A variant (SEQ ID NO: 90 or SEQ ID NO: 134), HiBiT Tag (SEQ ID NO: 337), and Nucleoplasmin NLS (SEQ ID NO: 33). Five additional variants of RT_C KD3 (SEQ ID NO: 329) were generated by mutating H145 and D146 with the goal of increasing dNTP affinity:Attorney Docket No.: 063586-545001 WOH145E, D146M, H145G, H145E D146M, and H145G D146M. These dNTP affinity mutants were fused to Nickase_A variant 4R+7C (SEQ ID NO: 134) in the same configuration as described above.The resulting set of mRNA encoding the 7 fusion proteins were delivered to cells with a template guide RNA and precise editing was measured as described in Example 1. The results are summarized in Table 35B.Table 35B: Precise Editing Activity of Nickase_A-RT_C Fusions in HEK293T Cells Delivered by Simultaneous Nucleofection of mRNA and gRNAThe use of the Nickase_A-RT_C variants generated approximately 1-5% precise editing with minimal imprecise edits. The addition of the H145E or D146M substitution in the RT_C KD3 variant increased precise editing, with D146M showing the highest levels of precise editing. The addition of both H145E and D146M substitutions to the RT_C KD3 variant led to the highest overall level of precise editing for the compact editor comprising the Nickase Variant 4R+7C (SEQ ID NO: 134) and the RT C variant (SEQ ID NO: 451).This Example demonstrates that high levels of editing can be achieved with an all RNA delivery of a compact gene editing system and compact RT editors comprising Nickase A and RT_C variants can lead to programmable precise edits in the human genome. The precise editing of the compact RT editors is maintained without introducing significant indels from double stranded DNA breaks when delivered as an all RNA editing strategy.Attorney Docket No.: 063586-545001 WOExample 15: Additional Engineering of CRISPR Nickase A (4R+7Q and RT C (KD3) Fusions to Increase RNA-Templated Editing in Human and Mouse cellsThis example describes additional variants of Nickase A and RT_C that increase the levels of precise editing in human cells relative to the fusion of the Nickase A 4R+7C variant (SEQ...

Claims

Attorney Docket No.: 063586-545001 WOWhat Is Claimed Is:

1. A fusion protein comprising (a) a nuclease polypeptide, and (b) a reverse transcriptase (RT) polypeptide; wherein the nuclease polypeptide comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; wherein each of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 comprises an HNH nuclease domain and an RuvC nuclease domain; and wherein the RT polypeptide comprises a polymerase domain, which 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: 70); and wherein the RT polypeptide has a length of about 250-350 amino acids.

2. The fusion protein of claim 1, wherein the nuclease polypeptide is a variant of SEQ ID NO: 1 comprising the following features relative to SEQ ID NO: 1 :(i) one or more mutations in the HNH nuclease domain or in the RuvC nuclease domain;(ii) one or more arginine and / or lysine substitutions (non-arginine / lysine);(iii) one or more deletions and / or insertions, optionally an N-terminal truncation;(iv) one or more non-arginine and / or lysine amino acid substitutions; or(v) a combination of (i), (ii), (iii); and / or (iv).

3. The fusion protein of claim 2, wherein the variant of SEQ ID NO: 1 comprises the one or more mutations in the HNH nuclease domain or in the RuvC nuclease domain; and wherein the one or more mutations are at one or more of positions D396, H397, N420, D24, E337, and D524 of SEQ ID NO: 1.

4. The fusion protein of claim 3, wherein the variant of SEQ ID NO: 1 comprises a mutation at position H397 of SEQ ID NO: 1, optionally wherein the mutation is H397A, H397K, H397R, H397W, or H397Y.

5. The fusion protein of any one of claims 2-4, wherein the variant of SEQ ID NO: 1 comprises or further comprises one or more arginine or lysine substitutions at one or more ofAttorney Docket No.: 063586-545001 WO positions D56, E59, G60, E63, 167, G71, T98, E102, E206, V210, R258, R259, R261, E490, E491, E495, W508, 1519, D533, D536, S564, D568, W593, T605, E655, E694, E706, G713, and R720 in SEQ ID NO: 1; optionally wherein the one or more arginine substitutions are at one or more of positions D56, E59, G60, E63, 167, G71, E206, E491, S564, D568, W593, T605, E655, E694, and E706, and optionally wherein the one or more lysine substitutions are at one or more of positions T98, E102, V210, R258, R259, R261, E490, E491, E495, W508, 1519, D533, D536, G713, and R720.

6. The fusion protein of claim 5, wherein the variant of SEQ ID NO: 1 comprises or further comprises arginine and / or lysine substitutions at the following positions relative to SEQ ID NO: 1:(a) 167, D568, and E706, optionally I67R, D568R, and E706R; or(b) 167, D568, W593, and E706, optionally I67R, D568R, W593R, and E706R7. The fusion protein of any one of claims 2-6, wherein the variant of SEQ ID NO: 1 comprises or further comprises the N-terminal truncation, which is a deletion within residues 1-15 of SEQ ID NO: 1; optionally wherein the N-terminal truncation is the deletion of residues 1-14 or 1-15 of SEQ ID NO: 1.

8. The fusion protein of claim 2, wherein the variant of SEQ ID NO: 1 comprises, relative to SEQ ID NO: 1 :(a) a mutation at position H397, optionally H397A, H397K, H397R, H397W, or H397Y; arginine and / or lysine substitutions at positions 167, D568, and E706, optionally I67R, D568R, and E706R; and a deletion of residues 1-15 of SEQ ID NO: 1; or(b) a mutation at position H397, optionally H397A, H397K, H397R, H397W, or H397Y; arginine and / or lysine substitutions at positions 167, D568, W593, and E706, optionally I67R, D568R, W593R, and E706R; and a deletion of residues 1-15 of SEQ ID NO: 1.

9. The fusion protein of any one of claims 2-8, wherein the variant of SEQ ID NO: 1 comprises or further comprises, relative to SEQ ID NO: 1 :(i) one or more non-arginine and / or lysine amino acid substitutions at one or more of positions Q72, K76, T83, N87, 196, T98, E102, Cl 11, S134, E137, E144,Attorney Docket No.: 063586-545001 WOA174, D225, L232, E241, T255, F265, F275, K288, E300, A307, 1406, V321, 1335, K348, E367, K366, E367, T391, 1406, E418, L434, H478, M479, E495, Q497, 1500, W508, D533, S548, T562, R567, R568, S570, N571, T574, P575, Y577, S587, L588, T596, E603, 1607, S608, Y611, K613, W622, N667, T673, L684, R699, S729, V733, R755, R757, and T766 of SEQ ID NO: 1; optionally wherein the non-arginine and / or lysine amino acid substitution(s) is Q72S, K76M or K76V, T83A, T83N, T83S, orN87S, I96T, T98N, Cl 11 A, S134D, E137D, E144A, A174L, D225N, L232I, E241L, T255E, F265E, F275D or F275G, K288E, E300Q, A307N, I406V, V321I, I335V, K348M, E367D, K366N, E367D, T391G or T391S, I406V, E418G, L434V, H478W, M479L, Q497V, I500T, S548N, T562S, R567M, R568I, R568M, S570G, N571S, T574S, P575A, Y577H, S587G, L588H, T596V, E603D, I607F, S608T, Y611D, K613E, W622F, N667D, T673S, L684P, R699S, S729G, R755S, R757S, V733I, T766V, and / or T766I;(ii) one or more deletions, optionally a deletion at position Q277 and / or positions E206-D207;(iii) one or more insertions, optionally an insertion between position 538 and 539 of SEQ ID NO: 1, optionally wherein the insertion is L between position 538 and 539 of SEQ ID NO: 1; or(iv) a combination of (i), (ii), and / or (iii).

10. The fusion protein of claim 8, wherein the variant of SEQ ID NO: 1 further comprises the non-arginine / lysine substitutions at the following positions relative to SEQ ID NO: 1: T83, N87, D225, R261, P575, W622, and S729; optionally wherein the non- arginine / lysine substitution(s) include T83N, N87S, D225N, R261K, P575A, W622F, and / or S729G.

11. The fusion protein of claim 10, wherein the variant of SEQ ID NO: 1 further comprises:(a) an arginine / lysine substitution at one or more of the following positions relative to SEQ ID NO: 1: E206, E490, E491, W508, 1519, and G713; optionally wherein the substitution(s) is E206R, E490K, E491K or E491R, W508K, I519K, and / or G713K;(b)one or more non-arginine / lysine substitutions at one or more of the following positions relative to SEQ ID NO: 1: Q72, K76, T255, F265, F275, K288, E300, E367, 1406,Attorney Docket No.: 063586-545001 WOE418, L434, H478, 1500, W508, T562, L588, E603, 1607, S608, Y611, N667, L684, and R699; optionally wherein the non-arginine / lysine substitution is Q72S, K76V, T255E, F265E, F275G, K288E, E300Q, E367D, I406V, E418G, L434V, H478W, I500T, W508K, T562S, L588H, E603D, I607F, S608T, Y611D, N667D, L684P, and / or R699S; or(c) a combination of (a) and (b).

12. The fusion protein of claim 2, wherein the variant of SEQ ID NO: 1 comprises:(a) a mutation in the RuvC nuclease domain, which is at position H397 of SEQ ID NO: 1; optionally wherein the mutation is H397A, H397K, H397R, H397W, or H397Y;(b) arginine or lysine substitutions at positions 167, D568, W593, and E706 of SEQ ID NO: 1, optionally I67R, D568R, W593R, and E706R;(c) amino acid substitutions at positions T83, N87, D225, R261, P575, W622, and S729 of SEQ ID NO: 1, optionally T83N, N87S, D225N, R261K, P575A, W622F, and S729G; and(d) deletion of residues 1-15 of SEQ ID NO: 1.

13. The fusion protein of claim 12, wherein the variant of SEQ ID NO: 1 further comprises one or more amino acid substitutions at one or more of positions E206, R258, R259, F275, K288, L434, E490, E491, L588, 1607, S608, Y611, N667, L684, and R699 of SEQ ID NO: 1; optionally wherein the one or more amino acid substitutions are E206R, R258K, R259K, F275G, K288E, L434V, E490K, E491R, L588H, I607F, S608T, Y611D, N667D, L684P, and R699S.

14. The fusion protein of claim 13, wherein the one or more amino acid substitutions are at one or more of positions E206, E490, E491, and R699; optionally wherein the one or more amino acid substitutions are E206R, E490K, E491R, and / or R699S.

15. The fusion protein of any one of claims 2-14, wherein the nuclease polypeptide comprises an amino acid sequence at least 95%, optionally at least 98%, identical to SEQ ID NO: 1; optionally wherein the nuclease polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 90, 134, 148, 153, 163, 428-443, and 469-480.Attorney Docket No.: 063586-545001 WO16. The fusion protein of claim 1, wherein the nuclease polypeptide is a variant of SEQ ID NO: 2 comprising the following feature relative to SEQ ID NO: 2:(i) one or more mutations in the HNH nuclease domain or in the RuvC nuclease domain of SEQ ID NO: 2;(ii) one or more arginine and / or lysine substitutions;(iii) one or more deletions, optionally an N-terminal truncation; or(iv) a combination of any one of (i)-(iii).

17. The fusion protein of claim 16, wherein the variant of SEQ ID NO: 2 comprises (i), and the one or more mutations are at positions D374, H375, and / or N398 in SEQ ID NO: 2.

18. The fusion protein of claim 17, wherein the mutation is at position H375, optionally H375A.

19. The fusion protein of any one of claims 17-18, wherein the variant of SEQ ID NO: 2 comprises or further comprises one or more arginine and / or lysine substitutions, optionally one or more arginine substitutions, at one or more of positions G42, D46, 153, F83, E128, E541, F570, E576, D582, C630, 1631, E683, S719, and T734 in SEQ ID NO: 2.

20. The fusion protein of claim 19, wherein the variant of SEQ ID NO: 2 comprises or further comprises arginine and / or lysine substitutions at the following positions relative to SEQ ID NO: 2: G42 and D582, optionally wherein the variant of SEQ ID NO: 2 comprise the G42R and D582R substitutions relative to SEQ ID NO: 2.

21. The fusion protein of claim 20, wherein the variant of SEQ ID NO: 2 comprises mutations H375A, G42R, and D582R relative to SEQ ID NO: 2.

22. The fusion protein of any one of claims 17-21, wherein the nuclease polypeptide comprises an amino acid sequence at least 95%, optionally 98%, identical to SEQ ID NO: 2; optionally wherein the nuclease polypeptide comprises the amino acid sequence of SEQ ID NO: 12.

23. The fusion protein of claim 1, wherein the nuclease polypeptide is a variant of SEQ ID NO: 3 comprising the following feature relative to SEQ ID NO: 3:Attorney Docket No.: 063586-545001 WO(i) one or more mutations in the HNH nuclease domain or in the RuvC nuclease of SEQ ID NO: 3;(ii) one or more arginine and / or lysine substitutions, optionally one or more arginine substitutions;(iii) one or more non-arginine / lysine amino acid substitutions;(iv) one or more deletions, optionally an N-terminal truncation; or(v) a combination of any one of (i)-(iv).

24. The fusion protein of claim 23, wherein the variant of SEQ ID NO: 3 comprises (i) and wherein the one or more mutations are at positions H231, H232, H255, D67, E176, and / or D329 of SEQ ID NO: 3.

25. The fusion protein of claim 24, wherein the variant of SEQ ID NO: 3 comprises a mutation at position H232 of SEQ ID NO: 3, optionally wherein the mutation is H232A.

26. The fusion protein of any one of claims 23-25, wherein the variant of SEQ ID NO: 3 comprises or further comprises one or more arginine and / or lysine substitutions, optionally one or more arginine substitutions, at one or more of positions R31, N34, R39, E99, Q102, E105, T106, T113, K123, R135, R151, M186, E191, Q194, 1206, N239, V256, 1272, A277, K241, L292, K318, E401, E432, and V433 in SEQ ID NO: 3; optionally wherein the one or more arginine substitutions are at one or more of positions E99, Q102, E105, T106, T113, K123, E191, 1206, N239, V256, 1272, A277, K241, K318, E401, E432, and V433; and optionally wherein the one or more lysine substitutions are at one or more of positions R31, N34, R39, R135, R151, Ml 86, QI 94, and L292.

27. The fusion protein of claim 26, wherein the variant of SEQ ID NO: 3 comprises or further comprises arginine and / or lysine substitutions at the following positions relative to SEQ ID NO: 3: QI 02, 1206, and V433, optionally wherein the variant of SEQ ID NO: 3 comprises the Q102R, I206R, and V433R substitutions.

28. The fusion protein of claim 27, wherein the variant of SEQ ID NO: 3 comprises mutations H232A, Q102R, I206R, and V433R relative to SEQ ID NO: 3.Attorney Docket No.: 063586-545001 WO29. The fusion protein of any one of claims 23-28, wherein the variant of SEQ ID NO: 3 comprises or further comprises one or more non-arginine / lysine substitutions at one or more of positions R2, N14, H18, N34, D70, C75, R84, V85, L88, H94, 1100, R102, T106, M109, Y116, 1128, H131, P133, E166, V168, 1182, 1190, R206, R207, A208, A222, P227, F228, Q229, Q238, N239, K241, K257, T260, K263, Q269, 1272, S290, L292, L296, G301, V305, S313, D323, N336, V339, T343, V350, K353, V379, E401, K428, K429, E437, N445, D448, S454, and C481; optionally wherein the one or more non-arginine / lysine substitutions are R2M, N14G, H18N, N34L, D70N, C75S, R84C, V85G, L88A, L88F, L88I, L88M, or L88V, H94G, HOOF, R102I or R102H, T106G, M109G, Y116N, I128E, H131Y, P133G, E166A, V168L, I182V, I190V, R207H, R207A, A208E, A222G, P227I, F228L, Q229E, Q238C, N239G, K241E, K241I, K241H, or K241Q, K257A, T260N, K263A, Q269L, I272N, S290P, L292N or L292Q, L296A, L296G, L296P, or L296V, G301P, V305I, S313T, D323P, N336C, V339I, T343V, V350F, K353E or K353N, V379I, E401 A, K428N or K428S, K429G, E437P, N445G, D448G, S454T, and / or C481 V.

30. The fusion protein of claim 23, wherein the variant of SEQ ID NO: 3 comprises (a) arginine and / or lysine substitutions at positions Q102, 1206, and V433 of SEQ ID NO: 3, optionally arginine substitutions Q102R, I206R, and V433R; (b) a non-arginine / lysine substitution at position L292 of SEQ ID NO: 3, optionally L292Q; and (c) optionally a substitution at position H232 of SEQ ID NO: 3, optionally H232A.

31. The fusion protein of any one of claims 23-30, wherein the nuclease polypeptide comprises an amino acid sequence at least 95%, optionally at least 98%, identical to SEQ ID NO: 3; optionally wherein the nuclease polypeptide comprises the amino acid sequence of SEQ ID NO: 166, or the nuclease polypeptide is a nickase variant of SEQ ID NO: 166 comprising the H232A substitution.

32. The fusion protein of any one of claims 1-31, wherein the nuclease polypeptide in the fusion protein is free of its native N-terminus methionine.

33. The fusion protein of any one of claims 1-32, wherein the RT polypeptide comprises an amino acid sequence at least 90%, optionally at least 95% or at least 98%, identical to SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.Attorney Docket No.: 063586-545001 WO34. The fusion protein of claim 33, wherein the RT polypeptide is a variant of SEQ ID NO: 4 comprising:(i) one or more arginine and / or lysine substitutions, optionally one or more arginine substitutions, 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: 4;(ii) one or more 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: 4; optionally wherein the substitutions are I20V, V92I, A100V, Q102T, VI 041, Fl 16L, SI 19C, Y121F, G122A, Q142L, H146W, Y155F, G157D, I159V, T163L, I172V, A188G, D189V, S190W, A191D, T192G, V202I, F220L, F238L, V240L, L241M, and / or L287F;(iii) one or more deletions comprise deletions within residues 1-14, optionally 1-12, of SEQ ID NO: 4, within residues 295-316 of SEQ ID NO: 4, or a combination thereof; or(iv) a combination of any one of (i)-(iii).

35. The fusion protein of claim 34, wherein the variant of SEQ ID NO: 4 comprises, relative to SEQ ID NO: 4:(i) one or more arginine and / or lysine substitutions at one or more of positions A24, N36, H84, and El 17 of SEQ ID NO: 4; optionally A24R, N36R, H84R, and / or El 17R; or(ii) a non-arginine / lysine substitution at position Q142 of SEQ ID NO: 4, optionally Q142L.

36. The fusion protein of claim 35, wherein the variant of SEQ ID NO: 4 further comprises the deletions of residues 1-12 and residues 298-316 of SEQ ID NO: 4.

37. The fusion protein of claim 34, wherein the variant of SEQ ID NO: 4 comprises the following features:(i) the substitution of A24R;(ii) the substitutions of A24R, N36R, H84R, and Q142L,; or(iii) the substitutions of A24R, N36R, H84R, and Q142L, and the deletions of residues 1-12 and residues 298-316 of SEQ ID NO: 4.Attorney Docket No.: 063586-545001 WO38. The fusion protein of claim 37, wherein the nuclease polypeptide is set forth in any one of claims 2-19 and 29.

39. The fusion protein of claim 34, wherein the variant of SEQ ID NO: 4 comprises mutation El 17R and the deletions of residues 1-12 and residues 298-316 of SEQ ID NO: 4.

40. The fusion protein of claim 39, wherein the nuclease polypeptide is set forth in any one of claims 20-29.

41. The fusion protein of any one of claims 34-40, wherein the RT polypeptide is the variant of SEQ ID NO: 4, the variant comprising (a) amino acid substitutions at positions A24, Y155, Al 88, V202, and L287 of SEQ ID NO: 4, optionally A24R, Y155F, A188G, V202I, and L287F; and (b) deletions of residues 1-12 and 298-316 of SEQ ID NO: 4.

42. The fusion protein of claim 34, wherein the variant of SEQ ID NO: 4 comprises the amino acid sequence of SEQ ID NO: 272.

43. The fusion protein of claim 33, wherein the RT polypeptide is a variant of SEQ ID NO: 5 comprising:(i) one or more arginine and / or lysine substitutions, optionally one or more arginine substitutions, at positions Q14, E26, Q30, Q32, Q71, N121, L122, N126, K130, C133, S193, E291, and A292 in SEQ ID NO: 5;(ii) one or more deletions comprise deletions within residues 1-5 of SEQ ID NO: 5, within residues 294-338 of SEQ ID NO: 5, or a combination thereof;(iii) one or more non-arginine / lysine amino acid residue substitutions; or(iv) a combination of (i), (ii), and / or (iii).

44. The fusion protein of claim 43, wherein the variant of SEQ ID NO: 5 comprises, relative to SEQ ID NO: 5: one or more arginine and / or lysine substitutions at one or more of positions Q32, N121, and E291, optionally Q32R, N121R, and / or E291R.

45. The fusion protein of claim 44, wherein the variant of SEQ ID NO: 5 further comprises the deletions of residues 1-5 and residues 294-338 of SEQ ID NO: 5.Attorney Docket No.: 063586-545001 WO46. The fusion protein of claim 44, wherein the variant of SEQ ID NO: 5 comprises the following features:(i) the substitution of E291R; or(ii) the substitution of E291R and the deletions of residues 1-5 and residues 294-338 of SEQ ID NO: 5.

47. The fusion protein of claim 46, wherein the nuclease polypeptide is set forth in any one of claims 2-19 and 29.

48. The fusion protein of claim 44, wherein the variant of SEQ ID NO: 5 comprises the following features:(i) the substitutions of Q32R, N121R, and E291R; or(ii) the substitutions of Q32R, N121R, and E291R and the deletions of residues 1-5 and residues 294-338 of SEQ ID NO: 5.

49. The fusion protein of claim 48, wherein the nuclease polypeptide is set forth in any one of claims 20-29.

50. The fusion protein of any one of claims 43-49, wherein the variant of SEQ ID NO: 5 comprises or further comprises one or more non-arginine / lysine amino acid residue substitutions at one or more of positions S63, Al 14, N121, and L257; optionally wherein the one or more non-arginine / lysine amino acid residue substitutions are S63G, Al 14C, N121G, and L257L.

51. The fusion protein of claim 43, wherein the RT polypeptide is a variant of SEQ ID NO: 5, the variant comprising arginine or lysine substitutions at positions Q32 and Q71 of SEQ ID NO: 5, optionally arginine substitutions Q32R and Q71R; and deletions of residues 1- 5 and 294-338 of SEQ ID NO: 5; optionally wherein the variant of SEQ ID NO: 5 comprises the amino acid sequence of SEQ ID NO: 290.

52. The fusion protein of claim 33, wherein the RT polypeptide is a variant of SEQ ID NO: 6 comprising:(i) one or more arginine and / or lysine substitutions, optionally one or more arginine substitutions, at one or more of positions E24, T32, A35, A28, K29, G31, T32, P34,Attorney Docket No.: 063586-545001 WOG36, V38, T51, K68, T70, E56, G73, A85, E88, N100, H102, N117, R122, H145, S218, F275, and P296 in SEQ ID NO: 6; optionally wherein the one or more arginine substitutions are at one or more of positions E24, K29, T32, A35, A28, G31, T32, P34, G36, V38, K68, T70, G73, A85, E88, H102, N117, H145, S218, and F275, and optionally wherein the lysine substitution(s) is at position N100, N117, R122, and / or P296;(ii) one or more deletions comprising deletions within residues 1-10, optionally 1-7 or 1-4, of SEQ ID NO: 6, within residues 297-350 of SEQ ID NO: 6, deletion of position 50 in SEQ ID NO: 6, or a combination thereof;(iii) one or more non-arginine / lysine amino acid substitutions; or(iv) a combination of (i), (ii), and / or (iii).

53. The fusion protein of claim 52, wherein the variant of SEQ ID NO: 6 comprises, relative to SEQ ID NO: 6, one or more arginine and / or lysine substitutions at one or more of positions K29, T32, K68, T70, N100, H102, N117, and R122, optionally K29R, T32R, K68R, T70R, N100K,Nl 17K or N117R, and / or R122K.

54. The fusion protein of any one of claims 52-53, wherein the variant of SEQ ID NO: 6 comprises or further comprises (a) the deletions of residues 1-4 and residues 297-350 of SEQ ID NO: 6; (b) the deletion of residues 297-325 of SEQ ID NO: 6; and / or (c) the deletion of position 50 of SEQ ID NO: 6.

55. The fusion protein of claim 52, wherein the variant of SEQ ID NO: 6 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: 6; or(iii) the substitution of T70R and the deletion of residues 297-325 of SEQ ID NO: 6.

56. The fusion protein of claim 55, wherein the nuclease polypeptide is set forth in any one of claims 2-19 and 29.

57. The fusion protein of claim 52, wherein the variant of SEQ ID NO: 6 comprises the following features:(i) the substitution of T32R; orAttorney Docket No.: 063586-545001 WO(ii) the substitution of T32R and the deletions of residues 1-4 and residues 297-350 of SEQ ID NO: 6.

58. The fusion protein of claim 57, wherein the nuclease polypeptide is set forth in any one of claims 20-29.

59. The fusion protein of any one of claims 52-58, wherein the variant of SEQ ID NO: 6 comprises or further comprises the one or more non-arginine / lysine amino acid substitutions at one or more of positions H8, F25, R43, E52, K48, G73, L83, A93, N100, M101, F103, N117, R110, L121, A127, H145, D146, G154, V170, M172, D180, K192, V195, 1199, V201, A202, M213, M234, L249, 1252, H266, V268, T286, V287, S297, P298, and P299 of SEQ ID NO: 6 optionally wherein the one or more non-arginine / lysine amino acid substitutions are one or more of H8N, F25Y, R43N or R43S, E52P, K66P, G73N, L83V, A93V, N100L, M101 A, F103I, N117E, N117M, or N117P, R110H or R1 ION, L121 V, A127E, H145E or H145S, D146A or D146M, G154D, M172F, V170Y, D180P, K192I, V195I, I199F, V201N, A202S, M213L, M234F, L249A, I252L, H266Y, V268M, T286D, V287D, S297G, P298S, and P299S.

60. The fusion protein of claim 59, wherein the variant of the SEQ ID NO: 6 comprises or further comprises, relative to SEQ ID NO: 6, one or more non-arginine / lysine substitutions at one or more of the following positions: H8, R43, M101, F103, N117, H145, D146, 1199, V201, A202, L249, V268, S297, P298, and / or P299; optionally wherein the non- arginine / lysine substitution(s) is H8N, R43N or R43S, M101 A, F103I, N117E, N117M, or N117P, H145S or H145E, D146A or D146M. I199F, V201N, A202S, L249A, V268M, S297G, P298S, and P299S.

61. The fusion protein of claim 52, wherein the RT polypeptide is a variant of SEQ ID NO: 6; and wherein the variant comprises, relative to SEQ ID NO: 6:(a) an amino acid substitution at position T70 of SEQ ID NO: 6, optionally T70R; or(b) amino acid substitutions at positions T32 and N117 of SEQ ID NO: 6, optionally T32R and N117E, N117K, N117M, N117Q, or N117R.Attorney Docket No.: 063586-545001 WO62. The fusion protein of any one of claims 52-61, wherein the variant of SEQ ID NO: 6 further comprises amino acid substitutions at positions S297, P298, and P299 of SEQ ID NO: 6, optionally S297G, P298S, and P299S; and the deletion of residues 300-327 of SEQ ID NO: 6.

63. The fusion protein of claim 52, wherein the variant of SEQ ID NO: 6 comprises the following features:(a) arginine or lysine substitutions at positions T32 and N117 of SEQ ID NO: 6, optionally T32R and N117R or N117K;(b) non-arginine / lysine substitutions at positions S297, P298, and P299 of SEQ ID NO: 6, optionally S297G, P298S, and P299S; and(c) deletion of residues 300-327 of SEQ ID NO: 6.

64. The fusion protein of claim 63, wherein the variant of SEQ ID NO: 6 further comprises:(i) arginine or lysine substitution(s) at position W7, K29, N100, and / or Hl 02, of SEQ ID NO: 6, optionally W7K, K29R, N100K, and / or H102R;(ii) non-arginine / lysine substitution(s) at position F25, R43, L83, M101, F103, H145, D146, V153, 1199, V201, L249, and / or T286 of SEQ ID NO: 6, optionally F25Y, R43E, L83I, M101 A, F103I, N117E, N117M, or N117P, H145S or H145E, D146A or D146M, V153T, I199F, V201N, L249A, and / or T286D; or(iii) a combination of (i) and (ii).

65. The fusion protein of claim 52, wherein the variant of SEQ ID NO: 6 comprises the following features:(a) arginine or lysine substitution at position T32 of SEQ ID NO: 6, optionally T32R;(b) non non-arginine / lysine substitutions at positions S297, P298, and P299 of SEQ ID NO: 6, optionally S297G, P298S, and P299S; and(c) deletion of residues 300-327 of SEQ ID NO: 6; wherein the variant of SEQ ID NO: 6 further comprises a non-arginine or lysine substitution at position N117 of SEQ ID NO: 6, which optionally is N117E, N117M, or N117P.Attorney Docket No.: 063586-545001 WO66. The fusion protein of claim 65, wherein the variant of SEQ ID NO: 6 further comprises:(i) arginine or lysine substitution(s) at position W7, K29, N100, and / or Hl 02, of SEQ ID NO: 6, optionally W7K, K29R, N100K, and / or H102R;(ii) non-arginine / lysine substitution(s) at position F25, R43, L83, M101, F103, H145, D146, V153, 1199, V201, L249, and / or T286 of SEQ ID NO: 6, optionally F25Y, R43E, L83I, M101A, F103I, , H145S or H145E, D146A or D146M, V153T, I199F, V201N, L249A, and / or T286D; or(iii)a combination of (i) and (ii).

67. The fusion protein of any one of claims 64-66, wherein the non-arginine / lysine substitutions are at: (a) positions M101 and F103 of SEQ ID NO: 6, optionally M101A and / or F103I; and (b) positions H145 and D146 of SEQ ID NO: 6, optionally H145S and D146A or H145E and D146M; optionally: wherein (a) is in combination with arginine / lysine substitution at position N100 and H102 of SEQ ID NO: 6, optionally N100K and H102R; and wherein (b) is in combination with arginine / lysine substitution at position N117 of SEQ ID NO: 6, optionally N117K.

68. The fusion protein of claim 63 or claim 65, wherein the variant of SEQ ID NO: 6 comprises the amino acid sequence of any one of SEQ ID NO: 26, 303, 329, 333, 444-466, and 481-491.

69. The fusion protein of claim 1, wherein the nuclease polypeptide comprises any one of the amino acid sequences listed in Sequence Table 1; and / or wherein the RT polypeptide comprises any one of the amino acid sequences listed in Sequence Table 2.

70. The fusion protein of claim 1, wherein the fusion protein comprises, from N- terminus to C-terminus:(i) the nuclease polypeptide comprising SEQ ID NO: 9 and the RT polypeptide comprising SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 26, or SEQ ID NO: 27;(ii) the nuclease polypeptide comprising SEQ ID NO: 12 and the RTAttorney Docket No.: 063586-545001 WO polypeptide comprising SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 26, or SEQ ID NO: 27; or(iii) the nuclease polypeptide comprising SEQ ID NO: 15 and the RT polypeptide comprising SEQ ID NO: 20, SEQ ID NO: 24, or SEQ ID NO: 28.

71. The fusion protein of claim 1, wherein the fusion protein comprises, from N- terminus to C-terminus:(i) the RT polypeptide comprising SEQ ID NO: 17, SEQ ID NO: 21, or SEQ ID NO: 25 and the nuclease polypeptide comprising SEQ ID NO: 9;(ii) the RT polypeptide comprising SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 29 and the nuclease polypeptide comprising SEQ ID NO: 10;(iii) the RT polypeptide comprising SEQ ID NO: 17, SEQ ID NO: 21, or SEQ ID NO: 25 and the nuclease polypeptide comprising SEQ ID NO: 12; or(iv) the RT polypeptide comprising SEQ ID NO: 20, SEQ ID NO: 24, or SEQ ID NO: 28 and the nuclease polypeptide comprising SEQ ID NO: 15.

72. The fusion protein of claim 1, comprising the following pairs of nuclease polypeptide and RT polypeptide:(1) the variant of SEQ ID NO: 1 set forth in any one of claims 10-12 and the variant of SEQ ID NO: 6 set forth in any one of claims 61-65;(2) the variant of SEQ ID NO: 1 set forth in any one of claims 10-12 and the variant of SEQ ID NO: 4 set forth in claim 41 or claim 42;(3) the variant of SEQ ID NO: 1 set forth in any one of claims 10-12 and the variant of SEQ ID NO: 5 set forth in claim 51;(4) the variant of SEQ ID NO: 2 set forth in claim 19 and the variant of SEQ ID NO: 6 set forth in any one of claims 61-65; or(5) the variant of SEQ ID NO: 3 set forth in claim 30 or claim 31 and the variant of SEQ ID NO: 6 set forth in any one of claims 61-65.

73. The fusion protein of claim 1, wherein the nuclease polypeptide and the RT polypeptide therein comprises the following pair of amino acid sequences, respectively:(1) SEQ ID NO: 90 and SEQ ID NO: 303;(2) SEQ ID NO: 134 and SEQ ID NO: 329;(3) SEQ ID NO: 134 and SEQ ID NO: 333;Attorney Docket No.: 063586-545001 WO(4) SEQ ID NO: 148 and SEQ ID NO: 329;(5) SEQ ID NO: 153 and SEQ ID NO: 329;(6) SEQ ID NO: 163 and SEQ ID NO: 329;(7) SEQ ID NO: 90 and SEQ ID NO: 290;(8) SEQ ID NO: 90 and SEQ ID NO: 272;(9) SEQ ID NO: 12 and SEQ ID NO: 26;(10) SEQ ID NO: 166 and SEQ ID NO: 303;(11) SEQ ID NO: 430 and SEQ ID NO: 329;(12) SEQ ID NO: 431 and SEQ ID NO: 444, and(13) SEQ ID NO: 431 and SEQ ID NO: 329.

74. The fusion protein of claim 1, wherein the nuclease polypeptide therein comprises any one of SEQ ID NOs: 134, 148, 143, 163, 428-443 and 469-480 and the RT polypeptide therein comprises any one of SEQ ID NOs: 329, 333, 444-466, and 481-491.

75. The fusion protein of any one of claims 1-74, wherein the fusion polypeptide further comprises one or more nuclear localization signals (NLSs); and / or one or more peptide linkers.

76. The fusion protein of any one of claims 1-75, which comprises, from N- terminus to C-terminus,(a) a first NLS, the nuclease polypeptide, a peptide linker, the RT polypeptide, and a second NLS; or(b) first NLS, the RT polypeptide, a peptide linker, the nuclease polypeptide, and a second NLS.

77. The fusion protein of claim 1, which comprises any of the amino acid sequences listed in Sequence Table 3.

78. A nucleic acid, comprising a nucleotide sequence encoding a fusion protein set forth in any one of claims 1-77.

79. The nucleic acid of claim 78, which is a vector, optionally an expression vector.Attorney Docket No.: 063586-545001 WO80. An adeno-associated viral (AAV) vector, comprising a transgene, which comprises a first nucleotide sequence encoding a fusion protein set forth in any one of claims 1-76, the encoding nucleotide sequence being in operable linkage to a promoter; wherein the transgene is flanked by a 5’ - inverted terminal repeat (ITR) sequence and a 3’-ITR sequence.

81. The AAV vector of claim 80, wherein the transgene further comprises a second nucleotide sequence encoding an editing template RNA.

82. An adeno-associated viral (AAV) particle, comprising an AAV genome, which comprises a transgene flanked by a 5’ - inverted terminal repeat (ITR) sequence and a 3 ’-ITR sequence; wherein the transgene comprises a first nucleotide sequence encoding a fusion protein set forth in any one of claims 1-77, the encoding nucleotide sequence being in operable linkage to a promoter; and optionally a second nucleotide sequence encoding an editing template RNA.

83. A gene editing system comprising:(a) a nuclease polypeptide or a nucleic acid encoding such, the nuclease polypeptide comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; wherein each of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 comprises an HNH nuclease domain and an RuvC nuclease domain;(b) a reverse transcriptase (RT) polypeptide or a nucleic acid encoding such, wherein the RT polypeptide comprises a polymerase domain, which 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: 70); and wherein the RT polypeptide has a length of about 250- 350 amino acids;(c) a guide RNA (gRNA) or a nucleic acid encoding such; and(d) a reverse transcription donor RNA (RT donor RNA) or a nucleic acid encoding such; wherein the gRNA comprises (i) a scaffold sequence recognizable by the nuclease polypeptide and (ii) a spacer sequence specific to a target sequence within a genomic site of interest, wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) specific to the nuclease polypeptide; and wherein the RT donor RNA comprises a primer binding site (PBS) and aAttorney Docket No.: 063586-545001 WO template sequence.

84. The gene editing system of claim 83, which comprises a fusion protein comprising the nuclease polypeptide of (a) and the RT polypeptide of (b), or a nucleic acid encoding the fusion protein.

85. The gene editing system of claim 83, wherein the fusion protein is set forth in any one of claims 1-76.

86. The gene editing system of any one of claims 83-85, which comprises a single editing template RNA comprising the gRNA of (c) and the RT donor RNA of (d), or a nucleic acid encoding the single editing template RNA.

87. The gene editing system of claim 86, wherein the single editing template RNA further comprises a linker and / or a 3’ extension.

88. The gene editing system of claim 87, wherein the single editing template RNA comprises, from 5’ to 3’, the spacer sequence, the scaffold sequence, the template sequence, the PBS, the linker, and the 3’ extension.

89. The gene editing system of any one of claims 83-88, which comprises an AAV vector set forth in claim 78 or claim 79, or an AAV particle set forth in claim 80.

90. The gene editing system of any one of claims 83-89, wherein the nuclease polypeptide is set forth in any one of claims 2-22; wherein the PAM is 5’-NGG-3’, in which N represents any nucleotide, and wherein the scaffold sequence comprises a nucleotide sequence at least 70% identical to SEQ ID NO: 63 or a fragment thereof; optionally wherein the scaffold sequence comprises the nucleotide sequence of any one of SEQ ID NOs: 63-66.

91. The gene editing system of any one of claims 83-89, wherein the nuclease polypeptide is set forth in any one of claims 2-22; wherein the PAM is 5’-NGG-3’, in which N represents any nucleotide, and wherein the scaffold sequence is a variant of SEQ ID NO: 66, comprising one or more deletions, one or more nucleotide substitutions, or a combination thereof, as compared with SEQ ID NO: 66; and wherein the scaffold sequence has a length ofAttorney Docket No.: 063586-545001 WO about 100-120 nucleotides.

92. The gene editing system of claim 91, wherein, relative to SEQ ID NO: 66, the scaffold sequence comprises:(i) a 3’ end deletion, optionally within nucleotides 126-179 of SEQ ID NO: 66;(ii) a deletion within nucleotides 8-31 of SEQ ID NO: 66; and(iii) one or more nucleotide substitutions.

93. The gene editing system of claim 92, wherein the scaffold sequence is at least 90% identical to SEQ ID NO: 65 and comprises one or more mutations relative to SEQ ID NO: 65.

94. The gene editing system of claim 93, wherein the scaffold sequence is Nuclease K Scaffold Variant 2, Nuclease K Scaffold Variant 7, Nuclease K Scaffold Variant 16, Nuclease K Scaffold Variant 28, Nuclease K Scaffold Variant 32, Nuclease K Scaffold Variant 33, Nuclease K Scaffold Variant 34, Nuclease K Scaffold Variant 36, Nuclease K Scaffold Variant 41, Nuclease K Scaffold Variant 42, or Nuclease K Scaffold Variant 43.

95. The gene editing system of any one of claims 83-89, wherein the nuclease polypeptide is set forth in any one of claims 23-31; wherein the PAM is 5’-RRT-3’, or 5’- NRT-3’, in which R represents A, and N represents any nucleotide; and wherein the scaffold sequence comprises a nucleotide sequence at least 70% identical to SEQ ID NO: 67; optionally wherein the scaffold sequence comprises the nucleotide sequence of any one of SEQ ID NOs: 67-69 and 78.

96. A pharmaceutical composition comprising the gene editing system of any one of claims 83-95.

97. A kit comprising the elements of the gene editing system set forth in any one of claims 83-95.

98. A gene editing method, comprising contacting the gene editing system of any one of claims 83-95 with host cells to allow for editing of a gene targeted by the gene editing system.Attorney Docket No.: 063586-545001 WO99. The gene editing method of claim 98, wherein the host cells are cultured in vitro.

100. The gene editing method of claim 99, wherein the host cells are located in a subject who needs gene editing of the target gene.

101. The gene editing method of any one of claims 98-100, wherein the gene editing system comprises the AAV particle set forth in claim 80.

102. A guide RNA, comprising a spacer sequence and a scaffold sequence, wherein the scaffold sequence is a variant of SEQ ID NO: 66, comprising one or more deletions, one or more nucleotide substitutions, or a combination thereof, as compared with SEQ ID NO: 66, provided that the scaffold sequence is not SEQ ID NO: 65; wherein the scaffold sequence has a length of about 100-120 nucleotides; and wherein the scaffold sequence is recognizable by Nuclease K and / or Nuclease A.

103. The guide RNA of claim 102, wherein, relative to SEQ ID NO: 66, the scaffold sequence comprises:(i) a 3’ end deletion, optionally within nucleotides 126-179 of SEQ ID NO: 66;(ii) a deletion within nucleotides 8-31 of SEQ ID NO: 66; and(iii) one or more nucleotide substitutions.

104. The guide RNA of claim 103, wherein the scaffold sequence is at least 90% identical to SEQ ID NO: 65 and comprises one or more mutations relative to SEQ ID NO: 65.

105. The guide RNA of claim 104, wherein the scaffold sequence is Nuclease K Scaffold Variant 2, Nuclease K Scaffold Variant 7, Nuclease K Scaffold Variant 16, Nuclease K Scaffold Variant 28, Nuclease K Scaffold Variant 32, Nuclease K Scaffold Variant 33, Nuclease K Scaffold Variant 34, Nuclease K Scaffold Variant 36, Nuclease K Scaffold Variant 41, Nuclease K Scaffold Variant 42, or Nuclease K Scaffold Variant 43.

106. A nuclease, which is a variant of SEQ ID NO: 1, wherein the nuclease is set forth in any one of claims 2-15, optionally set forth in any one of claims 11-15.Attorney Docket No.: 063586-545001 WO107. A reverse transcriptase (RT), which is a variant of SEQ ID NO: 6, wherein the RT is set forth in any one of 52-68, optionally set forth in any one of claims 63-68.

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