Nucleic acid-guided nickase fusion proteins

Abstract

The present disclosure provides compositions and methods useful for editing target nucleic acid molecules. The present disclosure provides MAD2019-H848A variant polypeptides, reverse transcriptases, and fusion proteins, and methods of using the MAD2019-H848A variant polypeptides, reverse transcriptases, and fusion proteins to edit nucleic acid molecules both in vivo and in vitro.

Description

[Technical field]

[0001] The present disclosure provides engineered nucleic acid-guided proteins (e.g., nickases) and fusion proteins that can be used to create targeted edits to nucleic acid molecules in living cells. [Background technology]

[0002] The ability to make precisely targeted changes to the genome of living cells has been a long-standing goal in biomedical research and development. In recent years, a variety of nucleases have been identified that allow for the manipulation of gene sequence, and therefore gene function.

[0003] The identified nucleases include nucleic acid-guided nucleases and nickases derived from nucleic acid-guided nucleases. Nickases generate single-strand breaks rather than double-strand breaks. The ability to cleave only one strand of DNA may increase the versatility of nucleic acid-guided nucleases for certain editing tasks.

[0004] One editing process that uses nickase is prime editing. Prime editing combines nickase with reverse transcriptase to create a fusion protein. The fusion protein forms a nucleoprotein complex with the prime editing guide RNA that defines the target site to be edited and codes for the desired editing. Prime editing can create all 12 types of insertion, deletion, and point mutation. Summary of the Invention [Problem to be solved by the invention]

[0005] There is a need in the art for novel nucleases and nickases derived from novel nickases that have altered activity in cells from different organisms, have different cleavage motifs, and / or have modified enzymatic fidelity. The novel polypeptides provided herein fulfill this unmet need. [Means for solving the problem]

[0006] In one aspect, the disclosure provides a MAD2019-H848A polypeptide. In one aspect, the disclosure provides a MAD2019-H848A variant polypeptide comprising an amino acid sequence at least 90% identical or similar to SEQ ID NO:1, wherein the MAD2019-H848A variant polypeptide comprises an alanine at position 848 according to SEQ ID NO:1.

[0007] In one aspect, the disclosure provides a fusion protein comprising a MAD2019-H848A variant polypeptide. In one aspect, the disclosure provides a fusion protein comprising a Tf1 reverse transcriptase that comprises an amino acid sequence at least 90% identical or similar to SEQ ID NO:13.

[0008] In one aspect, the disclosure provides a fusion protein comprising a Tf1 reverse transcriptase comprising the amino acid sequence of SEQ ID NO:14. In one aspect, the present disclosure provides a nucleoprotein complex comprising a MAD2019-H848A variant polypeptide.

[0009] In one aspect, the present disclosure provides a nucleoprotein complex comprising a fusion protein provided herein. In one aspect, the present disclosure provides a eukaryotic cell comprising a MAD2019-H848A variant polypeptide.In one aspect, the present disclosure provides a eukaryotic cell comprising a fusion protein provided herein.

[0010] In one aspect, the disclosure provides a method of providing a MAD2019-H848A variant polypeptide to a cell, the method comprising: (a) obtaining a cell; and (b) providing to the cell a MAD2019-H848 variant polypeptide or a nucleic acid molecule encoding a MAD2019-H848A variant polypeptide.

[0011] In one aspect, the disclosure provides a method of providing a fusion protein to a cell, the method comprising: (a) obtaining a cell; and (b) providing to the cell a fusion protein provided herein, or a nucleic acid molecule encoding the fusion protein.

[0012] In one aspect, the disclosure provides a method of editing at least one eukaryotic cell comprising: (a) introducing into the at least one eukaryotic cell a MAD2019-H848A variant polypeptide or a nucleic acid molecule encoding a MAD2019-H848A variant polypeptide; and (ii) into the at least one eukaryotic cell a guide RNA, the guide RNA or a nucleic acid molecule encoding the guide RNA comprising a nucleic acid sequence that is complementary to a target nucleic acid molecule in the genome of the eukaryotic cell; wherein the MAD2019-H848A variant polypeptide and the guide RNA form a nucleoprotein complex in the at least one eukaryotic cell, wherein the nucleoprotein complex cleaves one strand of the target nucleic acid molecule and at least one edit is made in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule; and (b) identifying the at least one eukaryotic cell comprising the at least one edit.

[0013] In one aspect, the disclosure provides a method of editing at least one eukaryotic cell, the method comprising: (a) introducing into the at least one eukaryotic cell a fusion protein or a nucleic acid molecule encoding the fusion protein as provided herein; and (ii) into the at least one eukaryotic cell a guide RNA, the guide RNA or the nucleic acid molecule encoding the guide RNA comprising a nucleic acid sequence complementary to a target nucleic acid molecule in the genome of the eukaryotic cell; the fusion protein and the guide RNA form a nucleoprotein complex in the at least one eukaryotic cell, the nucleoprotein complex cleaving one strand of the target nucleic acid molecule and at least one edit is made in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule; and (b) identifying the at least one eukaryotic cell comprising the at least one edit.

[0014] In one embodiment, the present disclosure provides a guide RNA (gRNA) comprising a scaffold region having a nucleic acid sequence at least 80% identical to SEQ ID NO: 24. The present disclosure also provides a nucleoprotein complex comprising a gRNA comprising a scaffold region having a nucleic acid sequence at least 85% identical to SEQ ID NO: 24.

[0015] A brief description of the sequence Table 1 provides a listing of the nucleic acid and amino acid sequences provided by the present disclosure. Table 1. Nucleic acid and amino acid sequences.

[0016] [Table 1-1]

[0017] [Table 1-2]

[0018] [Table 1-3]

[0019] [Table 1-4]

[0020] [Table 1-5]

[0021] [Table 1-6]

[0022] [Table 1-7]

[0023] [Table 1-8]

[0024] [Table 1-9]

[0025] [Table 1-10] [Brief description of the drawings]

[0026] [Figure 1] Without being limited by any scientific theory, FIG. 1 shows the mechanism for CREATE fusion editing. [Diagram 2] FIG. 2 shows an example of a workflow for screening nickases for cleavage activity and CREATE fusion activity. [Figure 3A] Figure 3A shows the results of editing with the nickase fusion enzyme MAD2019-H848A::reverse transcriptase in HEK293T cells. [Figure 3B] FIG. 3B shows the results of editing with the nickase fusion enzyme MAD2019-H848A::reverse transcriptase in induced pluripotent stem cells. [Figure 4] FIG. 4 shows the results of editing green fluorescent protein (GFP) into blue fluorescent protein (BFP) using MAD2019-H848A (SEQ ID NO: 1) fused to reverse transcriptase Tf1 (SEQ ID NO: 12) or MAD2019-H848A fused to reverse transcriptase Tf1-D364N (SEQ ID NO: 13). [Diagram 5] FIG. 5 shows the results of editing GFP into BFP using CFE19 (SEQ ID NO: 20) and CFE19 variants (for example, SEQ ID NOs: 21 to 23). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Unless otherwise defined, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Where a term is provided in the singular, the inventors also contemplate aspects of the present disclosure described by the plural of that term. In the event of discrepancies in terms and definitions used in references incorporated by reference, the terms used in this application shall have the definitions set forth herein. Other technical terms used have their ordinary meaning in the art in which they are used, as exemplified by various technical field dictionaries, such as "The American Heritage® Science Dictionary" (American Heritage Dictionaries editors, 2011, Houghton Mifflin Harcourt, Boston and New York), "McGraw-Hill Dictionary of Scientific and Technical Terms" (6th ed., 2002, McGraw-Hill, New York), or "Oxford Dictionary of Biology" (6th ed., 2008, Oxford University Press, Oxford and New York).

[0028] The implementation of the techniques described herein may use conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant technology), cell biology, biochemistry and sequencing technology, unless otherwise indicated, which are within the skill of the art. Such conventional techniques include polymer array synthesis and polynucleotide hybridization and ligation. Specific descriptions of suitable techniques can be obtained by reference to the examples herein. However, other equivalent conventional procedures can of course be used. Such routine techniques and explanations can be found in standard laboratory manuals, e.g., Green et al., eds., 1999, Genome Analysis: A Laboratory Manual Series, Vols. I-IV; Weiner, Gabriel, and Stephens, eds., 2007, Genetic Variation: A Laboratory Manual; Dieffenbach and Dveksler, eds., 2003, PCR Primers: A Laboratory Manual; Mount, 2004, Bioinformatics: Sequence and Genome Analysis; Sambrook and Russell, 2006, Condensed Protocols from Molecular Cloning: A Laboratory Manual. and Sambrook and Russell, 2002, Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press);Stryer, L., 1995, Biochemistry, 4th ed., WH Freeman, New York; Gait, Oligonucleotide Synthesis: A Practical Approach, 1984, IRL Press, London; Nelson and Cox, 2000, Lehninger, Principles of Biochemistry, 3rd ed., WH Freeman Pub., New York; Viral Vectors, Kaplift and Loewy, eds., Academic Press, 1995; all of which are incorporated herein by reference in their entireties for all purposes. For mammalian / stem cell culture and methods, see, e.g., Basic Cell Culture Protocols, 4th Edition (Helgason and Miller, eds., Humana Press, 2005); Culture of Animal Cells, 7th Edition (Freshney, eds., Humana Press, 2016); Microfluidic Cell Culture, 2nd Edition (Borenstein, Vandon, Tao, and Charest, eds., Elsevier Press, 2018); Human Cell Culture (Hughes, ed., Humana Press, 2011); 3D Cell Culture (Koledova, ed., Humana Press, 2013); Press), 2017);"Cell and Tissue Culture: Laboratory Procedures in Biotechnology" (Doyle and Griffiths, eds., John Wiley & Sons, 1998); "Essential Stem Cell Methods" (Lanza and Klimanskaya, eds., Academic Press, 2011); "Stem Cell Therapies: Opportunities for Ensuring the Quality and Safety of Clinical Offerings: Summary of a Joint Workshop" (Board on Health Sciences Policy, National Academies Press, 2014); "Essentials of Stem Cell Biology" (Cell and Tissue Culture: Laboratory Procedures in Biotechnology) ...Cell and Tissue Culture: Laboratory Procedures in Biotechnology) (Doyle and Griffiths, eds., John Wiley & Sons, 1998); "Essential Stem Cell Methods" (Cell and Tissue Culture: Laboratory Procedures in Biotechnology) (Doyle and Griffiths, eds., John Wiley & Sons, 1998); "Essential Stem Cell Methods" (Cell and Tissue Culture: Laboratory Procedures in Biotechnology) (Cell and Tissue Culture: Laboratory Procedures in Biotechnology) (Cell and Tissue Culture: Laboratory Procedures in Biotechnology) (Cell and Tissue Culture: Laboratory See, e.g., Genome Editing and Engineering from TALENs and CRISPRs to Molecular Surgery, Appasani and Church, 2018;and "CRISPR: Methods and Protocols," Lindgren and Charpentier, 2015.;

[0029] Any references cited herein, including, for example, all patents, published patent applications, and non-patent publications, are hereby incorporated by reference in their entirety. When a group of options is presented, each and every combination of the members of the group of options is specifically contemplated. For example, if an item is selected from the group consisting of A, B, C, and D, the inventors specifically contemplate each option individually (e.g., A alone, B alone, etc.) as well as combinations, e.g., A, B, and D; A and C; B and C, etc.

[0030] The term "and / or," when used in a list of two or more items, means any one of the listed items by itself or in combination with any one or more of the other listed items. For example, the phrase "A and / or B" is intended to mean either or both of A and B, e.g., A alone, B alone, or a combination of A and B. The phrase "A, B, and / or C" is intended to mean A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0031] When numerical ranges are provided herein, it is understood that the range includes the endpoints of the range and any number between the defined endpoints of the range. For example, "1 to 10" includes any number between 1 and 10, as well as the number 1 and the number 10.

[0032] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include a plurality of compounds, including mixtures thereof.

[0033] The nucleic acid-guided nickases provided herein are used to perform nucleic acid nickase fusion-directed genome editing to allow for the introduction of desired edits into living eukaryotic cells. The nucleic acid-guided nickases provided herein are also used to perform nucleic acid nickase fusion-directed genome editing to allow for the introduction of desired edits into target nucleic acid molecules in an in vitro environment.

[0034] As used herein, "nickase" refers to a nuclease that cleaves one strand of a double-stranded DNA molecule (e.g., the nickase "nicks" the DNA molecule). The nickase does not cleave both strands of the double-stranded DNA molecule. Examples of nickases or nucleic acid guided nucleases are described in U.S. Patent Nos. 9,982,279; 10,337,028; 10,435,714; 10,011,849; 10,626,416; 10,604,746; 10,665,114; 10,640,754; 10,689,669; 10,876,102; 10,883,077; 10,704,033; 10,745 ,678; 10,724,021; 10,767,169; 10,870,761; 10,883,077; 11,053,485; 11,085,030; 11,200,089; 11,268,078; 11,293,115; 11,306,298; and 11,332,742; as well as U.S. Patent Application Publication No. 2021 / 0214671.

[0035] Nickases can be derived from or engineered into nucleases that cleave both strands of a double-stranded DNA molecule. In some examples, the nickase is derived from a CRISPR-Cas enzyme. A non-limiting example of an engineered nickase is MAD2019 and variants including MAD2019-H848A (SEQ ID NO: 1) and variants provided in Table 2. In one embodiment, the nickase can be guided to a specific site within a target nucleic acid molecule by a nucleic acid molecule (e.g., a guide). A nickase that is guided by a nucleic acid molecule is referred to as a "nucleic acid-guided nickase."

[0036] The nucleic acid-guided nickases provided herein can be combined with reverse transcriptases to generate fusion enzymes (e.g., fusion proteins) that can both sequence-specifically bind and nick a target nucleic acid molecule and utilize a repair template (e.g., homology arms) to incorporate nucleotides into the target nucleic acid sequence at the site of the nick. Such enzymes can be referred to herein as "nucleic acid-guided nickase fusion enzymes," "CREATE fusion enzymes," or "CF enzymes." The process of editing a target nucleic acid molecule using such enzymes is referred to as "CREATE fusion editing."

[0037] FIG. 1 provides a simplified diagram of the process of CREATE fusion editing, including the steps of editing, flap balancing, flap removal and repair, and DNA replication and cell division. In addition to nucleic acid-guided nickases, various guide RNA scaffold sequences and enhancers of guide RNA (gRNA) have been identified for use with the nickases and fusion proteins provided herein to improve editing efficiency (see, e.g., U.S. Patent No. 11,268,078 and U.S. Patent Application No. 17 / 538,066, filed November 30, 2021).

[0038] Without being limited by any scientific theory, a nucleic acid-guided nickase fusion enzyme complexed with a guide nucleic acid in a cell can nick the genome of the cell within a target nucleic acid molecule. The guide nucleic acid assists the nucleic acid-guided nickase fusion enzyme in recognizing and cleaving one strand of the target nucleic acid molecule. By manipulating the nucleotide sequence of the guide nucleic acid molecule, the nucleic acid-guided nickase fusion enzyme can be programmed to target any DNA sequence for cleavage as long as a suitable protospacer adjacent motif (PAM) is located nearby. As is known in the art, the exact sequence and length requirements for the PAM vary between different nucleic acid-guided nucleases and nucleic acid-guided nickases. However, PAMs typically comprise 2-10 nucleotides in length (most typically 2-6 nucleotides), and they are usually adjacent to or within 10 nucleotides of the desired nick site. A non-limiting example of a PAM site is the sequence 5'-NGG-3'. The PAM can be located 5' or 3' of the desired nick site in the target nucleic acid molecule.

[0039] In one embodiment, the edit comprises an edit to the PAM. In one embodiment, the edit to the PAM results in the removal of the PAM from the target nucleic acid molecule. In one embodiment, the edit to the PAM results in the inactivation of the PAM in the target nucleic acid molecule.

[0040] CREATE fusion editing was used to modify the nickase MAD2019-H848A to identify "MAD2019-H848A variant polypeptides." The MAD2019-H848A variant polypeptides contain at least one amino acid change compared to SEQ ID NO:1, but they also maintain an alanine at position 848 according to the numbering of SEQ ID NO:1. In one embodiment, the MAD2019-H848A variant polypeptides cleave (e.g., nick) one strand of a double-stranded DNA molecule. In one embodiment, the MAD2019-H848A variant polypeptides do not cleave both strands of a double-stranded DNA molecule. While not intended to be limiting, specific examples of MAD2019-H848A variant polypeptides are provided in Table 2.

[0041] In one aspect, the disclosure provides a MAD2019-H848A variant polypeptide comprising an amino acid sequence at least 70% identical or similar to SEQ ID NO: 1, wherein the MAD2019-H848A variant polypeptide comprises an alanine at position 848 according to SEQ ID NO: 1. In one aspect, the disclosure provides a MAD2019-H848A variant polypeptide comprising an amino acid sequence at least 75% identical or similar to SEQ ID NO: 1, wherein the MAD2019-H848A variant polypeptide comprises an alanine at position 848 according to SEQ ID NO: 1. In one aspect, the disclosure provides a MAD2019-H848A variant polypeptide comprising an amino acid sequence at least 80% identical or similar to SEQ ID NO: 1, wherein the MAD2019-H848A variant polypeptide comprises an alanine at position 848 according to SEQ ID NO: 1. In one aspect, the disclosure provides a MAD2019-H848A variant polypeptide comprising an amino acid sequence at least 85% identical or similar to SEQ ID NO: 1, wherein the MAD2019-H848A variant polypeptide comprises an alanine at position 848 according to SEQ ID NO: 1. In one aspect, the disclosure provides a MAD2019-H848A variant polypeptide comprising an amino acid sequence at least 90% identical or similar to SEQ ID NO: 1, wherein the MAD2019-H848A variant polypeptide comprises an alanine at position 848 according to SEQ ID NO: 1. In one aspect, the disclosure provides a MAD2019-H848A variant polypeptide comprising an amino acid sequence at least 92.5% identical or similar to SEQ ID NO: 1, wherein the MAD2019-H848A variant polypeptide comprises an alanine at position 848 according to SEQ ID NO: 1. In one aspect, the disclosure provides a MAD2019-H848A variant polypeptide comprising an amino acid sequence at least 95% identical or similar to SEQ ID NO: 1, wherein the MAD2019-H848A variant polypeptide comprises an alanine at position 848 according to SEQ ID NO: 1. In one aspect, the disclosure provides a MAD2019-H848A variant polypeptide comprising an amino acid sequence at least 96% identical or similar to SEQ ID NO: 1, wherein the MAD2019-H848A variant polypeptide comprises an alanine at position 848 according to SEQ ID NO: 1.In one aspect, the disclosure provides a MAD2019-H848A variant polypeptide comprising an amino acid sequence at least 97% identical or similar to SEQ ID NO: 1, wherein the MAD2019-H848A variant polypeptide comprises an alanine at position 848 according to SEQ ID NO: 1. In one aspect, the disclosure provides a MAD2019-H848A variant polypeptide comprising an amino acid sequence at least 98% identical or similar to SEQ ID NO: 1, wherein the MAD2019-H848A variant polypeptide comprises an alanine at position 848 according to SEQ ID NO: 1. In one aspect, the disclosure provides a MAD2019-H848A variant polypeptide comprising an amino acid sequence at least 99% identical or similar to SEQ ID NO: 1, wherein the MAD2019-H848A variant polypeptide comprises an alanine at position 848 according to SEQ ID NO: 1. In one aspect, the disclosure provides a MAD2019-H848A variant polypeptide comprising an amino acid sequence at least 99.5% identical or similar to SEQ ID NO: 1, comprising an alanine at position 848 according to SEQ ID NO: 1. In one aspect, the disclosure provides a MAD2019-H848A variant polypeptide comprising an amino acid sequence 100% similar to SEQ ID NO: 1, comprising an alanine at position 848 according to SEQ ID NO: 1. In one aspect, the disclosure provides a MAD2019-H848A variant polypeptide comprising the amino acid sequence of SEQ ID NO: 1.

[0042] The term "percent identity" or "percent identical" when used herein with respect to two or more nucleotide or amino acid sequences is calculated by (i) comparing two optimally aligned sequences (nucleotide or amino acid) over a window of comparison (one or more regions "alignable"); (ii) determining the number of positions where identical nucleic acid bases (for nucleotide sequences) or amino acid residues (for proteins and polypeptides) occur in both sequences to obtain the number of matched positions; (iii) dividing the number of matched positions by the total number of positions in the window of comparison; and then (iv) multiplying this quotient by 100% to obtain the percent identity. When "percent identity" is calculated with respect to a reference sequence without specifying a specific comparison window, the percent identity is determined by dividing the number of matched positions over the region of alignment by the total length of the reference sequence. Thus, for the purposes of this application, when two sequences (query and subject) are optimally aligned (allowing for gaps in their alignment), the "percent identity" with respect to the query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions in the query sequence over its length (or comparison window) and then multiplied by 100%.

[0043] When percentage sequence identity is used with respect to amino acids, it is recognized that non-identical residue positions often differ by conservative amino acid substitutions, in which amino acid residues are substituted with other amino acid residues having similar chemical properties (e.g., charge or hydrophobicity), thus not altering the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity can be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity."

[0044] For optimal alignment of sequences to calculate percent sequence identity, various pairwise or multiple sequence alignment algorithms and programs are known in the art, such as ClustalW or Basic Local Alignment Search Tool® (BLAST™), which can be used to compare sequence identity or similarity between two or more nucleotide or amino acid sequences.Although other alignment and comparison methods are known in the art, the alignment and percent identity between two sequences (including the percent identity ranges listed above) may be determined by the ClustalW algorithm, see, e.g., Chenna et al., "Multiple sequence alignment with the Clustal series of programs," Nucleic Acids Research, 31:3497-3500, 2003; Thompson et al., "Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice," Nucleic Acids Research, 1999, 11:111-115, 2003, the entire contents and disclosures of which are incorporated herein by reference. Research, vol. 22, pp. 4673-4680, 1994; Larkin MA et al., "Clustal W and Clustal X version 2.0," Bioinformatics, vol. 23, pp. 2947-48, 2007; and Altschul et al., "Basic local alignment search tool." J. Mol. Biol., vol. 215, pp. 403-410, 1990.

[0045] The term "percent complementarity" or "percent complementary" as used herein in reference to two nucleotide sequences is similar to the concept of percent identity, but refers to the percentage of nucleotides in the query sequence that optimally base-pair or hybridize to the nucleotides of the subject sequence when the query and subject sequences are arranged linearly and optimally base-paired, without secondary folding structures, such as loops, stems or hairpins. Such percent complementarity can be between two DNA strands, two RNA strands, or between a DNA strand and an RNA strand. "Percent complementarity" can be calculated by (i) optimally base-pairing or hybridizing two nucleotide sequences in a linear, fully extended configuration (e.g., no folded or secondary structures) over a window of comparison, (ii) determining the number of base-pairing positions between the two sequences over the window of comparison to obtain the number of complementary positions, (iii) dividing the number of complementary positions by the total number of positions in the window of comparison, and (iv) multiplying this quotient by 100% to obtain the percent complementarity of the two sequences. Optimal base-pairing of two sequences can be determined based on known pairing of nucleotide bases through hydrogen bonds, e.g., GC, AT, and AU. When "percent complementarity" is calculated with respect to a reference sequence without specifying a specific window of comparison, the percent identity is determined by dividing the number of complementary positions between two linear sequences by the total length of the reference sequence. Thus, for the purposes of this application, when two sequences (query and subject) are optimally base-paired (allowing for mismatched or non-base-paired nucleotides), the "percent complementarity" for a query sequence is equal to the number of base-paired positions between the two sequences divided by the total number of positions in the query sequence over its length, then multiplied by 100%.

[0046] The use of the term "polynucleotide" or "nucleic acid molecule" is not intended to limit the present disclosure to polynucleotides that include deoxyribonucleic acid (DNA). For example, ribonucleic acid (RNA) molecules are also contemplated. Those skilled in the art will recognize that polynucleotides and nucleic acid molecules can include ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. Polynucleotides of the present disclosure also encompass all forms of sequence, including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-loop structures, and the like. In one embodiment, the nucleic acid molecules provided herein are DNA molecules. In another embodiment, the nucleic acid molecules provided herein are RNA molecules. In one embodiment, the nucleic acid molecules provided herein are single-stranded. In another embodiment, the nucleic acid molecules provided herein are double-stranded. In one embodiment, the nucleic acid molecules encode a polypeptide.

[0047] In one embodiment, the present disclosure provides a nucleic acid molecule encoding any MAD2019-H848A variant polypeptide provided herein. In one embodiment, the present disclosure provides a nucleic acid molecule encoding any fusion protein provided herein. In one embodiment, the present disclosure provides a nucleic acid molecule encoding any reverse transcriptase provided herein. In one embodiment, the present disclosure provides a nucleic acid molecule encoding any guide provided herein. In one embodiment, the present disclosure provides a nucleic acid molecule encoding any homology arm provided herein.

[0048] In one aspect, any nucleic acid molecule, fusion protein, or MAD2019-H848A variant polypeptide provided herein is provided for in vitro use. In one aspect, any nucleic acid molecule, fusion protein, or MAD2019-H848A variant polypeptide provided herein is provided for in vivo use. In one aspect, any nucleic acid molecule, fusion protein, or MAD2019-H848A variant polypeptide provided herein is provided for ex vivo use.

[0049] In one embodiment, the nucleic acid molecule comprises a promoter. In one embodiment, the promoter is operably linked to a nucleic acid molecule encoding a MAD2019-H848A variant polypeptide. In one embodiment, the promoter is operably linked to a nucleic acid molecule encoding a reverse transcriptase. In one embodiment, the promoter is operably linked to a nucleic acid molecule encoding a fusion protein. In one embodiment, the promoter is operably linked to a nucleic acid molecule encoding a guide. In one embodiment, the promoter is operably linked to a nucleic acid molecule encoding a homology arm. Any promoter suitable for expression in the cell of interest can be used.

[0050] As commonly understood in the art, the term "promoter" refers to a DNA sequence that contains an RNA polymerase binding site, a transcription initiation site, and / or a TATA box, and that supports or facilitates the transcription and expression of an associated transcribable polynucleotide sequence and / or gene (or transgene). Promoters can be synthetically produced, altered, or derived from known or naturally occurring promoter sequences or other promoter sequences. Promoters can also include chimeric promoters that contain a combination of two or more heterologous sequences. Thus, promoters in the present application can include variants of promoter sequences that are similar, but not identical, in composition to other promoter sequences known or provided herein.

[0051] As used herein, "operably linked" refers to a functional link between two or more elements. For example, an operably linked between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional link that allows expression of the polynucleotide of interest. The operably linked elements can be contiguous or non-contiguous. In one embodiment, a promoter is operably linked to a heterologous nucleic acid molecule.

[0052] In one embodiment, the promoter is an inducible promoter. As used herein, "inducible promoter" refers to a regulated promoter that becomes active in a cell in response to a specific stimulus (e.g., it drives the expression of an operably linked sequence).

[0053] In one embodiment, the promoter is a constitutive promoter. As used herein, "constitutive promoter" refers to a promoter that is always active in vivo. Typically, the activity of a constitutive promoter is limited only by the presence of a suitable concentration of a suitable RNA polymerase.

[0054] In one embodiment, the nucleic acid molecule comprises a transcription terminator. In one embodiment, the transcription terminator is operably linked to a nucleic acid molecule encoding a MAD2019-H848A variant polypeptide. In one embodiment, the transcription terminator is operably linked to a nucleic acid molecule encoding a reverse transcriptase. In one embodiment, the transcription terminator is operably linked to a nucleic acid molecule encoding a fusion protein. In one embodiment, the transcription terminator is operably linked to a nucleic acid molecule encoding a guide. In one embodiment, the transcription terminator is operably linked to a nucleic acid molecule encoding a homology arm. Any transcription terminator suitable for terminating transcription of a nucleic acid molecule in a cell of interest can be used.

[0055] As used herein, the term "polypeptide" refers to a chain of at least two covalently linked amino acids. A polypeptide can be encoded by a polynucleotide provided herein. A protein provided herein can be encoded by a nucleic acid molecule provided herein. A protein can include a polypeptide provided herein. As used herein, "protein" refers to a chain of amino acid residues that can provide structure or enzymatic activity to a cell. In one embodiment, the MAD2019-H848A variant polypeptide is a protein.

[0056] In one embodiment, the MAD2019-H848A variant polypeptide comprises an amino acid substitution of threonine at position 67 to glycine (T67G) compared to SEQ ID NO:1. In one embodiment, the MAD2019-H848A variant polypeptide comprises an amino acid substitution of serine to arginine at position 409 compared to SEQ ID NO:1 (S409R).

[0057] In one embodiment, the MAD2019-H848A variant polypeptide comprises an amino acid substitution of leucine to lysine at position 500 (L500K) compared to SEQ ID NO:1. In one embodiment, the MAD2019-H848A variant polypeptide comprises an amino acid substitution of leucine to arginine at position 500 (L500R) compared to SEQ ID NO:1.

[0058] In one embodiment, the MAD2019-H848A variant polypeptide comprises an amino acid substitution of glycine to phenylalanine at position 578 (G578F) compared to SEQ ID NO:1.

[0059] In one embodiment, the MAD2019-H848A variant polypeptide comprises an amino acid substitution of leucine to glutamine at position 624 (L624Q) compared to SEQ ID NO:1. In one embodiment, the MAD2019-H848A variant polypeptide comprises an amino acid substitution of asparagine at position 669 to serine (N669S) compared to SEQ ID NO:1.

[0060] In one embodiment, the MAD2019-H848A variant polypeptide comprises an amino acid substitution of aspartic acid at position 700 to alanine (D700A) compared to SEQ ID NO:1.

[0061] In one embodiment, the MAD2019-H848A variant polypeptide comprises an amino acid substitution of aspartic acid at position 701 to proline (D701P) compared to SEQ ID NO:1.

[0062] In one embodiment, the MAD2019-H848A variant polypeptide comprises an amino acid substitution of aspartic acid to asparagine at position 701 compared to SEQ ID NO:1 (D701N).

[0063] In one embodiment, the MAD2019-H848A variant polypeptide comprises an amino acid substitution of aspartic acid at position 701 to threonine (D701T) compared to SEQ ID NO:1.

[0064] In one embodiment, the MAD2019-H848A variant polypeptide comprises an amino acid substitution of lysine at position 720 to serine (K720S) compared to SEQ ID NO:1. In one embodiment, the MAD2019-H848A variant polypeptide comprises an amino acid substitution of leucine to arginine at position 1110 (L1110R) compared to SEQ ID NO:1.

[0065] In one embodiment, the MAD2019-H848A variant polypeptide comprises an amino acid substitution of isoleucine to arginine at position 1142 (I1142R) compared to SEQ ID NO:1.

[0066] In one embodiment, the MAD2019-H848A variant polypeptide comprises an amino acid substitution of isoleucine to lysine at position 1142 (I1142K) compared to SEQ ID NO:1.

[0067] In one embodiment, the MAD2019-H848A variant polypeptide comprises an amino acid substitution of valine to threonine at position 1143 (V1143T) compared to SEQ ID NO:1. In one embodiment, the MAD2019-H848A variant polypeptide comprises an amino acid substitution of alanine to histidine at position 1221 (A1221H) compared to SEQ ID NO:1.

[0068] In one embodiment, the MAD2019-H848A variant polypeptide comprises an amino acid substitution of lysine at position 1285 to arginine (K1285R) compared to SEQ ID NO:1. In one embodiment, the MAD2019-H848A variant polypeptide comprises an amino acid substitution of alanine to arginine at position 1321 compared to SEQ ID NO:1 (A1321R).

[0069] In one embodiment, the MAD2019-H848A variant polypeptide comprises an amino acid substitution of alanine to lysine at position 1321 (A1321K) compared to SEQ ID NO:1. In one embodiment, the MAD2019-H848A variant polypeptide comprises an amino acid substitution of serine to glutamine at position 1336 (S1336Q) compared to SEQ ID NO:1.

[0070] In one embodiment, the MAD2019-H848A variant polypeptide comprises an amino acid substitution of alanine to arginine at position 1339 compared to SEQ ID NO:1 (A1339R).

[0071] In one aspect, the MAD2019-H848A variant polypeptide comprises an amino acid substitution selected from the group consisting of T67G, S409R, L500K, L500R, G578F, L624Q, N669S, D700A, D701P, D701N, D701T, K720S, L1110R, D1139N, I1142R, I1142K, V1143T, A1221H, K1285R, A1321R, A1321K, S1136Q, and A1139R compared to SEQ ID NO:1. In one aspect, the MAD2019-H848A variant polypeptide comprises at least two amino acid substitutions selected from the group consisting of T67G, S409R, L500K, L500R, G578F, L624Q, N669S, D700A, D701P, D701N, D701T, K720S, L1110R, D1139N, I1142R, I1142K, V1143T, A1221H, K1285R, A1321R, A1321K, S1136Q, and A1139R compared to SEQ ID NO:1. In one aspect, the MAD2019-H848A variant polypeptide comprises at least three amino acid substitutions selected from the group consisting of T67G, S409R, L500K, L500R, G578F, L624Q, N669S, D700A, D701P, D701N, D701T, K720S, L1110R, D1139N, I1142R, I1142K, V1143T, A1221H, K1285R, A1321R, A1321K, S1136Q, and A1139R compared to SEQ ID NO:1. In one aspect, the MAD2019-H848A variant polypeptide comprises at least four amino acid substitutions selected from the group consisting of T67G, S409R, L500K, L500R, G578F, L624Q, N669S, D700A, D701P, D701N, D701T, K720S, L1110R, D1139N, I1142R, I1142K, V1143T, A1221H, K1285R, A1321R, A1321K, S1136Q, and A1139R compared to SEQ ID NO:1.In one aspect, the MAD2019-H848A variant polypeptide comprises at least five amino acid substitutions selected from the group consisting of T67G, S409R, L500K, L500R, G578F, L624Q, N669S, D700A, D701P, D701N, D701T, K720S, L1110R, D1139N, I1142R, I1142K, V1143T, A1221H, K1285R, A1321R, A1321K, S1136Q, and A1139R compared to SEQ ID NO:1. In one aspect, the MAD2019-H848A variant polypeptide comprises at least six amino acid substitutions selected from the group consisting of T67G, S409R, L500K, L500R, G578F, L624Q, N669S, D700A, D701P, D701N, D701T, K720S, L1110R, D1139N, I1142R, I1142K, V1143T, A1221H, K1285R, A1321R, A1321K, S1136Q, and A1139R compared to SEQ ID NO:1. In one aspect, the MAD2019-H848A variant polypeptide comprises at least seven amino acid substitutions selected from the group consisting of T67G, S409R, L500K, L500R, G578F, L624Q, N669S, D700A, D701P, D701N, D701T, K720S, L1110R, D1139N, I1142R, I1142K, V1143T, A1221H, K1285R, A1321R, A1321K, S1136Q, and A1139R compared to SEQ ID NO:1. In one aspect, the MAD2019-H848A variant polypeptide comprises at least eight amino acid substitutions selected from the group consisting of T67G, S409R, L500K, L500R, G578F, L624Q, N669S, D700A, D701P, D701N, D701T, K720S, L1110R, D1139N, I1142R, I1142K, V1143T, A1221H, K1285R, A1321R, A1321K, S1136Q, and A1139R compared to SEQ ID NO:1.

[0072] In one aspect, the MAD2019-H848A variant polypeptide comprises a L500R amino acid substitution, a D700A amino acid substitution, a D701P amino acid substitution, a K720S amino acid substitution, a I1142K amino acid substitution, and a V1143T amino acid substitution compared to SEQ ID NO:1.

[0073] In one aspect, the MAD2019-H848A variant polypeptide comprises a L500R amino acid substitution, a D700A amino acid substitution, a D701P amino acid substitution, a K720S amino acid substitution, and a V1143T amino acid substitution compared to SEQ ID NO:1.

[0074] In one embodiment, the MAD2019-H848A variant polypeptide comprises a L500K amino acid substitution and a V1143T amino acid substitution compared to SEQ ID NO:1. In one aspect, the MAD2019-H848A variant polypeptide comprises a S409R amino acid substitution, a L500K amino acid substitution, and a V1143T amino acid substitution compared to SEQ ID NO:1.

[0075] In one embodiment, the MAD2019-H848A variant polypeptide comprises a V1143T amino acid substitution and a A1221H amino acid substitution compared to SEQ ID NO:1. In one embodiment, the MAD2019-H848A variant polypeptide comprises a L500K amino acid substitution, a V1143T amino acid substitution, and an A1221H amino acid substitution compared to SEQ ID NO:1.

[0076] In one embodiment, the MAD2019-H848A variant polypeptide comprises a L500K amino acid substitution, a I1142R amino acid substitution, a V1143T amino acid substitution, and a A1221H amino acid substitution compared to SEQ ID NO:1.

[0077] In one embodiment, the MAD2019-H848A variant polypeptide comprises a L500K amino acid substitution, a D1139N amino acid substitution, a V1143T amino acid substitution, and an A1221H amino acid substitution compared to SEQ ID NO:1.

[0078] In one embodiment, the MAD2019-H848A variant polypeptide comprises a L500K amino acid substitution, a V1143T amino acid substitution, an A1221H amino acid substitution, and a K1285R amino acid substitution compared to SEQ ID NO:1.

[0079] In one embodiment, the MAD2019-H848A variant polypeptide comprises improved nicking efficiency compared to SEQ ID NO:1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency within 1% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency within 2.5% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency within 5% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency within 7.5% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency within 10% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency within 12.5% ​​of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency within 15% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency within 20% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency within 25% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency within 30% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency within 35% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency within 40% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency within 45% of the nicking efficiency of SEQ ID NO: 1.In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency within 50% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency within 55% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency within 60% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency within 65% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency within 70% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency within 75% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency within 80% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency within 90% of the nicking efficiency of SEQ ID NO: 1.

[0080] In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency of 1% to 90% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency of 1% to 75% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency of 1% to 60% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency of 1% to 50% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency of 1% to 40% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency of 1% to 30% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency of 1% to 20% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency of 1% to 10% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency of 10% to 70% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency of 10% to 60% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency of 10% to 50% of the nicking efficiency of SEQ ID NO: 1. In one embodiment, the MAD2019-H848A variant polypeptide nicks double stranded DNA molecules with an efficiency of 1% to 25% of the nicking efficiency of SEQ ID NO: 1.

[0081] Nicking efficiency can be measured using any suitable method in the art, see, for example, Wei et al., Scientific Reports, vol. 6, p. 32560, 2016.

[0082] In one embodiment, the MAD2019-H848A variant polypeptide further comprises at least one nuclear localization signal (NLS). In one embodiment, the fusion protein comprises at least one NLS. Nuclear localization signals are known in the art as short (e.g., but not limited to, typically less than 25 amino acids) amino acid sequences that "tag" a protein for import into the nucleus of a cell via nuclear transport. In one embodiment, the MAD2019-H848A variant polypeptide comprises at least two NLSs. In one embodiment, the MAD2019-H848A variant polypeptide comprises at least three NLSs. In one embodiment, the MAD2019-H848A variant polypeptide comprises at least four NLSs. In one embodiment, the MAD2019-H848A variant polypeptide comprises at least five NLSs. In one embodiment, the MAD2019-H848A variant polypeptide comprises at least six NLSs. In one embodiment, the MAD2019-H848A variant polypeptide comprises at least 7 NLSs. In one embodiment, the MAD2019-H848A variant polypeptide comprises at least 8 NLSs. In one embodiment, the MAD2019-H848A variant polypeptide comprises at least 9 NLSs. In one embodiment, the MAD2019-H848A variant polypeptide comprises at least 10 NLSs.

[0083] In one embodiment, the NLS is located before the N-terminus of the MAD2019-H848A variant polypeptide. In one embodiment, the NLS is located after the C-terminus of the MAD2019-H848A variant polypeptide. In one embodiment, the MAD2019-H848A variant polypeptide comprises a first NLS before its N-terminus and a second NLS after its C-terminus.

[0084] In one embodiment, the NLS is located before the N-terminus of the fusion protein. In one embodiment, the NLS is located after the C-terminus of the fusion protein. In one embodiment, the fusion protein comprises a first NLS before its N-terminus and a second NLS after its C-terminus.

[0085] In one embodiment, the NLS comprises 50 or fewer amino acids. In one embodiment, the NLS comprises 40 or fewer amino acids. In one embodiment, the NLS comprises 30 or fewer amino acids. In one embodiment, the NLS comprises 25 or fewer amino acids. In one embodiment, the NLS comprises 20 or fewer amino acids. In one embodiment, the NLS comprises 15 or fewer amino acids. In one embodiment, the NLS comprises 10 or fewer amino acids.

[0086] Any suitable NLS can be used with the MAD2019-H848A variant polypeptides provided herein. In one embodiment, the NLS comprises an amino acid sequence at least 70% identical or similar to SEQ ID NO: 15 or 19. In one embodiment, the NLS comprises an amino acid sequence at least 80% identical or similar to SEQ ID NO: 15 or 19. In one embodiment, the NLS comprises an amino acid sequence at least 85% identical or similar to SEQ ID NO: 15 or 19. In one embodiment, the NLS comprises an amino acid sequence at least 90% identical or similar to SEQ ID NO: 15 or 19. In one embodiment, the NLS comprises an amino acid sequence at least 92.5% identical or similar to SEQ ID NO: 15 or 19. In one embodiment, the NLS comprises an amino acid sequence at least 95% identical or similar to SEQ ID NO: 15 or 19. In one embodiment, the NLS comprises an amino acid sequence at least 97.5% identical or similar to SEQ ID NO: 15 or 19. In one embodiment, the NLS comprises an amino acid sequence at least 99% identical or similar to SEQ ID NO: 15 or 19. In one embodiment, the NLS comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 19.

[0087] In one aspect, the disclosure provides a nucleic acid sequence encoding an NLS. In one aspect, the disclosure provides a nucleic acid sequence encoding any of the MAD2019-H848A variant polypeptides provided herein. In one aspect, the disclosure provides a nucleic acid sequence encoding any one of the amino acid sequences of SEQ ID NOs: 1, 12, 13, and 15-23. In one aspect, the disclosure provides a nucleic acid sequence encoding an amino acid sequence at least 70% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 12, 13, and 15-23. In one aspect, the disclosure provides a nucleic acid sequence encoding an amino acid sequence at least 75% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 12, 13, and 15-23. In one aspect, the disclosure provides a nucleic acid sequence encoding an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 12, 13, and 15-23. In one aspect, the disclosure provides a nucleic acid sequence encoding an amino acid sequence at least 85% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 12, 13, and 15-23. In one embodiment, the present disclosure provides a nucleic acid sequence encoding an amino acid sequence at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 12, 13, and 15 to 23. In one embodiment, the present disclosure provides a nucleic acid sequence encoding an amino acid sequence at least 92.5% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 12, 13, and 15 to 23. In one embodiment, the present disclosure provides a nucleic acid sequence encoding an amino acid sequence at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 12, 13, and 15 to 23. In one embodiment, the present disclosure provides a nucleic acid sequence encoding an amino acid sequence at least 97.5% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 12, 13, and 15 to 23. In one embodiment, the present disclosure provides a nucleic acid sequence encoding an amino acid sequence at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 12, 13, and 15 to 23.In one aspect, the disclosure provides a nucleic acid sequence encoding an amino acid sequence at least 99.5% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 12, 13, and 15 to 23. In one aspect, the disclosure provides a nucleic acid sequence encoding an amino acid sequence at least 100% similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 12, 13, and 15 to 23.

[0088] In one embodiment, the present disclosure provides a nucleoprotein complex comprising any of the MAD2019-H848A variant polypeptides provided herein and a nucleic acid molecule. In one embodiment, the present disclosure provides a nucleoprotein complex comprising any of the fusion proteins provided herein and a nucleic acid molecule. As used herein, "nucleoprotein complex" refers to a protein conjugated with a nucleic acid molecule. When a nucleoprotein complex comprises an RNA molecule, it can be referred to as a ribonucleoprotein complex. When a nucleoprotein complex comprises a DNA molecule, it can be referred to as a deoxyribonucleoprotein complex. In one embodiment, the nucleoprotein complex provided herein is a ribonucleoprotein complex. In one embodiment, the nucleoprotein complex provided herein is a deoxyribonucleoprotein complex. In one embodiment, the nucleic acid molecule component of the nucleoprotein complex is an RNA molecule. In one embodiment, the nucleic acid molecule component of the nucleoprotein complex is a DNA molecule.

[0089] In one embodiment, the nucleic acid molecule provided herein encodes a guide. In one embodiment, the nucleic acid molecule provided herein comprises a guide. As used herein, "guide" refers to a nucleic acid molecule that can guide a protein to which it is complexed to a target nucleic acid molecule. Typically, the guide is complementary to the target nucleic acid molecule, but complete (e.g., 100%) complementarity is not required, and the guide can hybridize to the target nucleic acid molecule. In one embodiment, the guide is a DNA molecule. In one embodiment, the guide is an RNA molecule. In one embodiment, the guide comprises a DNA molecule and an RNA molecule. In one embodiment, the guide is single-stranded. In one embodiment, the guide is double-stranded. In one embodiment, the guide comprises one or more sections that are single-stranded and one or more regions that are double-stranded.

[0090] When the guide is an RNA molecule, it can be referred to as a "guide RNA" or "gRNA". In one embodiment, the nucleoprotein complex or ribonucleoprotein complex comprises a gRNA. In one embodiment, the gRNA can guide the MAD2019-H848A variant polypeptide to a target nucleic acid molecule. In one embodiment, the gRNA guides the MAD2019-H848A variant polypeptide to a target nucleic acid molecule. In one embodiment, the gRNA can guide a fusion protein to a target nucleic acid molecule. In one embodiment, the gRNA guides the fusion protein to a target nucleic acid molecule.

[0091] In one embodiment, the nucleoprotein complex comprises a MAD2019-H848A variant polypeptide and a gRNA. In one embodiment, the nucleoprotein complex comprises a fusion protein and a gRNA. In one embodiment, the nucleoprotein complex comprises a MAD2019-H848A variant polypeptide and a guide. In one embodiment, the nucleoprotein complex comprises a fusion protein and a guide. In one embodiment, the nucleoprotein complex comprises a MAD2019-H848A variant polypeptide, a homology arm, and a gRNA. In one embodiment, the nucleoprotein complex comprises a fusion protein, a homology arm, and a gRNA. In one embodiment, the nucleoprotein complex comprises a MAD2019-H848A variant polypeptide, a homology arm, and a guide. In one embodiment, the nucleoprotein complex comprises a fusion protein, a homology arm, and a guide.

[0092] In one embodiment, the guide comprises at least 5 nucleotides. In one embodiment, the guide comprises at least 10 nucleotides. In one embodiment, the guide comprises at least 15 nucleotides. In one embodiment, the guide comprises at least 20 nucleotides. In one embodiment, the guide comprises at least 25 nucleotides. In one embodiment, the guide comprises at least 30 nucleotides. In one embodiment, the guide comprises at least 35 nucleotides. In one embodiment, the guide comprises at least 40 nucleotides. In one embodiment, the guide comprises at least 45 nucleotides. In one embodiment, the guide comprises at least 50 nucleotides. In one embodiment, the guide comprises at least 60 nucleotides. In one embodiment, the guide comprises at least 70 nucleotides. In one embodiment, the guide comprises at least 80 nucleotides. In one embodiment, the guide comprises at least 90 nucleotides. In one embodiment, the guide comprises at least 100 nucleotides. In one embodiment, the guide comprises at least 125 nucleotides.

[0093] In one embodiment, the guide comprises 5 nucleotides to 150 nucleotides. In one embodiment, the guide comprises 5 nucleotides to 125 nucleotides. In one embodiment, the guide comprises 5 nucleotides to 100 nucleotides. In one embodiment, the guide comprises 5 nucleotides to 75 nucleotides. In one embodiment, the guide comprises 5 nucleotides to 50 nucleotides. In one embodiment, the guide comprises 5 nucleotides to 40 nucleotides. In one embodiment, the guide comprises 5 nucleotides to 30 nucleotides. In one embodiment, the guide comprises 5 nucleotides to 25 nucleotides. In one embodiment, the guide comprises 15 nucleotides to 30 nucleotides. In one embodiment, the guide comprises 15 nucleotides to 25 nucleotides. In one embodiment, the guide comprises 20 nucleotides to 150 nucleotides. In one embodiment, the guide comprises 20 nucleotides to 125 nucleotides. In one embodiment, the guide comprises 20 nucleotides to 100 nucleotides. In one embodiment, the guide comprises 20 nucleotides to 75 nucleotides. In one embodiment, the guide comprises 20 nucleotides to 50 nucleotides. In one embodiment, the guide comprises 40 nucleotides to 100 nucleotides. In one embodiment, the guide comprises between 50 and 150 nucleotides. In one embodiment, the guide comprises between 50 and 100 nucleotides.

[0094] In one embodiment, there are no mismatches between the guide or gRNA and the target nucleic acid molecule when optimally aligned. In one embodiment, there is no more than one mismatch between the guide or gRNA and the target nucleic acid molecule when optimally aligned. In one embodiment, there are no more than two mismatches between the guide or gRNA and the target nucleic acid molecule when optimally aligned. In one embodiment, there are no more than three mismatches between the guide or gRNA and the target nucleic acid molecule when optimally aligned. In one embodiment, there are no more than four mismatches between the guide or gRNA and the target nucleic acid molecule when optimally aligned. In one embodiment, there are no more than five mismatches between the guide or gRNA and the target nucleic acid molecule when optimally aligned. In one embodiment, there are no more than six mismatches between the guide or gRNA and the target nucleic acid molecule when optimally aligned. In one embodiment, there are no more than seven mismatches between the guide or gRNA and the target nucleic acid molecule when optimally aligned. In one embodiment, there are no more than eight mismatches between the guide or gRNA and the target nucleic acid molecule when optimally aligned. In one embodiment, there are 9 or fewer mismatches between the guide or gRNA and the target nucleic acid molecule when optimally aligned. In one embodiment, there are 10 or fewer mismatches between the guide or gRNA and the target nucleic acid molecule when optimally aligned. In one embodiment, there are 15 or fewer mismatches between the guide or gRNA and the target nucleic acid molecule when optimally aligned.

[0095] In one embodiment, the guide forms a nucleoprotein complex with the MAD2019-H848A variant polypeptide in the cell. In one embodiment, the gRNA forms a nucleoprotein complex with the MAD2019-H848A variant polypeptide in the cell. In one embodiment, the guide forms a nucleoprotein complex with the fusion protein in the cell. In one embodiment, the gRNA forms a nucleoprotein complex with the fusion protein in the cell.

[0096] In one embodiment, the guide forms a nucleoprotein complex with the MAD2019-H848A variant polypeptide. In one embodiment, the gRNA forms a nucleoprotein complex with the MAD2019-H848A variant polypeptide. In one embodiment, the guide forms a nucleoprotein complex with the fusion protein. In one embodiment, the gRNA forms a nucleoprotein complex with the fusion protein.

[0097] In one embodiment, the nucleoprotein complex comprises a fusion protein and a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence at least 80% identical to SEQ ID NO:24. In one embodiment, the nucleoprotein complex comprises a fusion protein and a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence at least 85% identical to SEQ ID NO:24. In one embodiment, the nucleoprotein complex comprises a fusion protein and a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence at least 90% identical to SEQ ID NO:24. In one embodiment, the nucleoprotein complex comprises a fusion protein and a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence at least 92.5% identical to SEQ ID NO:24. In one embodiment, the nucleoprotein complex comprises a fusion protein and a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence at least 95% identical to SEQ ID NO:24. In one embodiment, the nucleoprotein complex comprises a fusion protein and a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence at least 97.5% identical to SEQ ID NO:24. In one embodiment, the nucleoprotein complex comprises a fusion protein and a gRNA, wherein the gRNA comprises a scaffold region that comprises a nucleic acid sequence that is 100% identical to SEQ ID NO:24.

[0098] In one embodiment, a nucleoprotein complex comprises a MAD2019-H848A variant polypeptide and a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence at least 80% identical to SEQ ID NO:24. In one embodiment, a nucleoprotein complex comprises a MAD2019-H848A variant polypeptide and a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence at least 85% identical to SEQ ID NO:24. In one embodiment, a nucleoprotein complex comprises a MAD2019-H848A variant polypeptide and a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence at least 90% identical to SEQ ID NO:24. In one embodiment, a nucleoprotein complex comprises a MAD2019-H848A variant polypeptide and a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence at least 92.5% identical to SEQ ID NO:24. In one embodiment, a nucleoprotein complex comprises a MAD2019-H848A variant polypeptide and a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence at least 95% identical to SEQ ID NO: 24. In one embodiment, a nucleoprotein complex comprises a MAD2019-H848A variant polypeptide and a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence at least 97.5% identical to SEQ ID NO: 24. In one embodiment, a nucleoprotein complex comprises a MAD2019-H848A variant polypeptide and a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence 100% identical to SEQ ID NO: 24.

[0099] In one embodiment, the nucleoprotein complex comprises a nickase and a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence at least 80% identical to SEQ ID NO:24. In one embodiment, the nucleoprotein complex comprises a nickase and a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence at least 85% identical to SEQ ID NO:24. In one embodiment, the nucleoprotein complex comprises a nickase and a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence at least 90% identical to SEQ ID NO:24. In one embodiment, the nucleoprotein complex comprises a nickase and a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence at least 92.5% identical to SEQ ID NO:24. In one embodiment, the nucleoprotein complex comprises a nickase and a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence at least 95% identical to SEQ ID NO:24. In one embodiment, the nucleoprotein complex comprises a nickase and a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence at least 97.5% identical to SEQ ID NO:24. In one embodiment, the nucleoprotein complex comprises a nickase and a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence that is 100% identical to SEQ ID NO:24.

[0100] In one embodiment, the nucleoprotein complex comprises a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence at least 80% identical to SEQ ID NO:24. In one embodiment, the nucleoprotein complex comprises a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence at least 85% identical to SEQ ID NO:24. In one embodiment, the nucleoprotein complex comprises a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence at least 90% identical to SEQ ID NO:24. In one embodiment, the nucleoprotein complex comprises a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence at least 92.5% identical to SEQ ID NO:24. In one embodiment, the nucleoprotein complex comprises a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence at least 95% identical to SEQ ID NO:24. In one embodiment, the nucleoprotein complex comprises a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence at least 97.5% identical to SEQ ID NO:24. In one embodiment, the nucleoprotein complex comprises a gRNA, wherein the gRNA comprises a scaffold region comprising a nucleic acid sequence at least 100% identical to SEQ ID NO:24.

[0101] In one embodiment, the gRNA provided herein comprises at least one stem-loop structure. In one embodiment, the gRNA provided herein can bind to both strands of a target nucleic acid molecule. In one embodiment, the gRNA provided herein comprises a reverse transcriptase template. In one embodiment, the gRNA provided herein comprises an edit that is desired to be incorporated into a target nucleic acid molecule. In one embodiment, the gRNA provided herein comprises a primer binding site region. In one embodiment, the gRNA provided herein comprises a spacer region. In one embodiment, the gRNA provided herein comprises a scaffold region.

[0102] As used herein, "spacer region" refers to a subsection of a gRNA that hybridizes to a strand of a target nucleic acid molecule that is not cleaved by a MAD2019-H848A variant polypeptide provided herein. As used herein, "scaffold region" refers to a region of a gRNA that is located between the spacer region and the reverse transcriptase template. In one embodiment, the scaffold region includes at least one stem-loop structure. In one embodiment, the scaffold region includes at least two stem-loop structures. Without being limited by any scientific theory, the scaffold region may interact or complex with a protein (e.g., a MAD2019-H848A variant polypeptide). As used herein, "primer binding site region" refers to a subsection of a gRNA that hybridizes to a strand of a target nucleic acid molecule that is cleaved by a MAD2019-H848A variant polypeptide provided herein. Typically, the MAD2019-H848A variant polypeptide nicks the target nucleic acid molecule downstream of the primer binding site region. The primer binding site region is immediately upstream of the reverse transcriptase template, which itself is upstream of the scaffold region. In one embodiment, the reverse transcriptase reverse transcribes the reverse transcriptase template. In one embodiment, the reverse transcriptase template comprises at least one edit that is desired to be incorporated into the target nucleic acid molecule.

[0103] In one embodiment, the gRNA comprises a scaffold region having a nucleic acid sequence at least 80% identical to SEQ ID NO:24. In one embodiment, the gRNA comprises a scaffold region having a nucleic acid sequence at least 82.5% identical to SEQ ID NO:24. In one embodiment, the gRNA comprises a scaffold region having a nucleic acid sequence at least 85% identical to SEQ ID NO:24. In one embodiment, the gRNA comprises a scaffold region having a nucleic acid sequence at least 87.5% identical to SEQ ID NO:24. In one embodiment, the gRNA comprises a scaffold region having a nucleic acid sequence at least 90% identical to SEQ ID NO:24. In one embodiment, the gRNA comprises a scaffold region having a nucleic acid sequence at least 91% identical to SEQ ID NO:24. In one embodiment, the gRNA comprises a scaffold region having a nucleic acid sequence at least 92% identical to SEQ ID NO:24. In one embodiment, the gRNA comprises a scaffold region having a nucleic acid sequence at least 93% identical to SEQ ID NO:24. In one embodiment, the gRNA comprises a scaffold region having a nucleic acid sequence at least 94% identical to SEQ ID NO:24. In one embodiment, the gRNA comprises a scaffold region having a nucleic acid sequence at least 95% identical to SEQ ID NO:24. In one embodiment, the gRNA comprises a scaffold region having a nucleic acid sequence at least 96% identical to SEQ ID NO:24. In one embodiment, the gRNA comprises a scaffold region having a nucleic acid sequence at least 97% identical to SEQ ID NO:24. In one embodiment, the gRNA comprises a scaffold region having a nucleic acid sequence at least 98% identical to SEQ ID NO:24. In one embodiment, the gRNA comprises a scaffold region having a nucleic acid sequence at least 99% identical to SEQ ID NO:24. In one embodiment, the gRNA comprises a scaffold region having a nucleic acid sequence 100% identical to SEQ ID NO:24.

[0104] In one embodiment, the gRNA comprises at least 20 nucleotides. In one embodiment, the gRNA comprises at least 30 nucleotides. In one embodiment, the gRNA comprises at least 40 nucleotides. In one embodiment, the gRNA comprises at least 50 nucleotides. In one embodiment, the gRNA comprises at least 60 nucleotides. In one embodiment, the gRNA comprises at least 70 nucleotides. In one embodiment, the gRNA comprises at least 80 nucleotides. In one embodiment, the gRNA comprises at least 90 nucleotides. In one embodiment, the gRNA comprises at least 100 nucleotides. In one embodiment, the gRNA comprises at least 110 nucleotides. In one embodiment, the gRNA comprises at least 120 nucleotides. In one embodiment, the gRNA comprises at least 130 nucleotides. In one embodiment, the gRNA comprises at least 140 nucleotides. In one embodiment, the gRNA comprises at least 150 nucleotides. In one embodiment, the gRNA comprises at least 175 nucleotides. In one embodiment, the gRNA comprises at least 200 nucleotides. In one embodiment, the gRNA comprises at least 250 nucleotides.

[0105] In one embodiment, the gRNA comprises 20 to 500 nucleotides. In one embodiment, the gRNA comprises 20 to 400 nucleotides. In one embodiment, the gRNA comprises 20 to 300 nucleotides. In one embodiment, the gRNA comprises 20 to 200 nucleotides. In one embodiment, the gRNA comprises 20 to 150 nucleotides. In one embodiment, the gRNA comprises 20 to 100 nucleotides. In one embodiment, the gRNA comprises 50 to 250 nucleotides. In one embodiment, the gRNA comprises 50 to 200 nucleotides. In one embodiment, the gRNA comprises 50 to 150 nucleotides. In one embodiment, the gRNA comprises 75 to 250 nucleotides. In one embodiment, the gRNA comprises 100 to 250 nucleotides.

[0106] In one embodiment, the gRNA is a primed editing gRNA (pegRNA), which contains a sequence on its 5' end to guide a protein to a target nucleic acid molecule, a primer binding site region, and a reverse transcriptase template sequence on its 3' end that contains the desired edit.

[0107] In one embodiment, the nucleic acid molecule comprises a homology arm. In one embodiment, the nucleic acid molecule encodes a homology arm. As used herein, "homology arm" refers to a nucleic acid molecule that contains an edit desired to be incorporated into a target nucleic acid molecule, but is otherwise identical or complementary to a target nucleic acid molecule sequence. In one embodiment, the homology arm is incorporated into the target nucleic acid molecule by a reverse transcriptase. In one embodiment, the homology arm is incorporated into the target nucleic acid molecule by a fusion protein that includes a reverse transcriptase. In one embodiment, two homology arms are used to incorporate the desired edit into the target nucleic acid molecule. In one embodiment, one homology arm is used to incorporate the desired edit into the target nucleic acid molecule.

[0108] In one embodiment, the homology arm comprises DNA. In one embodiment, the homology arm comprises RNA. In one embodiment, the homology arm is single stranded. In one embodiment, the homology arm is double stranded. In one embodiment, the homology arm comprises at least 10 nucleotides. In one embodiment, the homology arm comprises at least 20 nucleotides. In one embodiment, the homology arm comprises at least 30 nucleotides. In one embodiment, the homology arm comprises at least 40 nucleotides. In one embodiment, the homology arm comprises at least 50 nucleotides. In one embodiment, the homology arm comprises at least 60 nucleotides. In one embodiment, the homology arm comprises at least 70 nucleotides. In one embodiment, the homology arm comprises at least 75 nucleotides. In one embodiment, the homology arm comprises at least 80 nucleotides. In one embodiment, the homology arm comprises at least 90 nucleotides. In one embodiment, the homology arm comprises at least 100 nucleotides. In one embodiment, the homology arm comprises at least 250 nucleotides. In one embodiment, the homology arm comprises at least 500 nucleotides. In one embodiment, the homology arms comprise at least 750 nucleotides. In one embodiment, the homology arms comprise at least 1000 nucleotides. In one embodiment, the homology arms comprise at least 1500 nucleotides.

[0109] In one embodiment, the homology arm comprises 10 nucleotides to 2500 nucleotides. In one embodiment, the homology arm comprises 10 nucleotides to 1000 nucleotides. In one embodiment, the homology arm comprises 10 nucleotides to 500 nucleotides. In one embodiment, the homology arm comprises 10 nucleotides to 400 nucleotides. In one embodiment, the homology arm comprises 10 nucleotides to 300 nucleotides. In one embodiment, the homology arm comprises 10 nucleotides to 250 nucleotides. In one embodiment, the homology arm comprises 10 nucleotides to 125 nucleotides. In one embodiment, the homology arm comprises 10 nucleotides to 100 nucleotides. In one embodiment, the homology arm comprises 10 nucleotides to 75 nucleotides. In one embodiment, the homology arm comprises 10 nucleotides to 50 nucleotides. In one embodiment, the homology arm comprises 50 nucleotides to 500 nucleotides. In one embodiment, the homology arm comprises 50 nucleotides to 400 nucleotides. In one embodiment, the homology arm comprises 50 nucleotides to 300 nucleotides. In one embodiment, the homology arm comprises 50 nucleotides to 200 nucleotides. In one embodiment, the homology arm comprises 50 nucleotides to 150 nucleotides. In one embodiment, the homology arm comprises 50 nucleotides to 100 nucleotides. In one embodiment, the homology arm comprises 100 nucleotides to 1000 nucleotides. In one embodiment, the homology arm comprises 100 nucleotides to 500 nucleotides. In one embodiment, the homology arm comprises 250 nucleotides to 1000 nucleotides. In one embodiment, the homology arm comprises 250 nucleotides to 500 nucleotides. In one embodiment, the homology arm comprises 500 nucleotides to 2000 nucleotides. In one embodiment, the homology arm comprises 500 nucleotides to 1000 nucleotides.

[0110] When referring to a desired edit, a "control sequence" (also referred to herein as a "control version of the target nucleic acid molecule") is used as a point of comparison. Any differences present in a homology arm compared to the control sequence should be considered as the "desired edit" or "edit" for that homology arm. A control sequence refers to an unedited sequence. A control sequence can be a naturally occurring or non-naturally occurring transgenic or synthetically produced (e.g., artificial) sequence. For example, a sequence encoding GFP inserted into the genome of a yeast cell can provide a control sequence for a transgene to be edited. A control version of a target nucleic acid molecule refers to an unedited target nucleic acid molecule. A control version of a target nucleic acid molecule can be a naturally occurring or non-naturally occurring transgenic or synthetically produced (e.g., artificial) sequence.

[0111] In one embodiment, the desired edit comprises a deletion of at least one nucleotide compared to the control sequence. In one embodiment, the desired edit comprises an insertion of at least one nucleotide compared to the control sequence. In one embodiment, the desired edit comprises a substitution of at least one nucleotide compared to the control sequence. In one embodiment, the desired edit comprises an insertion of at least two nucleotides compared to the control sequence.

[0112] In one embodiment, the editing comprises a deletion. In one embodiment, at least one nucleotide is deleted from the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least two nucleotides are deleted from the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least three nucleotides are deleted from the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least four nucleotides are deleted from the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least five nucleotides are deleted from the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least ten nucleotides are deleted from the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least fifteen nucleotides are deleted from the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least twenty nucleotides are deleted from the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least twenty-five nucleotides are deleted from the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least thirty nucleotides are deleted from the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least 40 nucleotides are deleted from the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least 50 nucleotides are deleted from the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least 75 nucleotides are deleted from the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least 100 nucleotides are deleted from the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least 250 nucleotides are deleted from the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least 500 nucleotides are deleted from the target nucleic acid molecule compared to a control version of the target nucleic acid molecule.

[0113] In one embodiment, the editing comprises an insertion. In one embodiment, at least one nucleotide is inserted into the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least two nucleotides are inserted into the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least three nucleotides are inserted into the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least four nucleotides are inserted into the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least five nucleotides are inserted into the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least ten nucleotides are inserted into the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least fifteen nucleotides are inserted into the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least twenty nucleotides are inserted into the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least twenty-five nucleotides are inserted into the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least thirty nucleotides are inserted into the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least forty nucleotides are inserted into the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least 50 nucleotides are inserted into the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least 75 nucleotides are inserted into the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least 100 nucleotides are inserted into the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least 250 nucleotides are inserted into the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, at least 500 nucleotides are inserted into the target nucleic acid molecule compared to a control version of the target nucleic acid molecule.

[0114] In one embodiment, the editing comprises a substitution. In one embodiment, the editing comprises a substitution of a single nucleotide in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the editing comprises a substitution of at least two nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. When two or more nucleotides are substituted in a nucleic acid molecule, the substitutions do not have to be adjacent to each other. The two or more nucleotide substitutions can be separated by a non-edited nucleotide. In one embodiment, the editing comprises a substitution of at least three nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the editing comprises a substitution of at least four nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the editing comprises a substitution of at least five nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the editing comprises a substitution of at least six nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the editing comprises a substitution of at least seven nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the editing comprises a substitution of at least eight nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the edit comprises a substitution of at least 9 nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the edit comprises a substitution of at least 10 nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the edit comprises a substitution of at least 15 nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the edit comprises a substitution of at least 20 nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the edit comprises a substitution of at least 30 nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the edit comprises a substitution of at least 40 nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the edit comprises a substitution of at least 50 nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule.

[0115] In one embodiment, the edit comprises an inversion. In one embodiment, the edit comprises an inversion of at least 2 nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the edit comprises an inversion of at least 3 nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the edit comprises an inversion of at least 4 nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the edit comprises an inversion of at least 5 nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the edit comprises an inversion of at least 10 nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the edit comprises an inversion of at least 20 nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the edit comprises an inversion of at least 30 nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the edit comprises an inversion of at least 40 nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the edit comprises an inversion of at least 50 nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the edit comprises an inversion of at least 75 nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the edit comprises an inversion of at least 100 nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the edit comprises an inversion of at least 150 nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the edit comprises an inversion of at least 200 nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the edit comprises an inversion of at least 500 nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule. In one embodiment, the edit comprises an inversion of at least 1000 nucleotides in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule.

[0116] In one embodiment, the editing comprises at least one deletion and at least one insertion. In one embodiment, the editing comprises at least one deletion and at least one substitution. In one embodiment, the editing comprises at least one deletion and at least one inversion. In one embodiment, the editing comprises at least one insertion and at least one substitution. In one embodiment, the editing comprises at least one insertion and at least one inversion. In one embodiment, the editing comprises at least one substitution and at least one inversion.

[0117] In one embodiment, the edits comprise at least one deletion, at least one insertion, and at least one substitution. In one embodiment, the edits comprise at least one deletion, at least one insertion, and at least one inversion. In one embodiment, the edits comprise at least one insertion, at least one substitution, and at least one inversion.

[0118] In one embodiment, the edits comprise at least one deletion, at least one insertion, at least one substitution, and at least one inversion. In one embodiment, the editing comprises the introduction of a premature stop codon into a nucleic acid sequence encoding a protein, hi one embodiment, the editing results in a loss-of-function mutation in the target nucleic acid molecule.

[0119] In one embodiment, the edit comprises one or more mutation types selected from the group consisting of nonsense edits, missense edits, frameshift edits, splice site edits, and any combination thereof. As used herein, "nonsense edits" refers to edits to a nucleic acid sequence that introduce a premature stop codon into the amino acid sequence encoded by the nucleic acid sequence. As used herein, "missense edits" refers to edits to a nucleic acid sequence that cause a substitution in the amino acid sequence encoded by the nucleic acid sequence. As used herein, "frameshift edits" refers to insertions or deletions to a nucleic acid sequence that shift the frame for translating the nucleic acid sequence into an amino acid sequence. "Splice site edits" refers to edits in a nucleic acid sequence that cause retention of an intron for protein translation or alternatively for exon exclusion from protein translation. Splice site edits can cause nonsense, missense, or frameshift edits.

[0120] Edits in the coding region of a gene (e.g., exon editing) can result in a truncated protein or polypeptide when the mutant messenger RNA (mRNA) is translated into a protein or polypeptide. In one embodiment, the present disclosure provides edits that result in truncation of a protein or polypeptide. As used herein, a "truncated" protein or polypeptide contains at least one less amino acid compared to an endogenous control protein or polypeptide. For example, if endogenous protein A contains 100 amino acids, a truncated version of protein A can contain 1 to 99 amino acids.

[0121] Without being limited by any scientific theory, one way to cause protein or polypeptide truncation is by the introduction of a premature stop codon in the mRNA transcript of an endogenous gene. In one aspect, the present disclosure provides an edit that results in a premature stop codon in the mRNA transcript of an endogenous gene. As used herein, a "stop codon" refers to a nucleotide triplet in an mRNA transcript that signals the end of protein translation. A "premature stop codon" refers to a stop codon that is located before (e.g., on the 5' side) the normal stop codon position in an endogenous mRNA transcript. Several stop codons are known in the art, including, but not limited to, "UAG", "UAA", "UGA", "TAG", "TAA", and "TGA".

[0122] As used herein, a "loss-of-function edit" refers to an edit that confers a complete loss of function for a protein encoded by a gene containing the edit, or alternatively, an edit that confers a complete loss of function for a small RNA encoded by a genomic locus. A loss-of-function edit can cause a loss of mRNA transcript production, a loss of small RNA transcript production, a loss of protein function, or a combination thereof.

[0123] When a protein or nucleoprotein complex "edits" a target nucleic acid molecule, the protein or nucleoprotein complex causes at least one deletion, insertion, substitution, or inversion in the target nucleic acid molecule sequence compared to a control version of the target nucleic acid molecule sequence. In one embodiment, the nucleoprotein complex edits the target nucleic acid molecule in a cell.

[0124] In one embodiment, the single nucleotide substitution comprises a transition, which is the substitution of one purine for another (e.g., an adenine for a guanine or vice versa) or one pyrimidine for another (e.g., a cytosine for a thymine or vice versa).

[0125] In one embodiment, the single nucleotide substitution comprises a transversion, which is the substitution of a purine for a pyrimidine or vice versa (e.g., adenine for cytosine or vice versa; adenine for thymine or vice versa; guanine for cytosine or vice versa; guanine for thymine or vice versa).

[0126] In one embodiment, the edit is located within an exon of the target nucleic acid molecule. In one embodiment, the edit is located within an intron of the target nucleic acid molecule. In one embodiment, the edit is located within the 5'-untranslated region (UTR) of the target nucleic acid molecule. In one embodiment, the edit is located within the 3'-UTR of the target nucleic acid molecule. In one embodiment, the edit is located within a non-coding region of the target nucleic acid molecule. In one embodiment, the edit is located in a coding region of the target nucleic acid molecule. The coding region of the target nucleic acid molecule may code for a protein or a non-coding RNA. In one embodiment, the edit is located within a gene in the target nucleic acid molecule. In one embodiment, the edit is located within a promoter. In one embodiment, the edit is located within a transcription terminator. In one embodiment, the edit is located within a polyadenylation site.

[0127] As used herein, a "target nucleic acid molecule" refers to any nucleic acid molecule that contains a nucleic acid sequence that is desired to be edited. In one embodiment, the target nucleic acid molecule is located in the genome of a cell. In one embodiment, the target nucleic acid molecule is located in the nuclear genome. In one embodiment, the target nucleic acid molecule is located in the mitochondrial genome. In one embodiment, the target nucleic acid molecule is located in the chloroplast genome. In one embodiment, the target nucleic acid molecule is located in a plasmid. In one embodiment, the target nucleic acid molecule is double-stranded. In one embodiment, the target nucleic acid molecule is a DNA molecule.

[0128] In one embodiment, the target nucleic acid molecule comprises a gene. In one embodiment, the target nucleic acid molecule comprises a promoter. In one embodiment, the target nucleic acid molecule comprises a protein coding sequence. In one embodiment, the target nucleic acid molecule comprises a sequence that codes for a non-coding RNA molecule. Non-coding RNA is an RNA molecule that is not translated into a protein. Non-limiting examples of non-coding RNA molecules include microRNA (miRNA), small interfering RNA (siRNA), Piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), extracellular RNA (exRNA), gRNA, etc.

[0129] In one embodiment, the target nucleic acid molecule comprises at least one exon. In one embodiment, the target nucleic acid molecule comprises at least one intron. In one embodiment, the target nucleic acid molecule comprises an untranslated region (e.g., 5'-UTR, 3'-UTR).

[0130] In one embodiment, the target nucleic acid molecule encodes a reporter gene. As used herein, "reporter gene" refers to any gene that can be used to assay the transcriptional activity of an operably linked promoter. Reporter gene activity can be detected by, but is not limited to, MRI, PET, bioluminescence or fluorescence visualization, color change, and whether cells can grow on a certain medium. Without being limited thereto, editing of the reporter gene can be used to determine the editing efficiency and / or effectiveness of the fusion protein or MAD2019-H848A variant polypeptide. See, for example, Example 1.

[0131] In one embodiment, the reporter gene encodes a fluorescent molecule. As used herein, "fluorescent molecule" refers to a molecule that can re-emit light upon excitation. Fluorescent molecules are also referred to in the art as fluorophores. In one embodiment, the fluorescent molecule is a green fluorescent protein (GFP). In one embodiment, the fluorescent molecule is a red fluorescent protein (RFP). In one embodiment, the fluorescent molecule is a yellow fluorescent protein (YFP). In one embodiment, the fluorescent molecule is a cyan fluorescent protein (CFP). In one embodiment, the fluorescent molecule is selected from the group consisting of mCherry, mOrange, mRaspberry, mKO, TagRFP, mKate, mRuby, FusionRed, mScarlet, and DsRed-Express.

[0132] In one embodiment, the reporter gene encodes a bioluminescent molecule, hi one embodiment, the bioluminescent molecule is luciferase. In one embodiment, the reporter gene encodes β-glucuronidase (GUS).

[0133] In one aspect, the disclosure provides a cell comprising any of the polypeptides, fusion proteins, DNA molecules, or RNA molecules provided herein. In one aspect, the disclosure provides a cell comprising any of the nucleoprotein complexes provided herein. In one aspect, the disclosure provides a cell comprising any of the MAD2019-H848A polypeptide variants provided herein. In one aspect, the disclosure provides a cell comprising any of the fusion proteins provided herein. In one aspect, the disclosure provides a cell comprising any of the guides provided herein. In one aspect, the disclosure provides a cell comprising any of the homology arms provided herein. In one aspect, the disclosure provides a cell comprising any of the reverse transcriptases provided herein.

[0134] In one embodiment, the cell is a prokaryotic cell. In one embodiment, the prokaryotic cell is a bacterial cell. In one embodiment, the prokaryotic cell is an archaeal cell. In one embodiment, the prokaryotic cell is an Escherichia coli cell.

[0135] In one embodiment, the cell is a eukaryotic cell. In one aspect, the disclosure provides a eukaryotic cell comprising any of the polypeptides, fusion proteins, DNA molecules, or RNA molecules provided herein. In one aspect, the disclosure provides a eukaryotic cell comprising any of the nucleoprotein complexes provided herein. In one aspect, the disclosure provides a eukaryotic cell comprising any of the MAD2019-H848A polypeptide variants provided herein. In one aspect, the disclosure provides a eukaryotic cell comprising any of the fusion proteins provided herein.

[0136] In one embodiment, the eukaryotic cell is an animal cell. In one embodiment, the eukaryotic cell is selected from the group consisting of a fish cell, a bird cell, a reptile cell, an amphibian cell, an insect cell, an arachnid cell, a flatworm cell, an annelid cell, and a crustacean cell. In one embodiment, the eukaryotic cell is a mammalian cell. In one embodiment, the mammalian cell is a primate cell. In one embodiment, the eukaryotic cell is a human cell. In one embodiment, the mammalian cell is selected from the group consisting of a feline cell, a dog cell, a rabbit cell, a rodent cell, an ungulate cell, a marsupial cell, and a bat cell.

[0137] In one embodiment, the eukaryotic cell is an in vivo cell. In one embodiment, the eukaryotic cell is an ex vivo cell. In one embodiment, the eukaryotic cell is not a human cell. In one embodiment, the eukaryotic cell is a non-human mammalian cell. In one embodiment, the eukaryotic cell is a non-human animal cell. In one embodiment, the human cell is an ex vivo cell.

[0138] In one embodiment, the human cell is an induced pluripotent stem cell. In one embodiment, the human cell is a HEK293T cell. In one aspect, the human cell is selected from the group consisting of bone cells, ligament cells, tendon cells, muscle cells, tongue cells, lip cells, salivary gland cells, pharyngeal cells, esophageal cells, stomach cells, small intestinal cells, large intestinal cells, rectal cells, liver cells, gallbladder cells, mesenteric cells, pancreatic cells, nasal cells, pharyngeal cells, laryngeal cells, tracheal cells, bronchial cells, bronchiolar cells, lung cells, kidney cells, ureter cells, bladder cells, urethral cells, reproductive system cells, pituitary cells, pineal gland cells, thyroid cells, parathyroid cells, adrenal cells, pancreatic cells, cardiac cells, arterial cells, venous cells, capillary cells, lymphatic vessel cells, lymph node cells, bone marrow cells, thymus cells, spleen cells, tonsil cells, interstitial cells, brain cells, spinal cord cells, nerve cells, eye cells, and ear cells.

[0139] In one embodiment, the eukaryotic cell is a fungal cell. In one embodiment, the eukaryotic cell is a yeast cell. In one embodiment, the eukaryotic cell is a Schizosaccharomyces pombe cell. In one embodiment, the eukaryotic cell is a Saccharomyces cerevisiae cell.

[0140] In one embodiment, the eukaryotic cell is a plant cell. In one aspect, the disclosure provides a fusion protein comprising a MAD2019-H848A variant polypeptide. In one aspect, the fusion protein comprises a MAD2019-H848A variant polypeptide and a reverse transcriptase. In one aspect, the disclosure provides a fusion protein comprising a Tf1 reverse transcriptase comprising an amino acid sequence at least 80% identical or similar to SEQ ID NO: 12. In one aspect, the disclosure provides a fusion protein comprising a Tf1 reverse transcriptase comprising an amino acid sequence at least 80% identical or similar to SEQ ID NO: 13, wherein the amino acid sequence comprises an asparagine at position 364 compared to SEQ ID NO: 13. In one aspect, the disclosure provides a fusion protein comprising a Tf1 reverse transcriptase comprising the amino acid sequence of SEQ ID NO: 13.

[0141] As used herein, "fusion protein" refers to a protein created by joining two or more polypeptide (or protein) amino acid sequences together. In one embodiment, a fusion protein is encoded by a single nucleic acid molecule. In one embodiment, a fusion protein comprises a nuclease and a reverse transcriptase. In one embodiment, a fusion protein comprises a nickase and a reverse transcriptase.

[0142] In one embodiment, the fusion protein comprises a nickase comprising SEQ ID NO:1 and an HIV-1 reverse transcriptase. In one embodiment, the fusion protein comprises a nickase comprising SEQ ID NO:1 and an M-MLV reverse transcriptase. In one embodiment, the fusion protein comprises a nickase comprising SEQ ID NO:1 and an AMV reverse transcriptase. In one embodiment, the fusion protein comprises a nickase comprising SEQ ID NO:16 and an HIV-1 reverse transcriptase. In one embodiment, the fusion protein comprises a nickase comprising SEQ ID NO:16 and an M-MLV reverse transcriptase. In one embodiment, the fusion protein comprises a nickase comprising SEQ ID NO:16 and an AMV reverse transcriptase.

[0143] In one embodiment, the fusion protein comprises a MAD2019-H848A variant polypeptide and a Tf1 reverse transcriptase. In one embodiment, the fusion protein comprises a MAD2019-H848A variant polypeptide and a Tf1-D364N reverse transcriptase. In one embodiment, the fusion protein comprises a MAD2019-H848A variant polypeptide and a HIV-1 reverse transcriptase. In one embodiment, the fusion protein comprises a MAD2019-H848A variant polypeptide and a M-MLV reverse transcriptase. In one embodiment, the fusion protein comprises a MAD2019-H848A variant polypeptide and a AMV reverse transcriptase.

[0144] In one embodiment, the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-23. In one embodiment, the fusion protein comprises a linker amino acid sequence. As used herein, "linker amino acid sequence" refers to amino acid residues disposed between two or more polypeptide sequences comprising a fusion protein. It is recognized that the linker amino acid sequence does not have enzymatic activity itself. In one embodiment, the linker amino acid sequence is located between the nickase and the reverse transcriptase. In one embodiment, the linker amino acid sequence is located between the MAD2019-H848A variant polypeptide and the reverse transcriptase. In one embodiment, the linker amino acid sequence is located between the MAD2019-H848A variant polypeptide and the Tf1 reverse transcriptase. In one embodiment, the linker amino acid sequence is located between the MAD2019-H848A variant polypeptide and the Tf1-D364N reverse transcriptase. In one embodiment, the linker amino acid sequence is located between the MAD2019-H848A variant polypeptide and the HIV-1 reverse transcriptase. In one embodiment, the linker amino acid sequence is located between the MAD2019-H848A variant polypeptide and the M-MLV reverse transcriptase. In one embodiment, the linker amino acid sequence is located between the MAD2019-H848A variant polypeptide and the AMV reverse transcriptase.

[0145] In one embodiment, the linker amino acid sequence is located between the NLS and the nickase. In one embodiment, the linker amino acid sequence is located between the NLS and the reverse transcriptase. In one embodiment, the linker amino acid sequence is located between the NLS and the MAD2019-H848A variant polypeptide. In one embodiment, the linker amino acid sequence is located between the NLS and the Tf1 reverse transcriptase. In one embodiment, the linker amino acid sequence is located between the NLS and the Tf1-D364N reverse transcriptase. In one embodiment, the linker amino acid sequence is located between the NLS and the HIV-1 reverse transcriptase. In one embodiment, the linker amino acid sequence is located between the NLS and the M-MLV reverse transcriptase. In one embodiment, the linker amino acid sequence is located between the NLS and the AMV reverse transcriptase.

[0146] In one embodiment, the linker amino acid sequence comprises at least 1 amino acid residue. In one embodiment, the linker amino acid sequence comprises at least 2 amino acid residues. In one embodiment, the linker amino acid sequence comprises at least 3 amino acid residues. In one embodiment, the linker amino acid sequence comprises at least 4 amino acid residues. In one embodiment, the linker amino acid sequence comprises at least 5 amino acid residues. In one embodiment, the linker amino acid sequence comprises at least 6 amino acid residues. In one embodiment, the linker amino acid sequence comprises at least 7 amino acid residues. In one embodiment, the linker amino acid sequence comprises at least 8 amino acid residues. In one embodiment, the linker amino acid sequence comprises at least 9 amino acid residues. In one embodiment, the linker amino acid sequence comprises at least 10 amino acid residues. In one embodiment, the linker amino acid sequence comprises at least 15 amino acid residues. In one embodiment, the linker amino acid sequence comprises at least 20 amino acid residues. In one embodiment, the linker amino acid sequence comprises at least 25 amino acid residues. In one embodiment, the linker amino acid sequence comprises at least 30 amino acid residues. In one embodiment, the linker amino acid sequence comprises at least 35 amino acid residues. In one embodiment, the linker amino acid sequence comprises at least 40 amino acid residues. In one embodiment, the linker amino acid sequence comprises at least 45 amino acid residues. In one embodiment, the linker amino acid sequence comprises at least 50 amino acid residues. In one embodiment, the linker amino acid sequence comprises at least 60 amino acid residues. In one embodiment, the linker amino acid sequence comprises at least 70 amino acid residues.

[0147] In one embodiment, the linker amino acid sequence comprises 1 to 75 amino acid residues. In one embodiment, the linker amino acid sequence comprises 1 to 50 amino acid residues. In one embodiment, the linker amino acid sequence comprises 1 to 40 amino acid residues. In one embodiment, the linker amino acid sequence comprises 1 to 30 amino acid residues. In one embodiment, the linker amino acid sequence comprises 1 to 20 amino acid residues. In one embodiment, the linker amino acid sequence comprises 1 to 10 amino acid residues. In one embodiment, the linker amino acid sequence comprises 10 to 50 amino acid residues. In one embodiment, the linker amino acid sequence comprises 10 to 40 amino acid residues. In one embodiment, the linker amino acid sequence comprises 20 to 50 amino acid residues. In one embodiment, the linker amino acid sequence comprises 20 to 40 amino acid residues.

[0148] As used herein, a "reverse transcriptase" is any enzyme capable of producing complementary DNA (cDNA) from an RNA template. Reverse transcriptases are classified under section 2.7.7.49 by Enzyme Number and CAS Registry Number® 9068-38-6. Non-limiting examples of reverse transcriptase amino acid sequences are provided as SEQ ID NOs: 12 and 13 (both are Tf1 reverse transcriptase).

[0149] In one embodiment, the reverse transcriptase is Tf1 reverse transcriptase. In one embodiment, the reverse transcriptase is derived from Tf1 reverse transcriptase. In one embodiment, the reverse transcriptase is human immunodeficiency virus-1 (HIV-1) reverse transcriptase. In one embodiment, the reverse transcriptase is derived from HIV-1 reverse transcriptase. In one embodiment, the reverse transcriptase is Moloney murine leukemia virus (M-MLV) reverse transcriptase. In one embodiment, the reverse transcriptase is derived from M-MLV reverse transcriptase. In one embodiment, the reverse transcriptase is avian myeloblastosis virus (AMV) reverse transcriptase. In one embodiment, the reverse transcriptase is derived from AMV reverse transcriptase. In one embodiment, the reverse transcriptase is selected from the group consisting of HIV-1 reverse transcriptase, M-MLV reverse transcriptase, and AMV reverse transcriptase. In one embodiment, the reverse transcriptase is derived from a reverse transcriptase selected from the group consisting of HIV-1 reverse transcriptase, M-MLV reverse transcriptase, and AMV reverse transcriptase.

[0150] In one embodiment, the reverse transcriptase comprises an amino acid sequence at least 80% identical or similar to SEQ ID NO: 12. In one embodiment, the reverse transcriptase comprises an amino acid sequence at least 85% identical or similar to SEQ ID NO: 12. In one embodiment, the reverse transcriptase comprises an amino acid sequence at least 90% identical or similar to SEQ ID NO: 12. In one embodiment, the reverse transcriptase comprises an amino acid sequence at least 91% identical or similar to SEQ ID NO: 12. In one embodiment, the reverse transcriptase comprises an amino acid sequence at least 92% identical or similar to SEQ ID NO: 12. In one embodiment, the reverse transcriptase comprises an amino acid sequence at least 93% identical or similar to SEQ ID NO: 12. In one embodiment, the reverse transcriptase comprises an amino acid sequence at least 94% identical or similar to SEQ ID NO: 12. In one embodiment, the reverse transcriptase comprises an amino acid sequence at least 95% identical or similar to SEQ ID NO: 12. In one embodiment, the reverse transcriptase comprises an amino acid sequence at least 96% identical or similar to SEQ ID NO: 12. In one embodiment, the reverse transcriptase comprises an amino acid sequence at least 97% identical or similar to SEQ ID NO: 12. In one embodiment, the reverse transcriptase comprises an amino acid sequence at least 98% identical or similar to SEQ ID NO: 12. In one embodiment, the reverse transcriptase comprises an amino acid sequence at least 99% identical or similar to SEQ ID NO: 12. In one embodiment, the reverse transcriptase comprises an amino acid sequence 100% identical or similar to SEQ ID NO: 12.

[0151] In one embodiment, the reverse transcriptase comprises an amino acid sequence at least 80% identical or similar to SEQ ID NO: 13, wherein the amino acid sequence comprises an asparagine at position 364 compared to SEQ ID NO: 13. In one embodiment, the reverse transcriptase comprises an amino acid sequence at least 85% identical or similar to SEQ ID NO: 13, wherein the amino acid sequence comprises an asparagine at position 364 compared to SEQ ID NO: 13. In one embodiment, the reverse transcriptase comprises an amino acid sequence at least 90% identical or similar to SEQ ID NO: 13, wherein the amino acid sequence comprises an asparagine at position 364 compared to SEQ ID NO: 13. In one embodiment, the reverse transcriptase comprises an amino acid sequence at least 91% identical or similar to SEQ ID NO: 13, wherein the amino acid sequence comprises an asparagine at position 364 compared to SEQ ID NO: 13. In one embodiment, the reverse transcriptase comprises an amino acid sequence at least 92% identical or similar to SEQ ID NO: 13, wherein the amino acid sequence comprises an asparagine at position 364 compared to SEQ ID NO: 13. In one embodiment, the reverse transcriptase comprises an amino acid sequence at least 93% identical or similar to SEQ ID NO: 13, wherein the amino acid sequence comprises an asparagine at position 364 compared to SEQ ID NO: 13. In one embodiment, the reverse transcriptase comprises an amino acid sequence at least 94% identical or similar to SEQ ID NO: 13, wherein the amino acid sequence comprises an asparagine at position 364 compared to SEQ ID NO: 13. In one embodiment, the reverse transcriptase comprises an amino acid sequence at least 95% identical or similar to SEQ ID NO: 13, wherein the amino acid sequence comprises an asparagine at position 364 compared to SEQ ID NO: 13. In one embodiment, the reverse transcriptase comprises an amino acid sequence at least 96% identical or similar to SEQ ID NO: 13, wherein the amino acid sequence comprises an asparagine at position 364 compared to SEQ ID NO: 13. In one embodiment, the reverse transcriptase comprises an amino acid sequence at least 97% identical or similar to SEQ ID NO: 13, wherein the amino acid sequence comprises an asparagine at position 364 compared to SEQ ID NO: 13. In one embodiment, the reverse transcriptase comprises an amino acid sequence at least 98% identical or similar to SEQ ID NO: 13, wherein the amino acid sequence comprises an asparagine at position 364 compared to SEQ ID NO: 13. In one embodiment, the reverse transcriptase comprises an amino acid sequence at least 99% identical or similar to SEQ ID NO: 13, wherein the amino acid sequence comprises an asparagine at position 364 compared to SEQ ID NO: 13.In one embodiment, the reverse transcriptase comprises an amino acid sequence 100% identical or similar to SEQ ID NO:13.

[0152] In one embodiment, the fusion protein comprising Tf1 reverse transcriptase further comprises a nickase. In one embodiment, the fusion protein comprising Tf1 reverse transcriptase further comprises a CRISPR-Cas nickase. In one embodiment, the fusion protein comprising Tf1 reverse transcriptase further comprises a type I CRISPR-Cas nickase. In one embodiment, the fusion protein comprising Tf1 reverse transcriptase further comprises a type II CRISPR-Cas nickase. In one embodiment, the fusion protein comprising Tf1 reverse transcriptase further comprises a type III CRISPR-Cas nickase. In one embodiment, the fusion protein comprising Tf1 reverse transcriptase further comprises a type IV CRISPR-Cas nickase. In one embodiment, the fusion protein comprising Tf1 reverse transcriptase further comprises a type V CRISPR-Cas nickase. In one embodiment, the fusion protein comprising Tf1 reverse transcriptase further comprises a type VI CRISPR-Cas nickase. In one embodiment, the fusion protein comprising Tf1 reverse transcriptase further comprises a Cas9 nickase. In one embodiment, the fusion protein comprising Tf1 reverse transcriptase further comprises MAD2019 nickase. In one embodiment, the fusion protein comprising Tf1 reverse transcriptase further comprises MAD2019-H848A polypeptide. In one embodiment, the fusion protein comprising Tf1 reverse transcriptase further comprises MAD2019-H848A variant polypeptide.

[0153] In one embodiment, the CRISPR-Cas nickase is a type I CRISPR-Cas induced nickase. In one embodiment, the CRISPR-Cas nickase is a type II CRISPR-Cas induced nickase. In one embodiment, the CRISPR-Cas nickase is a type III CRISPR-Cas induced nickase. In one embodiment, the CRISPR-Cas nickase is a type IV CRISPR-Cas induced nickase. In one embodiment, the CRISPR-Cas nickase is a type V CRISPR-Cas induced nickase. In one embodiment, the CRISPR-Cas nickase is a type VI CRISPR-Cas induced nickase. In one embodiment, the CRISPR-Cas nickase is a Cas9 nickase. In one embodiment, the CRISPR-Cas nickase is a Cas9 induced nickase. In one embodiment, the CRISPR-Cas nickase is a MAD2019 induced nickase. In one embodiment, the CRISPR-Cas nickase is a MAD2019-H848A polypeptide. In one embodiment, the CRISPR-Cas nickase is a MAD2019-H848A variant polypeptide.

[0154] CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein) enzymes are nucleases that use guides to recognize and cleave specific DNA targets. Without being limited by any scientific theory, nickases can be derived from nucleases (e.g., CRISPR-Cas enzymes) by mutating or editing the nuclease. In one embodiment, the nickase is derived from a CRISPR-Cas enzyme.

[0155] Non-limiting examples of type I CRISPR-Cas enzymes include Cas3, Cas5, Cas8a, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, and GSU0054. Non-limiting examples of type II CRISPR-Cas enzymes include Cas4, Cas9, and Csn2. Non-limiting examples of type III CRISPR-Cas enzymes include Cas10, Csm2, Cmr5, Csx10, and Csx11. A non-limiting example of a type IV CRISPR-Cas enzyme is Csf1. Non-limiting examples of type V CRISPR-Cas enzymes include Cas12, Cas12a (also known as Cpf1), Cas12b (also known as C2c1), Cas12c (also known as C2c3), Cas12d (also known as CasY), Cas12e (also known as CasX), Cas12f (also known as Cas14 or C2c10), Cas12g, Cas12h, Cas12i, Cas12k (also known as C2c5), C2c4, C2c8, and C2c9. Non-limiting examples of type VI CRISPR-Cas enzymes include Cas13, Cas13a (also known as C2c2), Cas13b, Cas13c, and Cas13d.

[0156] In one aspect, the disclosure provides a method of providing a MAD2019-H848A variant polypeptide to a cell, the method comprising: (a) obtaining a cell; and (b) providing to the cell a MAD2019-H848A variant polypeptide or a nucleic acid molecule encoding the MAD2019-H848A variant polypeptide. In one aspect, the disclosure provides a method of providing a fusion protein to a cell, the method comprising: (a) obtaining a cell; and (b) providing to the cell a fusion protein or a nucleic acid molecule encoding the fusion protein. In one aspect, the method further comprises transfecting the cell with a nucleic acid molecule encoding a guide. In one aspect, the method further comprises transfecting the cell with a nucleic acid molecule encoding a homology arm. In one aspect, the method further comprises transfecting the cell with a nucleic acid molecule encoding a guide and a homology arm. In one aspect, the method further comprises transfecting the cell with a nucleic acid molecule encoding (a) a guide; (b) a homology arm; or (c) a guide and a homology arm.

[0157] In one embodiment, the nucleic acid molecule provided herein is stably integrated into the genome in the cell. In one embodiment, the nucleic acid molecule provided herein is transiently introduced into the cell. In one embodiment, the nucleic acid molecule provided herein is located in a plasmid. As used herein, "plasmid" refers to a circular double-stranded DNA molecule. In one embodiment, the plasmid comprises an origin of replication. In one embodiment, the plasmid comprises a selection marker gene.

[0158] Methods are provided herein that include the transient transformation or stable integration of any nucleic acid molecule into a cell. As used herein, "stable integration" or "stably integrated" refers to the introduction of a nucleic acid molecule into the genome of a targeted cell, allowing the targeted cell to transfer the introduced nucleic acid molecule to the next generation of transformed organisms.

[0159] As used herein, "transiently introduced," "transiently transformed," or "transient transformation" refers to the introduction of DNA into a cell that is not introduced into subsequent generations of the transformed organism.

[0160] Numerous methods of transforming a cell with a nucleic acid molecule or a nucleoprotein complex (e.g., providing a cell with a nucleic acid molecule or a nucleoprotein complex) are known in the art and can be used in accordance with the methods of the present application. Any suitable method or technique for transforming a cell known in the art can be used in accordance with the methods. Non-limiting methods for transforming a cell include polyethylene glycol-mediated transformation, biolistic transformation, liposome-mediated transfection, viral transduction, the use of one or more delivery particles, and electroporation. In one embodiment, the method includes introducing one or more nucleic acid molecules or nucleoprotein complexes into a cell using a method selected from the group consisting of polyethylene glycol-mediated transformation, biolistic transformation, liposome-mediated transfection, viral transduction, the use of one or more delivery particles, and electroporation.

[0161] In one embodiment, the method comprises providing the nucleic acid molecule to the cell via polyethylene glycol-mediated transformation. In one embodiment, the method comprises providing the nucleic acid molecule to the cell via biolistic transformation. In one embodiment, the method comprises providing the nucleic acid molecule to the cell via liposome-mediated transfection. In one embodiment, the method comprises providing the nucleic acid molecule to the cell via viral transduction. In one embodiment, the method comprises providing the nucleic acid molecule to the cell via the use of one or more delivery particles. In one embodiment, the method comprises providing the nucleic acid molecule to the cell via microinjection. In one embodiment, the method comprises providing the nucleic acid molecule to the cell via electroporation. In one embodiment, the method comprises providing the nucleoprotein complex to the cell via microinjection. In one embodiment, the method comprises providing the nucleoprotein complex to the cell via electroporation.

[0162] Other methods of transformation, such as vacuum infiltration, pressure treatment, ultrasonic treatment, and silicon carbide fiber agitation, are also known in the art and are contemplated for use with any of the methods provided herein.

[0163] Methods for transforming cells are well known to those skilled in the art.For example, specific instructions for transforming plant cells (e.g., biolistic transformation) by microprojectile bombardment using particles coated with nucleic acid molecules can be found in U.S. Patent Nos. 5,550,318; 5,538,880; 6,160,208; 6,399,861; and 6,153,812.

[0164] Lipofection has been described, for example, in U.S. Patent Nos. 5,049,386; 4,946,787; and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, WO 91 / 17424; WO 91 / 16024.

[0165] Delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for expression of one or more elements of a nucleic acid molecule are those used in WO 2014 / 093622. In one embodiment, the method of providing a nucleic acid molecule or protein to a cell comprises delivery via a delivery particle. In one embodiment, the method of providing a nucleic acid molecule to a cell comprises delivery via a delivery vesicle. In one embodiment, the delivery vesicle is selected from the group consisting of an exosome and a liposome. In one embodiment, the method of providing a nucleic acid molecule to a cell comprises delivery via a viral vector. In one embodiment, the viral vector is selected from the group consisting of an adenoviral vector, a lentiviral vector, and an adeno-associated viral vector. In another embodiment, the method of providing a nucleic acid molecule to a cell comprises delivery via a nanoparticle. In one embodiment, the method of providing a nucleic acid molecule to a cell comprises microinjection. In one embodiment, the method of providing a nucleic acid molecule to a cell comprises a polycation. In one embodiment, the method of providing a nucleic acid molecule to a cell comprises a cationic oligopeptide.

[0166] In one embodiment, the delivery particle is selected from the group consisting of exosomes, adenoviral vectors, lentiviral vectors, adeno-associated viral vectors, nanoparticles, polycations, and cationic oligopeptides.In one embodiment, the method provided herein comprises the use of one or more delivery particles.In another embodiment, the method provided herein comprises the use of two or more delivery particles.In another embodiment, the method provided herein comprises the use of three or more delivery particles.

[0167] Suitable agents for facilitating the introduction of nucleic acid molecules into cells include agents that increase the permeability of cells to oligonucleotides or polynucleotides. Such agents for facilitating the introduction of compositions into cells include chemical agents, or physical agents, or combinations thereof. Chemical agents for conditioning include (a) detergents, (b) organic solvents, aqueous solutions, or aqueous mixtures of organic solvents, (c) oxidizing agents, (e) acids, (f) bases, (g) oils, (h) enzymes, or combinations thereof.

[0168] Agents for laboratory conditioning of cells for permeabilization with polynucleotides include, for example, application of chemical agents, enzymatic treatment, heating or refrigeration, treatment with positive or negative pressure, or sonication.

[0169] In one aspect, the disclosure provides a method of editing at least one eukaryotic cell comprising: (a) introducing into the at least one eukaryotic cell a MAD2019-H848A variant polypeptide, or a nucleic acid molecule encoding a MAD2019-H848A variant polypeptide; and (ii) into the at least one eukaryotic cell a guide RNA, the guide RNA or a nucleic acid molecule encoding the guide RNA comprising a nucleic acid sequence that is complementary to a target nucleic acid molecule in the genome of the eukaryotic cell; wherein the MAD2019-H848A variant polypeptide and the guide RNA form a nucleoprotein complex in the at least one eukaryotic cell, wherein the nucleoprotein complex cleaves one strand of the target nucleic acid molecule, and at least one edit is made in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule; and (b) identifying the at least one eukaryotic cell comprising the at least one edit. In one aspect, step (a) of the method further comprises introducing at least one homology arm, or a nucleic acid molecule encoding a homology arm, into a eukaryotic cell, wherein the at least one homology arm comprises a nucleic acid sequence that includes at least one edit.

[0170] In one aspect, the disclosure provides a method of editing at least one eukaryotic cell, comprising: (a) (i) introducing into the at least one eukaryotic cell a fusion protein or a nucleic acid molecule encoding the fusion protein; and (ii) introducing into the at least one eukaryotic cell a guide RNA, the guide RNA or the nucleic acid molecule encoding the guide RNA, comprising a nucleic acid sequence complementary to a target nucleic acid molecule in the genome of the eukaryotic cell; the fusion protein and the guide RNA form a nucleoprotein complex in the at least one eukaryotic cell, the nucleoprotein complex cleaving one strand of the target nucleic acid molecule, and at least one edit is made in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule; and (b) identifying at least one eukaryotic cell comprising at least one edit. In one aspect, step (a) of the method further comprises introducing into the eukaryotic cell at least one homology arm or a nucleic acid molecule encoding a homology arm, the at least one homology arm comprising a nucleic acid sequence comprising at least one edit.

[0171] The following examples of non-limiting embodiments are specifically contemplated: 1. A MAD2019-H848A variant polypeptide comprising an amino acid sequence at least 90% identical or similar to SEQ ID NO:1, wherein the MAD2019-H848A variant polypeptide comprises an alanine at position 848 according to SEQ ID NO:1.

[0172] 2. The MAD2019-H848A variant polypeptide of embodiment 1, comprising a V1143T amino acid substitution compared to SEQ ID NO:1. 3. The MAD2019-H848A variant polypeptide of embodiment 1, comprising a L500R amino acid substitution, a D700A amino acid substitution, a D701P amino acid substitution, a K720S amino acid substitution, a I1142K amino acid substitution, and a V1143T amino acid substitution compared to SEQ ID NO:1.

[0173] 4. The MAD2019-H848A variant polypeptide of embodiment 1, comprising a L500R amino acid substitution, a D700A amino acid substitution, a D701P amino acid substitution, a K720S amino acid substitution, and a V1143T amino acid substitution compared to SEQ ID NO:1.

[0174] 5. The MAD2019-H848A variant polypeptide of embodiment 1, comprising a L500K amino acid substitution and a V1143T amino acid substitution compared to SEQ ID NO:1. 6. The MAD2019-H848A variant polypeptide of embodiment 1, comprising a S409R amino acid substitution, a L500K amino acid substitution, and a V1143T amino acid substitution compared to SEQ ID NO:1.

[0175] 7. The MAD2019-H848A variant polypeptide of embodiment 1, comprising a V1143T amino acid substitution and a A1221H amino acid substitution compared to SEQ ID NO:1. 8. The MAD2019-H848A variant polypeptide of embodiment 1, comprising a L500K amino acid substitution, a V1143T amino acid substitution, and an A1221H amino acid substitution compared to SEQ ID NO:1.

[0176] 9. The MAD2019-H848A variant polypeptide of embodiment 1, comprising a L500K amino acid substitution, a I1142R amino acid substitution, a V1143T amino acid substitution, and a A1221H amino acid substitution compared to SEQ ID NO:1.

[0177] 10. The MAD2019-H848A variant polypeptide of embodiment 1, comprising a L500K amino acid substitution, a D1139N amino acid substitution, a V1143T amino acid substitution, and a A1221H amino acid substitution compared to SEQ ID NO:1.

[0178] 11. The MAD2019-H848A variant polypeptide of embodiment 1, comprising a L500K amino acid substitution, a V1143T amino acid substitution, an A1221H amino acid substitution, and a K1285R amino acid substitution compared to SEQ ID NO:1.

[0179] 12. The MAD2019-H848A variant polypeptide of embodiment 1, comprising an amino acid substitution selected from the group consisting of: T67G, S409R, L500K, L500R, G578F, L624Q, N669S, D700A, D701P, D701N, D701T, K720S, L1110R, D1139N, I1142R, I1142K, V1143T, A1221H, K1285R, A1321R, A1321K, S1136Q, and A1139R compared to SEQ ID NO:1.

[0180] 13. The MAD2019-H848A variant polypeptide of any one of embodiments 1 to 12, further comprising at least one nuclear localization signal. 14. A nucleic acid sequence encoding a MAD2019-H848A variant polypeptide according to any one of embodiments 1 to 13.

[0181] 15. A nucleoprotein complex comprising a MAD2019-H848A variant polypeptide and a nucleic acid molecule according to any one of embodiments 1 to 13. 16. The nucleoprotein complex of embodiment 15, wherein the nucleic acid molecule comprises: (a) a guide; (b) a homology arm; or (c) a guide and a homology arm.

[0182] 17. A nucleoprotein complex according to embodiment 15 or 16, wherein the nucleic acid molecule is an RNA molecule. 18. A eukaryotic cell comprising a MAD2019-H848A variant polypeptide according to any one of embodiments 1 to 13.

[0183] 19. A eukaryotic cell comprising a nucleic acid sequence according to embodiment 14. 20. A eukaryotic cell comprising a nucleoprotein complex according to any one of embodiments 15 to 17.

[0184] 21. A fusion protein comprising a MAD2019-H848A variant polypeptide according to any one of embodiments 1 to 12. 22. The fusion protein of embodiment 21, further comprising a reverse transcriptase.

[0185] 23. The fusion protein of embodiment 22, wherein the reverse transcriptase is a Tf1 reverse transcriptase comprising an amino acid sequence at least 90% identical or similar to SEQ ID NO:12. 24. The fusion protein of embodiment 23, wherein the Tf1 reverse transcriptase comprises a D362N amino acid substitution compared to SEQ ID NO: 12.

[0186] 25. The fusion protein of embodiment 23, wherein the Tf1 reverse transcriptase comprises SEQ ID NO: 13. 26. The fusion protein of embodiment 22, wherein the reverse transcriptase is derived from a reverse transcriptase selected from the group consisting of HIV-1 (human immunodeficiency virus) reverse transcriptase, M-MLV (Moloney murine leukemia virus) reverse transcriptase, and AMV (avian myeloblastosis virus) reverse transcriptase.

[0187] 27. A fusion protein according to any one of embodiments 21 to 26, further comprising at least one nuclear localization signal. 28. A fusion protein according to any one of embodiments 21 to 27, comprising a linker amino acid sequence.

[0188] 29. The fusion protein of embodiment 22, comprising a linker amino acid sequence located between the MAD2019-H848A variant polypeptide and the reverse transcriptase. 30. A fusion protein comprising a Tf1 reverse transcriptase that comprises an amino acid sequence at least 90% identical or similar to SEQ ID NO:12.

[0189] 31. The fusion protein of embodiment 30, wherein the Tf1 reverse transcriptase comprises a D362N amino acid substitution compared to SEQ ID NO: 12. 32. The fusion protein of embodiment 30, wherein the Tf1 reverse transcriptase comprises SEQ ID NO: 13.

[0190] 33. The fusion protein according to any one of embodiments 30 to 32, further comprising a nickase. 34. The fusion protein of embodiment 33, wherein the nickase is a CRISPR-Cas nickase.

[0191] 35. The fusion protein of embodiment 34, wherein the CRISPR-Cas nickase is a type I CRISPR-Cas nickase. 36. The fusion protein of embodiment 34, wherein the CRISPR-Cas nickase is a type II CRISPR-Cas nickase.

[0192] 37. The fusion protein of embodiment 34, wherein the CRISPR-Cas nickase is a type III CRISPR-Cas nickase. 38. The fusion protein of embodiment 34, wherein the CRISPR-Cas nickase is a type IV CRISPR-Cas nickase.

[0193] 39. The fusion protein of embodiment 34, wherein the CRISPR-Cas nickase is a type V CRISPR-Cas nickase. 40. The fusion protein of embodiment 34, wherein the CRISPR-Cas nickase is a type VI CRISPR-Cas nickase.

[0194] 41. The fusion protein of embodiment 34, wherein the CRISPR-Cas nickase is a Cas9 nickase. 42. The fusion protein of embodiment 34, wherein the CRISPR-Cas nickase is a MAD2019 polypeptide.

[0195] 43. The fusion protein of embodiment 34, wherein the CRISPR-Cas nickase is a MAD2019-H848A polypeptide. 44. The fusion protein of embodiment 34, wherein the CRISPR-Cas nickase is a MAD2019-H848A variant polypeptide.

[0196] 45. The fusion protein of any one of embodiments 30 to 32, further comprising a MAD2019-H848A variant polypeptide comprising an amino acid sequence at least 90% identical or similar to SEQ ID NO:1, wherein the MAD2019-H848A nickase comprises an alanine at position 848 according to SEQ ID NO:1.

[0197] 46. ​​The fusion protein of embodiment 45, wherein the MAD2019-H848A variant polypeptide comprises a V1143T amino acid substitution compared to SEQ ID NO:1. 47. The fusion protein of embodiment 45, wherein the MAD2019-H848A variant polypeptide comprises an L500R amino acid substitution, a D700A amino acid substitution, a D701P amino acid substitution, a K720S amino acid substitution, an I1142K amino acid substitution, and a V1143T amino acid substitution compared to SEQ ID NO:1.

[0198] 48. The fusion protein of embodiment 45, wherein the MAD2019-H848A variant polypeptide comprises an L500R amino acid substitution, a D700A amino acid substitution, a D701P amino acid substitution, a K720S amino acid substitution, and a V1143T amino acid substitution compared to SEQ ID NO:1.

[0199] 49. The fusion protein of embodiment 45, wherein the MAD2019-H848A variant polypeptide comprises a L500K amino acid substitution and a V1143T amino acid substitution compared to SEQ ID NO:1.

[0200] 50. The fusion protein of embodiment 45, wherein the MAD2019-H848A variant polypeptide comprises a S409R amino acid substitution, a L500K amino acid substitution, and a V1143T amino acid substitution compared to SEQ ID NO:1.

[0201] 51. The fusion protein of embodiment 45, wherein the MAD2019-H848A variant polypeptide comprises a V1143T amino acid substitution and an A1221H amino acid substitution compared to SEQ ID NO:1.

[0202] 52. The fusion protein of embodiment 45, wherein the MAD2019-H848A variant polypeptide comprises a L500K amino acid substitution, a V1143T amino acid substitution, and an A1221H amino acid substitution compared to SEQ ID NO:1.

[0203] 53. The fusion protein of embodiment 45, wherein the MAD2019-H848A variant polypeptide comprises a L500K amino acid substitution, a I1142R amino acid substitution, a V1143T amino acid substitution, and a A1221H amino acid substitution compared to SEQ ID NO:1.

[0204] 54. The fusion protein of embodiment 45, wherein the MAD2019-H848A variant polypeptide comprises a L500K amino acid substitution, a D1139N amino acid substitution, a V1143T amino acid substitution, and an A1221H amino acid substitution compared to SEQ ID NO:1.

[0205] 55. The fusion protein of embodiment 45, wherein the MAD2019-H848A variant polypeptide comprises a L500K amino acid substitution, a V1143T amino acid substitution, an A1221H amino acid substitution, and a K1285R amino acid substitution compared to SEQ ID NO:1.

[0206] 56. The fusion protein of embodiment 45, wherein the MAD2019-H848A variant polypeptide comprises an amino acid substitution selected from the group consisting of T67G, S409R, L500K, L500R, G578F, L624Q, N669S, D700A, D701P, D701N, D701T, K720S, L1110R, D1139N, I1142R, I1142K, V1143T, A1221H, K1285R, A1321R, A1321K, S1136Q, and A1139R compared to SEQ ID NO:1.

[0207] 57. A fusion protein according to any one of embodiments 30 to 56, further comprising at least one nuclear localization signal. 58. A fusion protein according to any one of embodiments 30 to 57, comprising a linker amino acid sequence.

[0208] 59. A fusion protein according to any one of embodiments 45 to 56, comprising a linker amino acid sequence located between the MAD2019-H848A variant polypeptide and the Tf1 reverse transcriptase.

[0209] 60. The fusion protein of embodiment 33, comprising a linker amino acid sequence located between the nickase and the Tf1 reverse transcriptase. 61. A nucleic acid sequence encoding a fusion protein according to any one of embodiments 21 to 60.

[0210] 62. A nucleoprotein complex comprising a fusion protein and a nucleic acid molecule according to any one of embodiments 21 to 60. 63. The nucleoprotein complex of embodiment 62, wherein the nucleic acid molecule comprises: (a) a guide; (b) a homology arm; or (c) a guide and a homology arm.

[0211] 64. A nucleoprotein complex according to embodiment 62 or 63, wherein the nucleic acid molecule is an RNA molecule. 65. A eukaryotic cell comprising a fusion protein according to any one of embodiments 21 to 60.

[0212] 66. A eukaryotic cell comprising a nucleic acid sequence according to embodiment 61. 67. A eukaryotic cell comprising a nucleoprotein complex according to any one of embodiments 62 to 64.

[0213] 68. A method for providing a MAD2019-H848A variant polypeptide to a cell, comprising: (a) obtaining cells; and (b) providing the cell with a MAD2019-H848A variant polypeptide according to any one of embodiments 1 to 13, or a nucleic acid molecule encoding the MAD2019-H848A variant polypeptide. The method includes:

[0214] 69. A method for providing a fusion protein to a cell, comprising: (a) obtaining cells; and (b) providing a cell with a fusion protein according to any one of embodiments 21 to 60, or a nucleic acid molecule encoding the fusion protein; The method includes:

[0215] 70. The method of embodiment 68 or 69, further comprising transfecting the cell with a nucleic acid molecule encoding: (a) a guide; (b) a homology arm; or (c) a guide and a homology arm.

[0216] 71. The method of embodiment 69, wherein the guide is an RNA molecule that forms a nucleoprotein complex with the MAD2019-H848A variant polypeptide in the cell. 72. The method of embodiment 71, wherein the guide comprises a sequence complementary to a target nucleic acid molecule in the cell.

[0217] 73. The method of embodiment 72, wherein the nucleoprotein complex edits a target nucleic acid molecule in a cell. 74. The method of embodiment 70, wherein the guide is an RNA molecule that forms a nucleoprotein complex with the fusion protein in the cell.

[0218] 75. The method of embodiment 74, wherein the guide comprises a sequence complementary to a target nucleic acid molecule in the cell. 76. The method of embodiment 75, wherein the nucleoprotein complex edits a target nucleic acid molecule in a cell.

[0219] 77. The method of any one of embodiments 72, 73, 75, and 76, wherein the target nucleic acid molecule is located within the nuclear genome of the cell. 78. The method of any one of embodiments 72, 73, 75, and 76, wherein the target nucleic acid molecule is located within the mitochondrial genome of the cell.

[0220] 79. The method of any one of embodiments 68-76, comprising a method selected from the group consisting of polyethylene glycol-mediated transformation, biolistic transformation, liposome-mediated transfection, viral transduction, use of one or more delivery particles, microinjection, and electroporation.

[0221] 80. A method for editing at least one eukaryotic cell, comprising: (a) (i) injecting into at least one eukaryotic cell a MAD2019-H848A variant polypeptide according to any one of embodiments 1 to 13, or a nucleic acid molecule encoding a MAD2019-H848A variant polypeptide; and (ii) administering to at least one eukaryotic cell a guide RNA, the guide RNA comprising a nucleic acid sequence complementary to a target nucleic acid molecule in the genome of the eukaryotic cell, or a nucleic acid molecule encoding the guide RNA; To introduce; the MAD2019-H848A variant polypeptide and the guide RNA form a nucleoprotein complex in at least one eukaryotic cell, the nucleoprotein complex cleaving one strand of the target nucleic acid molecule and at least one edit being made in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule; and (b) identifying at least one eukaryotic cell that contains at least one edit; The method includes:

[0222] 81. A method for editing at least one eukaryotic cell, comprising: (a) (i) injecting into at least one eukaryotic cell a fusion protein according to any one of embodiments 21 to 60, or a nucleic acid molecule encoding the fusion protein; and (ii) administering to at least one eukaryotic cell a guide RNA, the guide RNA comprising a nucleic acid sequence complementary to a target nucleic acid molecule in the genome of the eukaryotic cell, or a nucleic acid molecule encoding the guide RNA; To introduce; the fusion protein and the guide RNA form a nucleoprotein complex in at least one eukaryotic cell, the nucleoprotein complex cleaving one strand of the target nucleic acid molecule and at least one edit being made in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule; and (b) identifying at least one eukaryotic cell that contains at least one edit; The method includes:

[0223] 82. The method of embodiment 80 or 81, wherein step (a) of the method further comprises introducing at least one homology arm, or a nucleic acid molecule encoding a homology arm, into a eukaryotic cell, wherein at least one homology arm comprises a nucleic acid sequence comprising at least one edit.

[0224] 83. The method of any one of embodiments 80 to 82, wherein at least one edit includes an insertion. 84. The method of any one of embodiments 80 to 82, wherein at least one edit comprises a deletion.

[0225] 85. The method of any one of embodiments 80 to 82, wherein at least one edit comprises a substitution. 86. The method of any one of embodiments 80 to 82, wherein at least one edit comprises a substitution of a single nucleotide.

[0226] 87. The method of embodiment 86, wherein the single nucleotide substitution comprises a transision. 88. The method of embodiment 86, wherein the single nucleotide substitution comprises a transversion.

[0227] 89. A guide RNA (gRNA) comprising a scaffold region having a nucleic acid sequence at least 80% identical to SEQ ID NO:24. 90. The gRNA described in embodiment 89, wherein the nucleic acid sequence is at least 90% identical to SEQ ID NO: 24.

[0228] 91. The gRNA described in embodiment 89, wherein the nucleic acid sequence is at least 95% identical to SEQ ID NO: 24. 92. The gRNA described in embodiment 89, wherein the nucleic acid sequence is 100% identical to SEQ ID NO: 24.

[0229] 93. A nucleoprotein complex comprising a fusion protein and a gRNA according to any one of embodiments 89 to 92. 94. The nucleoprotein complex of embodiment 93, wherein the fusion protein comprises a MAD2019-H848A variant polypeptide.

[0230] 95. The nucleoprotein complex of embodiment 93 or 94, wherein the fusion protein comprises a reverse transcriptase. 96. A nucleoprotein complex comprising a MAD2019-H848A variant polypeptide and a gRNA according to any one of embodiments 89 to 92.

[0231] 97. A nucleoprotein complex comprising a nickase and a gRNA according to any one of embodiments 89 to 92. 98. A nucleoprotein complex comprising a gRNA according to any one of embodiments 89 to 92.

[0232] Having now generally described the disclosure, it will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended as limitations of the disclosure unless specified. EXAMPLES

[0233] Example 1. Work procedure overview MAD2019 variant polypeptides provided herein based on MAD2019-H848A (SEQ ID NO: 1) were identified using the method shown in FIG. 2. First, a pool of variants (Seq 1 in FIG. 2; Seq 1 refers to SEQ ID NO: 1 variants with amino acid histidine at position 848 instead of alanine) was generated by site saturation mutagenesis for all residues of SEQ ID NO: 1. Another pool of variants of Seq 1 with H848 was generated by site saturation mutagenesis for all residues. Screening was then performed to identify SEQ ID NO: 1 variants with improved cleavage efficiency compared to fusion proteins containing SEQ ID NO: 1 (Screening 1 in FIG. 2) or variants with improved CREATE fusion editing efficiency compared to Seq 1 or fusion proteins containing Seq 1 (Screening 2 in FIG. 2). See, e.g., U.S. Pat. No. 11,268,078.

[0234] After identification of candidates for improved cleavage efficiency, variants are introduced into MAD2019-H848A (Figure 2) for further evaluation. A second screening round to identify MAD2019-H848A variant polypeptides with improved CREATE fusion editing, generated by site saturation mutagenesis for all residues. The pool of candidates from both screens (the collection of candidates in Figure 2) is further validated to measure the improvement of CREATE fusion editing through multiple assays (e.g., validation). Table 2 provides a list of MAD2019-H848A variants that showed improved CREATE fusion editing compared to MAD2019-H848A in at least one assay (e.g., assay T21, assay T22, assay T23, assay T24 in Figure 2).

[0235] Screening for cleavage efficiency involves detecting fluorescence generated from expression of a single copy of synthetic green fluorescent protein (GFP) randomly integrated into the genome of the cells. If no cleavage occurs, the GFP signal is similar to GFP expression from control cells grown under similar conditions. If a double-stranded break is generated that interrupts normal expression or activity of GFP, a reduction in GFP signal is observed. However, if precise editing occurs, GFP can be converted to emit blue wavelength fluorescence (BFP), which can be detected and quantified. See, e.g., Heim et al., Proc. Natl. Sci. USA 91:12501-12504, 1994; and Glaser et al., Mol. Ther. Nucleic Acids 5:e334, 2016.

[0236] Table 2. MAD2019-H848A variant polypeptides with improved CREATE fusion editing in at least one assay compared to MAD2019-H848A (SEQ ID NO: 1). The MAD2019-H848A polypeptide variant column indicates the amino acid residue change and the location of the change for each variant. For example, T67G refers to a change from threonine (T) to glycine (G) at position 67 of SEQ ID NO: 1. Values ​​in the Assay column refer to fold activity compared to SEQ ID NO: 1, with a value of 1.0 referring to activity equal to SEQ ID NO: 1. Assay refers to the guide sequences provided in Table 3.

[0237] [Table 2]

[0238] Example 2. Vector constructs and guide expression plasmids for MADzyme MAD2019 variant polypeptides were cloned and expressed in HEK293T (human kidney) cells under the control of the CMV promoter. MAD2019 variant polypeptides were also cloned and expressed in induced pluripotent stem cells (PGP168) under the EF1-alpha promoter. The HEK293T cell line contained a single copy of synthetic green fluorescent protein (GFP) randomly integrated into its genome. For the CREATE fusion editing assay, MAD2019 polypeptides and MAD2019-H848A polypeptide variants were fused with reverse transcriptase and expressed as a single fusion construct.

[0239] Guide RNAs targeting the GFP locus were cloned under the control of the human U6 promoter with a single guide RNA (sgRNA) scaffold (SEQ ID NO: 24) and a guide sequence located at the 5' end of each sgRNA (see Table 3). CREATE fusion homology arms were located at the 3' end of each sgRNA (see Table 3) when the CREATE fusion editing assay was performed with an additional RNA stabilizing sequence element (e.g., SEQ ID NO: 25) at the 3' end of the final sequence followed by a transcription terminator (e.g., 5'-TTTTTTT-3').

[0240] Table 3. Guide and homology arm sequences used in the CREATE fusion assay. PAM refers to protospacer adjacent motif.

[0241] [Table 3]

[0242] Example 3. Transfection in HEK293T cells 1 μL of PolyFect™ transfection reagent was mixed with a plasmid (50 ng) expressing the guide or pUC19 as a negative control and a plasmid (50 ng) expressing the MAD2019-H848A variant polypeptide fused to M-MLV reverse transcriptase (see Table 2) and diluted in 35 μL of OptiMem™. This mixture was added to 25000 TrypLE monoclonalized HEK293T host cells containing a synthetic GFP target locus in 100 μL of DMEM (Dulbecco's modified Eagle's medium) for reverse transfection.

[0243] Six days after transfection, HEK293T cells were harvested and analyzed using a flow cytometer to detect depletion of GFP signal, evidence of double-strand breaks that prevented GFP expression, or BFP signal, evidence of precise genome editing (see Figure 3A).

[0244] Example 4. Transfection in induced pluripotent stem cells 0.75 μL of Lipofectamine™ Stem Transfection Reagent was mixed with a plasmid (25 ng) expressing the guide or pUC19 as a negative control, and a plasmid (25 ng) expressing the MAD2019-H848A variant polypeptide fused to M-MLV reverse transcriptase (see Table 2) and diluted in OptiMem™. Forward transfections were performed on pre-plated induced pluripotent stem cells.

[0245] Six days after transfection, the induced pluripotent stem cells were harvested and analyzed using a flow cytometer to detect depletion of GFP signal, evidence of double-strand breaks that prevented GFP expression, or BFP signal, evidence of precise genome editing (see Figure 3B).

[0246] Example 5. Vector constructs and guide expression plasmids for MADzymes A fusion protein of MAD2019-H848A or a MAD2019-H848A polypeptide variant listed in Table 2 with Schizosaccharomyces pombe reverse transcriptase Tf1 (SEQ ID NO: 12) or Tf1 variant Tf1-D364N (SEQ ID NO: 13) was cloned into a plasmid under the control of a CMV promoter for expression in mammals.

[0247] Guide RNAs targeting the GFP locus were cloned under the control of the human U6 promoter with a single guide RNA (sgRNA) scaffold (SEQ ID NO: 14) and a guide sequence located at the 5' end of each sgRNA (see Table 3). A CREATE fusion homology arm was located at the 3' end of each sgRNA (see Table 3).

[0248] The construct is intended to be introduced into HEK293T cells that contain a single copy of GFP randomly integrated into the cell's genome. Example 6. Transfection in HEK293T cells Two groups of plasmid sets are prepared and transfected into HEK293T cells.

[0249] The first group contained plasmids (50 ng) expressing the guide, or pUC19 as a negative control, and plasmids (50 ng) expressing MAD2019-H848A::Tf1 (SEQ ID NOs: 1 and 12, respectively), mixed with 1 μL of PolyFect™ transfection reagent and diluted in 35 μL of OptiMem™. This mixture was added to 25,000 TrypLE monoclonalized HEK293T host cells containing a synthetic GFP target locus in 100 μL of DMEM for reverse transfection.

[0250] The second group contained plasmids (50 ng) expressing the guide, or pUC19 as a negative control, and plasmids (50 ng) expressing MAD2019-H848A::Tf1-D364N (SEQ ID NOs: 1 and 13, respectively), mixed with 1 μL of PolyFect™ transfection reagent and diluted in 35 μL of OptiMem™. This mixture was added to 25,000 TrypLE monoclonalized HEK293T host cells containing a synthetic GFP target locus in 100 μL of DMEM for reverse transfection.

[0251] Six days after transfection, the induced pluripotent stem cells were harvested and analyzed using a flow cytometer to detect depletion of GFP signal, evidence of double-strand breaks that prevented GFP expression, or to detect BFP signal, evidence of precise genome editing (see Figure 4).

[0252] Example 7. Additional transfections in HEK293T cells Two additional groups of plasmid sets are prepared and transfected into HEK293T cells.

[0253] The first group contains 23 unique mixtures. Each mixture contains a plasmid expressing a guide (50 ng), or pUC19 as a negative control, and one of the 23 MAD2019-H848A polypeptide variants provided in Table 2::Tf1 (50 ng). Each of the 23 mixtures is independently mixed with 1 μL of PolyFect™ transfection reagent and diluted in 35 μL of OptiMem™. Each of the mixtures is added to a single independent aliquot of 25000 TrypLE monoclonalized HEK293T host cells containing a synthetic GFP target locus in 100 μL of DMEM for reverse transfection, resulting in 23 unique sets of plasmid / cell combinations.

[0254] The second group contains 23 unique mixtures. Each mixture contains a plasmid expressing a guide (50 ng), or pUC19 as a negative control, and one of the 23 MAD2019-H848A polypeptide variants provided in Table 2::Tf1-D364N (50 ng). Each of the 23 mixtures is independently mixed with 1 μL of PolyFect™ transfection reagent and diluted in 35 μL of OptiMem™. Each of the mixtures is added to a single independent aliquot of 25000 TrypLE monoclonalized HEK293T host cells containing a synthetic GFP target locus in 100 μL of DMEM for reverse transfection, resulting in 23 unique sets of plasmid / cell combinations.

[0255] Six days after transfection, the induced pluripotent stem cells were harvested and analyzed using a flow cytometer to detect depletion of GFP signal, evidence of double-stranded breaks that prevented GFP expression, or BFP signal, evidence of precise genome editing.

[0256] Example 8. Generation of additional MAD2019-H848A variant polypeptides CREATE Fusion Enzyme 19 (CFE19) comprises a nuclear localization signal (SEQ ID NO: 15), MAD2019-H848A nickase (SEQ ID NO: 16), a linker (SEQ ID NO: 17), MLV reverse transcriptase (SEQ ID NO: 18) and a second nuclear localization signal (SEQ ID NO: 19). The full length amino acid sequence for CFE19 is provided in SEQ ID NO: 20.

[0257] CFE19 is subjected to editing to generate combinatorial MAD2019-H848A variant polypeptides. Table 4 provides a summary of the variants produced. Table 4. CFE19 variants

[0258] [Table 4]

[0259] Each of the CFE19 variants provided in Table 4 is expressed from a plasmid under the control of the EF1-alpha promoter after introduction into induced pluripotent stem cells. Expression of each CFE19 variant is monitored via the mCherry reporter linked by a T2A linker.

[0260] Forward transfection was performed on pre-plated induced pluripotent stem cells containing a single copy of the GFP coding sequence randomly inserted. Six days after transfection, the induced pluripotent stem cells were harvested and analyzed using a flow cytometer to detect depletion of GFP signal, evidence of double-strand breaks that prevented GFP expression, or BFP signal, evidence of precise genome editing (see Figure 5). Notably, CFE19v3 showed improved overall performance for multiple targets.

Claims

1. A MAD2019-H848A variant polypeptide comprising an amino acid sequence at least 90% identical or similar to SEQ ID NO:1, wherein the MAD2019-H848A variant polypeptide comprises an alanine at position 848 according to SEQ ID NO:

1.

2. 2. The MAD2019-H848A variant polypeptide of claim 1, comprising a V1143T amino acid substitution compared to SEQ ID NO:

1.

3. 2. The MAD2019-H848A variant polypeptide of claim 1, comprising a L500R amino acid substitution, a D700A amino acid substitution, a D701P amino acid substitution, a K720S amino acid substitution, a I1142K amino acid substitution, and a V1143T amino acid substitution compared to SEQ ID NO:

1.

4. 2. The MAD2019-H848A variant polypeptide of claim 1, comprising a L500R amino acid substitution, a D700A amino acid substitution, a D701P amino acid substitution, a K720S amino acid substitution, and a V1143T amino acid substitution compared to SEQ ID NO:

1.

5. 2. The MAD2019-H848A variant polypeptide of claim 1, comprising a L500K amino acid substitution and a V1143T amino acid substitution compared to SEQ ID NO:

1.

6. 2. The MAD2019-H848A variant polypeptide of claim 1, comprising a S409R amino acid substitution, a L500K amino acid substitution, and a V1143T amino acid substitution compared to SEQ ID NO:

1.

7. 2. The MAD2019-H848A variant polypeptide of claim 1, comprising a V1143T amino acid substitution and a A1221H amino acid substitution compared to SEQ ID NO:

1.

8. 2. The MAD2019-H848A variant polypeptide of claim 1, comprising a L500K amino acid substitution, a V1143T amino acid substitution, and an A1221H amino acid substitution compared to SEQ ID NO:

1.

9. 2. The MAD2019-H848A variant polypeptide of claim 1, comprising a L500K amino acid substitution, a I1142R amino acid substitution, a V1143T amino acid substitution, and a A1221H amino acid substitution compared to SEQ ID NO:

1.

10. 2. The MAD2019-H848A variant polypeptide of claim 1, comprising a L500K amino acid substitution, a D1139N amino acid substitution, a V1143T amino acid substitution, and an A1221H amino acid substitution compared to SEQ ID NO:

1.

11. 2. The MAD2019-H848A variant polypeptide of claim 1, comprising a L500K amino acid substitution, a V1143T amino acid substitution, an A1221H amino acid substitution, and a K1285R amino acid substitution compared to SEQ ID NO:

1.

12. 2. The MAD2019-H848A variant polypeptide of claim 1, comprising an amino acid substitution selected from the group consisting of: T67G, S409R, L500K, L500R, G578F, L624Q, N669S, D700A, D701P, D701N, D701T, K720S, L1110R, D1139N, I1142R, I1142K, V1143T, A1221H, K1285R, A1321R, A1321K, S1136Q, and A1139R compared to SEQ ID NO:

1.

13. 13. The MAD2019-H848A variant polypeptide of any one of claims 1 to 12, further comprising at least one nuclear localization signal.

14. A nucleic acid sequence encoding the MAD2019-H848A variant polypeptide of any one of claims 1 to 13.

15. A nucleoprotein complex comprising a MAD2019-H848A variant polypeptide according to any one of claims 1 to 13 and a nucleic acid molecule.

16. The nucleoprotein complex of claim 15, wherein the nucleic acid molecule comprises: (a) a guide; (b) a homology arm; or (c) a guide and a homology arm.

17. The nucleoprotein complex of claim 15 or 16, wherein the nucleic acid molecule is an RNA molecule.

18. 14. A eukaryotic cell comprising the MAD2019-H848A variant polypeptide of any one of claims 1 to 13.

19. A eukaryotic cell comprising the nucleic acid sequence of claim 14.

20. A eukaryotic cell comprising a nucleoprotein complex according to any one of claims 15 to 17.

21. A fusion protein comprising the MAD2019-H848A variant polypeptide of any one of claims 1 to 12.

22. 22. The fusion protein of claim 21 further comprising a reverse transcriptase.

23. 23. The fusion protein of claim 22, wherein the reverse transcriptase is a Tf1 reverse transcriptase comprising an amino acid sequence at least 90% identical or similar to SEQ ID NO:

12.

24. 24. The fusion protein of claim 23, wherein the Tf1 reverse transcriptase comprises a D364N amino acid substitution compared to SEQ ID NO:

12.

25. 24. The fusion protein of claim 23, wherein the Tf1 reverse transcriptase comprises SEQ ID NO:

13.

26. 23. The fusion protein of claim 22, wherein the reverse transcriptase is derived from a reverse transcriptase selected from the group consisting of HIV-1 (human immunodeficiency virus) reverse transcriptase, M-MLV (Moloney murine leukemia virus) reverse transcriptase, and AMV (avian myeloblastosis virus) reverse transcriptase.

27. The fusion protein of any one of claims 21 to 26, further comprising at least one nuclear localization signal.

28. A fusion protein according to any one of claims 21 to 27, comprising a linker amino acid sequence.

29. 23. The fusion protein of claim 22, comprising a linker amino acid sequence located between the MAD2019-H848A variant polypeptide and the reverse transcriptase.

30. A fusion protein comprising Tf1 reverse transcriptase, the amino acid sequence of which is at least 90% identical or similar to SEQ ID NO:

12.

31. 31. The fusion protein of claim 30, wherein the Tf1 reverse transcriptase comprises a D362N amino acid substitution compared to SEQ ID NO:

12.

32. 31. The fusion protein of claim 30, wherein the Tf1 reverse transcriptase comprises SEQ ID NO:

13.

33. The fusion protein of any one of claims 30 to 32, further comprising a nickase.

34. The fusion protein of claim 33, wherein the nickase is a CRISPR-Cas nickase.

35. The fusion protein of claim 34, wherein the CRISPR-Cas nickase is a type I CRISPR-Cas nickase.

36. The fusion protein of claim 34, wherein the CRISPR-Cas nickase is a type II CRISPR-Cas nickase.

37. The fusion protein of claim 34, wherein the CRISPR-Cas nickase is a type III CRISPR-Cas nickase.

38. The fusion protein of claim 34, wherein the CRISPR-Cas nickase is a type IV CRISPR-Cas nickase.

39. The fusion protein of claim 34, wherein the CRISPR-Cas nickase is a type V CRISPR-Cas nickase.

40. The fusion protein of claim 34, wherein the CRISPR-Cas nickase is a type VI CRISPR-Cas nickase.

41. 35. The fusion protein of claim 34, wherein the CRISPR-Cas nickase is a Cas9 nickase.

42. 35. The fusion protein of claim 34, wherein the CRISPR-Cas nickase is a MAD2019 polypeptide.

43. The fusion protein of claim 34, wherein the CRISPR-Cas nickase is a MAD2019-H848A polypeptide.

44. 35. The fusion protein of claim 34, wherein the CRISPR-Cas nickase is a MAD2019-H848A variant polypeptide.

45. 33. The fusion protein of any one of claims 30 to 32, further comprising a MAD2019-H848A variant polypeptide comprising an amino acid sequence at least 90% identical or similar to SEQ ID NO:1, wherein said MAD2019-H848A nickase comprises an alanine at position 848 according to SEQ ID NO:

1.

46. 46. ​​The fusion protein of claim 45, wherein the MAD2019-H848A variant polypeptide comprises a V1143T amino acid substitution compared to SEQ ID NO:

1.

47. 46. ​​The fusion protein of claim 45, wherein the MAD2019-H848A variant polypeptide comprises a L500R amino acid substitution, a D700A amino acid substitution, a D701P amino acid substitution, a K720S amino acid substitution, a I1142K amino acid substitution, and a V1143T amino acid substitution compared to SEQ ID NO:

1.

48. 46. ​​The fusion protein of claim 45, wherein the MAD2019-H848A variant polypeptide comprises a L500R amino acid substitution, a D700A amino acid substitution, a D701P amino acid substitution, a K720S amino acid substitution, and a V1143T amino acid substitution compared to SEQ ID NO:

1.

49. 46. ​​The fusion protein of claim 45, wherein the MAD2019-H848A variant polypeptide comprises a L500K amino acid substitution and a V1143T amino acid substitution compared to SEQ ID NO:

1.

50. 46. ​​The fusion protein of claim 45, wherein the MAD2019-H848A variant polypeptide comprises a S409R amino acid substitution, a L500K amino acid substitution, and a V1143T amino acid substitution compared to SEQ ID NO:

1.

51. 46. ​​The fusion protein of claim 45, wherein the MAD2019-H848A variant polypeptide comprises a V1143T amino acid substitution and an A1221H amino acid substitution compared to SEQ ID NO:

1.

52. 46. ​​The fusion protein of claim 45, wherein the MAD2019-H848A variant polypeptide comprises a L500K amino acid substitution, a V1143T amino acid substitution, and an A1221H amino acid substitution compared to SEQ ID NO:

1.

53. 46. ​​The fusion protein of claim 45, wherein the MAD2019-H848A variant polypeptide comprises a L500K amino acid substitution, a I1142R amino acid substitution, a V1143T amino acid substitution, and a A1221H amino acid substitution compared to SEQ ID NO:

1.

54. 46. ​​The fusion protein of claim 45, wherein the MAD2019-H848A variant polypeptide comprises a L500K amino acid substitution, a D1139N amino acid substitution, a V1143T amino acid substitution, and an A1221H amino acid substitution compared to SEQ ID NO:

1.

55. 46. ​​The fusion protein of claim 45, wherein the MAD2019-H848A variant polypeptide comprises a L500K amino acid substitution, a V1143T amino acid substitution, an A1221H amino acid substitution, and a K1285R amino acid substitution compared to SEQ ID NO:

1.

56. 46. ​​The fusion protein of claim 45, wherein the MAD2019-H848A variant polypeptide comprises an amino acid substitution selected from the group consisting of T67G, S409R, L500K, L500R, G578F, L624Q, N669S, D700A, D701P, D701N, D701T, K720S, L1110R, D1139N, I1142R, I1142K, V1143T, A1221H, K1285R, A1321R, A1321K, S1136Q, and A1139R compared to SEQ ID NO:

1.

57. The fusion protein of any one of claims 30 to 56, further comprising at least one nuclear localization signal.

58. A fusion protein according to any one of claims 30 to 57, comprising a linker amino acid sequence.

59. 57. The fusion protein of any one of claims 45 to 56, comprising a linker amino acid sequence located between the MAD2019-H848A variant polypeptide and the Tf1 reverse transcriptase.

60. 34. The fusion protein of claim 33, comprising a linker amino acid sequence located between the nickase and the Tf1 reverse transcriptase.

61. A nucleic acid sequence encoding a fusion protein according to any one of claims 21 to 60.

62. A nucleoprotein complex comprising a fusion protein according to any one of claims 21 to 60 and a nucleic acid molecule.

63. The nucleoprotein complex of claim 62, wherein the nucleic acid molecule comprises: (a) a guide; (b) a homology arm; or (c) a guide and a homology arm.

64. The nucleoprotein complex of claim 62 or 63, wherein the nucleic acid molecule is an RNA molecule.

65. A eukaryotic cell comprising a fusion protein according to any one of claims 21 to 60.

66. 62. A eukaryotic cell comprising the nucleic acid sequence of claim 61.

67. A eukaryotic cell comprising a nucleoprotein complex according to any one of claims 62 to 64.

68. 1. A method of providing a MAD2019-H848A variant polypeptide to a cell, comprising: (a) obtaining a cell; and (b) providing said cell with a MAD2019-H848A variant polypeptide according to any one of claims 1 to 13, or a nucleic acid molecule encoding said MAD2019-H848A variant polypeptide. The method includes:

69. 1. A method for providing a fusion protein to a cell, comprising: (a) obtaining a cell; and (b) providing said cell with a fusion protein according to any one of claims 21 to 60, or a nucleic acid molecule encoding said fusion protein. The method includes:

70. 70. The method of claim 68 or 69, further comprising transfecting the cell with a nucleic acid molecule encoding: (a) a guide; (b) a homology arm; or (c) a guide and a homology arm.

71. 70. The method of claim 69, wherein the guide is an RNA molecule that forms a nucleoprotein complex with the MAD2019-H848A variant polypeptide in the cell.

72. 72. The method of Claim 71, wherein the guide comprises a sequence complementary to a target nucleic acid molecule in the cell.

73. 73. The method of Claim 72, wherein said nucleoprotein complex edits the target nucleic acid molecule in the cell.

74. 71. The method of claim 70, wherein the guide is an RNA molecule that forms a nucleoprotein complex with the fusion protein in the cell.

75. 75. The method of Claim 74, wherein the guide comprises a sequence complementary to a target nucleic acid molecule in the cell.

76. 76. The method of Claim 75, wherein said nucleoprotein complex edits the target nucleic acid molecule in the cell.

77. 77. The method of any one of claims 72, 73, 75, and 76, wherein the target nucleic acid molecule is located within the nuclear genome of the cell.

78. 77. The method of any one of claims 72, 73, 75, and 76, wherein the target nucleic acid molecule is located within the mitochondrial genome of the cell.

79. 77. The method of any one of claims 68 to 76, wherein said providing comprises a method selected from the group consisting of polyethylene glycol mediated transformation, biolistic transformation, liposome mediated transfection, viral transduction, use of one or more delivery particles, microinjection, and electroporation.

80. 1. A method for editing at least one eukaryotic cell, comprising: (a) (i) administering to said at least one eukaryotic cell a MAD2019-H848A variant polypeptide according to any one of claims 1 to 13, or a nucleic acid molecule encoding said MAD2019-H848A variant polypeptide; and (ii) administering to the at least one eukaryotic cell a guide RNA, the guide RNA comprising a nucleic acid sequence complementary to a target nucleic acid molecule in the genome of the eukaryotic cell, or a nucleic acid molecule encoding the guide RNA. To introduce; the MAD2019-H848A variant polypeptide and the guide RNA form a nucleoprotein complex in the at least one eukaryotic cell, the nucleoprotein complex cleaving one strand of the target nucleic acid molecule and at least one edit being made in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule; and (b) identifying at least one eukaryotic cell containing said at least one edit. The method includes:

81. 1. A method for editing at least one eukaryotic cell, comprising: (a) (i) administering to said at least one eukaryotic cell a fusion protein according to any one of claims 21 to 60, or a nucleic acid molecule encoding said fusion protein; and (ii) administering to the at least one eukaryotic cell a guide RNA, the guide RNA comprising a nucleic acid sequence complementary to a target nucleic acid molecule in the genome of the eukaryotic cell, or a nucleic acid molecule encoding the guide RNA. To introduce; the fusion protein and the guide RNA form a nucleoprotein complex in the at least one eukaryotic cell, the nucleoprotein complex cleaving one strand of the target nucleic acid molecule and at least one edit being made in the target nucleic acid molecule compared to a control version of the target nucleic acid molecule; and (b) identifying at least one eukaryotic cell containing said at least one edit. The method includes:

82. 82. The method of Claim 80 or 81, wherein step (a) of the method further comprises introducing at least one homology arm, or a nucleic acid molecule encoding said homology arm, into said eukaryotic cell, wherein said at least one homology arm comprises a nucleic acid sequence comprising said at least one edit.

83. The method of any one of claims 80 to 82, wherein the at least one edit comprises an insertion.

84. 83. The method of any one of claims 80 to 82, wherein the at least one edit comprises a deletion.

85. 83. The method of any one of claims 80 to 82, wherein the at least one edit comprises a substitution.

86. 83. The method of any one of claims 80 to 82, wherein the at least one edit comprises a substitution of a single nucleotide.

87. 87. The method of claim 86, wherein said substitution of a single nucleotide comprises a transition.

88. 87. The method of claim 86, wherein said substitution of a single nucleotide comprises a transversion.

89. A guide RNA (gRNA) comprising a scaffold region having a nucleic acid sequence at least 80% identical to SEQ ID NO:

24.

90. 90. The gRNA of Claim 89, wherein the nucleic acid sequence is at least 90% identical to SEQ ID NO:

24.

91. 90. The gRNA of Claim 89, wherein the nucleic acid sequence is at least 95% identical to SEQ ID NO:

24.

92. 90. The gRNA of Claim 89, wherein the nucleic acid sequence is 100% identical to SEQ ID NO:

24.

93. A nucleoprotein complex comprising a fusion protein and a gRNA according to any one of claims 89 to 92.

94. The nucleoprotein complex of claim 93, wherein the fusion protein comprises a MAD2019-H848A variant polypeptide.

95. 95. The nucleoprotein complex of claim 93 or 94, wherein the fusion protein comprises a reverse transcriptase.

96. A nucleoprotein complex comprising a MAD2019-H848A variant polypeptide and a gRNA according to any one of claims 89 to 92.

97. A nucleoprotein complex comprising a nickase and a gRNA according to any one of claims 89 to 92.

98. A nucleoprotein complex comprising a gRNA according to any one of claims 89 to 92.

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