Reverse transcription-mediated gene editing systems and uses thereof

Abstract

A gene editing system comprising (a) a fusion polypeptide comprising a CRISPR nuclease and a reverse transcriptase, or a nucleic acid encoding the fusion polypeptide, and (b) an RNA molecule comprising a guide RNA and a reverse transcription donor RNA, or a nucleic acid encoding the RNA molecule. Also provided herein are methods of using the gene editing system for modifying target genes of interest.

Description

Attorney Docket No.: 063586-523001WO REVERSE TRANSCRIPTION-MEDIATED GENE EDITING SYSTEMS AND USES THEREOF CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing dates of U.S. Provisional Application No.63 / 581,354, filed September 8, 2023, the content of which is incorporated by reference herein in its entire. SEQUENCE LISTING The instant application contains a Sequence Listing which has been filed electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on September 6, 2024, is named “Sequence_Listing_ST26063586-523001WO” and is 236.5 KB in size. BACKGROUND Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR- associated (Cas) genes, collectively known as CRISPR-Cas or CRISPR / Cas systems, are adaptive immune systems in archaea and bacteria that defend particular species against foreign genetic elements. Reverse transcriptases (RTs) are enzymes that generate a strand of DNA that is complementary to an RNA or DNA template. The combination of reverse transcriptases and CRISPR / Cas systems has shown great potentials in genetic editing. CRISPR-guided reverse transcription allows for introduction of desired nucleotide substitutions at a genomic site. It is therefore of great interest to develop efficient and accurate reverse transcriptase- CRISPR gene editing systems for use in disease treatment. SUMMARY OF THE PRESENT DISCLOSURE The present disclosure provides reverse transcriptase-CRISPR-mediated gene editing systems, which successfully introduced designed nucleotide substitutions into target genetic sites. In some embodiments, the gene editing systems disclosed herein involve a fusion polypeptide comprising a CRISPR nuclease fragment and a reverse transcriptase (RT) fragment, and optionally one or more nuclear localization sequences (NLS) and / or peptide linkers. The CRISPR nuclease polypeptide can be genetically engineered to possess advantageous enzymatic activities (e.g., high indel activities and / or DNA cleavage activities,Attorney Docket No.: 063586-523001WO and precise gene editing as designed). Accordingly, the gene editing systems provided herein would be expected to show superior effectiveness in inserting desired base substitutions at a genomic site of interest. Accordingly, one aspect of the present disclosure features a gene editing system comprising: (a) a fusion polypeptide comprising a CRISPR nuclease polypeptide and a reverse transcriptase (RT) polypeptide, or a first nucleic acid encoding the fusion polypeptide; and (b) an RNA molecule comprising a guide RNA (gRNA) and a reverse transcription donor RNA (RT donor RNA), or a second nucleic acid encoding the RNA molecule. The CRISPR nuclease polypeptide may comprise the amino acid sequence of SEQ ID NO:1. Alternatively, it may be a variant of SEQ ID NO:1. The gRNA in the RNA molecule of (b) comprises a scaffold sequence recognizable by the CRISPR nuclease and a spacer sequence specific to a target sequence within a genomic site of interest, the target sequence being upstream to a protospacer adjacent motif (PAM). The RT donor RNA in the RNA molecule of (b) comprises a primer binding site (PBS) and a template sequence. In some embodiments, the fusion polypeptide may further comprise one or more nuclear localization sites (NLS), which may be located upstream or downstream to the CRISPR nuclease polypeptide, the RT polypeptide, or both. In some examples, the fusion polypeptide, from N-terminus to C-terminus, may comprise a first NLS, the CRISPR nuclease polypeptide, the RT polypeptide, and a second NLS. Alternatively or in addition, the fusion polypeptide further comprises a peptide linker between the CRISPR nuclease polypeptide and the RT polypeptide. In some embodiments, the CRISPR nuclease polypeptide in the gene editing system disclosed herein can be a variant of SEQ ID NO:1. In some instances, such a variant may comprise: (i) one or more mutations in the HNH nuclease domain or in the RuvC nuclease domain of SEQ ID NO:1 that reduce or eliminate the nuclease activity thereof; (ii) one or more arginine and / or lysine substitutions, optionally one or more arginine substitutions; or (iii) a combination of (i) and (ii). For example, the CRISPR nuclease polypeptide may comprise one or more mutations in the HNH nuclease domain at positions E425, H426, and / or N449 relative to SEQ ID NO:1. In some examples, the mutation is at position E425. In some examples, the mutation at E425 is an amino acid substitution, e.g., E425A, E425G, E425L, or E425S. In some examples, the mutation at H426 is an amino acid substitution, e.g., H426A, H426G, H426L, or H426S. InAttorney Docket No.: 063586-523001WO some examples, the mutation at N449 is an amino acid substitution, e.g., N449A, N449G, N449L, or N449S. Alternatively, the CRISPR nuclease polypeptide comprises one or more mutations in the RuvC nuclease domain at positions D10 (e.g., of D10A, D10G, D10L, or D10S), E362 (E362A, E362G, E362L, or E362S), and / or D487 (e.g., D487A, D487G, D487L, or D487S) relative to SEQ ID NO:1. In some instances, the mutation is at position T549, for example, T549P. In some examples, the CRISPR nuclease polypeptide may comprise one or more amino acid residue substitutions relative to SEQ ID NO: 1. In some instances, the amino acid residue substitutions may comprise arginine and / or lysine substitutions, optionally arginine substitutions. In some instances, the amino acid residue substitutions such as arginine / lysine substitutions may be located in a bridge helix (BH) domain, a nucleic acid recognition (REC) domain, a phosphate lock loop (PLL) domain, a wedge (WED) domain, and / or a PAM- interacting (PID) domain of the nuclease. In some instances, the CRISPR nuclease polypeptide contains up to 20 arginine and / or lysine substitutions relative to the reference CRISPR nuclease. In some examples, the engineered CRISPR nuclease polypeptide contains up to 15 arginine and / or lysine substitutions relative to the reference CRISPR nuclease. In specific examples, the one or more arginine and / or lysine substitutions are at positions A65, V85, L134, V117, A657, N691, S695, D707, S925, and / or E715 in SEQ ID NO:1. Exemplary combinations of arginine and / or lysine substitutions may be at the positions listed in Table 5 below. Alternatively or in addition, the amino acid residue substitutions may comprise or further comprise one or more non-arginine / lysine substitutions (i.e., the native amino acid residue is replaced by an amino acid residue that is not arginine or lysine). In one specific example, the non-arginine / lysine substitution can be at position T549 of SEQ ID NO: 1, for example, T549P. Positions for other non-arginine / lysine substitutions can be found in Table 28. In some examples, the CRISPR nuclease polypeptide provided herein may comprise a combination of mutations set forth in any one of Tables 27-29. In one specific example, the CRISPR nuclease polypeptide comprises arginine and / or lysine substitutions (optionally arginine substitutions) at positions A65 (e.g., A65R), A657 (e.g., A657R), and S925 (e.g., S925R), and an amino acid substitution at position E425 (e.g., E425A) in SEQ ID NO: 1. In another specific example, the CRISPR nuclease polypeptide comprises animo acidAttorney Docket No.: 063586-523001WO substitutions at positions A65 (e.g., A65R), V85 (e.g., V85R), L134 (e.g., L134R), E425 (e.g., E425A), T549 (e.g., T549P), A657 (e.g., A657R), and S925 (e.g., S925R) relative to SEQ ID NO: 1. In yet another specific example, the CRISPR nuclease polypeptide comprises animo acid substitutions at positions A65 (e.g., A65R), V85 (e.g., V85R), L134 (e.g., L134R), E425 (e.g., E425A), A657 (e.g., A657R), D707 (e.g., D707R), and S925 (e.g., S925R) relative to SEQ ID NO: 1. Any of the CRISPR nuclease polypeptide for use in the gene editing system provided herein may comprise an amino acid sequence at least 90% identical to SEQ ID NO: 1. In some examples, the CRISPR nuclease polypeptide may comprise an amino acid sequence at least 95% identical to SEQ ID NO: 1. In further examples, the CRISPR nuclease polypeptide may comprise an amino acid sequence at least 98% identical to SEQ ID NO: 1. Exemplary CRISPR nuclease polypeptides are provided in Table 1 and Table 6, each of which is within the scope of the present disclosure. Further, the CRISPR nuclease polypeptides provided herein may comprise any of the mutations set forth in any of Tables 5 and 26-29 relative to SEQ ID NO: 1. In any of the gene editing systems, the RT polypeptide can be a reverse transcriptase provided in Table 10, for example, Moloney Murine Leukemia Virus (MMLV)-RT or a variant thereof. In some examples, the MMLV-RT comprises the amino acid sequence of SEQ ID NO: 49. Exemplary fusion polypeptides comprising the CRISPR nuclease and the RT are provided in Table 11 and Table 12, each of which is within the scope of the present disclosure. In some embodiments, the gene editing system disclosed herein comprises the fusion polypeptide. Alternatively, the gene editing system comprises the first nucleic acid encoding the fusion polypeptide. Such a nucleic acid may be located on a vector, which optionally is a viral vector. Alternatively, the nucleic acid can be a messenger RNA (mRNA). The RNA molecule in any of the gene editing systems comprise a gRNA and an RT donor RNA. The gRNA comprises a spacer sequence, which may be 15-30-nucleotide in length. In some examples, the spacer sequence is about 15-20-nucleotide in length. The spacer sequence is specific to a target sequence within a genomic site of interest, the target sequence being upstream to a protospacer adjacent motif (PAM). In some embodiments, the PAM is 5’-NRV-3’, in which N represents any nucleotide, R represents A or G, and V represents A, C, or G. In some examples, the PAM is 5’-NRR’3’ (e.g., 5’-NRG-3’ or 5’-Attorney Docket No.: 063586-523001WO NGG-3’), for example 5’-CAG-3'. In specific examples, the PAM is 5’-RRR-3’, for example, 5’-AAA-3’ or 5’-GAG-3’. The gRNA also comprises a scaffold sequence, which is recognizable by the CRISPR nuclease polypeptide disclosed herein. In some examples, the scaffold sequence comprises a nucleotide sequence at least 70% (e.g., at least 75%, at least 80%, or at least 85%) identical to SEQ ID NO:2. In some examples, the scaffold sequence comprises the nucleotide sequence of SEQ ID NO:2. In other examples, the scaffold sequence may be a truncated variant of SEQ ID NO: 2 having a truncation within residues 17-43 of SEQ ID NO: 2. Such a truncated variant may have 145-155 nucleotides in length. In one example, the truncated variant of SEQ ID NO: 2 comprises (e.g., consists of) the nucleotide sequence of SEQ ID NO: 80. In another example, the truncated variant of SEQ ID NO: 2 comprises (e.g., consists of) the nucleotide sequence of SEQ ID NO: 81. The RT donor RNA comprises a primer binding site (PBS) and a template sequence (a.k.a., RTT sequence). In some embodiments, the PBS in the RT donor RNA can be about 5- 50 nucleotides in length; for example, about 5-20 nucleotides in length. In one example, the PBS is 8-nucleotides in length. In other examples, the PBS is 12-nucleotides in length. In some examples, the PBS binds a PBS-targeting site that is adjacent to or overlaps with the target sequence. In some instances, the PBS-targeting site is adjacent to the 5’ of the PAM. For example, the 3’end nucleotide of the PBS-targeting site can be about 2-15 nucleotides upstream to the PAM. The template sequence in the RT donor RNA can be about 5-100 nucleotides in length, for example, about 15-25 nucleotides in length. In one example the template sequence is about 21 nucleotides in length. In some examples, the template sequence in the RT donor RNA is homologous to the genomic site of interest and comprises one or more nucleotide variations relative to the genomic site of interest. In some instances, at least one nucleotide variation is located within the target sequence. Alternatively or in addition, at least one nucleotide variation is located in the PAM. Any of the RNA molecules provided herein may further comprise a 3’ end extension and optionally a linker, which may be between the 3’ end extension and the rest of the molecule. Alternatively or in addition, the RNA molecule in the gene editing system disclosed herein may further comprise a 5’ end protection fragment, a 3’ protection fragment, one or more linkers, or a combination thereof. Such a protection fragment may form aAttorney Docket No.: 063586-523001WO secondary structure, for example, a hairpin, a pseudoknot, a circularization, or a triplex structure. In some examples, the RNA molecule of the gene editing system disclosed herein may comprise, from 5’ to 3’: the spacer sequence, the scaffold sequence, the template sequence, and the PBS. Alternatively, the RNA molecule may comprise, from 5’ to 3’: the spacer sequence, the scaffold sequence, the template sequence, the PBS, and the 3’ extension. In yet other examples, the RNA molecule may comprise, from 5’ to 3’: the spacer sequence, the scaffold sequence, the template sequence, the PBS, the linker, and the 3’ extension. In some embodiments, the gene editing system disclosed herein may comprise any of the RNA molecules disclosed herein. Alternatively, the gene editing system may comprise a nucleic acid encoding the RNA molecule. Such a nucleic acid may be located on a vector, which optionally can be a viral vector. In some instances, the gene editing system disclosed herein may comprise one or more lipid nanoparticles (LNPs) associated with the fusion polypeptide, the RNA molecule, or both. Alternatively, the gene editing system may comprise one or more viral vectors encoding the fusion polypeptide and / or the RNA molecule. In some examples, the one or more vectors may be viral vectors such as adeno-associated viral (AAV) vectors. Also provided herein is a pharmaceutical composition comprising the gene editing system disclosed herein, as well as a kit comprising the fusion polypeptide and / or the RNA molecule of the gene editing system. In other aspects, the present disclosure features a gene editing method, comprising delivering any of the gene editing systems disclosed herein to a host cell to edit a genomic site targeted by the gRNA of the gene editing system. In some embodiments, the host cell is cultured in vitro. In other embodiments, the host cell is located in a subject who needs the gene editing and method comprises administering the gene editing system to the subject via a suitable route. In addition, the present disclosure provides a fusion polypeptide, comprising any of the CRISPR nuclease polypeptides disclosed herein and a reverse transcriptase polypeptide as also disclosed herein. Exemplary fusion polypeptides are provided in Table 11 or Table 12. Also provided herein are nucleic acids encoding such fusion polypeptides. Examples are also provided in Table 11 or Table 12. In some instances, the nucleic acid can be a vector such as an expression vector. In other instances, the nucleic acid encoding the fusion polypeptide provided herein may be a messenger RNA (mRNA).Attorney Docket No.: 063586-523001WO In yet other aspects, provided herein is a nucleic acid (e.g., an RNA molecule) comprising a truncated scaffold sequence relative to SEQ ID NO:2. Such a truncated scaffold sequence may have 145-155 nucleotides in length. In some instances, the truncated scaffold sequence may comprise a truncation within residues 17-43 of SEQ ID NO: 2. In some instances, the truncated scaffold sequence comprises a nucleotide sequence at least 78% identical to SEQ ID NO: 80. In one example, the truncated scaffold sequence comprises (e.g., consists of) the nucleotide sequence of SEQ ID NO: 80. In another example, the truncated scaffold sequence comprises (e.g., consists of) the nucleotide sequence of SEQ ID NO: 81. In some instances, any of the nucleic acids disclosed above may further comprise a spacer sequence. In addition, the nucleic acid may further comprise a reverse transcription donor RNA (RT donor RNA), which comprises a template sequence and a primer binding site (PBS). In some instances, the nucleic acid may further comprise a 3’ extension, a 5’ protection fragment, or both. In one example, the nucleic acid provided herein may comprise, from 5’ to 3’: a spacer sequence, any of the truncated scaffold sequences provided herein (e.g., SEQ ID NO: 80 or 81), a template sequence, and a primer binding site (PBS). In another example, the nucleic acid provided herein may comprise, from 5’ to 3’: a spacer sequence, any of the truncated scaffold sequences provided herein (e.g., SEQ ID NO: 80 or 81), a template sequence, and a primer binding site (PBS), and a 3’ extension. In yet another example, the nucleic acid provided herein may comprise, from 5’ to 3’: a spacer sequence, any of the truncated scaffold sequences provided herein (e.g., SEQ ID NO: 80 or 81), a template sequence, and a primer binding site (PBS), a linker, and a 3’ extension. The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawing in combination with the detailed description of specific embodiments presented herein. FIG.1 is a diagram showing percentages of NGS reads comprising indels at sixAttorney Docket No.: 063586-523001WO genetic loci as indicated in the presence or absence of the CRISPR nuclease of SEQ ID NO: 1. FIGs.2A-2D include gel images and quantification of nuclease activity. FIG.2A: Gel image captured using an 800 nm channel showing in vitro cleavage of the target strand (labelled on the 5’ end with an IR800 dye) of the target DNA substrate by the reference CRISPR nuclease, putative HNH-knockout nickases, or putative RuvC-knockout nickases. FIG.2B: Gel image captured using a 700 nm channel showing in vitro cleavage of the non- target strand (labelled on the 5’ end with an IR700 dye) of a target DNA substrate by the reference CRISPR nuclease, putative HNH-knockout nickases, or putative RuvC-knockout nickases. FIG.2C: Overlaid images captured using 800 nm and 700 nm channels of FIG.2A and FIG.2B. FIG.2D: Quantification of the percent of cleaved target and non-target DNA generated by the reference CRISPR nuclease, the putative HNH-knockout nickases, and the putative RuvC-knockout nickases tested. FIGs.3A- 3C include diagrams showing predicted secondary structures of scaffold sequences. FIG.3A: Reference Scaffold. FIG.3B: Truncated Reference Scaffold. FIG.3C: Scaffold 2. DETAILED DESCRIPTION OF THE INVENTION The present disclosure provides a gene editing system involving both a CRISPR nuclease polypeptide and a reverse transcriptase (RT) polypeptide, as well as a guide RNA, which directs gene editing at a desired genomic site, and an RT donor RNA, which serves as the RNA template for the RT polypeptide to synthesize DNA strands carrying desired base substitutions. In some embodiments, the gene editing system provided herein may comprise a fusion polypeptide comprising the CRISPR nuclease fragment and the RT fragment, or a nucleic acid encoding the fusion polypeptide. Alternatively or in addition, the gene editing system may comprise a single RNA molecule comprising the guide RNA and the RT donor RNA, or a nucleic acid encoding the single RNA molecule. The gene editing system provided herein has shown successful substitution of nucleotides at target sites. See Examples below. Such gene editing systems are expected to be effective in introducing desired nucleotide substitutions at genetic sites of interests, thereby achieving desired therapeutic effects (e.g., correcting genetic defects). The gene editing system provided herein can also be used in other areas, for example, in breeding and genomic functional studies of animals and plants.Attorney Docket No.: 063586-523001WO I. RT-CRISPR Mediated Gene Editing System In some aspects, provided herein is an RT-CRISPR mediated gene editing system, which involves at least two protein components, i.e., a CRISPR nuclease polypeptide and an RT polypeptide, and at least two RNA components, i.e., a guide RNA and an RT donor RNA. In specific embodiments, the two protein components can be located on a fusion polypeptide. Alternatively or in addition, the two RNA components may be located on a single RNA molecule. In some instances, the gene editing system may comprise the protein components and / or the RNA components. In other instances, the gene editing system may comprise nucleic acid(s) encoding the protein components, and / or nucleic acid(s) encoding the RNA components. In some instances, the RT-CRISPR mediated gene editing system comprises a CRISPR nuclease having nickase activity as disclosed herein. Such a gene editing system is expected to achieve precise gene editing at a desired genomic target site. A. Protein Components The gene editing systems provided herein involve at least two enzymes, a CRISPR nuclease and an RT. In some embodiments, the gene editing system comprises the two enzymes. In specific examples, the gene editing system may comprise a fusion polypeptide comprising the two enzyme components. Alternatively, the gene editing system may comprise one or more nucleic acids encoding the two enzyme components. For example, the gene editing system may comprise one or more expression vectors (e.g., viral vectors such as retroviral vectors, adenoviral vectors, or adeno-associated viral vectors) capable of expressing the CRISPR nuclease, the RT, or the fusion polypeptide comprising such. In other examples, the gene editing system may comprise one or more mRNA molecules coding for the CRISPR nuclease, the RT, or the fusion polypeptide comprising such. In some embodiments, the CRISPR nuclease polypeptide and the RT polypeptide as disclosed herein may form a complex, which may be a heterodimer of the two protein components via a dimerization domain (e.g., a leucine zipper), an antibody, a nanobody, or an aptamer. (i) CRISPR Nuclease PolypeptidesAttorney Docket No.: 063586-523001WO The CRISPR nuclease polypeptide for use in the gene editing systems disclosed herein may be a CRISPR nuclease comprising the amino acid sequence of SEQ ID NO:1 (the reference CRISPR nuclease), or a variant thereof. As used herein, the term “CRISPR nuclease” refers to an RNA-guided effector that is capable of binding a nucleic acid and introducing a single-stranded break or double-stranded break. A CRISPR nuclease typically comprises multiple functional domains, e.g., nuclease domains (e.g., RuvC and HNH), bridge helix (BH) domain, nucleic acid recognition (REC) domain, phosphate lock loop (PLL), wedge domain (WED), PAM-interacting domain (PID), or a combination thereof. As used herein, the term “domain” refers to a distinct functional and / or structural unit of a polypeptide. In some instances, a functional domain may be linear. In other instances, a functional domain can be discontinuous and conformational. In some embodiments, a domain may comprise a conserved amino acid sequence across different CRISPR nucleases. The reference CRISPR nuclease of SEQ ID NO:1 (see Table 1 below) is a CRISPR nuclease that comprises both a RuvC nuclease domain (located at residues 1-39, 323-363, and 530-655 of SEQ ID NO:1) and a HNH domain (located at residues 364-529 of SEQ ID NO:1). The RuvC nuclease domain and the HNH nuclease domain coordinate cleavage of the DNA strand adjacent to the 5’-NRV-3’, 5’-NRR-3', or 5’-RRR-3' motif, in which N represents any nucleotide, R represents A or G, and V represents A, C, or G. Positions D10, E362, and D587 are deemed the active sites in the RuvC domain and positions E425, H426, and N449 are deemed the active sites in the HNH domain. R329 and H338 may also be important for the nuclease activity of the RuvC domain. In addition to the nuclease domains, the reference CRISPR nuclease of SEQ ID NO:1 also includes a BH domain (residues 40-78 of SEQ ID NO:1), a REC domain (residues 79-322 of SEQ ID NO:1), a PLL domain (residues 656-670), a WED domain (residues 671-819), and a PID domain (residues 820-946). In some embodiments, the gene editing system disclosed herein comprises a variant CRISPR nuclease polypeptide derived from SEQ ID NO:1, e.g., a variant CRISPR nuclease polypeptide comprising one or more arginine or lysine substitutions, one or more mutations in one of the nuclease domains such as in the HNH nuclease domain or the RuvC nuclease domain, or a combination thereof, as relative to the reference CRISPR nuclease of SEQ ID NO:1. Such a protein component may form a complex with the gRNA(s) in the same gene editing system.Attorney Docket No.: 063586-523001WO Variants of CRISPR Nuclease Polypeptide In some embodiments, the CRISPR nuclease polypeptide in the gene editing system disclosed herein is a variant of the reference CRISPR nuclease of SEQ ID NO:1, e.g., via introducing one or more mutations to the reference CRISPR nuclease to modulate (e.g., enhance or reduce) one or more activities of the nuclease. As used herein, the term “variant CRISPR nuclease polypeptide” refers to a CRISPR nuclease polypeptide comprising an alteration, e.g., a substitution, insertion, deletion and / or fusion, at one or more residue positions, compared to the reference CRISPR nuclease (SEQ ID NO:1). The variant CRISPR nuclease polypeptides may comprise one or more mutations (e.g., arginine substitutions) relative to the reference CRISPR nuclease. In other instances, the variant CRISPR nuclease polypeptide may comprise one or more non-arginine / lysine substitutions (i.e., the amino acid residue in the reference CRISPR nuclease polypeptide of SEQ ID NO: 1 is replaced with a residue other than arginine or lysine). Alternatively or in addition, the variant CRISPR nuclease polypeptide may comprise one or more mutations in either the RuvC nuclease domain or the HNH nuclease domain. Such mutations may reduce or eliminate the nuclease activity of either the RuvC or the HNH nuclease domain, leading to a variant exhibiting nickase activity. The variant CRISPR nuclease polypeptides may share a high sequence homology relative to the reference CRISPR nuclease (e.g., at least 85% sequence identity). As used herein, the term “nickase” refers to an enzyme that cuts one strand of a double-stranded DNA at a specific recognition nucleotide sequence (e.g., the target sequence disclosed herein). A nickase may interact with one strand of the DNA duplex to produce DNA molecules that are cut at one strand (a.k.a., nicked). In some embodiments, a nickase is a variant of a CRISPR nuclease that comprises a deactivated HNH domain. In some embodiments, a nickase is a variant of a CRISPR nuclease that comprises a deactivated RuvC domain. The variant CRISPR nuclease polypeptides may share a high sequence homology relative to the reference CRISPR nuclease (e.g., at least 85% sequence identity). The variant CRISPR nuclease polypeptides provided herein are expected to possess advantageous features relative to the reference CRISPR nuclease, for example, exhibiting nickase activity and / or higher nuclease activity, etc. As such, the variant CRISPR nuclease polypeptides disclosed herein would be expected to exhibit improved gene editing relative toAttorney Docket No.: 063586-523001WO the reference CRISPR nuclease, e.g., higher efficiency and accuracy in gene editing involving strand replacement. In some embodiments, the variant CRISPR nuclease polypeptides provided herein, relative to the reference CRISPR nuclease SEQ ID NO:1, comprises one or more mutations in either the RuvC nuclease domain or the HNH nuclease domain (e.g., in the HNH nuclease domain) to reduce or eliminate the nuclease activity, and / or comprises one or more arginine and / or lysine substitutions to improve nuclease features suitable for use in gene editing. The variant CRISPR nuclease polypeptides provided herein are expected to exhibit one or more modulated activities (e.g., enhanced or reduced) relative to the reference CRISPR nuclease. As used herein, the term “activity” refers to a biological activity. In some embodiments, activity includes enzymatic activity, e.g., catalytic ability of an effector. For example, activity can include nuclease activity. In some embodiments, activity includes nickase activity. For example, the variant CRISPR nuclease polypeptides may cut substantially at only one strand of the target DNA duplex. In some embodiments, activity includes binding activity, e.g., binding of an effector (e.g., a CRISPR nuclease) to an RNA guide and / or target nucleic acid. In some examples, the variant CRISPR nuclease polypeptides disclosed herein have an enhanced binding to a cognate guide RNA (gRNA) as compared with the reference CRISPR nuclease, e.g., having a binding activity at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, or greater than that of the reference CRISPR nuclease. A cognate gRNA refers to a gRNA having a scaffold recognizable by the CRISPR nuclease. In some examples, the variant CRISPR nuclease polypeptides disclosed herein have an enhanced enzymatic activity relative to the reference CRISPR nuclease, e.g., having an enzymatic activity at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, or greater than that of the reference CRISPR nuclease. In other examples, the variant CRISPR nuclease polypeptides disclosed herein have a decreased enzymatic activity (e.g., the enzymatic activity for cleaving both strands of a target DNA duplex) relative to the reference CRISPR nuclease, e.g., having an enzymatic activity at least 20%, 30%, 40%, 50%, 60%, or 70% lower than that of the reference CRISPR nuclease. In some instances, the decreased enzymatic activity is achieved by reducing or diminishing the nuclease activity of the RuvC domain. In other instances, the decreasedAttorney Docket No.: 063586-523001WO enzymatic activity is achieved by reducing or diminishing the nuclease activity of the HNH domain. In some instances, the variant CRISPR nuclease polypeptides disclosed herein have enhanced indel activity relative to the reference CRISPR nuclease. As used herein, the term “indel activity” refers to the ability of a CRISPR nuclease to introduce an indel (insertion / deletion) into a sequence (e.g., a genomic target). For example, the CRISPR nuclease may, in some instances, introduce a double-strand break into a sequence (e.g., a genomic target in a cell) and through DNA repair mechanisms, an indel is created. In some embodiments, the variant CRISPR nuclease polypeptide provided herein share a high sequence homology relative to the reference CRISPR nuclease. For example, the variant CRISPR nuclease polypeptide may comprise an amino acid sequence at least 70% (e.g., at least 80%, 85%, 90%, 95%, or higher) identical to SEQ ID NO:1. In some instances, the variant CRISPR nuclease polypeptide may comprise an amino acid sequence at least 90% identical to SEQ ID NO:1. In some instances, the variant CRISPR nuclease polypeptide may comprise an amino acid sequence at least 95% identical to SEQ ID NO:1. In other instances, the variant CRISPR nuclease polypeptide may comprise an amino acid sequence at least 97% (e.g., 98%, 99%, 99.5%, or greater) identical to SEQ ID NO:1. The “percent identity” (a.k.a., sequence identity) of two nucleic acids or of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol.215:403-10, 1990. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength-12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, word length=3 to obtain amino acid sequences homologous to the protein molecules of the invention. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res.25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. The variant CRISPR nuclease polypeptide provided herein can contain one or more alterations relative to the reference CRISPR nuclease of SEQ ID NO:1, e.g., one or moreAttorney Docket No.: 063586-523001WO amino acid residue substitutions, one or more deletions, one or more insertions, fusion, or a combination thereof. In some instances, the alterations may be introduced into the BH domain, the PLL domain, the WED domain, the PID domain, or a combination thereof. In some embodiments, the variant CRISPR nuclease polypeptide provided herein may comprise one or more arginine substitutions relative to SEQ ID NO:1. “Arginine substitutions” or “lysine substitution” refers to the replacement of a non-arginine or non- lysine residue in SEQ ID NO:1 with an arginine or lysine residue. In some examples, the variant CRISPR nuclease polypeptide may contain up to 20 arginine and / or lysine substitutions, e.g., up to 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 arginine and / or lysine substitutions. In specific examples, the variant CRISPR nuclease polypeptide may contain 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 arginine and / or lysine substitutions. In some instances, one or more of the substituting arginine residues may be replaced by a conservative amino acid residue such as lysine or histidine. In some embodiments, the variant CRISPR nuclease polypeptide provided herein may comprise one or more arginine substitutions, one or more lysine substitutions, or a combination thereof. In some instances, the arginine substitutions may be located in the BH domain, in the PLL domain, in the WED domain, in the PID domain, or in any of the combination thereof. In some examples, the variant CRISPR nuclease polypeptide may contain one or more arginine and / or lysine substitutions at one or more of positions: K14, L27, L37, D39, S44, K45, E48, V51, M56, T59, K60, K64, A65, A68, H69, E71, L75, S76, V84, V85, V88, E102, P106, 108, E109, E112, E113, E114, A115, E116, V117, F118, F120, E124, S127, N131, L134, E135, E139, L142, I143, H151, D156, A173, A177, D189, 206, S210, D211, L214, V235, S238, Q245, L248, F260, S262, G263, L264, A265, Q266, D267, L268, P269, E270, E271, T272, I273, E274, E277, T280, N288, E291, Q292, S293, G294, E277, A305, Y309, G314, T324, S326, A380, D382, E386, Q389, Q390, Y394, G395, K397, L400, K405, N408, K416, P417, E419, T420, N432, K455, A456, S461, D475, D447, A460, S468, A478, D479, L499, S457, K480, K482, T483, E518, G521, V523, G529, P532, Q543, V548, Q564, V577, G579, T581, T599, 602, A603, N605, W609, I610, S612, A613, V615, K617, S620, A621, Q624, Q627, A632, H635, Q648, A657, H665, S666, L679, E680, A684, A688, A690, N691, A694, S695, N698, K701, E702, K703, P705, D707, K710, E711, E715, V717, V718, A726, T733, E739, S740, A742,Attorney Docket No.: 063586-523001WO A743, I746, S747, K751, D759, S764, Q769, A770, A772, D777, D780, G783, E784, A785, A788, I794, A801, Q811, Q814, Q826, L833, G834, S835, K843, E848, A854, V856, P857, G858, F859, A861, S863, E864, Q871, H890, A891, Q894, S895, A896, E897, S905, H911, S925, K930, G947, I950, P951, and I953. In some examples, the arginine and / or lysine substitutions (e.g., arginine substitutions) are at positions A65, V85, L134, V117, A657, S695, N691, D707, E715, and / or S925 relative to SEQ ID NO:1. For example, the engineered CRISPR nuclease polypeptide may comprise A65R, V85R, L134R, V117R, A657R, S695R, N691R, D707R, E715R, and / or S925R relative to SEQ ID NO:1. Alternatively, the engineered CRISPR nuclease polypeptide may comprise A65K, V85K, L134K, V117K, A657K, S695K, N691K, D707K, E715K, and / or S925K relative to SEQ ID NO:1. In some instances, the variant CRISPR nuclease polypeptide may comprise a combination of at least two arginine and / or lysine substitutions (e.g., 2, 3, 4, 5, 6, 7, or 8) relative to the reference nuclease of SEQ ID NO:1. For example, the substitutions may be at two or more positions of A65, V85, L134, V117, A657, S695, N691, D707, E715, and / or S925. See Tables 5 and 26-28 below, each of which is within the scope of the present disclosure. Specific examples include: (1) A65, E715, and S925; (2) A65, A657 and S925; (3) A657, S695, and S925 (4) N691, S695, and S925; (5) A65, N691, and S925; (6) V117, S695, and S925; (7) N691, E715, and S925; (8) V117, E715, and S925; (9) S695, E715, and S925; (10) A65, S695, and S925; (11) A657, N691, and S925; (12) A657, N691, and S695; (13) A65, V117, and E715; (14) V117, A657, and S925; (15) V117, S695, and E715; (16) S695, and S925; (17) A65, A657, and E715; and (18) E715 and S925. See also Example 3 below. In some examples, the arginine and / or lysine (optionally arginine) substitutions are at positions A65, A657 and S925 relative to SEQ ID NO:1; e.g., A65R, A657R, and S925R. In some instances, additional arginine and / or lysine substitutions may be added on top of the A65 / A657 / S925 combination. Examples can be found in Tables 26-28 and Example 10 below. In one example, the additional arginine / lysine substitution is at positions V85 (e.g., V85R) and L134 (e.g., L134R). In another example, the additional arginine / lysine substitution is at positions V85 (e.g., V85R), L134 (e.g., L134R), and D707 (e.g., D707R). Alternatively or in addition, the variant CRISPR nuclease polypeptide provided herein may comprise one or more mutations within either the RuvC or the HNH nucleaseAttorney Docket No.: 063586-523001WO domain to reduce or eliminate the nuclease activity of the target domain, thereby producing a variant with nickase activity. Such mutations may be deletions, insertions, amino acid substitutions, or a combination thereof. In some embodiments, the mutations within either the RuvC or the HNH nuclease domain are amino acid substitutions, of which the substituting amino acid residue is not a conservative substitution of the native amino acid residue at the position of the mutation. For example, if the native amino acid residue is R, the substituting residue can be any amino acid residue except for K. Similarly, if the native amino acid residue is K, the substituting residue can be any amino acid residue except for R. Groups of conservative amino acid residue substitutions are provided herein. In some instances, the one or more mutation may be within the HNH nuclease domain, for example, at E425, H426, and / or N449 of SEQ ID NO:1. In some examples, the mutations may be amino acid residue substitutions and the native amino acid residues in SEQ ID NO:1 may be replaced by an amino acid residue not of the same type as the native residues. For example, a positively charged residue may be replaced by a non- charged amino acid residue, or vice versa. In some examples, the amino acid residue substitution at E425 may be E425G, E425A, E425L, or E425S. In one specific example, the mutation can be E425A. In another example, the amino acid residue substitution at H426 may be H426G, H426A, H426I, H426L, H426M, H426V, or H426S. In one specific example, the mutation at position H426 can be H426A. Alternatively or in addition, the amino acid residue substitution may be at position N449, for example, N449G, N449A, N449L, or N449S. In one example, the mutation at position N449 is N449A. In some instances, the one or more mutation may be with the RuvC nuclease domain, for example, at position D10, E362, D487, T549, or a combination thereof, of SEQ ID NO:1 (e.g., at position E362 and / or D487, or at position T549). In some examples, the mutations may be amino acid residue substitutions and the native amino acid residues in SEQ ID NO:1 may be replaced by an amino acid residue not of the same type as the native residues. For example, a positively charged residue may be replaced by a non-charged amino acid residue, or vice versa. In some examples, the amino acid residue substitution at D10 may be D10G, D10A, D10L, or D10S. In some examples, the amino acid residue substitution at E362 may be E362G, E362A, E362L, or E362S. Alternatively or in addition, the amino acid residue substitution at D487 may be D487G, D487A, D487L, or D487S. Further, the amino acid residue substitution at position T549 can beAttorney Docket No.: 063586-523001WO T549P. In some examples, the variant CRISPR nuclease polypeptide provided herein may be a nickase variant, which comprises one or more mutations in one nuclease domain (e.g., the HNH nuclease domain such as at position E425, e.g., E425A). Exemplary nickase variants are provided in Tables 6 below. Such a nickase variant may further comprise one or more arginine or lysine substitutions (e.g., arginine substitutions) for enhancing certain features, such as indel activities. Exemplary arginine and / or lysine substitutions are provided herein, for example, at one or more of positions A65, V85, V117, L134, A657, S695, N691, D707, E715, and / or S925 relative to SEQ ID NO:1 (e.g., at positions A65, A657 and S925 in SEQ ID NO:1). In some instances, the variant CRISPR nuclease polypeptide may further comprise one or more non-arginine / lysine substitutions or mutations. Examples can be found in Table 28, for example, V51A, F96Y, A454G, T549P, I561L, E642G, F651Y, V719I, E809D, E897G, deletion of D267, deletion of D707-R708, and deletion of E702-R708. In some examples, the variant CRISPR nuclease polypeptide may comprise both arginine and / or lysine substitutions and one or more mutations in either the RuvC or HNH nuclease domain. For example, the variant CRISPR nuclease polypeptide may comprise (a) arginine and / or lysine (e.g., arginine) substitutions at one or more of positions A65, V117, A657, S695, N691, E715, and / or S925 relative to SEQ ID NO:1 (e.g., at positions A65, A657 and S925 in SEQ ID NO:1) and (b) one or more mutations such as amino acid substitutions at position E425, H426 or N449 in SEQ ID NO:1. Alternatively, the variant CRISPR nuclease polypeptide may comprise (a) arginine and / or lysine (e.g., arginine) substitutions at one or more of positions A65, V117, A657, S695, N691, E715, and / or S925 relative to SEQ ID NO:1 (e.g., at positions A65, A657 and S925 in SEQ ID NO:1) and (b) one or more mutations such as amino acid substitutions at position D10, E362, D487, and T549 in SEQ ID NO:1. In some instances, the arginine and / or lysine substitutions may be at positions shown in Tables 5 and 26-28 below. In one specific example, the variant CRISPR nuclease polypeptide may comprise arginine and / or lysine (e.g., arginine) substitutions at positions A65, A657 and S925, and an amino acid substitution at position E425 (e.g., E425G, E425A, E425L, or E425S, optionally E425A) in SEQ ID NO:1. In another example, the variant CRISPR nuclease polypeptide may comprise arginine and / or lysine (e.g., arginine) substitutions at positions A65, V85, L134,Attorney Docket No.: 063586-523001WO A657, and S925 and amino acid substitutions at position E425 (e.g., E425G, E425A, E425L, or E425S, optionally E425A) and T549 (e.g., T549P) in SEQ ID NO:1. In yet another example, the variant CRISPR nuclease polypeptide may comprise arginine and / or lysine (e.g., arginine) substitutions at positions A65, V85, L134, A657, D707, and S925 and an amino acid substitution at position E425 (e.g., E425G, E425A, E425L, or E425S, optionally E425A). Exemplary variant CRISPR nuclease polypeptides (relative to SEQ ID NO: 1) include, but are not limited to: (i) A65R / A657R / S925R / E425A, (ii) A65R / L134R / E425A / A657R / S925R; (iii) A65R / V85R / L134R / E425A / A657R / S925R; (iv) A65R / L134R / E425A / A657R / D707R / S925R; (v) A65R / V85R / L134R / E425A / A657R / S925R; (vi) A65R / V85R / L134R / E425A / A657R / D707R / S925R; and (vii) A65R / V85R / L134R / E425A / T549P / A657R / S925R. In one specific example, the variant CRISPR nuclease polypeptide comprises the mutations of (vi) relative to SEQ ID NO: 1. In another specific example, the variant CRISPR nuclease polypeptide comprises the mutations of (vii) relative to SEQ ID NO: 1. In some examples, the variant CRISPR nuclease polypeptide that contains one or more arginine substitutions may share a sequence identity at least 90% (e.g., 95%, 97%, 98%, 99%, 99.5%, or greater) with SEQ ID NO:1. In some instances, the variant CRISPR nuclease polypeptide may comprise one or more conservative amino acid residue substitutions, in addition to the mutations in the HNH or RuvC nuclease domain, and / or the arginine / lysine substitutions. As used herein, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids includeAttorney Docket No.: 063586-523001WO substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Exemplary CRISPR nuclease polypeptides for use in the gene editing systems provided herein are disclosed in Tables 1 and 6 below, each of which is within the scope of the present disclosure. Combinations of mutations contained in exemplary CRISPR nuclease polypeptide variants (relative to reference nuclease of SEQ ID NO: 1) are listed in Tables 26- 28, each of which is within the scope of the present disclosure. In some embodiments, the CRISPR nuclease polypeptide in the gene editing systems disclosed herein (e.g., the reference nuclease of SEQ ID NO:1 or any of the variants thereof as disclosed herein) may be a fusion polypeptide comprising a CRISPR nuclease and one or more additional functional moieties. As used herein, the terms “fusion” and “fused” refer to the joining of at least two nucleotide or protein molecules. For example, “fusion” and “fused” can refer to the joining of at least two polypeptide domains that are encoded by separate genes in nature. The fusion can be an N-terminal fusion, a C-terminal fusion, or an intramolecular fusion. In some aspects, the domains are transcribed and translated to produce a single polypeptide. Exemplary functional moieties to include in the fusion polypeptide include a peptide tag, a fluorescent protein, a base-editing domain, a DNA methylation domain, a histone residue modification domain, a localization factor, a transcription modification factor, a light-gated control factor, a chemically inducible factor, a chromatin visualization factor, or a combination thereof. In some embodiments, the additional functional moiety may comprise a nuclear localization site (NLS), a nuclear export signal (NES), or a combination thereof. In some examples, the fusion polypeptide may comprise an NLS, which may be located at either the N-terminus or the C-terminus. In specific examples, the fusion polypeptide may comprise a first NLS located at the N-terminus and a second NLS located at the C- terminus. The first and second NLS fragments may be identical. Alternatively, the two NLS fragments may be different. In some embodiments, the fusion polypeptide may comprise an NLS near the N-terminus and / or near the C-terminus (e.g., within about 1, 2, 3, 4, or 5 of the first amino acid or last amino acid of the CRISPR nuclease). In some embodiments, the fusion polypeptide may comprise an NLS within a flexible loop of the CRISPR nuclease.Attorney Docket No.: 063586-523001WO In some embodiments, the gene editing system provided herein may comprise the CRISPR nuclease polypeptide, which may form a ribonucleoprotein (RNP) complex with the cognate guide RNA. As used herein, the term “complex” refers to a grouping of two or more molecules. In some embodiments, the complex comprises a polypeptide and a nucleic acid molecule interacting with (e.g., binding to, coming into contact with, adhering to) one another. In other embodiments, the gene editing system provided herein may comprise a nucleic acid encoding the CRISPR nuclease polypeptide. In some examples, the nucleotide sequence encoding the CRISPR nuclease polypeptide described herein can be codon- optimized for use in a particular host cell or organism. For example, the nucleic acid can be codon-optimized for any non-human eukaryote including mice, rats, rabbits, dogs, livestock, or non-human primates. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at the world wide web site of kazusa.orjp / codon / and these tables can be adapted in a number of ways. See Nakamura et al. Nucl. Acids Res.28:292 (2000), which is incorporated herein by reference in its entirety. Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA). In some examples, the nucleic acid encoding the CRISPR nuclease polypeptides as disclosed herein can be an mRNA molecule, which can be codon optimized. Exemplary codon-optimized nucleotide sequences encoding exemplary CRISPR nuclease polypeptides can be found in Tables 1 and 6 below, any of which is within the scope of the present disclosure. In some examples, the gene editing system may comprise a vector (e.g., a viral vector such as an AAV vector, an AdV vector, or a retroviral vector) encoding the CRISPR nuclease polypeptide. (ii) Reverse Transcriptase Polypeptides The gene editing system disclosed herein also comprise a reverse transcriptase (RT) polypeptide, which may be a wild-type RT or a variant thereof. In some instances, the RT polypeptide and the CRISPR nuclease polypeptide disclosed herein may form a fusion protein. As used herein, the terms “reverse transcriptase” or “RT” refer to a multi-functional enzyme that typically has three enzymatic activities including RNA- and DNA-dependent DNA polymerization activity and an RNase H activity that catalyzes the cleavage of RNA in RNA-DNA hybrids. A reverse transcriptase can generate DNA from an RNA template.Attorney Docket No.: 063586-523001WO In some embodiments, the reverse transcriptase polypeptide is any wild-type reverse transcriptase obtained from any naturally-occurring organism or virus, or obtained from a commercial or non-commercial source. The reverse transcriptase polypeptide may also be a variant reverse transcriptase polypeptide. The reverse transcriptase polypeptide can be obtained from a number of different sources. For instance, the gene may be obtained from eukaryotic cells which are infected with retrovirus or from a plasmid that comprises either a portion of or the entire retrovirus genome. In addition, RNA that comprises the reverse transcriptase gene can be obtained from retroviruses. In some embodiments, the reverse transcriptase is expressed or otherwise provided as an individual component, i.e., not as a fusion protein with the CRISPR nuclease polypeptide provided herein. A person of ordinary skill in the art will recognize that reverse transcriptases are known in the art, including, but not limited to, Moloney Murine Leukemia Virus (MMLV) reverse transcriptase, Human Immunodeficiency Virus (HIV) reverse transcriptase, and avian Sarcoma-Leukosis Virus (ASLV) reverse transcriptase, which includes but is not limited to Rous Sarcoma Virus (RSV) reverse transcriptase, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Avian Erythroblastosis Virus (AEV) Helper Virus MCAV reverse transcriptase, Avian Myelocytomatosis Virus MC29 Helper Virus MCAV reverse transcriptase, Avian Reticuloendotheliosis Virus (REV-T) Helper Virus REV-A reverse transcriptase, Avian Sarcoma Virus UR2 Helper Virus UR2AV reverse transcriptase, Avian Sarcoma Virus Y73 Helper Virus YAV reverse transcriptase, Rous Associated Virus (RAV) reverse transcriptase, and Myeloblastosis Associated Virus (MAV) reverse transcriptase may be suitably used in the composition described herein. In some embodiments, the reverse transcriptase is MMLV-RT, MarathonRT from Eubacterium rectale, or RTX reverse transcriptase or a variant of MMLV-RT, MarathonRT, or RTX reverse transcriptase. In some embodiments, the reverse transcriptase is a sequence shown in Table 10, a variant thereof, or an ortholog thereof. Table 10. Exemplary Reverse Transcriptase Sequences. Reverse Sequence SEQ IDAttorney Docket No.: 063586-523001WO RDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQY VDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYL LKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAAttorney Docket No.: 063586-523001WO SLLGDLLEERQKIKKRMKATIDPIERKLLDYRQRAIKILANSLYGYYGYA RARWYCKECAESVIAWGREYLTMTIKEIEEKYGFKVIYSDTDGFFATIPG ADAETVKKKAMEFLKYINAKLPGALELEYEGFYKRGLFVTKKKYAVIDEEAttorney Docket No.: 063586-523001WO HIV-1 RT p51MPISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKEGKISK60 IGPENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAG LKKKKSVTVLDVGDAYFSVPLDEDFRKYTAFTIPSINNETPGIRYQYNVLso e e o e s, e eve se a sc p ase po ypep e s a e o -p o e reverse transcriptase variant. Error-prone reverse transcriptases that are known and / or available in the art may be used. It will be appreciated that reverse transcriptases naturally do not have any proofreading function; thus, the error rate of reverse transcriptases is generally higher than DNA polymerases comprising a proofreading activity. In some embodiments, the reverse transcriptase is considered to be “error-prone” if it has an error rate that is less than one error in about 15,000 nucleotides synthesized. In some embodiments, the reverse transcriptase polypeptide has a mutation or mutations in the RNase H domain. In some embodiments, the reverse transcriptase polypeptide does not comprise an RNase H domain (e.g., the RNase H domain has been removed from the reverse transcriptase polypeptide). In some embodiments, the RNase H domain is truncated in a reverse transcriptase polypeptide. In some embodiments, the reverse transcriptase polypeptide has a mutation or mutations in the RNA-dependent DNA polymerase domain. In some embodiments, the reverse transcriptase polypeptide is a variant that has altered thermostability characteristics. The ability of a reverse transcriptase to withstand high temperatures is an important aspect of cDNA synthesis. Elevated reaction temperatures help denature RNA with strong secondary structures and / or high GC content, allowing reverse transcriptases to read through the sequence. As a result, reverse transcription at higher temperatures enables full-length cDNA synthesis and higher yields. Wild-type M- MLV reverse transcriptase typically has an optimal temperature in the range of 37-48ºC;Attorney Docket No.: 063586-523001WO however, mutations may be introduced that allow for the reverse transcription activity at higher temperatures of over 48ºC, including 49ºC, 50ºC, 51ºC, 52ºC, 53ºC, 54ºC, 55ºC, 56ºC, 57ºC, 58ºC, 59ºC, 60ºC, 61ºC, 62ºC, 63ºC ¸64ºC¸ 65ºC ¸66ºC, and higher. Variant reverse transcriptase polypeptides used herein may be at least about 20% identical, at least about 25% identical, at least about 30% identical, at least about 35% identical, at least about 40% identical, at least about 45% identical, at least about 50% identical, at least about 55% identical, at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference reverse transcriptase polypeptide, including any wild-type reverse transcriptase, mutant reverse transcriptase, or fragment of a reverse transcriptase, or other reverse transcriptase variant disclosed or contemplated herein or known in the art. In some embodiments, a reverse transcriptase variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or up to 100, or up to 200, or up to 300, or up to 400, or up to 500 or more amino acid changes compared to a reference reverse transcriptase. In some embodiments, the reverse transcriptase variant comprises a fragment of a reference reverse transcriptase, such that the fragment is at least about 20% identical, at least about 25% identical, at least about 30% identical, at least about 35% identical, at least about 40% identical, at least about 45% identical, at least about 50% identical, at least about 55% identical, at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of the reference reverse transcriptase. Variant reverse transcriptases, including error-prone reverse transcriptases, thermostable reverse transcriptases, and reverse transcriptases with increased processivity, can be engineered by various routine strategies, including mutagenesis or evolutionary processes. In some cases, the variants can be produced by introducing a single mutation. In other cases, the variants may require more than one mutation. For those mutants comprisingAttorney Docket No.: 063586-523001WO more than one mutation, the effect of a given mutation may be evaluated by introduction of the identified mutation to the wild-type gene by site-directed mutagenesis in isolation from the other mutations borne by the particular mutant. Screening assays of the single mutant thus produced will then allow the determination of the effect of that mutation alone. In some embodiments, the reverse transcriptase polypeptides comprise or is fused to a domain to improve extension rates and / or efficiency of the reverse transcriptase. In some embodiments, the reverse transcriptase polypeptide is fused to an Sso7d polypeptide such as an Sso7d polypeptide from Sulfolobus solfataricus. See, e.g., Wang et al., Nucleic Acids Res. 32(3): 1197-207 (2004). In some embodiments, the reverse transcriptase as in any one of the embodiments described herein interacts with a ligase, an integrase, and / or a recombinase. In some embodiments, the reverse transcriptase as in any one of the embodiments described herein is fused to a ligase, an integrase, and / or a recombinase. In some embodiments, the ligase, integrase, and / or recombinase is fused to the N-terminus or C-terminus of the reverse transcriptase. In some embodiments, the ligase, integrase, and / or recombinase is fused internally to the reverse transcriptase. In some embodiments, the integrase is a serine integrase. In some embodiments, the integrase is a Bxb1, TP901, or PhiBT1 integrase. In some embodiments, the recombinase is a serine recombinase or a tyrosine recombinase. In some embodiments, the recombinase is a CRE recombinase. In some embodiments, a reverse transcriptase that interacts with or is fused to a ligase, integrase, and / or recombinase further interacts with or is fused to the CRISPR nuclease polypeptide disclosed herein. In other embodiments, the gene editing system provided herein may comprise a nucleic acid encoding the RT polypeptide. In some examples, the nucleotide sequence encoding the RT polypeptide described herein can be codon-optimized for use in a particular host cell or organism. In some examples, the nucleic acid encoding the RT polypeptides can be an mRNA molecule, which can be codon optimized. Exemplary codon-optimized nucleotide sequences encoding exemplary RT polypeptides can be found in Table 9 below, which is within the scope of the present disclosure. In some examples, the gene editing system may comprise a vector (e.g., a viral vector such as an AAV vector, an AdV vector, or a retroviral vector) encoding the RT polypeptide. (iii) Fusion Polypeptides In some embodiments, the gene editing system provided herein comprise a fusionAttorney Docket No.: 063586-523001WO polypeptide that includes both the CRISPR nuclease polypeptide and the RT polypeptide as also disclosed herein. Alternatively, the gene editing system may comprise a nucleic acid (e.g., a vector such as an expression vector) encoding the fusion polypeptide. As used herein, the terms “fusion” and “fused” refer to the joining of at least two nucleotide or protein molecules. For example, “fusion” and “fused” can refer to the joining of at least two polypeptide domains that are encoded by separate genes (e.g., the CRISPR nuclease polypeptide and the reverse transcriptase polypeptide provided herein) in nature. The fusion can be an N-terminal fusion, a C-terminal fusion, or an intramolecular fusion. In some embodiments, the fusion polypeptide may comprise the reverse transcriptase polypeptide at its N-terminus and the CRISPR nuclease polypeptide downstream to the RT polypeptide. In other embodiments, the fusion polypeptide may comprise the CRISPR nuclease polypeptide at its N-terminus and the RT polypeptide downstream to the CRISPR nuclease polypeptide. In some embodiments, the RT polypeptide may be fused with the CRISPR nuclease polypeptide at an intramolecular position within the RT polypeptide, for example, the CRISPR nuclease polypeptide may be within a loop of the reverse transcriptase polypeptide. In some embodiments, any of the CRISPR nuclease-RT fusion polypeptides disclosed herein may comprise one or more additional functional elements, e.g., those provided herein. In some instances, the additional functional elements may be one or more NLS elements. In some examples, the fusion polypeptide may comprise an NLS at its N-terminus, at its C- terminus, or both. Alternatively or in addition, the additional functional elements may be a flexible peptide linker, which can be located between the CRISPR nuclease polypeptide and the RT polypeptide. Suitable peptide linkers include, but are not limited to, G / S rich peptide linkers and XTEN peptide linkers. Examples of NLS and peptide linkers are provided in Table 1 below. See also Example 1. In some examples, the CRISPR nuclease-RT polypeptide disclosed herein may comprise, from N-terminus to C-terminus, a first NLS, the CRISPR nuclease, a peptide linker, the RT polypeptide, and a second NLS. In other examples, the CRISPR nuclease-RT polypeptide disclosed herein may comprise, from N-terminus to C-terminus, a first NLS, the RT nuclease, a peptide linker, the CRISPR nuclease polypeptide, and a second NLS (which may be identical to the first NLS), the CRISPR nuclease polypeptide, a first peptide linker, a second NLS (which may be identical to the first NLS), a second peptide linker, the RT nuclease, a third peptide linker (which may be identical to the first peptide linker), a thirdAttorney Docket No.: 063586-523001WO NLS (which may be identical to the first NLS), a fourth peptide linker, and a fourth NLS. Examples of the CRISPR nuclease-RT polypeptides are provided in Tables 11 and 12 below, all of which are within the scope of the present disclosure. In other embodiments, the gene editing system provided herein may comprise a nucleic acid encoding the CRISPR nuclease-RT fusion polypeptide. In some examples, the nucleotide sequence encoding the fusion polypeptide described herein can be codon- optimized for use in a particular host cell or organism. In some examples, the nucleic acid encoding the fusion polypeptides as disclosed herein can be an mRNA molecule, which can be codon optimized. Exemplary codon-optimized nucleotide sequences encoding exemplary CRISPR nuclease-RT fusion polypeptides can be found in Tables 11 and 12 below, any of which is within the scope of the present disclosure. See also Example 6 and 7 below. In some examples, the gene editing system may comprise a vector (e.g., a viral vector such as an AAV vector, an AdV vector, or a retroviral vector) encoding the CRISPR nuclease-RT fusion polypeptide. (iv) Preparation of Protein Components The CRISPR nuclease polypeptide, the RT polypeptide, or the CRISPR nuclease-RT fusion polypeptide disclosed herein may be prepared by conventional methods or the methods disclosed herein. For example, the CRISPR nuclease polypeptide, the RT polypeptide, or the CRISPR nuclease-RT fusion polypeptide can be prepared by culturing host cells such as bacteria cells or mammalian cells, capable of producing the nuclease polypeptides, isolating the nuclease polypeptides thus produced, and optionally, purifying the nuclease polypeptides. The CRISPR nuclease polypeptide, the RT polypeptide, or the CRISPR nuclease-RT fusion polypeptide can be also prepared by an in vitro coupled transcription-translation system. Host cells that can be used for preparation of the CRISPR nuclease polypeptide, the RT polypeptide, or the CRISPR nuclease-RT fusion polypeptide are not particularly limited as long as they can produce the CRISPR nuclease polypeptide, the RT polypeptide, or the CRISPR nuclease-RT fusion polypeptide. Some nonlimiting examples of the host cells include bacteria cells (e.g., E. coli cells), yeast cells, insect cells, or mammalian cells. Vectors The present disclosure provides vectors for expressing the CRISPR nuclease polypeptide, the RT polypeptide, or the CRISPR nuclease-RT fusion polypeptide. In someAttorney Docket No.: 063586-523001WO embodiments, a vector disclosed herein includes a nucleotide sequence encoding CRISPR nuclease polypeptide, the RT polypeptide, or the CRISPR nuclease-RT fusion polypeptide. In some embodiments, the vector comprises a Pol II promoter or a Pol III promoter. Expression of natural or synthetic polynucleotides is typically achieved by operably linking a polynucleotide encoding the CRISPR nuclease polypeptide, the RT polypeptide, or the CRISPR nuclease-RT fusion polypeptide to a promoter and incorporating the construct into an expression vector. The expression vector is not particularly limited as long as it includes a polynucleotide encoding the CRISPR nuclease polypeptide, the RT polypeptide, or the CRISPR nuclease-RT fusion polypeptide and can be suitable for replication and integration in eukaryotic cells. Typical expression vectors include transcription and translation terminators, initiation sequences, and promoters useful for expression of the desired polynucleotide. For example, plasmid vectors carrying a recognition sequence for RNA polymerase (pSP64, pBluescript, etc.). may be used. Vectors including those derived from retroviruses such as lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Examples of vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. The expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and described in a variety of virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to phage viruses, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. The kind of the vector is not particularly limited, and a vector that can be expressed in host cells can be appropriately selected. To be more specific, depending on the kind of the host cell, a promoter sequence to ensure the expression of the polypeptide(s) from the polynucleotide is appropriately selected, and this promoter sequence and the polynucleotide are inserted into any of various plasmids etc. for preparation of the expression vector. Additional promoter elements, e.g., enhancing sequences, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. Depending on the promoter, it appears thatAttorney Docket No.: 063586-523001WO individual elements can function either cooperatively or independently to activate transcription. Further, the disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the disclosure. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter. The expression vector to be introduced can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate transcriptional control sequences to enable expression in the host cells. Examples of such a marker include a dihydrofolate reductase gene and a neomycin resistance gene for eukaryotic cell culture; and a tetracycline resistance gene and an ampicillin resistance gene for culture of E. coli and other bacteria. By use of such a selection marker, it can be confirmed whether the polynucleotide encoding the polypeptide(s) of the present invention has been transferred into the host cells and then expressed without fail. The preparation method using recombinant expression vectors is not particularly limited, and examples thereof include methods using a plasmid, a phage or a cosmid. Methods of Expression The present disclosure includes a method for protein expression, comprising translating the CRISPR nuclease polypeptide, the RT polypeptide, or the CRISPR nuclease- RT fusion polypeptide described herein. In some embodiments, a host cell described herein is used to express the CRISPR nuclease polypeptide, the RT polypeptide, or the CRISPR nuclease-RT fusion polypeptide. The host cell is not particularly limited, and various known cells can be preferably used. Specific examples of the host cell include bacteria such as E. coli, yeasts (budding yeast, Saccharomyces cerevisiae, and fission yeast, Schizosaccharomyces pombe), nematodes (Caenorhabditis elegans), Xenopus laevis oocytes, and animal cells (for example, CHO cells,Attorney Docket No.: 063586-523001WO COS cells and HEK293 cells). The method for transferring the expression vector described above into host cells, i.e., the transformation method, is not particularly limited, and known methods such as electroporation, the calcium phosphate method, the liposome method and the DEAE dextran method can be used. After a host is transformed with the expression vector, the host cells may be cultured, cultivated or bred, for production the CRISPR nuclease polypeptide, the RT polypeptide, or the CRISPR nuclease-RT fusion polypeptide. After expression, the host cells can be collected and the CRISPR nuclease polypeptide, the RT polypeptide, or the CRISPR nuclease-RT fusion polypeptide purified from the cultures etc. according to conventional methods (for example, filtration, centrifugation, cell disruption, gel filtration chromatography, ion exchange chromatography, etc.). A variety of methods can be used to determine the level of production of a mature CRISPR nuclease polypeptide, a mature RT polypeptide, or a mature CRISPR nuclease-RT fusion polypeptide in a host cell. Such methods include, but are not limited to, for example, methods that utilize either polyclonal or monoclonal antibodies specific for the proteins or a labeling tag as described elsewhere herein. Exemplary methods include, but are not limited to, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), fluorescent immunoassays (FIA), and fluorescent activated cell sorting (FACS). These and other assays are well known in the art (See, e.g., Maddox et al., J. Exp. Med.158:1211

[1983] ). The present disclosure provides methods of in vivo expression of the CRISPR nuclease polypeptide, the RT polypeptide, or the CRISPR nuclease-RT fusion polypeptide (and optionally the gRNA and / or the RT donor RNA in the gene editing system disclosed herein). Such a method may comprise providing a polyribonucleotide encoding the CRISPR nuclease polypeptide, the RT polypeptide, or the CRISPR nuclease-RT fusion polypeptide to a host cell in a subject (e.g., a human subject) wherein the polyribonucleotide encodes the CRISPR nuclease polypeptide, the RT polypeptide, or the CRISPR nuclease-RT fusion polypeptide and expressing the CRISPR nuclease polypeptide, the RT polypeptide, or the CRISPR nuclease-RT fusion polypeptide from the cell. B. RNA Components The gene editing systems provided herein also involve at least two RNA components, a guide RNA (gRNA), which directs gene editing at a desired genetic site, and an RT donor RNA, which serves as the RNA template for the RT polypeptide in reverse transcription. TheAttorney Docket No.: 063586-523001WO RT donor RNA comprises desired nucleotide substitutions to be inserted into the genetic site of interest. In some embodiments, the gene editing system comprises the two RNA molecules. In specific examples, the gene editing system may comprise a single RNA molecule comprising the gRNA and the RT donor RNA. Alternatively, the gene editing system may comprise one or more nucleic acids encoding the two RNA components. For example, the gene editing system may comprise one or more expression vectors (e.g., viral vectors such as retroviral vectors, adenoviral vectors, or adeno-associated viral vectors) capable of producing the gRNA, the RT donor RNA, or the single RNA molecule comprising such. In some embodiments, the gRNA and the RT donor RNA as disclosed herein may form a complex. (i) Guide RNAs The gene editing system disclosed herein further comprises one or more gRNAs or nucleic acid(s) encoding such. As used herein, the terms “RNA guide”, “RNA guide sequence,” or “guide RNA (gRNA)” refer to an RNA molecule or a modified RNA molecule that facilitates the targeting of a CRISPR nuclease described herein to a genomic site of interest. For example, an RNA guide can be a molecule that comprises a spacer sequence and a scaffold sequence. The spacer sequence recognizes (e.g., binds to) a site in a non-PAM strand that is complementary to a target sequence in the PAM strand, e.g., designed to be complementary to a specific nucleic acid sequence. The scaffold sequence contains a nuclease binding sequence for binding to the CRISPR nuclease. In some embodiments, the scaffold is an RNA sequence. In some instances, the gRNA disclosed herein may further comprise a linker sequence, a 5’ end and / or 3’ end protection fragment, or a combination thereof. Spacer Sequences As used herein, the term “spacer” and “spacer sequence” (a.k.a., a DNA-binding sequence) is a portion in an RNA guide that is the RNA equivalent of the target sequence (a DNA sequence). The spacer contains a sequence capable of binding to the non-PAM strand via base-pairing at the site complementary to the target sequence (which is in the PAM strand). Such a spacer is also known as specific to the target sequence. In some instances, the spacer may be at least 75% identical to the target sequence (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%), except for the RNA-DNA sequenceAttorney Docket No.: 063586-523001WO difference. In some instances, the spacer may be 100% identical to the target sequence except for the RNA-DNA sequence difference. The gene editing system disclosed herein comprises one or more gRNAs, each comprising a spacer for targeting a genomic site of interest and a scaffold, which is recognizable by the variant CRISPR nuclease polypeptide contained in the gene editing system. The target sequence can be adjacent to a protospacer adjacent motif (PAM) of 5’- NRV-3’, in which N represents any nucleotide, R represents A or G, and V represents A, C, or G. In some examples, the PAM is 5’-NRR’3’ (e.g., 5’-NRG-3’ or 5’-NGG-3’), for example 5’-CAG-3'. In specific examples, the PAM is 5’-RRR-3’, for example, 5’-AAA-3’ or 5’-GAG-3’. The PAM motif is located on the 3’ end of the target sequence. As used herein, the term “protospacer adjacent motif” or “PAM sequence” refers to a DNA sequence adjacent to a target sequence. In some embodiments, a PAM sequence is required for binding of the CRISPR nuclease and / or indel activity. In a double-stranded DNA molecule, the strand containing the PAM motif is called the “PAM-strand” and the complementary strand is called the “non-PAM strand.” The gRNA binds to a site in the non-PAM strand that is complementary to a target sequence disclosed herein, and the PAM sequence as described herein is present in the PAM-strand. The PAM motif can be located upstream to the target sequence. As used herein, the term “adjacent to” refers to a nucleotide or amino acid sequence in close proximity to another nucleotide or amino acid sequence. In some embodiments, a nucleotide sequence is adjacent to another nucleotide sequence if no nucleotides separate the two sequences (i.e., immediately adjacent). In some embodiments, a nucleotide sequence is adjacent to another nucleotide sequence if a small number of nucleotides separate the two sequences (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides). A spacer sequence as disclosed herein may have a length of from about 15 nucleotides to about 30 nucleotides. For example, the spacer sequence can have a length of from about 15 nucleotides to about 20 nucleotides, from about 15 nucleotides to about 25 nucleotides, from about 20 nucleotides to about 25 nucleotides, or from about 20 nucleotides to about 30 nucleotides. In some embodiments, the spacer in the gRNA may be generally designed to have a length of between 15 and 25 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25) and be complementary to a specific target sequence. In some embodiments, theAttorney Docket No.: 063586-523001WO spacer sequence may be designed to have a length of between 18-22 nucleotides (e.g., 20 nucleotides). In some embodiments, the spacer sequence may have at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to a target sequence as described herein and is capable of binding to the complementary region of the target sequence via base- pairing. In some embodiments, the spacer sequence comprises only RNA bases. In some embodiments, the spacer sequence comprises a DNA base (e.g., the spacer comprises at least one thymine). In some embodiments, the spacer sequence comprises RNA bases and DNA bases (e.g., the DNA-binding sequence comprises at least one thymine and at least one uracil). Scaffold Sequence The scaffold sequence in the gRNA is recognizable by the variant CRISPR nuclease polypeptide also in the gene editing system. In some instances, the scaffold sequence comprises SEQ ID NO:2, which is the cognate scaffold for the reference CRISPR nuclease of SEQ ID NO:1. GUUUCAGCUACCCUGAGUUUCUCAACUCCGUUGAGACUUCCUCA GGUUCAAGCUGAAAAGAGCAUCUGUCCGGAAGGACCACUCCGGG UUAGGGCAGACCCGGCUCUUGGGCUUCCUUGGUCUUUGACUCUG GAGGAAACCUUCCGGCGUGUCGCAAGACAAGAUGUGAGCCUG (SEQ ID NO: 2) In other instances, the scaffold sequence may be a variant derived from SEQ ID NO:2. Such a variant scaffold sequence may comprise a nucleotide sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, or greater) identical to SEQ ID NO:2. Alternatively or in addition, the variant scaffold sequence may comprise deletions, nucleotide substitutions, or a combination thereof. The variant CRISPR nuclease polypeptide may have increased binding to the variant scaffold sequence as compared with the scaffold of SEQ ID NO:2. In some examples, the variant scaffold may be a fragment of SEQ ID NO:2 or a variant thereof as disclosed herein. For example, the variant scaffold may be a truncatedAttorney Docket No.: 063586-523001WO version of SEQ ID NO: 2. Such a truncated version may have a length ranging from 120-160 nucleotides, for example, 130-160 nucleotides, 140-160 nucleotides, or 145-155 nucleotides. In some instances, the truncated version has one or more deletions relative to SEQ ID NO: 2, for example, in the P1c stem of SEQ ID NO: 2 (see FIG.3A). Alternatively or in addition, the truncated version may have a shortened P1b stem relative to SEQ ID NO:2 (see FIG. 3A). In some examples, the truncated version may have one or more deletions within the residues 17-43 of SEQ ID NO: 2. Such a truncated version may comprise (e.g., consists of) SEQ ID NO: 80. In some instances, the truncated version may further comprise one or more mutations (e.g., nucleotide substitutions, insertions, and / or deletions) relative to SEQ ID NO: 2. In some examples, the truncated version may comprise (e.g., consists of) a nucleotide sequence at least 75% (e.g., at least 78%, 80%, 85%, or greater) identical to SEQ ID NO: 80. In one example, the truncated version comprises (e.g., consists of) the nucleotide sequence of SEQ ID NO: 81. In a gRNA, the scaffold may be located at the 3’ end of the spacer. In some instances, the scaffold and spacer are connected directly. In other instances, the scaffold and spacer may be connected via a nucleotide linker. (ii) RT Donor RNA As used herein, the terms “reverse transcription donor RNA” or “RT donor RNA” refer to an RNA molecule comprising a reverse transcription template sequence (RTT sequence) and a primer binding site (PBS). An RT donor RNA may be fused to an RNA guide at either the 5’ end or 3’ end of the RNA guide. In some instances, an RT donor RNA may comprise additional elements such as a linker and / or an end protection motif. Any of the RT donor RNAs disclosed herein comprises: (i) a primer binding site (PBS), and (ii) an RTT sequence. In some instances, the RT donor RNA may further comprise: (iii) a nucleotide linker sequence, (iv) a 5’ end and / or 3’ end protection fragment (see disclosures herein), or a combination thereof. In some examples, the 5’ end or 3’ end protection fragment (e.g., 3’ extension) may comprise a pseudoknot motif to protect against 3’ exonuclease activity. In some embodiments, a RT donor RNA comprises an aptamer. In some embodiments, the aptamer recruits a reverse transcriptase polypeptide.Attorney Docket No.: 063586-523001WO Primer Binding Site (PBS) In some embodiments, the PBS in an RT donor RNA as disclosed herein is an RNA sequence capable of binding to a DNA strand via base-paring. The DNA strand has been or can be nicked or cleaved by the CRISPR nuclease polypeptide of the gene editing system disclosed herein. In some embodiments, the PBS comprises an RNA sequence capable of binding to a DNA strand (a PBS-targeting site) via base-pairing. The DNA strand may have a free 3’ end or a 3’ free end can be generated via cleavage by the CRISPR nuclease polypeptide contained in the same gene editing system. In some examples, the PBS-targeting site may be located on the same DNA strand as the PAM sequence (the PAM strand). In some embodiments, the PBS may be about 5-50 nucleotides in length. For example, the PBS may be about 5-40, 5-30, or 5-20 nucleotides in length. In specific examples, the PBS may be about 5-20 (e.g., 7-17 such as 8 or 12) nucleotides in length. In some examples, the PBS may contain 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, or 20 nucleotides. As used herein, the term “PBS-targeting site” refers to the region to which a PBS binds. The PBS-targeting site may be adjacent to (e.g., upstream to) the PAM. In a gene editing system comprising a CRISPR nuclease polypeptide that is a nickase variant (e.g., comprises a disrupted HNH nuclease domain as disclosed herein), the PBS in the RT donor RNA may bind to a region (the PBS-targeting site) on the PAM strand. In some embodiments, the PBS-targeting site may partially or completely overlap with the target sequence. In some instances, the PBS-targeting site may be located upstream to the PAM sequence. For example, the PBS-targeting site may be up to 100 nucleotides upstream to the PAM sequence, for example, up to 50 nucleotides, up to 30 nucleotides, up to 25 nucleotides, up to 20 nucleotides, up to 15 nucleotides, up to 10 nucleotides, or up to 5 nucleotides upstream to the PAM sequence. In specific examples, the PBS-targeting site may start about 3 nucleotides to about 10 nucleotides upstream of the PAM sequence (i.e., the 5’-most nucleotide of the PBS may bind about 3 nucleotides, 4, nucleotides, 5, nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides upstream of the PAM.) In specific examples, the PBS-targeting site may start 1 nucleotide, 1-2 nucleotides, 1- 3 nucleotides, 1-4 nucleotides, or 1-5 nucleotides, upstream of the PAM sequence. When a free 3’ end is generated by the CRISPR nuclease polypeptide in the gene editing systemAttorney Docket No.: 063586-523001WO within or nearby the target sequence, the PBS binding to the PAM strand at a site upstream to the PAM sequence could efficiently facilitate DNA synthesis by the RT polypeptide in the gene editing system, starting from the free 3’ end generated in the PAM strand. Reverse Transcription Template (RTT) Sequence The reverse transcription template sequence (RTT sequence) serves as the template for the reverse transcription mediated by the RT polypeptide in the gene editing system disclosed herein. In some embodiments, the RTT sequence comprises a sequence with at least one encoded edit. In some embodiments, the RTT sequence comprises sequence homology to a target sequence or its complementary region with at least one encoded edit. In some embodiments, the RTT sequence is at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length. In some embodiments, the RTT sequence is about 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 110 nucleotides, or 120 nucleotides in length or any length in between. In some embodiments, the RTT sequence is about 10 nucleotides. In some embodiments, the RTT sequence is about 11 nucleotides. In some embodiments, the RTT sequence is about 12 nucleotides. In some embodiments, the RTT sequence is about 13 nucleotides. In some embodiments, the RTT sequence is about 14 nucleotides. In some embodiments, the RTT sequence is about 15 nucleotides. In some embodiments, the RTT sequence is about 16 nucleotides. In some embodiments, the RTT sequence is about 17 nucleotides. In some embodiments, the RTT sequence is about 18 nucleotides. In some embodiments, the RTT sequence is about 19 nucleotides. In some embodiments, the RTT sequence is about 20 nucleotides. In some embodiments, the RTT sequence is about 21 nucleotides. In some embodiments, the RTT sequence is about 22 nucleotides. In some embodiments, the RTT sequence is about 23 nucleotides. In some embodiments, the RTTAttorney Docket No.: 063586-523001WO sequence is about 24 nucleotides. In some embodiments, the RTT sequence is about 25 nucleotides. In some embodiments, the RTT sequence is about 26 nucleotides. In some embodiments, the RTT sequence is about 27 nucleotides. In some embodiments, the RTT sequence is about 28 nucleotides. In some embodiments, the RTT sequence is about 29 nucleotides. In some embodiments, the RTT sequence is about 30 nucleotides. In some embodiments, the reverse transcription template sequence comprises at least one encoded edit (e.g., at least two) relative to a target sequence. In some embodiments, the at least one encoded edit comprises at least one substitution, insertion, and / or deletion. In some embodiments, the edit in the target sequence comprises a substitution, an insertion, and / or a deletion relative to the sequence of a target sequence. In some embodiments, the reverse transcription template sequence comprises at least one LoxP site. In some embodiments, the edit can be a single or multi-nucleotide substitution, such as a G to T substitution, a G to A substitution, a G to C substitution, a T to G substitution, a T to A substitution, a T to C substitution, a C to G substitution, a C to T substitution, a C to A substitution, an A to T substitution, an A to G substitution, or an A to C substitution. In some embodiments, the change in sequence can convert a G:C base pair to a T:A base pair, a G:C base pair to an A:T base pair, a G:C base pair to C:G base pair, a T:A base pair to a G:C base pair, a T:A base pair to an A:T base pair, a T:A base pair to a C:G base pair, a C:G base pair to a G:C base pair, a C:G base pair to a T:A base pair, a C:G base pair to an A:T base pair, an A:T base pair to a T:A base pair, an A:T base pair to a G:C base pair, or an A:T base pair to a C:G base pair. In some embodiments, a template sequence described herein may further introduce one or more silent mutations. As used herein, a silent mutation refers to a mutation that does not change the amino acid residue encoded by the codon comprising the mutation. The RTT sequence can be transcribed into DNA by the reverse transcriptase of the gene editing system described herein. In some embodiments, the RTT sequence is transcribed from 5’ to 3’ into DNA of the PAM strand. In some embodiments, the RTT sequence is 5’ of the PBS. In some embodiments, the RTT sequence is 3’ of the PBS. In some instances, the PBS and the RTT sequence in the RT donor RNA provided herein may be connected via a linker sequence to avoid steric hindrance between the two RNA components. In some embodiments, the PBS and an end protection fragment (e.g., a 3’ end protection fragment) may be connected via a linker sequence to avoid steric hindrance between the two RNA components.Attorney Docket No.: 063586-523001WO (iii) Single RNA Molecule In some embodiments, the gene editing system provided herein comprises a single RNA molecule, which includes both the gRNA and the RT donor RNA, or a nucleic acid encoding the single RNA molecule. Such a single RNA molecule is capable of mediating cleavage at a target sequence within a genomic site of interest by the CRISPR nuclease polypeptide and synthesis of a DNA fragment from a free 3’end of a free DNA strand generated by the CRISPR nuclease polypeptide cleavage based on the RTT sequence in the single RNA molecule. In some embodiments, the single RNA molecule may comprise the RNA guide linked to the RT donor RNA, optionally via a linker. In some examples, the single RNA molecule, from 5’ to 3’ end, comprises a spacer sequence, a scaffold sequence recognizable by the CRISPR nuclease polypeptide, a RTT sequence, and a PBS. In specific examples, the single RNA molecule may comprise, from 5’ to 3’, a spacer sequence, a scaffold sequence, an RTT sequence, a PBS, and a protection fragment. Any of the single RNA molecules provided herein may further comprise a linker, which may be located between a scaffold sequence and an RTT or following a PBS. In some examples, the linker may comprise a hairpin structure. In some examples, the linker may comprise an aptamer domain. In some examples, the 5’ end and / or the 3’ end of the single RNA molecule, or the gRNA and / or RT donor RNA, may contain a protection fragment, which may enhance resistance of the RNA molecule to exonuclease activity. In some instances, the end protection fragment may comprise a nucleotide sequence capable of forming a secondary structure, such as hairpin, a circularization, a pseudoknot, or a triplex structure. In other instances, the end protection fragment may comprise the sequence of an exoribonuclease-resistant RNA (xrRNA), a transfer RNA (tRNA), or a tRNA-like sequence. In some embodiments, the modification is a Zika-like pseudoknot, a murine leukemia virus pseudoknot (MLV-PK) sequence, a red clover necrotic mosaic virus (RCNMV) sequence, a sweet clover necrotic mosaic virus (SCNMV) sequence, a carnation ringspot virus (CRSV) sequence, a preQ1 aptamer sequence, a truncated preQ1 aptamer sequence, a boxB RNA sequence, or an RNA bacteriophage MS2 sequence. In some examples, the 5’ end of the single RNA molecule (or the 5’ end of the gRNA and / or the RT donor RNA when separate RNA molecules are used) may contain a 5’ extension motif, which can be any of the protection fragments disclosed herein. In someAttorney Docket No.: 063586-523001WO specific examples, the 3’ end of the single RNA molecule (or the 3’ end of the gRNA and / or the RT donor RNA when separate RNA molecules are used) may contain a 3’ extension motif, which can be any of the protection fragments disclosed herein. (iv) Modifications of Nucleic Acids Any of the RNA components in a gene editing system as disclosed herein, e.g., the single RNA molecule, the gRNA, and / or the RT donor RNA, may include one or more modifications. Exemplary modifications can include any modification to the sugar, the nucleobase, the internucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage / to the phosphodiester backbone), and any combination thereof. Some of the exemplary modifications provided herein are described in detail below. Any of the RNA components disclosed herein may include any useful modification, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage / to the phosphodiester backbone). One or more atoms of a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). In some instances, one or more atoms of a purine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). In certain embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and the internucleoside linkage. Modifications may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additional modifications are described herein. In some embodiments, any of the RNA components in a gene editing system as disclosed herein comprises an abasic site (i.e., a location that does not have a purine or a pyrimidine). In some embodiments, the abasic site (also referred to as an apurinic / apyrimidinic site) is present in an editing template RNA. For example, an abasic site can be present in the RTT of an editing template RNA. In some embodiments, activity of a reverse transcriptase is halted at or near an abasic site. In some embodiments, the modification may include a chemical or cellular induced modification. For example, some nonlimiting examples of intracellular RNA modificationsAttorney Docket No.: 063586-523001WO are described by Lewis and Pan in “RNA modifications and structures cooperate to guide RNA-protein interactions” from Nat Reviews Mol Cell Biol, 2017, 18:202-210. Different sugar modifications, nucleotide modifications, and / or internucleoside linkages (e.g., backbone structures) may exist at various positions in the sequence. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of the sequence, such that the function of the sequence is not substantially decreased. The sequence may include from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e. any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%>, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). In some embodiments, sugar modifications (e.g., at the 2’ position or 4’ position) or replacement of the sugar at one or more ribonucleotides of the sequence may, as well as backbone modifications, include modification or replacement of the phosphodiester linkages. Specific examples of a sequence include, but are not limited to, sequences including modified backbones or no natural internucleoside linkages such as internucleoside modifications, including modification or replacement of the phosphodiester linkages. Sequences having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this application, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, a sequence will include ribonucleotides with a phosphorus atom in its internucleoside backbone. Modified sequence backbones may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates such as 3’-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates such as 3’-amino phosphoramidate andAttorney Docket No.: 063586-523001WO aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3’-5’ linkages, 2’-5’ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3’-5’ to 5’-3’ or 2’-5’ to 5’-2’. Various salts, mixed salts and free acid forms are also included. In some embodiments, the sequence may be negatively or positively charged. The modified nucleotides, which may be incorporated into the sequence, can be modified on the internucleoside linkage (e.g., phosphate backbone). Herein, in the context of the polynucleotide backbone, the phrases “phosphate” and “phosphodiester” are used interchangeably. Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates). The α-thio substituted phosphate moiety is provided to confer stability to RNA and DNA polymers through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment. In specific embodiments, a modified nucleoside includes an alpha-thio-nucleoside (e.g., 5’-O-(1-thiophosphate)-adenosine, 5’-O-(1-thiophosphate)-cytidine (a-thio-cytidine), 5’-O-(1-thiophosphate)-guanosine, 5’-O-(1-thiophosphate)-uridine, or 5’-O-(1- thiophosphate)-pseudouridine).Other internucleoside linkages that may be employed according to the present invention, including internucleoside linkages which do not contain a phosphorous atom, are described herein. In some embodiments, the sequence may include one or more cytotoxic nucleosides. For example, cytotoxic nucleosides may be incorporated into sequence, such as bifunctional modification. Cytotoxic nucleoside may include, but are not limited to, adenosineAttorney Docket No.: 063586-523001WO arabinoside, 5-azacytidine, 4’-thio-aracytidine, cyclopentenylcytosine, cladribine, clofarabine, cytarabine, cytosine arabinoside, 1-(2-C-cyano-2-deoxy-beta-D-arabino- pentofuranosyl)-cytosine, decitabine, 5-fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafur ((RS)-5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine- 2,4(1H,3H)-dione), troxacitabine, tezacitabine, 2’-deoxy-2’-methylidenecytidine (DMDC), and 6-mercaptopurine. Additional examples include fludarabine phosphate, N4-behenoyl-1- beta-D-arabinofuranosylcytosine, N4-octadecyl-1-beta-D-arabinofuranosylcytosine, N4- palmitoyl-1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl) cytosine, and P-4055 (cytarabine 5’-elaidic acid ester). In some embodiments, the sequence includes one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, poly-A sequence, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol groups and tyrosine residues, etc.). The one or more post-transcriptional modifications can be any post-transcriptional modification, such as any of the more than one hundred different nucleoside modifications that have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197) In some embodiments, the first isolated nucleic acid comprises messenger RNA (mRNA). In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of pyridin-4-one ribonucleoside, 5- aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl- pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1- taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl- pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2- thio-pseudouridine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl- pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5- methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1- methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-Attorney Docket No.: 063586-523001WO zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy- cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1- methyl-pseudoisocytidine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 2-aminopurine, 2, 6-diaminopurine, 7- deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2- aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1- methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine. In some embodiments, mRNA comprises at least one nucleoside selected from the group consisting of inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza- guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7- deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6- methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8- oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio- guanosine, and N2,N2-dimethyl-6-thio-guanosine. The sequence may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotides (e.g., naturally-occurring nucleotides, purine or pyrimidine, or any one or more or all of A, G, U, C, I, pU) may or may not be uniformly modified in the sequence, or in a given predetermined sequence region thereof. In some embodiments, the sequence includes a pseudouridine. In some embodiments, the sequence includes an inosine, which may aid in the immune system characterizing the sequence as endogenous versus viral RNAs. The incorporation of inosine may also mediate improved RNA stability / reduced degradation. See for example, Yu, Z. et al. (2015) RNA editing by ADAR1 marks dsRNA as “self”. Cell Res.25, 1283-1284, which is incorporated by reference in its entirety. In some embodiments, any RNA sequence described herein, such as an editing template RNA, may comprise an end modification (e.g., a 5’ end modification or a 3’ end modification). In some embodiments, the end modification is a chemical modification. In some embodiments, the end modification is a structural modification. See disclosures herein.Attorney Docket No.: 063586-523001WO When a gene editing system disclosed herein comprises nucleic acids encoding the CRISPR nuclease and / or the RT polypeptide, e.g., mRNA molecules, such nucleic acid molecules may contain any of the modifications disclosed herein, where applicable. C. Exemplary Gene Editing Systems Exemplary gene editing systems described herein, meant to be illustrative only, may comprise: (a) a fusion polypeptide or a nucleic acid encoding such, wherein the fusion polypeptide comprises any of the CRISPR nuclease polypeptides, any of the RT polypeptides, and optionally one or more NLSs, which may be located at the N-terminus and / or the C-terminus, one or more peptide linkers, or a combination thereof; and (b) a single RNA molecule, comprising a guide RNA, an RT donor RNA, and optionally one or more nucleotide linkers, one or more 5’ end or 3’ end protection elements, or a combination thereof. In some embodiments, the CRISPR nuclease polypeptide in the fusion polypeptide may be a nickase variant (e.g., those provided in Table 6 below, such as the nickase variant containing the E425 mutation, e.g., E425A. Alternatively or in addition, the CRISPR nuclease polypeptide in the fusion polypeptide may comprise one or more arginine and / or lysine substitutions (e.g., arginine substitutions), for example, at the positions disclosed herein (e.g., A65, V85, V117, L134, A657, S695, N691, D707, E715, and / or S925 in SEQ ID NO:1). In specific examples, the arginine and / or lysine substitutions (e.g., arginine substitutions) are at positions A65, A657 and S925, optionally further at positions V85 and L134 in SEQ ID NO:1. In some instances, the arginine and / or lysine substitutions may further comprise the D707 position. Additional examples can be found in Tables 26-28. In some embodiments, the RT polypeptide in the fusion polypeptide may be an MMLV variant, for example, SEQ ID NO: 49 provided in Examples 6 and 7 below. In some examples, the fusion polypeptide provided herein may comprise an N- terminal CRISPR nuclease polypeptide at the N-terminus and a C-terminal RT polypeptide. Optionally, the fusion polypeptide may comprise a peptide linker (e.g., a G / S rich linker or an XTEN peptide linker) between the CRISPR nuclease polypeptide and the RT polypeptide. Alternatively or in addition, the fusion polypeptide may comprise an NLS at the N-terminus and / or the C-terminus. In some examples, the fusion polypeptide may comprise two different NLS sites, one at the N-terminus and the other one at the C-terminus.Attorney Docket No.: 063586-523001WO Exemplary fusion polypeptides for use in the gene editing system disclosed herein are provide in Tables 11 and 12. The single RNA molecule contained in the exemplary gene editing system may comprise the guide RNA and the RT donor RNA in any orientation. Optionally, the single RNA molecule may contain one or more nucleotide linkers between the gRNA and the RT donor RNA, and / or between the functional domains in the gRNA (e.g., between the spacer and the scaffold sequences) and / or in the RT donor RNA (e.g., between the PBS and the RTT sequences). In some examples, the single RNA molecule may further comprise a protection fragment (e.g., those disclosed herein) at the 5’ and / or 3’ end. In specific examples, the single RNA molecule contained in the exemplary gene editing system may comprise, from 5’ to 3’, a spacer sequence, a scaffold sequence, an RTT, a PBS, and a 3’ extension, which may have a pseudoknot motif. In some examples, an exemplary gene editing system provided herein may comprise any of the CRISPR-RT fusion polypeptides provided in Table 11 or Table 12 below, and a single RNA molecule provided herein (e.g., comprising, from 5’ to 3’, a spacer sequence, a scaffold sequence, an RTT, a PBS, and optionally a 3’ extension, which may have a pseudoknot motif). In other examples, an exemplary gene editing system provided herein may comprise any of the CRISPR-RT fusion polypeptides provided in Table 11 or Table 12 below, and a nucleic acid (e.g., a vector such as a viral vector) coding for a single RNA molecule provided herein (e.g., comprising, from 5’ to 3’, a spacer sequence, a scaffold sequence, an RTT, a PBS, and optionally a 3’ extension, which may have a pseudoknot motif). In yet other examples, an exemplary gene editing system provided herein may comprise a nucleic acid encoding any of the CRISPR-RT fusion polypeptides provided in Table 11 or Table 12 below, and a single RNA molecule provided herein (e.g., comprising, from 5’ to 3’, a spacer sequence, a scaffold sequence, an RTT, a PBS, and optionally a 3’ extension, which may have a pseudoknot motif). The nucleic acid may comprise an encoding nucleotide sequence that is codon optimized, for example, those provided in Table 11 or Table 12. In still other examples, an exemplary gene editing system provided herein may comprise a nucleic acid encoding any of the CRISPR-RT fusion polypeptides provided in Table 11 or Table 12 below, and a nucleic acid (e.g., a vector such as a viral vector) encoding a single RNA molecule provided herein (e.g., comprising, from 5’ to 3’, a spacerAttorney Docket No.: 063586-523001WO sequence, a scaffold sequence, an RTT, a PBS, and optionally a 3’ extension, which may have a pseudoknot motif). The nucleic acid encoding the fusion polypeptide may be codon optimized, for example, those provided in Table 11 or Table 12. In some instances, the gene editing system may comprise two vectors, one encoding the fusion polypeptide and the other one encoding the single RNA molecule. Alternatively, the gene editing system may comprise one vector encoding both the fusion polypeptide and the single RNA molecule. III. Genetic Editing Methods Any of the gene editing systems can be used to genetically modify (edit) a target nucleic acid, which can be a genetic site of interest, e.g., a genetic site where genetic editing is needed, for example, to fix a genetic mutation, to introduce a protective mutation, to introduce modifications for modulating expression of a gene, etc. A. Delivery of Gene Editing System to Target Cells Components of any of the gene editing systems disclosed herein may be formulated, for example, including a carrier, such as a carrier and / or a polymeric carrier, e.g., a liposome, and delivered by known methods to a cell (e.g., a mammalian cell). Such methods include, but not limited to, transfection (e.g., lipid-mediated, cationic polymers, calcium phosphate, dendrimers); electroporation or other methods of membrane disruption (e.g., nucleofection), viral delivery (e.g., lentivirus, retrovirus, adenovirus, adeno-associated virus (AAV)), microinjection, microprojectile bombardment (“gene gun”), fugene, direct sonic loading, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, exosome- mediated transfer, lipid nanoparticle-mediated transfer, and any combination thereof. In some examples, the delivery method involves the use of lipid nanoparticles to mediate delivery of one or more components of the gene editing system disclosed herein. In some embodiments, the method comprises delivering one or more nucleic acids (e.g., nucleic acids encoding the CRISPR nuclease polypeptide, the RT polypeptide, or the fusion polypeptide comprising both, the RNA guide, the RT donor RNA, or the single RNA molecule comprising both, etc.), one or more transcripts thereof, and / or a pre-formed RNA guide / CRISPR nuclease polypeptide / RT polypeptide complex to a cell, where a ternary complex is formed. In some embodiments, an RNA guide and / or RT donor RNA, or a fusion thereof, and an RNA encoding a CRISPR nuclease polypeptide or a RT polypeptide, or a fusion polypeptide comprising both, are delivered together in a single composition. In someAttorney Docket No.: 063586-523001WO embodiments, an RNA guide and an RNA encoding a CRISPR nuclease polypeptide are delivered in separate compositions. In some embodiments, an RNA guide / RT donor RNA and an RNA encoding a CRISPR nuclease polypeptide / RT polypeptide delivered in separate compositions are delivered using the same delivery technology. In some embodiments, an RNA guide / RT donor RNA and an RNA encoding a CRISPR nuclease polypeptide / RT polypeptide delivered in separate compositions are delivered using different delivery technologies. In some embodiments, one or more of the protein components and one or more of the RNA components are delivered together. For example, the CRISPR nuclease and / or RT polypeptide and the RNA guide and / or RT donor RNA are packaged together in a single AAV particle. In another example, the CRISPR nuclease and / or RT polypeptide and the RNA guide and / or RT donor RNA are delivered together via lipid nanoparticles (LNPs). In some embodiments, the CRISPR nuclease and / or RT polypeptides and the RNA guide and / or RT donor RNA are delivered separately. For example, the CRISPR nuclease and / or RT polypeptides and the RNA guide and / or RT donor RNA are packaged into separate AAV particles. In another example, the CRISPR nuclease and / or RT polypeptides is delivered by a first delivery mechanism and the RNA guide and / or RT donor RNA is delivered by a second delivery mechanism. Exemplary intracellular delivery methods, include, but are not limited to: viruses, such as AAV, or virus-like agents; chemical-based transfection methods, such as those using calcium phosphate, dendrimers, liposomes, or cationic polymers (e.g., DEAE-dextran or polyethylenimine); non-chemical methods, such as microinjection, electroporation, cell squeezing, sonoporation, optical transfection, impalefection, protoplast fusion, bacterial conjugation, delivery of plasmids or transposons; particle-based methods, such as using a gene gun, magnectofection or magnet assisted transfection, particle bombardment; and hybrid methods, such as nucleofection. In some embodiments, a lipid nanoparticle comprises an mRNA encoding a CRISPR nuclease-RT fusion polypeptide, an editing template RNA, or an mRNA encoding such. In some embodiments, the present application further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells.Attorney Docket No.: 063586-523001WO B. Genetically Modified Cells Any of the gene editing systems disclosed herein can be delivered to a variety of cells (e.g., to mammalian cells such as a mouse cell, a non-human primate cell, or a human cell). In some embodiments, the cell is in cell culture or a co-culture of two or more cell types. In some embodiments, the cell is ex vivo. In some embodiments, the cell is obtained from a living organism and maintained in a cell culture. In some embodiments, the cell is derived from a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, 293T, MF7, K562, HeLa, CHO, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, the cell is an immortal or immortalized cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is a stem cell such as a totipotent stem cell (e.g., omnipotent), a pluripotent stem cell, a multipotent stem cell, an oligopotent stem cell, or an unipotent stem cell. In some embodiments, the cell is an induced pluripotent stem cell (iPSC) or derived from an iPSC. In some embodiments, the cell is a differentiated cell. In some embodiments, the cell is a mammalian cell, e.g., a human cell or a murine cell. In some embodiments, the murine cell is derived from a wild-type mouse, an immunosuppressed mouse, or a disease-specific mouse model. In some embodiments, the cell is a cell within a living tissue, organ, or organism. Any of the genetically modified cells produced using any of the gene editing system disclosed herein is also within the scope of the present disclosure. Such modified cells may comprise a disrupted target gene. Any of the gene editing systems, compositions comprising such, vectors, nucleic acids, RNA guides and cells disclosed herein may be used in therapy. Gene editing systems, compositions, vectors, nucleic acids, RNA guides and cells disclosed herein may be used in methods of treating a disease or condition in a subject. Any suitable delivery or administration method known in the art may be used to deliver compositions, vectors, nucleic acids, RNA guides and cells disclosed herein. Such methods may involve contacting a target sequence with a composition, vector, nucleic acid, or RNA guide disclosed herein. Such methods may involve a method of editing a target sequence as disclosed herein. In some embodiments, a cell engineered using an RNA guide disclosed herein is used for ex vivo gene therapy.Attorney Docket No.: 063586-523001WO IV. Therapeutic Applications Any of the gene editing systems or modified cells generated using such a gene editing system as disclosed herein may be used for treating a disease that is associated with the target gene, for example, a genetic defect in the target gene. In some embodiments, provided herein is a method for treating a target disease as disclosed herein comprising administering to a subject (e.g., a human patient) in need of the treatment any of the gene editing systems disclosed herein. The gene editing system may be delivered to a specific tissue or specific type of cells where the gene edit is needed. The gene editing system may comprise LNPs encompassing one or more of the components, one or more vectors (e.g., viral vectors) encoding one or more of the components, or a combination thereof. Components of the gene editing system may be formulated to form a pharmaceutical composition, which may further comprise one or more pharmaceutically acceptable carriers. In some embodiments, modified cells produced using any of the gene editing systems disclosed herein may be administered to a subject (e.g., a human patient) in need of the treatment. The modified cells may comprise a substitution, insertion, and / or deletion described herein. In some examples, the modified cells may include a cell line modified by the CRISPR nuclease polypeptide, the RT polypeptide, or the CRISPR nuclease-RT fusion polypeptide, and the RNA guide and RT donor RNA or the single RNA molecule comprising both. In some instances, the modified cells may be a heterogenous population comprising cells with different types of gene edits. Alternatively, the modified cells may comprise a substantially homogenous cell population (e.g., at least 80% of the cells in the whole population) comprising one particular gene edit in the target gene. In some examples, the cells can be suspended in a suitable media. In some embodiments, provided herein is a composition comprising the gene editing system or components thereof. Such a composition can be a pharmaceutical composition. A pharmaceutical composition that is useful may be prepared, packaged, or sold in a formulation suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, intra- lesional, buccal, ophthalmic, intravenous, intra-organ or another route of administration. A pharmaceutical composition of the disclosure may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition (e.g., the gene editing system or components thereof), which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.Attorney Docket No.: 063586-523001WO A formulation of a pharmaceutical composition suitable for parenteral administration may comprise the active agent (e.g., the gene editing system or components thereof or the modified cells) combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such a formulation may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Some injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Some formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Some formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. The pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the cells, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulation may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or saline. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which that are useful include those which may comprise the cells in a packaged form, in a liposomal preparation, or as a component of a biodegradable polymer system. Some compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt. V. Kits and Uses Thereof The present disclosure also provides kits that can be used, for example, to carry out a method described herein for genetical modification of a target gene. In some embodiments, the kits include an RNA guide and an RT donor RNA, or a single RNA molecule comprising both, a CRISPR nuclease polypeptide, and an RT polypeptide, or a fusion polypeptide thereof. In some embodiments, the kits include the single RNA molecule and the CRISPR nuclease-RT fusion polypeptide. In some embodiments, the kits include a polynucleotide that encodes the CRISPR nuclease polypeptide, the RT polypeptide, or the CRISPR nuclease-RTAttorney Docket No.: 063586-523001WO fusion polypeptide, and optionally the polynucleotide is comprised within a vector, e.g., as described herein. In some embodiments, the kits include a polynucleotide that encodes the RNA components disclosed herein. The CRISPR nuclease polypeptide, the RT polypeptide, or a fusion polypeptide thereof (or polynucleotide encoding such) and the RNA components (e.g., as a ribonucleoprotein) can be packaged within the same or other vessel within a kit or can be packaged in separate vials or other vessels, the contents of which can be mixed prior to use. The CRISPR nuclease polypeptide, the RT polypeptide, and the RNA components can be packaged within the same or other vessel within a kit or can be packaged in separate vials or other vessels, the contents of which can be mixed prior to use. The kits can additionally include, optionally, a buffer and / or instructions for use of the RNA components, the CRISPR nuclease polypeptide, and the RT polypeptide, or the fusion polypeptide thereof. General techniques The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed.1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed.1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds.1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E.Attorney Docket No.: 063586-523001WO Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed.1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds.(1985»; Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984»; Animal Cell Culture (R.I. Freshney, ed. (1986»; Immobilized Cells and Enzymes (lRL Press, (1986»; and B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.). Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present disclosure to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the present disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein. EXAMPLES The following examples are provided to further illustrate some embodiments of the present disclosure but are not intended to limit the scope of the present disclosure; it will be understood by their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used. Example 1: CRISPR Nuclease Mediated Editing of Human Target Sequences in HEK293T Cells This Example describes the genomic editing of the AAVS1, EMX1, and VEGFA genes by the CRISPR nuclease of SEQ ID NO: 1 introduced into cells by lipid-based transient transfection into the HEK293T cell line. The CRISPR nuclease was tagged with an N-terminal SV40 nuclear localization sequence (NLS) and a C-terminal XTEN linker directly upstream of a nucleoplasmin NLS, and its coding sequence was converted to a human codon-optimized DNA sequence, synthesized, and cloned into a pcDNA3.1 vector (Invitrogen), containing a CMV promoter for expression. The reference and NLS-tagged sequences used are in Table 1. Plasmids were received from GenScript.Attorney Docket No.: 063586-523001WO Table 1. Amino Acid Sequences Sequence Nucleotide sequence Amino acid sequence (codon optimized for human cells) Reference at actcaacacacacact ccatc atttc ca caa aacatc MSSTHTLAIDFGSKNIGIAL D R H F E L G A L R T Q L R A G N N P R V T S V N E G A V Q L G A S PAttorney Docket No.: 063586-523001WO cgtgatcggcgtgagaatccggtacccctggctgaaggccaacatgg LSGRVIPNIRI (SEQ ID NO: acctgcaccgggtggaacggcagacaggccagatcgtgcacagata 1) tgtggccgaccccgctaaccaggccctgatcgttgcctacctgggcag S N L E L R E F Q KI K E K L I P C R A L E D HAttorney Docket No.: 063586-523001WO agatgctgagaaaggaatttaacggcctttgcgcctactgcggta ALDAMILACDLPDVTALEA aacctagcgaaaccatcgtggagagagagcacatcctgccgaa RNLPPWIISAWVDKVRSAA PQAGQEGIPAIPHPDFAVPEF N E A V P S L G Y Q L Y S E S Q e 5’-Attorney Docket No.: 063586-523001WO NRV-3’ PAM sequences (the PAM sequence is on the 3’ end of the target sequence). The U6 PolIII promoter uses a +1 G at the start of the transcript (i.e., the 5’ end of the RNA) for more efficient transcription that is excluded from the sequences described here. See all RNA guide sequences in Table 2. Plasmids were purified using a midiprep kit. Table 2. Target and RNA Guide Sequences Guide PAM Target RNA Guide Sequence* u gu u u uu gu u u u gu u u u gu u u u gu u uAttorney Docket No.: 063586-523001WO VEGFA- GAG CCCAGTATGGGC CCCAGUAUGGGCAAAGGAAAguuucagcuacccugaguu T6 AAAGGAAA ucucaacuccguugagacuuccucagguucaagcugaaaagagcaucugu ccggaaggaccacuccggguuagggcagacccggcucuugggcuuccuu uApproximately 16 hours prior to transfection, 25,000 HEK293T cells in DMEM / 10%FBS+Pen / Strep (D10 media) were plated into each well of a 96-well plate. On the day of transfection, the cells were 50-90% confluent. For each well to be transfected, a mixture of Lipofectamine 2000™ (ThermoFisher Scientific) and Opti-MEM™ (ThermoFisher Scientific) was prepared and incubated at room temperature for 5 minutes (Solution 1). After incubation, the Lipofectamine 2000™:Opti-MEM™ mixture was added to a separate mixture containing the CRISPR nuclease plasmid (NLS-tagged), RNA guide plasmid, and Opti-MEM™ (Solution 2). In the case of negative controls, the CRISPR nuclease plasmid was excluded. Solutions 1 and 2 were mixed by pipetting up and down, then incubated at room temperature for 25 minutes. Following incubation, the Solution 1 and 2 mixture was added dropwise to each well of a 96-well plate containing the cells. Approximately 72 hours post transfection, cells were trypsinized by adding TrypLE™ (Thermo Fisher Scientific) to the center of each well and incubating at 20-37ºC for approximately 5 minutes. D10 media was then added to each well and mixed to resuspend cells. The resuspended cells were centrifuged for 10 minutes to obtain a pellet, and the supernatant was discarded. The cell pellet was then resuspended in QuickExtract™ buffer (Lucigen®), and cells were incubated at 65ºC for 15 minutes, 68ºC for 15 minutes, and 98ºC for 10 minutes. Next Generation Sequencing (NGS) samples were prepared by two rounds of PCR. Three technical replicates were analyzed per target for the reference and each variant. The first round (PCR1) was used to amplify specific genomic regions depending on the target. Round 2 PCR (PCR2) was performed to add Illumina adapters and indices. Reactions were then pooled and purified by column purification. Sequencing runs were performed using a 150 Cycle NextSeq 500 / 550 Mid or High Output v2.5 Kit or a 200 Cycle NovaSeq 6000 SP or S1 Reagent Kit v1.5.Attorney Docket No.: 063586-523001WO For NGS analysis, the indel mapping function used a sample’s fastq file, the amplicon reference sequence, and the forward primer sequence. For each read, a kmer-scanning algorithm was used to calculate the edit operations (match, mismatch, insertion, deletion) between the read and the reference sequence. In order to remove small amounts of primer dimer present in some samples, the first 30 nt of each read was required to match the reference and reads where over half of the mapping nucleotides are mismatches were filtered out as well. Up to 50,000 reads passing those filters were used for analysis, and reads were counted as an indel read if they contained an insertion or deletion. The QC standard for the minimum number of reads passing filters was 10,000. For each target, indel ratios, referring to the percentage of NGS reads comprising indels, were calculated for each sample and its cognate no protein control. Targets comprising a higher percentage of indels when the CRISPR nuclease was included in the transfection were indicative of DNA editing outcomes in the cell. As shown in FIG.1, each of the six targets tested demonstrated a greater level of indels observed when the CRISPR nuclease plasmid was present. This Example thus shows that the CRISPR nuclease of SEQ ID NO: 1 edited human genes. Example 2: Effectiveness of Variant CRISPR Nucleases for Targeting of Mammalian Genes This Example describes indel assessment on mammalian targets using CRISPR nuclease variants transfected into HEK293T cells. Arginine scanning mutagenesis was performed to individually substitute each non- arginine residue of the reference CRISPR nuclease (SEQ ID NO: 1) to arginine. SEQ ID NO: 1 is referred to herein as the reference sequence. This resulted in 858 single arginine substitution variants. Nucleic acids encoding the reference CRISPR nuclease and each CRISPR nuclease variant were then individually cloned into a pcDNA3.1 backbone (InvitrogenTM), and the plasmids were maxi-prepped and diluted. The plasmids comprised a CMV promoter, a first NLS (KRTADGSEFESPKKKRKV; SEQ ID NO: 3) upstream of the coding sequence, an XTEN linker (SGGSSGGSSGSETPGTSESATPESSGGSSGGSS; SEQ ID NO:5), and a second NLS (KRPAATKKAGQAKKKK; SEQ ID NO: 4) downstream of the coding sequence. See also Example 1 above. The RNA guide and target sequences are shown in Table 3. RNA guides were cloned into a pUC19 backbone (New England Biolabs®) following the U6 PolIII promoter andAttorney Docket No.: 063586-523001WO terminated with a 6x polyT sequence. The plasmids were then maxi-prepped and diluted. The RNA guide sequences shown in Table 3 exclude a +1 G that is utilized by the U6 PolIII promoter at the start of the transcript (i.e., the 5’ end of the RNA) for more efficient transcription. Table 3. Mammalian Targets and Corresponding crRNAs Target PAM Target Sequence RNA Guide Sequence Identifier Sequence U C C G C U C A U C C G C U C A U C C G C U C ACells were transfected, and samples were prepared for NGS as described in Example 1. Indel ratios, referring to the percentage of NGS reads comprising indels, were calculated for the reference and for each variant. The indel ratios used for fold change calculations were the average of two technical replicates. To then calculate fold change in indel ratios, the indel ratio for each variant was divided by the indel ratio for the reference. Table 4 shows fold change in indel ratios for the target. Numbering is relative to the reference nuclease of SEQ ID NO: 1 (i.e., without an NLS).Attorney Docket No.: 063586-523001WO As shown in Table 4, 7 variants with single arginine substitutions (left column) were characterized as yielding at least a 2X increase in indel ratio relative to the reference indel ratio, when averaged across all targets (right column). Table 4. Fold Change in Indel Ratios* bstitutionAAVAverage Fold SuS1 EMX1 VEGFAChangey g os, when averaged across all targets: A801R, E114R, A861R, E702R, E116R, D479R, A65R, L134R, V51R, A854R, E102R, F118R, G294R, V717R, E277R, S905R, T483R, E711R, A380R, N131R, A173R, N698R, D707R, I794R, V85R, F859R, E386R, A177R, N288R, Q292R, E271R, S44R, E270R, E113R, A115R, D382R, E112R, E124R, E48R, L37R, Q769R, A694R, E139R, D267R, A68R, A478R, D475R, A743R, A770R, P106R, S76R, S127R, S747R, S666R, Q826R, S326R, V523R, L268R, E739R, A788R, Q811R, N605R, I143R, A896R, E518R, A742R, V856R, G108R, K930R, I746R, G783R, M56R, T272R, L833R, S764R, Q266R, Q245R, I273R, A265R, G858R, P417R, E71R, H69R, E897R, Q206R, A785R, A690R, K701R, S620R, P269R, S612R, K60R, T280R, T599R, G529R, Q389R, T733R, E864R, L75R, L264R, E109R, T59R, Q627R, S895R, S863R, E419R, K617R, V577R, I953R, H911R, N432R, D759R, A603R, F260R, L248R, V88R, D777R, A688R, P532R, V548R, A613R, G947R, K751R, V718R, I610R, S835R, H151R, S468R, A684R, Q390R, S238R, L142R, Y394R, S210R, E135R, H635R, D39R, A621R, G579R, T420R, K455R, E680R, A891R, K710R, A456R, G263R, E602R, D189R, K45R, L214R, K405R, N408R, P705R, Q814R, V84R, K64R, V235R, K703R, L679R, Q624R, A460R, T324R, E274R, G395R, P951R, P857R, W609R, S457R, H665R, I950R, Q871R, F120R, K397R, Q543R, S740R, D780R, D211R, A305R, E291R, A632R, K416R, D447R, E784R, K843R, Q894R, K480R, L400R, S293R, V615R, A726R, A772R, Q564R, T581R, G834R, Q648R, K482R, G314R, D156R, K14R, S461R, Y309R, G521R, L27R, L499R, H890R, andAttorney Docket No.: 063586-523001WO S262R. The remaining variants (645 variants) resulted in decreased indel ratios relative to the reference indel ratios (fold change in indel ratios of less than 1.0). The following substitutions, which resulted in at least a 2X-increase in indel ratios relative to the reference, were selected to engineer combination variants: S925R, E715R, S695R, N691R, A657R, V117R, and A65R. Example 3: Effectiveness of Combination CRISPR Nuclease Variants for Targeting of Mammalian Genes This Example describes indel assessment on mammalian targets using CRISPR nuclease variants comprising two or more substitutions identified as increasing indel activity in Example 2.56 combination CRISPR variants were tested. Each CRISPR nuclease variant was cloned as described in Example 2. HEK293T cells were further transfected with the CRISPR nuclease variant and an RNA guide of Table 3 above, followed by NGS analysis, as described in Example 2. For each target, indel ratios, referring to the percentage of NGS reads comprising indels, were calculated for the reference CRISPR nuclease (SEQ ID NO: 1) and for each variant CRISPR nuclease. The indel ratios shown in Table 5 were calculated as the average of two bioreplicates, each of which contained two technical replicates. Table 5. Fold Change in Indel Ratios* Substitutions Average AAVS1 EMX1 VEGFA FoldAttorney Docket No.: 063586-523001WO E715R, A657R, A65R 2.96 4.30 3.29 3.52 S925R, E715R 2.77 4.25 3.50 3.51Attorney Docket No.: 063586-523001WO As shown in Table 5, all 56 CRISPR nuclease variants with combinations of amino acid substitutions exhibited higher indel activity than the reference CRISPR nuclease (SEQ ID NO: 1).18 variants were analyzed as yielding at least a 3.5X increase in indel ratio relative to the reference indel ratio, when averaged across all targets: a) S925R, E715R, and A65R, b) S925R, A657R, and A65R, c) S925R, S695R, and A657R, d) S925R, S695R, and N691R, e) S925R, N691R, and A65R, f) S925R, S695R, and V117R, g) S925R, E715R, and N691R, h) S925R, E715R, and V117R, i) S925R, E715R, and S695R, j) S925R, S695R, and A65R, k) S925R, N691R, and A657R, l) S695R, N691R, and A657R, m) E715R, V117R, and A65R, n) S925R, A657R, and V117R, o) E715R, S695R, and V117R, p) S925R and S695R, q) E715R, A657R, and A65R, and r) S925R and E715R. The remaining variants (38) were analyzed as having indel ratios 2X-3.5X of the reference indel ratios, when averaged across all targets. The two CRISPR nuclease variants that resulted in the highest increase in indel ratio relative to the reference indel ratio were selected for further experimentation. These CRISPR nuclease variants comprised the following substitutions: a) S925R, E715R, and A65R and b) S925R, A657R, and A65R. Example 4: Engineering and Effectiveness of CRISPR Nickase Variants for Targeting Mammalian Genes This Example describes introducing mutations into the CRISPR nuclease of SEQ ID NO: 1 that disrupt either the HNH or RuvC domains to produce a functional nickase. E425, H426, and N449 were identified as putative catalytic residues of the HNH domain. D10, E362, and D487 positions were identified as putative catalytic residues of the RuvC domain. These positions were identified by analyzing models generated with AlphaFold2 (Jumper et al., Nature 596: 583-9 (2021)) for structural regions resembling known HNH and RuvC active sites and / or by performing sequence alignments to other nucleases for which candidate positions had been previously identified. Examples of reference structures used to identify the HNH and RuvC active sites are represented with the following PDB IDs: 5h0m, 7eu9, 6ltu, 7odf, 7lys, 8dc2, 4cmp, 4oo8, 7z4j, 5axw, 5b2o, 6kc8, 7utn, 8csz, 8ctl, 8dmb. The coding sequence of the reference CRISPR nuclease was converted into an E. coli- codon optimized DNA sequence, synthesized, and cloned into a pET-28a(+) vector (Novagen) containing lac and T7 RNA polymerase promoters for gene expression. To test for nickase activity, individual alanine mutants were cloned for each of the positions identified asAttorney Docket No.: 063586-523001WO putative active site residues of the HNH and RuvC domains. A leucine mutant was also cloned for position H426. Research grade plasmids were received from GenScript. The engineered nickase sequences are shown in Table 6. The codon encoding the substituted residue is capitalized, bold, and underlined in the nucleotide sequence, and the substituted residue is shown in bold and underlined in the amino acid sequence. The putative HNH- knockout nickases were anticipated to cleave the non-target strand but not the target strand. The putative RuvC-knockout nickases were anticipated to cleave the target strand but not the non-target strand. Table 6. CRISPR Nuclease and Nickase Sequences CRISPR Nucleotide sequence Amino acid sequence Nuclease (codon optimized for E. coli cells)Attorney Docket No.: 063586-523001WO gctcgctacctgtgtgaaaagctccgtcaacgttacgatcgtgtgacccaga tcgcatttcgttctggtagacacacggcgatctggcgtcaagcggcttaccc R LS K V EP SA E R A DP LR E K N PF LW ER Y L RA PD ES LF E RAttorney Docket No.: 063586-523001WO aaggacgatctggaaatgctgcgcaaggaattcaacggtttgtgcgcatact PLARYLCEKLRQRYDRVTQIA gcggtaagccgagcgagaccattgttgaacgcGCGcacattctgccga FRSGRHTAIWRQAAYPDFDKA KEKVSGGTINHALDAMILACD V PD KL G A V ES AR GT M PA P EV WQ Q Y R R LS K V EP SA E R A DP LR E K N PF LWAttorney Docket No.: 063586-523001WO aaattcctgtctggcttagcccaggacctgccggaagaaacgatcgaaccg RYIEARVLPLAPAGIDRVVVER aaagaggcctggacccatattcgtcgtaacctgatgaatctgattgagcaga TAFDMLAGSRKQRQKVADDY LEEMYQQGPRYGFKDDLEML RE PD ES LF E R A KA D V PD KL G A V ES AR GT M PA P EV WQ Q Y R R LS K V EP SA E RAttorney Docket No.: 063586-523001WO agccgtgaggaattcttcagcgcgttagagaacgaactgctggagcgcctg GASRCAWDGCNSVTPRRDNA ccggaagagaagcttatcccggttctagacctctgcgaacacgggttgaac LRDALAQFVFTVYAQKVRSDP DLYEKIQRMLDQLAQLGKRLR E K N PF LW ER DY L RE PD ES LF E PR IA KA D V PD KL G A V ES AR GT M PA P EV WQ Q Y GRAttorney Docket No.: 063586-523001WO gctccccgctgtccggccgcgttattccgaatatccgcatc (SEQ ID NO: 28) N449A MSSTHTLAIDFGSKNIGIALVR LS K V EP SA E R A DP LR E K N PF LW ER Y L RE PD ES LF E R A KA D V PD KL G A V ES AR GT M PA P EV WQ Q Y RAttorney Docket No.: 063586-523001WO accgcgtagagcgccaaaccggtcaaattgtgcaccgctacgtggctgatc cggcgaaccaggccctcatcgtcgcgtacctgggcagcaatggtaaggtg R LS K V EP SA E R A DP LR E K N PF LW ER Y L RE PD ES LF E R A KA D V PD KL G A V ES AR GT M PA P EV WQ QAttorney Docket No.: 063586-523001WO caagatgcatacggctggtgtggtctggaggacgtgccggctaagcgcatc GCVVHYEDGSERYIRNFSSSY gccgcggcgaatctggcggcgtccctccgcaacgcagataaggagaaaa GFKKSLFKGMVGVRRSPLSGR VIPNIRI (SEQ ID NO: 33) R LS K V EP SA E R A DP LR E K N PF LW AR Y L RE PD ES LF E R A KA D V PD KL G A VAttorney Docket No.: 063586-523001WO tggctcgctacctgtgtgaaaagctccgtcaacgttacgatcgtgtgaccca VHPRLRAVLEAANTGERPGES gatcgcatttcgttctggtagacacacggcgatctggcgtcaagcggcttac SAAALISWLRKAVKGSLKDAR LSSHPADQARAKALRDFADGT M PA P EV WQ Q Y R R LS K V EP SA E R A DP LR E K N PF LW ER Y L RE PD ES LF E RAttorney Docket No.: 063586-523001WO aaggacgatctggaaatgctgcgcaaggaattcaacggtttgtgcgcatact PLARYLCEKLRQRYDRVTQIA gcggtaagccgagcgagaccattgttgaacgcgaacacattctgccgaaa FRSGRHTAIWRQAAYPDFDKA KEKVSGGTINHALAAMILACD V PD KL G A V ES AR GT M PA P EV WQ Q Y GRA linear DNA template sequence encoding an RNA guide was designed and ordered (IDT) with a T7 promoter upstream and a T7Te terminator sequence downstream of the guide. The RNA guide was designed to be specific to a previously tested target sequence, described in Example 1, within the coding exon of VEGFA with a 5’-NRV-3’ PAM sequence (the PAM is 3’ of the target sequence). The T7 promoter uses a +1 G at the start of the transcript (i.e., the 5’ end of the RNA) for more efficient transcription that is shown for SEQ ID NO: 40. The sequence of the encoded RNA guide and its individual components are shown in Table 7.Attorney Docket No.: 063586-523001WO Table 7. RNA Sequences CRISPR RNA sequence SEQ ID Nuclease NOA DNA target was designed and ordered as a synthesized linear DNA fragment. The target sequence from VEGFA and 10 bases upstream and downstream within the exon was flanked by 100 bases of unrelated sequence upstream and 200 bases of unrelated sequence downstream. The extra sequence was added so that the cleaved and uncleaved products would separate well on a gel. The target and non-target strands were labelled with 5’ IR800 and 5’ IR700 labels, respectively, through PCR amplification using labelled primers. The sequences of the DNA target, the individual components of the DNA target, and the labelled PCR primers are in Table 8. Table 8. Target gBlock and Primer Sequences CRISPR DNA sequence SEQ ID lAttorney Docket No.: 063586-523001WO Full target gblock gctcaaagctttagactggaggtaccgtagtactatgagcttaatcgcgctgagggtgat 45 ttgggtgttcgtctcatatcttcgattatcggacagtagtTTCCTTGGTCCCCA TAT AAA AAA A TA A Ahe putative nickases was assessed using in vitro cleavage assays. Each polypeptide was individually co-expressed with the RNA guide in vitro by incubating the plasmid encoding the protein of interest from Table 6 and linear DNA template for the T7 transcribed VEGFA- T6 sgRNA from Table 7 in a PURExpress®solution (NEB) containing SUPERase•In™ RNase Inhibitor (Invitrogen) for 2 hours at 37ºC. The unpurified polypeptide / RNA solution was then diluted into a solution of 1X NEB Buffer 2 (NEB) containing approximately 1 ng / µl of the labelled DNA target amplicon. The solution was then incubated for 1 hour at 37ºC. Reactions were stopped by incubating with RNase Cocktail™ (Invitrogen; approximately 1 U / µl final concentration) at 37ºC for 15 minutes, followed by incubating with Proteinase K (NEB; approximately 0.04 U / µl final concentration) at 55ºC for 30 minutes. The DNA was then purified using CleanNGS DNA & RNA Clean-Up Magnetic Beads (Bulldog Bio). The cleaved and uncleaved products of the target and non-target strands were separated by running the samples on a 10% TBE-Urea PAGE gel. The gel was imaged using a LI-COR Odyssey M imaging system using the 800 nm and 700 nm channels to visualize the 5’ IR800 and 5’ IR700 labels on the target and non-target strands of the target DNA substrate. Band intensities were quantified using ImageJ software. Gel images are shown in FIGs.2A-2C, and quantification of the percent of cleaved target and non-target strands are shown in FIG.2D. The uncleaved, HNH-cleaved, and RuvC-cleaved strands are indicated. FIG.2A is a gel image captured using the 800 nm channel showing cleavage of the target strand. FIG.2B is a gel image captured using the 700 nm channel showing cleavage of the non-target strand. FIG.2C is an overlay of the gel images from FIG.2A and FIG.2B. As shown in FIG.2A-D, the reference CRISPR nuclease (SEQ ID NO: 1) cleaved both the target strand and the non-target strand, as expected. Each of the four HNH-knockout nickase constructs (E425A, H426A, H426L, and N449A) showed significantly decreased activity on the target strand while retaining activity that is similar toAttorney Docket No.: 063586-523001WO or greater than the reference protein on the non-target strand. Each of the three RuvC- knockout nickase constructs (D10A, E362A, and D487A) showed significantly decreased activity on the non-target strand while retaining activity on the target strand (FIG.2A-2D). This Example thus shows that HNH-knockout nickases and RuvC-knockout nickases were successfully engineered. Variants containing the E425A nickase mutation were utilized to install edits into human gene targets, as described in Examples 7, 9, and 10. Example 5: Fusion of CRISPR Nuclease and CRISPR Nickase to a Reverse Transcriptase In this Example, a reverse transcriptase polypeptide was fused to the C-terminus of the CRISPR nuclease of SEQ ID NO: 1 or is fused to an E425A nickase variant of the CRISPR nuclease (SEQ ID NO: 25). A sequence encoding the CRISPR nuclease-reverse transcriptase fusion polypeptide was cloned into a pcDNA3.1 vector (Invitrogen) comprising a CMV promoter. The fusion comprised the following components arranged from N- to C-termini: 1) the SV40 NLS (SEQ ID NO: 7), 2) the CRISPR nuclease of SEQ ID NO: 1, 3) the XTEN linker (SEQ ID NO: 5), 4) a variant Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (SEQ ID NO: 49), and 5) the nucleoplasmin NLS (SEQ ID NO: 4). The nucleotide and amino acid sequences of the SV40 NLS, CRISPR nuclease of SEQ ID NO: 1, XTEN linker, and nucleoplasmin NLS are shown in Table 1 in Example 1, and the nucleotide and amino acid sequences of the variant MMLV are shown in Table 9 below. The variant MMLV reverse transcriptase was a human codon-optimized DNA sequence. Research grade plasmids were received from GenScript. Table 9. Exemplary Reverse Transcriptase Sequences Description Nucleotide Sequence Amino Acid Sequence V G L E S F E PAttorney Docket No.: 063586-523001WO ccttacaacctcttgagcgggctcccaccgtcccaccagtggta TLFNEALHRDLADFRIQHPD cactgtgcttgatttaaaggatgcctttttctgcctgagactccacc LILLQYVDDLLLAATSELDC QQGTRALLQTLGNLGYRAS K T F L L E RP L G D D E D S VI L R T FL A P O:The CRISPR nuclease-reverse transcriptase fusion polypeptide plasmid DNA is used to install an E425A nickase mutation (see Example 4) using a site-directed mutagenesis kit (New England Biolabs®). The nucleotide and amino acid sequences of the CRISPR nuclease-reverse transcriptase fusion polypeptide and CRISPR nickase-reverse transcriptase fusion polypeptide are shown in Table 11 and comprise the following components arranged from N- to C-termini: 1) the SV40 NLS (SEQ ID NO: 3), 2) the CRISPR nickase (SEQ IDAttorney Docket No.: 063586-523001WO NO: 25), 3) the XTEN linker (SEQ ID NO: 5), 4) a variant Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (SEQ ID NO: 49), and 5) the nucleoplasmin NLS (SEQ ID NO: 4). See Example 1 and Table 1 above. The codon encoding the substituted E425A residue is capitalized, bold, and underlined in the nucleotide sequence, and the substituted residue is shown in bold and underlined in the amino acid sequence. The sequence-verified plasmid is purified using a Qiagen Maxiprep kit or received from GenScript. Table 11. Exemplary Fusion Polypeptides Description Nucleotide Sequence Amino acid Sequence (codon optimized for human cells) R GI T Q A V E F E G R V L K F A I S V F D G F M S P K L L S K M P Q EAttorney Docket No.: 063586-523001WO acctagacctttggctcgctacctgtgcgagaaactgagacagcggta FEKVLQGNFIETDLAKLN cgaccgggtgacccagatcgcctttagaagcggacggcacaccgcc WNRKHSRVQRQDAYGW CGLEDVPAKRIAAANLA K V A R F Y T I L R A Y K G S S P M Q K N S A W P D R K E Q F L Y L G S G P L P I P G A TAttorney Docket No.: 063586-523001WO gcctcttcatccctgggtttgcagaaatggcagcccccctgtaccctct QALKMAEGKKLNVYTD caccaaaccggggactctgtttaattggggcccagaccaacaaaagg SRYAFATAHIHGEIYRRR GWLTSEGKEIKNKDEILA H Q IE K R GI T Q A V E F E G R V L K F A I S V F D G FAttorney Docket No.: 063586-523001WO cagggtggtcgtggaaagaacagccttcgacatgctggccggatctc FDSYLNMVPACPDCNRM ggaaacagaggcagaaagtggccgacgactacctggaagaaatgta WKAKASPGASGLRINES AYQAYSDYLADKFKTRP K L L RS K M P Q E N W A K V A R F Y T I L R A Y K G S S P M Q K N S A W P D R K E Q F L Y LAttorney Docket No.: 063586-523001WO cttgagcgggctcccaccgtcccaccagtggtacactgtgcttgattta TKPFELFVDEKQGYAKG aaggatgcctttttctgcctgagactccaccccaccagtcagcctctctt VLTQKLGPWRRPVAYLS KKLDPVAAGWPPCLRM G P L P I G A T D R A H Q IE KThis Example thus describes how the CRISPR nuclease-reverse transcriptase fusion polypeptide was cloned as well as how the CRISPR nickase (E425A) variant-reverse transcriptase fusion polypeptide construct is cloned. The CRISPR nuclease-reverse transcriptase fusion polypeptide was used to install edits in human target genes as described in Example 7.Attorney Docket No.: 063586-523001WO Example 6: Fusion of Variant CRISPR Nickase to a Reverse Transcriptase In this Example, a reverse transcriptase polypeptide was fused to the C-terminus of a variant CRISPR nickase comprising the S925R, A657R, and A65R substitutions (Example 3) and the E425A (Example 4) substitution. A sequence encoding the variant CRISPR nickase-reverse transcriptase fusion polypeptide was cloned as described in Example 5. The nucleotide and amino acid sequences of the variant CRISPR nickase-reverse transcriptase fusion polypeptide is shown in Table 12. The codon encoding the substituted E425A residue is capitalized, bold, and underlined in the nucleotide sequence, and the substituted residue is shown in bold and underlined in the amino acid sequence. The sequence-verified plasmid was purified using a Qiagen Maxiprep kit or received from GenScript. Table 12. Exemplary Fusion Polypeptide Description Nucleotide Sequence Amino acid Sequence (codon optimized for human cells) K N G E E D S A K K E Y D I R G F RAttorney Docket No.: 063586-523001WO ttcaaggatgacctggagatgctgagaaaggaatttaacggcctttgc AHILPKANFFFDSYLNM gcctactgcggtaaacctagcgaaaccatcgtggagagaGCCca VPACPDCNRMWKAKA SPGASGLRINESAY AY H L D S D T V N L H G K Y C R R D S T L C V L F RAttorney Docket No.: 063586-523001WO tccaccccaccagtcagcctctcttcgcctttgagtggagagatccag GTRALLQTLGNLGYRA agatgggaatctcaggacaattgacctggaccagactcccacaggg SAKKAQICQKQVKYLG YLLKE R LTEARKE E E L L K D I L D P D L Q V E L I K E AThis Example thus describes how the variant CRISPR nickase-reverse transcriptase fusion polypeptide was cloned. This construct was used to install edits in human target genes, as described in Examples 7, 9, and 10.Attorney Docket No.: 063586-523001WO Example 7: RNA-Templated Editing of Human Genes in HEK293T Cells using CRISPR Nuclease-Reverse Transcriptase and Variant CRISPR Nickase-Reverse Transcriptase Fusion Polypeptides This Example shows genetic modification of human genes utilizing the CRISPR nuclease-reverse transcriptase and variant CRISPR nickase (S925R / A657R / A65R / E425A)- reverse transcriptase fusion polypeptides constructed in Example 5 and Example 6, respectively. Specifically, the fusion polypeptides were used to install sequence substitutions of 6 nucleotides into the human target genes. Editing template RNAs were designed to be specific to the target sequences shown in Table 13 and cloned into a pUC19 plasmid comprising a U6 PolIII promoter and a 6x polyT terminator sequence. The editing templates were synthesized by GenScript and comprised the following four components from 5’ to 3’: 1) spacer sequence, 2) scaffold motif, 3) reverse transcription template (RTT) encoding a 6- nucleotide substitution, 4) primer binding site (PBS). The RTT was 21 nucleotides in length. Two different PBS sequences were tested and varied by length: 8 nucleotides (editing template 1) and 12 nucleotides (editing template 2). The sequences of the individual components of the editing template RNAs are shown in Table 14, and the full-length editing template RNA sequences are shown in Table 15. The U6 PolIII promoter uses a +1 G at the start of the transcript (i.e., the 5’ end of the RNA) for more efficient transcription that is excluded from the sequences described in Table 14. Table 13. Target Sequences Target PAM Target Sequence SEQ ID NO Ta. q g p p Editing Target Spacer Scaffold RTT PBS t l t AAttorney Docket No.: 063586-523001WO UCUGGAGGAAACCUUC CGGCGUGUCGCAAGAC AAGAUGUGAGCCUG ATable 15. Full-Length Editing Template RNA Sequences Editing Target Sequences template U UApproximately 16 hours prior to transfection, 25,000 HEK293T cells in DMEM / 10%FBS+Pen / Strep (D10 media) were plated into each well of a 96-well plate. On the day of transfection, the cells were 50-90% confluent. For each well to be transfected, a mixture of Lipofectamine 2000™ (ThermoFisher Scientific) and Opti-MEM™ (ThermoFisher Scientific) was prepared and incubated at room temperature for 5 minutesAttorney Docket No.: 063586-523001WO (Solution 1). After incubation, the Lipofectamine 2000™: Opti-MEM™ mixture was added to a separate mixture containing the CRISPR nuclease-reverse transcriptase fusion polypeptide or variant CRISPR nickase-reverse transcriptase fusion polypeptide, editing template RNA, and Opti-MEM™ (Solution 2) was added. Solutions 1 and 2 were mixed by pipetting up and down, then incubated at room temperature for 25 minutes. Following incubation, the Solution 1 and 2 mixture was added dropwise to each well of a 96-well plate containing the cells. Approximately 72 hours post transfection, cells were trypsinized by adding TrypLE™ (Thermo Fisher Scientific) to the center of each well and incubating at 20- 37ºC for approximately 5 minutes. D10 media was then added to each well and mixed to resuspend cells. The resuspended cells were centrifuged for 10 minutes to obtain a pellet, and the supernatant was discarded. The cell pellet was then resuspended in Quick Extract™ buffer (Lucigen®), and cells were incubated at 65ºC for 15 minutes, 68ºC for 15 minutes, and 98ºC for 10 minutes. Samples were prepared for NGS and analyzed as described in Example 1. For each target, the fraction of NGS reads containing indels were calculated for each sample and its cognate no protein control. To determine the percentage of edits installed in the target genes, sequencing reads comprising the 6-nucleotide substitution encoded by the editing template RNAs were analyzed and quantified. The percentages of NGS reads comprising indels and the 6-nucleotide edits using the CRISPR nuclease-reverse transcriptase fusion polypeptide are shown in Table 16, and the percentages of NGS reads comprising indels and the 6-nucleotide edits using the variant CRISPR nickase-reverse transcriptase fusion polypeptide are shown in Table 17. The data in Table 16 and Table 17 are the average of three technical replicates. Table 16. Editing Efficiencies with CRISPR Nuclease-Reverse Transcriptase Fusion Polypeptide Editing Template Average % Indel Average % 6-nt substitutionTable 17. Editing Efficiencies with Variant CRISPR Nickase (S925R / A657R / A65R / E425A)-Reverse Transcriptase Fusion Polypeptide Editing Template Average % Indel Average % 6-nt substitutionAttorney Docket No.: 063586-523001WO 1 0.057 0.32 2 0.070 0.62CRISPR nickase-reverse transcriptase fusion polypeptide introduced substitutions encoded by the tested editing template RNAs at the VEGFA target locus. Indels induced by the CRISPR nuclease-reverse transcriptase fusion polypeptide and editing templates 1 and 2 exceeded those induced by the variant CRISPR nickase (S925R / A657R / A65R / E425A)-reverse transcriptase fusion polypeptide and editing templates 1 and 2. Conversely, incorporation of the 6-nucleotide substitution encoded by editing template RNAs 1 and 2 with the variant CRISPR nickase-reverse transcriptase fusion polypeptide exceeded incorporation of the 6- nucleotide substitution encoded by editing template RNAs 1 and 2 with the CRISPR nuclease-reverse transcriptase fusion polypeptide. Editing efficiency mediated by the CRISPR nuclease-reverse transcriptase fusion polypeptide was further tested with editing template RNAs with PBS lengths of 4, 6, 10, and 12 nucleotides as well as RTT lengths of 26 and 31 nucleotides. Overall, the editing template RNAs with a PBS length of 8 or 12 nucleotides and an RTT length of 21 nucleotides were found to incorporate the 6-nucleotide substitution with a slightly higher efficiency than the other tested editing template RNAs. Editing efficiency mediated by the variant CRISPR nickase (S925R / A657R / A65R / E425A)-reverse transcriptase fusion polypeptide was further tested with editing template RNAs with PBS lengths of 4, 6, 10, and 12 nucleotides as well as RTT lengths of 26 and 31 nucleotides. Overall, the editing template RNAs with a PBS length of 8 or 12 nucleotides and an RTT length of 21 nucleotides were found to incorporate the 6- nucleotide substitution with a slightly higher efficiency than the other tested editing template RNAs. Conversely, controls consisting of the CRISPR nuclease of SEQ ID NO: 1 with the RNA guides of Table 3 or the editing templates of Table 15 induced indel formation but did not incorporate the 6-nucleotide substitution encoded by the editing template RNAs. Furthermore, controls consisting of the CRISPR nuclease-reverse transcriptase fusion polypeptide with the RNA guides of Table 3 did not result in incorporation of the 6- nucleotide substitution encoded by the editing template RNAs.Attorney Docket No.: 063586-523001WO Overall, this Example shows that the CRISPR nuclease-reverse transcriptase fusion polypeptide and variant CRISPR nickase (S925R / A657R / A65R / E425A)-reverse transcriptase fusion polypeptide incorporated substitutions encoded by editing template RNAs into human target genes. Example 8 – Editing of Human Target Genes by a CRISPR Nuclease in HEK293T Cells and Editing Template RNAs with Alternative Scaffold Sequences This Example describes the genomic editing of the exemplary target genes by the reference CRISPR nuclease of SEQ ID NO: 1 and / or the S925R / A657R / A65R variant CRISPR nuclease described in Example 3 when combined with editing template RNAs comprising alternative scaffold sequences. RNA guides were designed using the spacer sequences and scaffold sequences of Table 18 and cloned into a pUC19 plasmid with a U6 PolIII promoter and a 6x polyT sequence. Plasmid preparation was performed by IDT, Azenta, or Genscript. The full-length RNA guide sequences and their corresponding target sequences are shown in Table 19. The U6 PolIII promoter uses a +1 G at the start of the transcript (i.e., the 5’ end of the RNA) for more efficient transcription that is excluded from the sequences described in Table 19. Table 18. RNA guide component sequences Component Sequence g cg c uAttorney Docket No.: 063586-523001WO Table 19. Mammalian Targets and Corresponding RNA Guide Sequences Description PAM Target Sequence RNA Guide Sequence* Sequence Truncated CAC AAATTACCCCCCAAGTC AAAUUACCCCCCAAGUCCCU uuuc u cc a ga c u cc a ga u a ac g ug c u cc a ga uc u cc a ga u a ac g ug u a ac g ug c a c u ga c aAttorney Docket No.: 063586-523001WO gcauccguccggaagggccacuccgggguggggc agccccggcacuugggccgcuuggcuucggcucu gcggcagcccucuuccggcgcgucuucggacagga u g g c g c a c u ga uc a c u ga u g g c g u g g c gThe AAVS1-T3 and VEGFA-T6 truncated reference scaffold RNA guides from Table 19 were first tested. The RNA guides were combined with either the reference CRISPR nuclease of SEQ ID NO: 1 or the S925R / A657R / A65R variant CRISPR nuclease described in Example 3 and were introduced into HEK293T cells by lipid-based transient transfection as described in Example 1. The reference scaffold AAVS1-T3 and VEGFA-T6 RNA guides from Example 1 were tested for comparison. Genomic DNA was recovered approximately 72 hours post-transfection, and samples were prepared for NGS and analyzed as described in Example 1. The percentage of NGS reads comprising indels shown in Tables 20 and 21 were calculated as the average of two biological replicates, which each comprised two technical replicates, unless otherwise noted.Attorney Docket No.: 063586-523001WO Table 20. Indel activity of the Reference CRISPR Nuclease Average Indel Average Indel RNA Guide Percentage PercentageTable 21. S925R / A657R / A65R Variant CRISPR Nuclease Indel Activity Average Indel Average IndelPercentageAs shown in Table 20, RNA guides comprising the truncated reference scaffold exhibited similar or higher indel activity than the RNA guides comprising the reference scaffold when paired with the reference CRISPR nuclease. As shown in Table 21, RNA guides containing the truncated reference scaffold exhibited higher indel activity than the equivalent RNA guides containing the reference scaffold when paired with the I67R / D568R / E706R variant CRISPR nuclease. In all cases, the average percent indel observed was significantly higher than background controls comprising either the reference CRISPR nuclease or the I67R / D568R / E706R variant CRISPR nuclease in the absence of an RNA guide.Attorney Docket No.: 063586-523001WO Next, RNA guides from Table 19 comprising the truncated reference scaffold or scaffold 2 were individually transfected with the I67R / D568R / E706R variant CRISPR nuclease as described above. The percentage of NGS reads comprising indels shown in Table 22 were calculated as the average of three technical replicates, unless otherwise noted. Table 22. S925R / A657R / A65R Variant CRISPR Nuclease Indel Activity RNA Guide Average Indel Average Indel Percentage Percentage n10,000 NGS reads were generated As shown in Table 22, five of the seven RNA guides comprising scaffold 2 exhibited higher indel activity than the corresponding RNA guides comprising the truncated reference scaffold; indel activity was similar for the remaining RNA guides.Attorney Docket No.: 063586-523001WO This Example thus demonstrates that the truncated reference scaffold is capable of being recognized by the reference CRISPR nuclease and variants thereof; indel activity using the truncated reference scaffold is similar to or exceeds the indel activity observed when using the reference scaffold. This Example further shows that scaffold 2 is capable of being recognized by variants of the reference CRISPR nuclease; indel activity using scaffold 2 exceeds the indel activity observed when using the truncated reference scaffold. The truncated reference scaffold and scaffold 2 were used to install edits into human gene targets in Example 9 below. Scaffold 2 was further used to install edits in human gene targets in Examples 10 and 11 below. Example 9: Evaluation of RNA Scaffold Variants for Mammalian Gene Targeting and RNA-Templated Editing of Human Genes This Example shows genetic modification of human genes utilizing the variant CRISPR nickase (S925R / A657R / A65R / E425A)-reverse transcriptase fusion polypeptide of SEQ ID NO: 67 constructed in Example 6. Editing template RNAs were designed to be specific to the target sequences shown in Table 23 and cloned into a pUC19 plasmid comprising a U6 PolIII promoter and a 6x polyT terminator sequence. Cloning and plasmid preparation was performed by GenScript. The editing templates comprised the following six components, from 5’ to 3’: 1) spacer sequence, 2) scaffold motif, 3) reverse transcription template (RTT) encoding 6 nucleotide substitutions, 4) primer binding site (PBS), 5) an 8-nucleotide linker, and 6) a 3’ extension motif. The RTT sequences were 20-nucleotides in length for twelve of the fourteen template RNAs and 21-nucleotides in length for the remaining editing template RNAs (editing template RNAs 8 and 15). The PBS sequences were 10-nucleotides in length for all editing template RNAs. The 3’ extension motif contained a pseudoknot to protect against 3’ exonuclease activity of the editing template RNA. The linker sequence was present to prevent steric clashes between the PBS and the pseudoknot motif. Editing template RNAs targeting AAVS1, EMX1, and VEGFA loci were used in this Example. The PAM, target, and spacer sequences are shown in Table 19 above. The sequences of each editing template RNA component and the full-length editing template RNA sequences are shown in Tables 23 and 24 below. The U6 PolIII promoter uses a +1 G at the start of the transcript (i.e., the 5’ end of the RNA) for more efficient transcription that is excluded from the full-length sequences described in Table 24.Attorney Docket No.: 063586-523001WO Table 23. Sequences of Editing Template RNA Components Editing Spacer Scaffold RTT* PBS Linker 3’ Extension Template RNAs C C C CAttorney Docket No.: 063586-523001WO Editing Spacer Scaffold RTT* PBS Linker 3’ Extension Template RNAs C C CAttorney Docket No.: 063586-523001WO Editing Spacer Scaffold RTT* PBS Linker 3’ Extension Template RNAs C C C CAttorney Docket No.: 063586-523001WO Editing Spacer Scaffold RTT* PBS Linker 3’ Extension Template RNAs C C CAttorney Docket No.: 063586-523001WO Table 24. Full-length Editing Template RNA Sequences Editing Target Scaffold ID Sequence* template RNA A AC G AA G G A AC G AA A G AA AC G AA CU C A AC G AA CU C A AC G AA GA C AA AC G AA U CAttorney Docket No.: 063586-523001WO Editing Target Scaffold ID Sequence* template RNA AA AC G AA CC C A G G GU G G A G G GU A G AA G G GU AA U A G G GU GA U AA G G GU A GAttorney Docket No.: 063586-523001WO Editing Target Scaffold ID Sequence* template RNA A GG G GU U AG AA G G GU C GThe editing template RNAs were transfected with the variant CRISPR nickase (S925R / A657R / A65R / E425A)-reverse transcriptase fusion polypeptide of SEQ ID NO: 67 from Table 12 into HEK293T cells by lipid-based transient transfection as described in Example 7. Genomic DNA was recovered approximately 72 hours post-transfection, and samples were prepared for NGS and analyzed as described in Example 7. The percentage of NGS reads comprising indels and the 6-nucleotide edits are shown in Table 25. The data in Table 25 is the average of three technical replicates, unless otherwise noted. Table 25. Editing Efficiencies with Variant CRISPR Nickase (S925R / A657R / A65R / E425A)-Reverse Transcriptase Fusion Polypeptide Edi i B k d A % 6 Background 6- on *Attorney Docket No.: 063586-523001WO 8TruncatedReferenceVEGFA-T6 0.07 0.08 2.22 <0.01 Td- reverse transcriptase fusion polypeptide of SEQ ID NO: 67 installed the encoded substitutions for 12 of the 14 tested editing template RNAs. These edits were above the background rate of <0.01% observed for all control samples containing the editing template RNA without a CRISPR nickase-reverse transcriptase fusion polypeptide. Five of the six editing template RNAs containing scaffold 2 resulted in higher installation of the encoded edit compared to the equivalent editing template RNAs containing the truncated reference scaffold. Editing template RNA 15, which targeted VEGFA-T6 and utilized Scaffold 2, resulted in the highest average percentage of NGS reads comprising the encoded substitution at 3.64% of NGS reads. This Example thus shows that variant CRISPR nickase-reverse transcriptase fusion polypeptides were capable of recognizing editing template RNAs comprising the truncated reference scaffold or scaffold 2 and installing substitutions into human genes. Example 10: Effectiveness of Additional CRISPR Nickase-Reverse Transcriptase Fusion Polypeptide Variants for RNA-Templated Editing of Human Genes in HEK293T Cells This Example describes the evaluation of variant CRISPR nickase-reverse transcriptase fusion polypeptides for precise genetic modification of human genes. Variant CRISPR nickase-reverse transcriptase fusion polypeptides were cloned and evaluated in successive engineering rounds. Cloning was performed either through mutagenesis or subcloning (to introduce multiple distal mutations) of a parent construct.Attorney Docket No.: 063586-523001WO Cloning and plasmid preparation was performed by GenScript. The variant CRISPR nickase (S925R / A657R / A65R / E425A)-reverse transcriptase fusion polypeptide from Example 6 was used as the parent construct for the first engineering round. The top-performing constructs from each engineering round were used for the subsequent round. In each engineering round, variant CRISPR nickase-reverse transcriptase fusion polypeptides were transfected with editing template RNAs 11 and / or 15 from Table 24 and into HEK293T cells by lipid-based transient transfection as described in Example 7. Genomic DNA was recovered approximately 72 hours post-transfection, and samples were prepared for NGS and analyzed as described in Example 7. The percentage of NGS reads comprising indels and the 6-nucleotide edits are shown in Tables 26, 27, and 28. The data in Tables 26, 27, and 28 is the average of three technical replicates, unless otherwise noted. Table 26. Editing Efficiencies with Variant CRISPR Nickase-Reverse Transcriptase Fusion Polypeptides from Engineering Round 1 Construct Initial Nickase Mutations Additional Average %substitution ID Nuclease / Nickase installationAttorney Docket No.: 063586-523001WO 22 A65R, E425A, A657R, S925R N691R 2.86 23 A65R, E425A, A657R, S925R S695R 3.59Table 27. Editing Efficiencies with Variant CRISPR Nickase-Reverse Transcriptase Fusion Polypeptides from Engineering Round 2 Average Average %substitution %substitutionAttorney Docket No.: 063586-523001WO A65R, L134R, 47 E425A, A657R, Q292R 0.80 3.51 S925R 0,000 NGSTable 28. Editing Efficiencies with Variant CRISPR Nickase-Reverse Transcriptase Fusion Polypeptides from Engineering Round 3 Average Average %substitution %substitutionAttorney Docket No.: 063586-523001WO Average Average %substitution %s struct Init ubstitution Con ial Additional installation installationAttorney Docket No.: 063586-523001WO Average Average %substitution %s struct Init ubstitution Con ial Additional installation installationAttorney Docket No.: 063586-523001WO Average Average I %substitution %substitution Construct nitial Additional installation installation 000 NGS**Two technical replicates from one biological replicate were excluded because fewer than 10,000 NGS reads were generated In engineering round 1, activity of the parent construct (construct 1) and variant constructs 2-44 was measured using editing template RNA 15. Nineteen variants were identified that installed the encoded substitution at a level that was above the parent construct in this assay. Fifteen of these 19 variants were carried into the next engineering round. The selected constructs contained the following substitutions in addition to the four substitutions that were present in the parent CRISPR nickase variant (S925R / A657R / A65R / E425A): V51R (construct 2), V85R (construct 3), L134R (construct 10) Q292R (construct 16), D479R (construct 20), S695R (construct 23), E702R (construct 25), D707R (construct 26), E711R (construct 27), E715R (construct 28), V717R (construct 29), A854R (construct 33), A861R (construct 35), and S905R (construct 36). Construct 10 yielded the highest substitution level and was selected as the parent construct for the next set of variants. In engineering round 2, parent construct 2 (construct 10) and variant constructs 45-57 were individually transfected with editing template RNAs 11 and 15. Construct 1 was also tested for comparison. As shown in Table 27, each of the engineered variants installed the encoded substitutions at levels that were above background (<0.01%) for both targets. Twelve of the thirteen variants demonstrated substitution levels that were greater than those obtained with construct 10 when transfected with editing template RNA 15. Of these, four (constructs 46 and 50-52) also demonstrated substitution levels that were greater than thoseAttorney Docket No.: 063586-523001WO obtained with construct 10 when transfected with editing template RNA 11. Following this round, constructs 46 and 51 were selected as parent constructs for the next set of variants. In engineering round 3, parent constructs 3 and 4 (constructs 46 and 51, respectively) and the variant constructs (constructs 58-90) were paired with editing template RNAs 11 and 15 in separate transfections. Constructs 1 and 10 were also tested for comparison. As shown in Table 28, each of the engineered variants installed the encoded substitutions at levels that were above background (<0.01%). Sixteen of the thirty-three variants demonstrated substitution levels that were greater than those obtained with their respective parent construct when transfected with editing template RNA 15. Of these sixteen variants, three variants (constructs 63, 65, and 78) also demonstrated substitution levels that were greater than those obtained with their respective parent construct when paired with editing template RNA 11. After three engineering rounds, constructs 78 and 63 were identified as the top- performing variant CRISPR nickase-reverse transcriptase fusion polypeptides. Construct 78 contains the following mutations in the CRISPR nuclease domain: A65R, V85R, L134R, E425A, T549P, A657R, and S925R. Construct 63 contains the following mutations in the CRISPR nuclease domain: A65R, V85R, L134R, E425A, A657R, D707R, and S925R. Example 11: Effectiveness of Combination CRISPR Nuclease Variants for Targeting of Mammalian Genes This Example describes indel assessment on mammalian targets using additional CRISPR nuclease variants comprising four or more substitutions identified as increasing indel activity in Example 2. Fourteen additional combination CRISPR variants were tested. The CRISPR nuclease variant comprising the following substitutions: A65R, A657R, S925R and each additional CRISPR nuclease variant was cloned as described in Example 2. Cloning and plasmid preparation were performed by GenScript. The variant CRISPR nucleases were individually transfected with the AAVS1-T2b and VEGFA-T6 Scaffold 2 RNA guides from Table 19 into HEK293T cells by lipid-based transient transfection as described in Example 1. Genomic DNA was recovered approximately 72 hours post- transfection, and samples were prepared for NGS and analyzed as described in Example 1. The percentage of NGS reads comprising indels shown in Table 29 were calculated as the average of three technical replicates.Attorney Docket No.: 063586-523001WO Table 29. Indel Frequencies of Variant CRISPR Nucleases Nuclease substitutions %Indel (AAVS1-T2b) %Indel (VEGFA-T6) NGSAs shown in Table 29, each CRISPR nuclease variant introduced indels at both target sites above the background rates observed for the guide-only control. Indel activity of each of the fourteen variants was similar to or exceeded indel activity observed with the A65R, A657R, S925R variant from Example 3.

Claims

Attorney Docket No.: 063586-523001WO WHAT IS CLAIMED IS:

1. A gene editing system comprising: (a) a fusion polypeptide comprising a CRISPR nuclease polypeptide and a reverse transcriptase (RT) polypeptide, or a first nucleic acid encoding the fusion polypeptide; wherein the CRISPR nuclease polypeptide comprises the amino acid sequence of SEQ ID NO:1, or is a variant of SEQ ID NO:1, the variant comprising: (i) one or more mutations in the HNH nuclease domain or in the RuvC nuclease domain of SEQ ID NO:1, the one or more mutations reducing or eliminating the nuclease activity of the HNH nuclease domain or the RuvC nuclease domain; (ii) one or more arginine and / or lysine substitutions, optionally one or more arginine substitutions; or (iii) a combination of (i) and (ii); and (b) an RNA molecule comprising a guide RNA (gRNA) and a reverse transcription donor RNA (RT donor RNA), or a second nucleic acid encoding the RNA molecule; wherein the gRNA comprises a scaffold sequence recognizable by the CRISPR nuclease polypeptide and a spacer sequence specific to a target sequence within a genomic site of interest, the target sequence being upstream to a protospacer adjacent motif (PAM); and wherein the RT donor RNA comprises a primer binding site (PBS) and a template sequence.

2. The gene editing system of claim 1, wherein the fusion polypeptide further comprises one or more nuclear localization sites (NLS) upstream or downstream to the CRISPR nuclease polypeptide, the RT polypeptide, or both.

3. The gene editing system of claim 2, wherein the fusion polypeptide, from N- terminus to C-terminus, comprises a first NLS, the CRISPR nuclease polypeptide, the RT polypeptide, and a second NLS; optionally wherein the fusion polypeptide further comprises a peptide linker between the CRISPR nuclease polypeptide and the RT polypeptide.Attorney Docket No.: 063586-523001WO 4. The gene editing system of any one of claims 1-3, wherein the CRISPR nuclease polypeptide is the variant of SEQ ID NO:

1.

5. The gene editing system of claim 4, wherein the CRISPR nuclease polypeptide comprises one or more mutations in the HNH nuclease domain at positions E425, H426, and / or N449 relative to SEQ ID NO:1; optionally wherein the mutation is at position E425.

6. The gene editing system of claim 5, wherein: (a) the mutation at E425 is an amino acid substitution of E425A, E425G, E425L, or E425S; (b) the mutation at H426 is an amino acid substitution of H426A, H426G, H426L, or H426S; and (c) the mutation at N449 is an amino acid substitution of N449A, N449G, N449L, or N449S.

7. The gene editing system of claim 4, wherein the CRISPR nuclease polypeptide comprises one or more mutations in the RuvC nuclease domain at positions D10, E362, and / or D487 relative to SEQ ID NO:1; optionally wherein: (a) the mutation at D10 is an amino acid substitution of D10A, D10G, D10L, or D10S; (b) the mutation at E362 is an amino acid substitution of E362A, E362G, E362L, or E362S; (c) the mutation at D487 is an amino acid substitution of D487A, D487G, D487L, or D487S.

8. The gene editing system of any one of claims 4-7, wherein the CRISPR nuclease polypeptide comprises a bridge helix (BH) domain, a nucleic acid recognition (REC) domain, a phosphate lock loop (PLL) domain, a wedge (WED) domain, and a PAM- interacting (PID) domain; and wherein the one or more arginine and / or lysine substitutions, optionally arginine substitutions, are located in the BH domain, in the REC domain, in the PLL domain, in the WED domain, in the PID domain, or a combination thereof.Attorney Docket No.: 063586-523001WO 9. The gene editing system of any one of claims 1-8, wherein the CRISPR nuclease polypeptide contains up to 20 arginine and / or lysine substitutions relative to the reference CRISPR nuclease; optionally wherein the engineered CRISPR nuclease polypeptide contains up to 15 arginine and / or lysine substitutions relative to the reference CRISPR nuclease.

10. The gene editing system of claim 9, wherein the one or more arginine and / or lysine substitutions are at positions A65, V85, L134, V117, A657, N691, S695, D707, S925, and / or E715 in SEQ ID NO:1; optionally wherein the CRISPR nuclease polypeptide comprises arginine and / or lysine substitutions at a combination of positions listed in Table 5.

11. The gene editing system of any one of claims 4-10, wherein the variant further comprises a non-arginine / lysine substitution, which optionally is at position T549 of SEQ ID NO: 1, optionally wherein the non-arginine / lysine substitution is T549P.

12. The gene editing system of claim 4, wherein the CRISPR nuclease polypeptide comprises arginine and / or lysine substitutions at positions A65, A657, and S925, and an amino acid substitution at position E425 in SEQ ID NO:

1.

13. The gene editing system of claim 4, wherein the CRISPR nuclease polypeptide comprises a combination of mutations set forth in any one of Tables 27-29; optionally wherein the CRISPR nuclease polypeptide comprises animo acid substitutions at the following positions relative to SEQ ID NO: 1: (a) A65, V85, L134, E425, T549, A657, and S925, optionally A65R, V85R, L134R, E425A, T549P, A657R, and S925R; or (b) A65, V85, L134, E425, A657, D707, and S925, optionally A65R, V85R, L134R, E425A, A657R, D707R, and S925R.

14. The gene editing system of any one of claims 4-13, wherein the CRISPR nuclease polypeptide comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1.Attorney Docket No.: 063586-523001WO 15. The engineered CRISPR nuclease polypeptide of claim 14, wherein the CRISPR nuclease polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO:

1.

16. The engineered CRISPR nuclease polypeptide of claim 15, wherein the CRISPR nuclease polypeptide comprises an amino acid sequence at least 98% identical to SEQ ID NO:

1.

17. The engineered CRISPR nuclease polypeptide of claim 1, wherein the CRISPR nuclease polypeptide is set forth in Table 1 and Table 6 or comprises the mutations set forth in any one of Tables 5 and 26-29 relative to SEQ ID NO:

1.

18. The gene editing system of any one of claims 1-17, wherein the RT polypeptide is Moloney Murine Leukemia Virus (MMLV)-RT, optionally the MMLV-RT comprises the amino acid sequence of SEQ ID NO:

49.

19. The gene editing system of claim 1, wherein the fusion polypeptide is set forth in Table 11 and Table 12.

20. The gene editing system of any one of claims 1-19, wherein the system comprises the fusion polypeptide.

21. The gene editing system of any one of claims 1-19, wherein the system comprises the first nucleic acid encoding the fusion polypeptide.

22. The gene editing system of claim 21, wherein the first nucleic acid is located on a vector, which optionally is a viral vector.

23. The gene editing system of claim 21, wherein the first nucleic acid is a messenger RNA (mRNA).Attorney Docket No.: 063586-523001WO 24. The gene editing system of any one of claims 1-21, wherein the spacer sequence in the gRNA of (b) is 15-30-nucleotide in length; optionally 15-20-nucleotide in length.

25. The gene editing system of any one of claims 1-24, wherein the PAM is NRV- 3’, in which N represents any nucleotide, R represents A or G, and V represents A, C, or G; optionally wherein the PAM is 5’-NRR-3’ or 5’-RRR’3’, in which each of N and R is defined herein.

26. The gene editing system of any one of claims 1-25, wherein the scaffold sequence comprises a nucleotide sequence at least 70% identical to SEQ ID NO:

2.

27. The gene editing system of claim 26, wherein the scaffold sequence comprises the nucleotide sequence of SEQ ID NO:

2.

28. The gene editing system of claim 26, wherein the scaffold sequence is a truncated variant of SEQ ID NO: 2 having a truncation within residues 17-43 of SEQ ID NO: 2; optionally wherein the truncated variant is 145-155-nucleotide in length.

29. The gene editing system of claim 28, wherein the truncated variant of SEQ ID NO: 2 comprises the nucleotide sequence of SEQ ID NO: 80 or SEQ ID NO:

81.

30. The gene editing system of any one of claims 1-29, wherein the PBS in the RT donor RNA is 5-50 nucleotides in length; optionally 5-20 nucleotides in length.

31. The gene editing system of any one of claims 1-30, wherein the PBS binds a PBS-targeting site that is adjacent to or overlaps with the target sequence.

32. The gene editing system of claim 31, wherein the PBS-targeting site is adjacent to the 5’ of the PAM, optionally wherein the 3’end nucleotide of the PBS-targeting site is about 2-15 nucleotides upstream to the PAM.Attorney Docket No.: 063586-523001WO 33. The gene editing system of any one of claims 1-32, wherein the template sequence in the RT donor RNA is 5-100 nucleotides in length; optionally 15-25 nucleotides in length.

34. The gene editing system of any one of claims 1-33, wherein the template sequence in the RT donor RNA is homologous to the genomic site of interest and comprises one or more nucleotide variations relative to the genomic site of interest.

35. The gene editing system of claim 34, wherein at least one nucleotide variation is located within the target sequence; and / or wherein at least one nucleotide variation is located in the PAM.

36. The gene editing system of any one of claims 1-35, wherein the RNA molecule of (b) further comprises a 3’ end extension, a linker, or a combination thereof.

37. The gene editing system of any one of claims 1-36, wherein the RNA molecule of (b) further comprises a 5’ end protection fragment, a 3’ protection fragment, or both, each of the 5’ end protection fragment and the 3’ end protection fragment forming a secondary structure, which optionally is a hairpin, a pseudoknot, a circularization, or a triplex structure.

38. The gene editing system of claim 36, wherein the RNA molecule of (b) comprises, from 5’ to 3’: (i) the spacer sequence, the scaffold sequence, the template sequence, and the PBS; (ii) the spacer sequence, the scaffold sequence, the template sequence, the PBS, and the 3’ extension; or (iii) the spacer sequence, the scaffold sequence, the template sequence the PBS, the linker, and the 3’ extension.

39. The gene editing system of any one of claims 1-38, wherein the system comprises the RNA molecule of (b).Attorney Docket No.: 063586-523001WO 40. The gene editing system of any one of claims 1-38, wherein the system comprises the second nucleic acid encoding the RNA molecule.

41. The gene editing system of claim 40, wherein the nucleic acid is located on a vector, which optionally is a viral vector.

42. The gene editing system of any one of claims 1-41, wherein the system comprises one or more lipid excipients associated with one or more of elements (a) and (b); optionally wherein the one or more lipid excipients form nanoparticles (LNPs).

43. The gene editing system of any one of claims 1-22 and 24-41, wherein the system comprises one or more vectors, optionally one or more viral vectors, encoding one or more of elements (a) and (b).

44. A pharmaceutical composition comprising the gene editing system of any one of claims 1-43.

45. A kit comprising the elements (a) and (b) of the gene editing system set forth in any one of claims 1-43.

46. A gene editing method, comprising delivering the gene editing system of any one of claims 1-43 to a host cell to edit a genomic site targeted by the gRNA of the gene editing system.

47. The gene editing method of claim 46, wherein the host cell is cultured in vitro.

48. The gene editing method of claim 47, wherein the host cell is located in a subject who needs the gene editing.

49. A fusion polypeptide, comprising a CRISPR nuclease polypeptide set forth in any one of claims 1-17 and a reverse transcriptase polypeptide set forth in claim 1 or claim 18.Attorney Docket No.: 063586-523001WO 50. The fusion polypeptide of claim 49, which comprises an amino acid sequence listed in Table 11 or Table 12.

51. A nucleic acid encoding the fusion polypeptide of claim 49 or claim 50.

52. The nucleic acid of claim 51, which comprises a nucleotide sequence listed in Table 11 or Table 12.

53. The nucleic acid of claim 51 or claim 52, which is a vector, optionally an expression vector.

54. The nucleic acid of claim 51 or claim 52, which is a messenger RNA (mRNA).

55. A nucleic acid comprising a truncated scaffold sequence relative to SEQ ID NO:2, optionally wherein the truncated scaffold sequence has 145-155 nucleotides in length.

56. The nucleic acid of claim 55, wherein the truncated scaffold sequence comprises a truncation within residues 17-43 of SEQ ID NO:

2.

57. The nucleic acid of claim 55 or claim 56, wherein the truncated scaffold sequence comprises a nucleotide sequence at least 78% identical to SEQ ID NO: 80; optionally wherein the truncated scaffold sequence comprises the nucleotide sequence of SEQ ID NO: 80 or SEQ ID NO:

81.

58. The nucleic acid of any one of claims 55-57, which further comprises a spacer sequence.

59. The nucleic acid of claim 58, which further comprises a reverse transcription donor RNA (RT donor RNA); wherein the RT donor RNA comprises a template sequence and a primer binding site (PBS).Attorney Docket No.: 063586-523001WO 60. The nucleic acid of claim 59, which further comprises a 3’ extension, a 5’ protection fragment, a linker, or a combination thereof.

61. The nucleic acid of any one of claims 55-60, which comprises, from 5’ to 3’: (i) a spacer sequence, the truncated scaffold sequence, a template sequence, and a primer binding site (PBS); (ii) a spacer sequence, the truncated scaffold sequence, a template sequence, a primer binding site (PBS), and a 3’ extension; or (iii) a spacer sequence, the truncated scaffold sequence, a template sequence, a primer binding site (PBS), a linker and a 3’ extension.