Dual nuclease systems for gene editing, methods of producing and using same

Abstract

Provided herein are dual cleaving chimeric nucleases comprising an I-TevI domain and a RNA-guided domain such as Cas9 for use in gene editing methods, as well as chimeric nuclease systems comprising a single nucleic acid that encodes said chimeric nuclease, one or more guide RNAs (gRNAs), one or more tRNAs, and optionally one or more donor polynucleotides. Also provided herein are methods of making and using such chimeric nucleases, chimeric nuclease systems, and nucleic acids encoding such chimeric nucleases and chimeric nuclease systems.

Description

DUAL NUCLEASE SYSTEMS FOR GENE EDITING, METHODS OF PRODUCING AND USING SAMECROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional App. Ser. No. 63 / 631,166 filed on April 8, 2024, and U.S. Provisional App. Ser. No. 63 / 590,771 filed on October 16, 2023, and U.S. Provisional App. Ser. No. 63 / 578,032 filed on August 22, 2023, which are incorporated by reference herein in their entirety.BACKGROUND

[0002] Existing gene editing technologies, such as RNA-programmable gene editors (CRISPR- Cas9 and Cas9 fusions), meganucleases, zinc finger proteins, type IIS restriction endonucleases (FokI and FokI fusions), and TALENS are limited in the ability to introduce gene deletions of a specific length or to precisely insert a DNA sequence in a sufficient number of cells. For therapeutic gene editing it is important to deliver the gene editor to the target tissue or cell in an organism and to efficiently and precisely modify the target site. To disrupt genes using RNA- programmable gene editors, it is important to co-deliver the gene editor and one or more guide RNAs to a cell and for the gene editor to result in predictable deletions or insertions of small and / or large sequences to achieve the desired editing outcome. To insert new sequences using RNA-programmable gene editors, it is important to co-deliver the gene editor, one or more guide RNAs and one or more repair templates to the cells and for the gene editor to replace or insert the sequence in the target site with minimal collateral effects at the target site such as additional insertions and deletions to achieve the desired repair outcomes.

[0003] Current gene editing technologies often rely on inefficient repair pathways in the cell such as error-prone non-homologous end joining (NHEJ), homology directed repair (HDR) or base excision repair (BER) to modify the target site in the genome leading to low target site repair, cell cycle specific repair or unpredictable editing outcomes.

[0004] Several technologies have been developed to deliver gene editors and repair templates in vivo for example viral delivery and non-viral delivery. However, gene editors are often too large to co-package into a single adeno-associated viral vector (AAV) together with all the regulatory elements required to express and stabilize the gene editor, guide RNAs, and donorDNA sequences or repair templates. Similarly, for non-viral delivery with messenger RNA, coexpression of all the necessary elements for efficient and predictable target site disruption and repair suffers limitations. For ribonucleoprotein (RNP) delivery, current versions of gene editors are limited to being able to co-deliver the donor nucleic acid sequences together with the protein version of the gene editor to the same cell to ensure high precision repair. In the case of gene editors expressed in a cell after viral delivery, controlling expression of the nuclease over time to control the therapeutic duration and mitigate any unwanted editing due to constitutive expression is imperative.SUMMARY

[0005] Each of the aspects and embodiments described herein are capable of being used together, unless excluded either explicitly or clearly from the context of the embodiment or aspect.

[0006] In one aspect, a nucleic acid is provided comprising (i) a polynucleotide encoding a chimeric nuclease comprising an I-TevI domain and a RNA-guided nuclease domain; (ii) a polynucleotide encoding a first guide RNA (gRNA); and (iii) a polynucleotide encoding a tRNA; wherein the polynucleotides in (ii) - (iii) are in sequential order.

[0007] In one aspect, a nucleic acid is provided comprising (i) a polynucleotide encoding a chimeric nuclease comprising a GIY-YIG nuclease domain and a RNA-guided nuclease domain; (ii) a polynucleotide encoding a first guide RNA (gRNA); and (iii) a polynucleotide encoding a tRNA; wherein the polynucleotides in (ii) - (iii) are in sequential order.

[0008] In some embodiments, the nucleic acid further comprises (iv) an RNA stabilizing polynucleotide located downstream of (i).

[0009] In one aspect, a nucleic acid is provided comprising (i) a polynucleotide encoding a first guide RNA (gRNA); and (ii) a polynucleotide encoding a tRNA; wherein the polynucleotides in (i) - (ii) are in sequential order.

[0010] In some embodiments, the nucleic acid further comprises two or more donor polynucleotides positioned in tandem; and a ribozyme polynucleotide; wherein the donor polynucleotides are in sequential order and the donor polynucleotide located upstream comprises a ribozyme cleavage site sequence at the 3’ end.

[0011] In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the DNA is circular plasmid DNA, linear double-strand DNA, single strand DNA, or chimeric RNA and DNA. In some embodiments, the RNA is mRNA. In some embodiments, the mRNA comprises nucleic acid mimetics selected from the group of peptide nucleic acid (PNA), morpholino nucleic acid, cyclohexenyl nucleic acid (CeNAs), and locked nucleic acid (LNA). In some embodiments, the mRNA comprises modified sugar moieties, optionally wherein the modified sugar moiety is selected from the group of N1 -methylpseudouridine, 9-Methyladenine, 2'-O-(2-methoxyethyl), 2'-dimethylaminooxyethoxy, 2'-dimethylaminoethoxyethoxy, 2'-O- methyl, and 2'-fluoro. In some embodiments, the mRNA comprises a modified nucleobase, optionally wherein the modified nucleobase is selected from the group of a 5-methylcytosine; a 5 -hydroxymethyl cytosine; a xanthine; a hypoxanthine; a 2-aminoadenine; a 6-methyl derivative of adenine; a 6-methyl derivative of guanine; a 2-propyl derivative of adenine; a 2-propyl derivative of guanine; a 2-thiouracil; a 2-thiothymine; a 2-thiocytosine; a 5-halouracil; a 5- halocytosine; a 5-propynyl uracil; a 5-propynyl cytosine; a 6-azo uracil; a 6-azo cytosine; a 6-azo thymine; a pseudouracil; a 4-thiouracil; an 8-halo; an 8-amino; an 8-thiol; an 8-thioalkyl; an 8- hydroxyl; a 5-halo; a 5-bromo; a 5 -trifluoromethyl; a 5-substituted uracil; a 5-substituted cytosine; a 7-methylguanine; a 7-methyladenine; a 2-F-adenine; a 2-amino-adenine; an 8- azaguanine; an 8-azaadenine; a 7-deazaguanine; a 7-deazaadenine; a 3-deazaguanine; a 3- deazaadenine; a tricyclic pyrimidine; a phenoxazine cytidine; a phenothiazine cytidine; a substituted phenoxazine cytidine; a carbazole cytidine; a pyridoindole cytidine; a 7-deaza- adenine; a 7-deazaguanosine; a 2-aminopyridine; a 2-pyridone; a 5-substituted pyrimidine; a 6- azapyrimidine; an N-2, N-6 or 0-6 substituted purine; a 2-aminopropyladenine; a 5- propynyluracil; and a 5-propynylcytosine. In some embodiments, the mRNA comprises a non- naturally occurring or a non-natural internucleoside linkage selected from the group of a phosphorothioate, a phosphoramidate, a non-phosphodiester, a heteroatom, a chiral phosphorothioate, a phosphorodithioate, a phosphotriester, an aminoalkylphosphotriester, a 3'- alkylene phosphonates, a 5'-alkylene phosphonate, a chiral phosphonate, a phosphinate, a 3'- amino phosphoramidate, an aminoalkylphosphoramidate, a phosphorodiamidate, a thionophosphoramidate, a thionoalkylphosphonate, a thionoalkylphosphotriester, a selenophosphate, and a boranophosphate.

[0012] In some embodiments, the RNA-guided nuclease is selected from the group of Staphylococcus aureus Cas9 (“saCas9”), Streptococcus pyogenes Cas9, Acidaminococcus Casl2 and Deltaproteobacteria CasX, and Eubacterium rectale Casl2a. In some embodiments, the Cas is a deactivated Cas (dCas). In some embodiments, the Cas is a nickase or a dCas.

[0013] In some embodiments, the I-TevI is a nickase. In some embodiments, the I-TevI nickase domain comprises mutations at amino acid residues R27A, V117F, K135R, and N140S. In some embodiments, the I-TevI is deactivated. In some embodiments, the I-TevI deactivating mutation is the R27A mutation.

[0014] In some embodiments, wherein the ribozyme polynucleotide is located within the tRNA polynucleotide.

[0015] In some embodiments, the nucleic acid further comprises a polynucleotide encoding a second gRNA, optionally wherein the second gRNA polynucleotide is located 3’ of the tRNA polynucleotide.

[0016] In some embodiments, the order of the polynucleotides is chimeric nuclease, RNA stabilizing polynucleotide, guide RNA, tRNA, and guide RNA. In some embodiments, the order of the polynucleotides is chimeric nuclease, RNA stabilizing polynucleotide, guide RNA, tRNA / ribozyme, guide RNA, donor polynucleotide 1, and donor polynucleotide 2.

[0017] In some embodiments, the RNA stabilizing polynucleotide comprises a metastasis- associated lung adenocarcinoma transcript 1 (MALAT) 3' sequence, or a 3 ’-end of the multiple endocrine neoplasia beta transcript (MEN P). In some embodiments, the RNA stabilizing sequence comprises a triple helix RNA structure or a 3 ’-end of an RNA transcript lacking a canonical poly adenylation signal.

[0018] In some embodiments, the donor polynucleotide is single stranded or double stranded. In some embodiments, the donor polynucleotide is DNA or RNA. In some embodiments, one strand of the double stranded donor polynucleotide is DNA and one strand is RNA. In some embodiments, the donor polynucleotide comprises a cis-acting single-strand RNA polynucleotide annealed to a complementary single-strand DNA polynucleotide. In some embodiments, the single or double stranded donor polynucleotide comprises a 2 to 18 nucleotide overhang at the 3’ end. In some embodiments, the single or double stranded donor polynucleotide comprises a 14 nucleotide overhang at the 3’ end. In some embodiments, the overhang at the 3’ end is a singlestranded RNA polynucleotide.

[0019] In some embodiments, the nucleic acid additionally comprises a second guide RNA capable of targeting a region 5’ to the donor polynucleotide target site. In some embodiments, the first guide RNA is capable of targeting a first chimeric nuclease to a first I-TevI or Cas9 target site and cleaving at the first I-TevI or Cas9 target site in a genome of a cell and the second guide RNA is capable of targeting a second chimeric nuclease to a second I-TevI target site in the genome of the cell and cleaving at the second I-TevI target site, wherein the cleavage creates a nucleotide overhang at the second I-TevI target site.

[0020] In some embodiments, the 3’ end of the donor polynucleotide is complementary to the overhang created by the cleavage of second I-TevI at the second I-TevI target site.

[0021] In some embodiments, the guide RNA and the donor polynucleotide target mutations in the CFTR gene. In some embodiments, the guide RNA and donor polynucleotide target and replace the CFTR c.l521_1523del (p.Phe508del), C.1624G>T (p.Gly542Ter), C.1652G>A (p.Gly551Asp), C.1657C>T (p.Arg553Ter) or c.3846G>A (p.Trpl282Ter) mutations.

[0022] In some embodiments, the guide RNA and the donor polynucleotide target mutations in the SERPINA1 gene. In some embodiments, the guide RNA and donor polynucleotide target and replace the SERPINA1 c.lO96G>A (p.Glu342Lys) mutation.

[0023] In some embodiments, the nucleic acid further comprises a promoter. In some embodiments, the promoter is selected from the group of CMV promoter, SV40 promoter, minimal cytomegalovirus (CMV) promoter, and a human elongation factor- 1 alpha (EFla) promoter. In some embodiments, the promoter is selected from the group of muscle-specific synthetic promoter SPc5-12 neuronal-specific promoter hSYNl, aldhlLl, cTNT, alpha-MHC, SPc5-12, MUC2, Ksp-cadherin, Albumin, HAS, insulin, rhodopsin, rNSE, and Cone-opsin promoters.

[0024] In some embodiments, the tRNA comprises a glycine arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine tRNA.In some embodiments, the ribozyme comprises a hammerhead ribozyme or a hepatitis delta virus (HDV) ribozyme. In some embodiments, the donor polynucleotide comprises a trans-acting double-stranded RNA polynucleotide with 3’- or 5 ’-overhanging nucleotides; a cis-acting singlestrand RNA polynucleotide with sequence similarity to the target strand of the nuclease; a cis- acting single-strand RNA polynucleotide with sequence similarity to the non-target strand of thenuclease; one or more binding sites for a genome modifying factor optionally a binding site for a site-specific recombinase, such as serine recombinases or LoxP target sites; a repair template for a protein coding sequence ;\one or more exons with splice acceptor and donor sequences one or more selectable sequences selected from the group of NeoR, BsdR, HygR, PuroR, and BleoR genes; one or more drug-inducible regulatory sequences for controlled gene expression; and / or a 2 nucleotide overhang at the 3’ end.

[0025] In some embodiments, the nucleic acid further comprises a polyadenylation signal. In some embodiments, the polyadenylation signal comprises a simian virus 40 (SV40), a globin, P- globin, human growth hormone (hGH), bovine growth hormone (BGH), herpes simplex virus type 1 thymidine kinase (HSV TK), or synthetic polyadenylation (Synt poly A) polyadenylation signal.

[0026] In some embodiments, the nucleic acid further comprises a self-inactivating sequence.

[0027] In some embodiments, the nucleic acid is about 5kb in length. In some embodiments, the nucleic acid is less than 5kb in length.

[0028] In some embodiments, the nucleic acid is packaged in a virus. In some embodiments, the virus is a lentivirus, adeno-associated virus (AAV), adenovirus, retrovirus, or modified Herpes Simplex Virus (HSV). In some embodiments, the AAV is a AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV7, AAV8, AAV9, AAV10, AAV-DJ, AAV2.5T or AAVmyo.

[0029] In one aspect, a vector comprising a nucleic acid of the disclosure is provided.

[0030] In one aspect, a viral vector comprising a nucleic acid of the disclosure is provided.

[0031] In one aspect, an AAV virus comprising a nucleic acid of the disclosure is provided.

[0032] In one aspect, a cell comprising a nucleic acid of the disclosure, a vector of the disclosure, a viral vector of the disclosure, or a AAV of the disclosure is provided.

[0033] In one aspect, a composition comprising a chimeric nuclease polypeptide comprising an I-TevI domain and an RNA-guided nuclease domain and a nucleic acid of the disclosure is provided.

[0034] In one aspect, a composition comprising a chimeric nuclease nucleic acid encoding a chimeric nuclease comprising an I-TevI domain and a RNA-guided nuclease domain and a nucleic acid of the disclosure is provided.

[0035] In some embodiments, the chimeric nuclease nucleic acid is an mRNA.

[0036] In one aspect, an LNP composition comprising a nucleic acid of the disclosure or the composition of the disclosure is provided.

[0037] In one aspect, a pharmaceutical composition comprising a nucleic acid of the disclosure, a vector of the disclosure, a viral vector of the disclosure, or a AAV of the disclosure, the composition of the disclosure, or the LNP composition of the disclosure, and an excipient is provided.

[0038] In one aspect, a method of delivering messenger RNA encoding a chimeric nuclease comprising an LTevI domain and an RNA-guided nuclease domain to a cell is provided, the method comprising contacting the cell with a polynucleotide encoding one or more guide RNAs and polynucleotide donor.

[0039] In one aspect, a method of genetically modifying the genome of a cell is provided, the method comprising contacting the cell with a nucleic acid of the disclosure, a vector o of the disclosure, a viral vector of the disclosure, or a AAV of the disclosure, the composition of the disclosure, or the LNP composition of the disclosure.

[0040] In some embodiments, the modification comprises an insertion, deletion, substitution, or mutation of the genome. In some embodiments, the insertion of the donor polynucleotide into the genome of the cell results in removal of sequences between a LTevI target site and a Cas9 target site. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0041] In one aspect, a method of inserting or replacing a sequence at a chimeric nuclease target site in a genome in a cell is provided, the method comprising: contacting the cell with a nucleic acid comprising one or more nucleic acid sequences encoding a chimeric nuclease comprising a Cas9 domain and a LTevI domain; and a nucleic acid comprising a guide polynucleotide and a donor polynucleotide; wherein the guide polynucleotide and the chimeric nuclease form a complex, and the complex binds and cleaves the genomic DNA at a Cas9 target site and a LTevI target site; wherein the 3’ end of the donor polynucleotide comprises at least 2 bases of complementarity over the 5’ end of the LTevI target site after LTevI cleavage; and wherein the donor polynucleotide is incorporated into the chimeric nuclease target site at a position 5’ to the Cas9 target site.

[0042] In some embodiments, the 3’ end of the guide polynucleotide and the 5’ end of the donor polynucleotide are connected.

[0043] In some embodiments, a cellular polymerase is targeted to the chimeric nuclease target site. In some embodiments, the cellular polymerase is polymerase theta.

[0044] In one aspect, a method of replacing at least a portion of a CFTR gene in the genome in a cell is provided, the method comprising contacting the nucleic acid of the disclosure, a vector of the disclosure, a viral vector of the disclosure, or a AAV of the disclosure, the composition of the disclosure, or the LNP composition of the disclosure to the cell. In some embodiments, the guide RNA and donor polynucleotide target mutations in the CFTR gene.

[0045] In some embodiments, the guide RNA and donor polynucleotide target and replace the CFTR c.l521_1523del (p.Phe508del), c,1624G>T (p.Gly542Ter), c,1652G>A (p.Gly551Asp), C.1657C>T (p.Arg553Ter), or c.3846G>A (p.Trpl282Ter) mutations.

[0046] In one aspect, a method of treating cystic fibrosis in a patient in need thereof is provided, the method comprising, administering a nucleic acid of the disclosure, a vector of the disclosure, a viral vector of the disclosure, or a AAV of the disclosure, the composition of the disclosure, or the LNP composition of the disclosure to the patient.

[0047] In some embodiments, the guide RNA and donor polynucleotide target mutations in the CFTR gene. In some embodiments, the guide RNA and donor polynucleotide target and replace the CFTR c,1521_1523del (p.Phe508del), c,1624G>T (p.Gly542Ter), c,1652G>A (p.Gly551Asp), C.1657C>T (p.Arg553Ter) or c.3846G>A (p.Trpl282Ter) mutations.

[0048] A method of replacing at least a portion of a SERPINA1 gene in the genome in a cell is provided, the method comprising contacting a nucleic acid of the disclosure, a vector of the disclosure, a viral vector of the disclosure, or a AAV of the disclosure, the composition of the disclosure, or the LNP composition of the disclosure to the cell.

[0049] In some embodiments, the guide RNA and donor polynucleotide target mutations in the SERPINA1 gene. In some embodiments, the guide RNA and donor polynucleotide target and replace the SERPINA1 c.lO96G>A (p.Glu342Lys) mutation.

[0050] In one aspect, a method of treating alpha- 1 -antitrypsin deficiency in a patient in need thereof is provided, the method comprising, administering a nucleic acid of the disclosure, a vector of the disclosure, a viral vector of the disclosure, or a AAV of the disclosure, the composition of the disclosure, or the LNP composition of the disclosure to the patient. In some embodiments, the guide RNA and donor polynucleotide target mutations in the SERPINA1 gene.In some embodiments, the guide RNA and donor polynucleotide target and replace the SERPINA1 c,1096G>A (p.Glu342Lys) mutation.

[0051] In one aspect, a method of replacing at least a portion of a DMPK gene in the genome in a cell is provided, the method comprising contacting a nucleic acid of the disclosure, a vector of the disclosure, a viral vector of the disclosure, or a AAV of the disclosure, the composition of the disclosure, or the LNP composition of the disclosure to the cell. In some embodiments, the one or more guide RNAs target mutations in the DMPK gene. In some embodiments, the one or more guide RNAs target CAG triplet polynucleotide sequences in the 3’ untranslated region of the DMPK gene.

[0052] In one aspect, a method of treating myotonic dystrophy type 1 in a patient in need thereof is provided, the method comprising, administering a nucleic acid of the disclosure, a vector of the disclosure, a viral vector of the disclosure, or a AAV of the disclosure, the composition of the disclosure, or the LNP composition of the disclosure to the patient.

[0053] In one aspect, a method of replacing at least a portion of a C9ORF72 gene in the genome in a cell is provided, the method comprising contacting a nucleic acid of the disclosure, a vector of the disclosure, a viral vector of the disclosure, or a AAV of the disclosure, the composition of the disclosure, or the LNP composition of the disclosure to the cell. In some embodiments, the one or more guide RNAs target mutations in the C9ORF72 gene. In some embodiments, the one or more guide RNAs target GGGGCC hexanucleotide repeat sequences between Exon la and Exon lb of the C9ORF72 gene.

[0054] In one aspect, a method of treating amyotrophic lateral sclerosis or frontotemporal dementia in a patient in need thereof is provided, the method comprising, administering a nucleic acid of the disclosure, a vector of the disclosure, a viral vector of the disclosure, or a AAV of the disclosure, the composition of the disclosure, or the LNP composition of the disclosure patient.BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0056] FIG. 1 shows a schematic of an exemplary AAV cassette encoding elements for target site disruption or repair comprising a CMV promoter, Dualase, a MALAT sequence, gRNAl, tRNA’, gRNA2, repair templates (RT), and a Poly A sequence (synt[A]). The minimal CMV promoter drives the transcription of this exemplary 3-in-l construct containing Dualase, long non-coding RNA MALAT- 1, synthetic transfer RNA containing sequence-specific ribozyme (tRNA’), gRNA fused with repair template (RT), and synthetic poly A signal sequence (synt[A]). Upon transcription, RNA maturation at 3 ’-end of MALAT and both ends of tRNA’ result in the separation of Dualase-MALAT RNA, tRNA, and gRNA. The U-rich repeat on MALAT can help protect Dualase RNA from degradation while tRNA’ will recognize and cleave at the region between RTs as well as between RT and synt[A] sequence. After translation, the Dualase can form a complex with gRNA (RNP) and cleave the DNA at the intended target site. The presence of in-place repair template fused to the 3 ’ end of gRNA (cis-sense and antisense) or abundant free-floating repair template (trans-antisense and trans-sense) serve as a bridge between two cleavage sites and local reference template for the cellular repair machinery.

[0057] FIG. 2 shows a schematic of an exemplary mRNA cassette encoding elements for efficient accurate target site disruption or repair. The T7 promoter drives the transcription of an exemplary 3-in-l construct containing Dualase, long non-coding RNA MALAT- 1, synthetic transfer RNA containing sequence-specific ribozyme (tRNA’), gRNA fused with repair template (RT), and synthetic poly A signal sequence (synt[A]). Upon cellular delivery, RNA maturation at the 3’ end of MALAT and both ends of the tRNA’ result in the separation of the mRNA encoding for Dualase-MALAT RNA, tRNA, and gRNA. The U-rich repeat on the MALAT sequence can protect the Dualase RNA from degradation while the tRNA’ can recognize and cleave the region between the RTs as well as between RT and synt[A] sequence. After translation, the Dualase can form a complex with gRNA (RNP) and cleave at the intended target site. Optionally, a GFP coding sequence is included to track cell uptake and is translated and separated from the Dualase protein in the cell by a N-terminal T2A (thosea asigna virus 2A) peptide sequence which is skipped by the ribosome. The presence of an in-place repair template fused to the 3 ’ end of gRNA (cis-sense and antisense) or abundant free-floating repair template (trans-antisense and trans-sense) serve as a bridge between two cleavage sites and local reference template for cell repair machinery.

[0058] FIG. 3 shows a schematic of an exemplary dual-cleaving nuclease ribonucleoprotein (RNP) complex (Dualase) with an all-in-one guide RNA repair cassette. The Dualase and 2-in- 1 gRNA-RT are incubated to form ribonucleoprotein complex (RNP) and delivered into target cells where they can interact and cleave the intended target site after translocation into the nucleus.

[0059] FIGs. 4A and 4B show exemplary Tracking of Indels by DEcomposition (TIDE) data of the outcome of genome editing with an AAV-Dualase- A V.S7 Dualase. Portions of deletions and insertions at defined length as well as unmodified reads are characterized as bar graph. (FIG. 4A). Dotted line P <0.01. (FIG. 4B) shows chromatograms of sequenced samples treated with AAV-Dualase-AA VS1 or reagents only in comparison with a reference sequence where differences in peaks are used to determine the repaired sequence.

[0060] FIGs. 5A - 5C show bar graphs and sequence plots of the results from next generation sequencing (NGS) of cells only, cis-antisense, cis-sense, and trans-RT. NGS data was analyzed using two bioinformatics tools (FIG. 5A) CRIS.Py and Geneious alignment platforms, as well as (FIG. 5B) CRISPRESSO2. “Indels” represent repair events resulting in an insertion or deletion. “Precise repair” represents sequencing reads with complete alignment to the repaired sequence. “Repair + SNP” represents sequence alignments with repair and another nucleotide change. And “Other” represents other sequence modifications in the sample. Total and aligned reads were used to identify editing outcome. The alignment of NGS sequencing reads of cis-antisense repaired sequence is shown in FIG. 5C with the number and percentage of reads indicated. A dotted line indicates reads below the generally accepted limits of detection of sequencing and the I-TevI and Cas9 target sites are indicated above the sequences.

[0061] FIGs. 6A - 6C show pictures of gels depicting the editing efficiency by restriction enzyme (RE) in HEK293 cells treated with AAV expressing Dualase and gRNA-RT targeting AAVS1 (all-in-one) as well as a control of AAV expressing Dualase targeting the AAVS1 codelivered with the indicated repair template (co-delivery) by lipofection in the presence of increasing doses of inhibitors blocking the (FIG. 6A) non-homologous end-joining (NHEJ), (FIG. 6B) homologous-directed repair (HDR), or (FIG. 6C) Rad52-dependent pathways. Samples were analyzed for editing efficiency by restriction enzyme (RE) digestion efficiency using a unique RE at the inserted repair template site. NHEJ (DNA) = duplex DNA withouthomology, HDR (DNA) = duplex DNA with homology arms & NHEJ (RNA) = single-strand RNA without homology.

[0062] FIGs. 7A-7B show pictures of gels depicting the directional insertion of repair template with Dualase ribonucleoprotein (RNP) complex formed with an AAVS1 -targeting guide RNA combined with RNA repair templates by directional polymerase chain reaction (PCR). (FIG. 7A) shows the results of a restriction enzyme digestion of HEK293 cells lipofected with Dualase together with guide RNA and repair template delivered separately (co-delivery) or Dualase with guide RNA and RNA repair template fused (“All-in-one”), lipofection reagent only (’’Reagent only”) or cells only (“Mock”). Shown for each reaction are the undigested PCR amplicon of the AA VS1 site (“Sub”) and digested PCR amplicon of the AAVS1 site (“Digested”). (FIG. 7B) shows PCR product present in the correct orientation “Right oriented RT” only if the repair template is inserted correctly and a PCR product is present in the incorrect orientation (“Wrong oriented RT”) if the repair template inserted incorrectly.

[0063] FIG. 8 shows a picture of a gel depicting the directional insertion of repair template with AAVS1 -targeting Dualase all-in-one mRNA with cis-sense or cis-antisense repair templates by directional polymerase chain reaction (PCR). Shown are the results of a restriction enzyme digestion (Bglll digest) or PCR reactions of HEK293 cells lipofected with Dualase (Tev[VKN]- SaCas9[WT] or Tev[VKN]-SaCas9[D10E]), SaCas9[WT] or lipofection reagent only. Shown for each reaction are the undigested PCR amplicon of the AAVS1 site (“Full length”), digested PCR amplicon of the AAVS1 site (“Bglll digest”), PCR product if the repair template inserted only in the correct orientation (“Right oriented RT”) and PCR product if the repair template inserted only in the incorrect orientation (“Wrong oriented RT”). Control cells treated with Dualase mRNA targeting the AA VS J site with co-delivered repair template (co-delivery) is also shown.

[0064] FIG. 9 shows a picture of a gel depicting cellular repair after Dualase cleavage and treatment with NHEJ and Rad52 inhibitors and trans dsRNA repair template. HEK293 cells were treated with AAV expressing Dualase and gRNA-RT targeting AAVS1 (all-in-one) as well as a control of AAV expressing Dualase targeting the AA VS J co-delivered with the indicated repair template (co-delivery) by lipofection in the presence of increasing doses of inhibitors blocking the non-homologous end-joining (NHEJ inhibitor) or Rad52 pathway (“Rad52inhibitor”). NHEJ (DNA) = duplex DNA without homology, HDR (DNA) = duplex DNA with homology arms & NHEJ (RNA) = single-strand RNA without homology.

[0065] FIG. 10A shows a schematic for precise removal of large repeat sequences using a dual-guide TevCas9 nuclease in which the guide RNAs target opposite strands of the duplex DNA to orient two I-TevI domains head-to-head. A first TevCas9 nuclease (1) containing the inactivating D10A+H557A mutation but an active I-TevI domain (2) is targeted to the 5 ’-end of a repeated sequence (3) using a guide RNA. A second TevCas9 nuclease (4) containing the inactivating D10A+H557A mutation but an active I-TevI domain is targeted to 3 ’-end of the repeated sequence but on the opposing strand such that the I-TevI domains of both nucleases are pointed towards the repeat sequence. Upon binding and cleavage (5) by the I-TevI nuclease domains, two complementary 3’ 2-nucleotide overhangs remain (6 and 7). Through the non- homologous end joining pathway, the cell can repair these complementary overhangs (8) and the large repeated sequence is removed (9) leaving a defined number of repeats in the genomic DNA (10).

[0066] FIG. 10B shows a schematic of precise removal of large repeat sequences using a dualguide TevCas9 nuclease in which the guide RNAs target the same strands of the duplex DNA to orient two I-TevI domains in tandem. A first TevCas9 nuclease (1) containing the inactivating D10A+H557A mutation but an active I-TevI domain (2) is targeted to the 5’-end of a repeated sequence (3) using a first guide RNA. A second TevCas9 nuclease (4) containing the inactivating D10A+H557A mutation but an active I-TevI domain is targeted downstream of the 3 ’-end of the repeat sequence on the same strand such that the I-TevI domains of both chimeric nucleases are pointed in the same direction. Upon binding and cleavage (5) by the two I-TevI nuclease domains, two complementary 3’ 2-nucleotide overhangs remain (6 and 7). Through the non-homologous end joining pathway, the cell can repair these complementary overhangs (8) and the large repeated sequence is removed (9) leaving a defined number of repeats in the genomic DNA (10).

[0067] FIGs. 11A - G show the results for the removal of CAG triplet repeat expansion in DMPK (Dystrophia myotonica protein kinase, (also labelled DmpkI) 3’-UTR using an all-in-one AAV encoding TevCas9 together with dual guides targeting the 5’- and 3 ’-ends of the repeat expansion. FIG. 11A shows a schematic of the active I-TevI domains and inactivated Cas9 domains with the D10A+H557A mutations targeted by two guide RNAs to orient the I-TevIdomains into the CAG repeat sequence and downstream of the CAG repeat. CAG repeats of greater than 75 result in skeletal muscular diseases such as myotonic dystrophy. FIG. 11B shows a schematic of the expression cassette encoding the dual guided TevfKTQ]- SaCas9[D10A+H557A] and conditionally cleavable guide RNAs when the expressed MiniCMV is the promoter sequence, HHribo is the hammer head ribozyme sequence, sgRNAl is the 5’guide RNA targeting sequence, Gly tRNA is a glycine tRNA sequence, sgRNA2 is the 3’ guide RNA targeting sequence, HDVribo is the hepatitis delta virus ribozyme sequence and polyA is the poly adenylation sequence (SEQ ID NO: 118). FIG. 11C shows the schematic of the expected products of an in vitro cleavage reaction using purified TevfKTQ]- saCas9[D10A+H557A] protein complexed with the dual guides in equimolar ratios targeting the DMPK CAG repeats. Shown on the right is an agarose gel with the products of an in vitro cleavage reaction using DMPK CAG repeat DNA substrate mixed with TevfKTQ]- saCas9[D10A+H557A] dual guide ribonucleoprotein complex. The expected products of the reaction by size are indicated next to the gel image. FIG. 11D shows the alignment of Sanger sequencing reads of clonal amplicons 1- 10 generated from cells transduced with an all-in-one AAV encoding TevCas9 together with dual guides together with the expected cleavage.[CAG]si+ indicates the repeat sequence is present and lowercase letters indicate the precise number of CAG repeats after TevCas9 cleavage. FIG. HE show the workflow of the experiment to quantify repeat collapse using repeat-primed PCR in DMPK^^ CAG repeat cells transduced with all-in-one AAV TevCas9 and SaCas9 dual guides. Transduced cells are harvested and genomic DNA extracted for PCR-amplification using a primer outside and inside the repeat expansion. The results PCR products are analyzed on an Agilent Bioanalyzer and peaks corresponding to the repeat are quantified relative to DMPK^[VVXCAG repeat cells only. More than 65% of the repeat was collapsed in the TevCas9 treated cells relative whereas -30% of the repeat was collapsed in the SaCas9 treated cells. FIG. HF shows a graph for the fold change of DMPK in transduced cells subjected to quantitative RT-PCR to quantify the levels of DMPK^^1and DMPKWTtranscripts after each treatment using primers specific for each transcript. Statistical significance of the changes are indicated with the horizontal bars labels as ns = nosignificant change, * p = <0.05, ** = <0.01 or *** = p <0.001. FIG. 11G shows a graph for the fold change of a mis-spliced variant of the CLCN 1 gene in Exon 6 (E6) in TevCas9 dual guide (TevCas9dg), TevCas9 single guide (TevCas9sg) and Cas9 dual guide (Cas9dg) transduced cellsrelative to untreated cells (“Cell Only”). Mis-splicing of the human C1C-1 chloride channel causes myotonic dystrophy. Both TevCas9 dual guide and TevCas9 single guide significantly reduced the mis-splicing of CLCN 1 Exon 6, whereas Cas9 did not significantly change the mis- splicing. Statistical significance of the changes are indicated with the horizontal bars labels as ns = no-significant change, ** = <0.01 or *** = p <0.001. FIG. 11H-T show the results for the removal of a GGGGCC hexanucleotide repeat expansion between Exon la and lb of the C9ORF72 gene using an all-in-one AAV TevCas9 together with dual guide targeting the 5’- and 3’-ends of the repeat expansion. FIG. 11H shows a schematic of the active I-TevI domain and inactivated Cas9 domains with the D10A+H557A mutations targeted by two guide RNAs to orient the I-TevI domains into the GGGGCC repeat sequence. GGGGCC repeats of greater than 24 result in motor neuron diseases such as ALS. FIG. Ill shows the schematic of the expected products of an in vitro cleavage reaction using purified Tev[VKN]-saCas9[D10A+H557A] protein complexed with the dual guides in equimolar ratios targeting C9ORF72 GGGGCC repeats. Shown on the right is an agarose gel with the products of an in vitro cleavage reaction using C9ORF72 GGGGCC repeat DNA substrate mixed with TevfVKNJ- saCas9[D10A+H557A] dual guide ribonucleoprotein complex. The expected products of the reaction by size are indicated next to the gel image. FIG. 11 J shows micrographs of the maturation of C9ORF72 repeat expansion motor neuron progenitors into motor neurons. Shown also is confirmation of motor neuron maturation by Western blot of the motor neuron-specific marker ISL1 compared to the housekeeping gene beta-tubulin. FIG. 11K shows the summary results of repeat-primed PCR to quantify the percentage of large repeat products in motor disease motor neurons transduced with an all-in-one AAV encoding TevCas9 or Cas9 together with dual guides. FIG. 11L shows the results of sequencing single clones of collapsed repeat sequences from TevCas9 AAV-transduced motor neurons indicating the precise collapsed repeat products in 4 of 9 sequences indicated by an asterix (*). FIG. 11M shows a summary of percent editing as determined by deep sequencing analysis of potential off-target sites in motor neuron cells transduced AAV expressing the TevSaCas9 dual guide. The hashed line indicated the limits of detection of deep sequencing for potential off-target effect with no off-target detected in transduced cells. FIG. UN shows a summary of the percent editing as determined by deep sequencing analysis of potential off-target sites in motor neuron cells transduced AAV expressing the SaCas9 dual guide. An off-target (OT24) with active SaCas9 in Chromosome 11is indicated. Statistical significance of the changes is label as nd = no-significant difference, or * = p <0.001. FIG. 11O shows the results of a Western Blot on the left using an anti-C9ORF72 antibody and anti-GAPDH house keeping gene control antibody in motor neurons transduced with AAV expressing the TevCas9 dual guide (TevCas9dg), TevCas9 single guide (TevCas9sg) and Cas9 dual guide (Cas9dg) as well as a cell only control. Shown on the right is a summary of quantifying the fold C9ORF72 expression change in three replicates of the Western Blots normalized to the GAPDH housekeeping gene expression. Treatment of motor neurons with AAV expression TevCas9 and C9ORF72 dual guides significantly increased expression >2-fold. Statistical significance of the changes is label a * = p <0.01. FIG. IIP show a representative anti-poly GR dipeptide dot blot on the left of cell lysate from motor neurons transduced with AAV expressing the TevCas9 dual guide (TevCas9dg), TevCas9 single guide (TevCas9sg) and Cas9 dual guide (Cas9dg) as well as a mock treated cells. Shown on the right is a summary quantification of the relative intensity of the dot blots to the mock treated cells with TevCas9 c9orf72 dual guide showing a reduction in the amount of poly GR detected. FIG. 11Q shows a schematic of an intracranial (ICV) injection of two doses of AAV (Low = 1 x 1013viral genomes and High = 2 x 1013) expressing dual guided TevCas9 into mice that have copies of GGGGCC repeat expanded human C9ORF72. The right shows the results of RT-qPCR to detect the expression of TevCas9 in samples of the cerebellum of injected mice 28 days after injection. Control mice were injected with phosphate buffered saline (PBS), reverse transcriptase quantitative PCR (RT-qPCR) shows dose-dependent expression of TevCas9 in bulk cerebellum tissue relative to the PBS injected control mice (n = 3). FIG. HR shows the results of a PCR to detect the size of the GGGGCC repeats in the cerebellum of humanized mice injected with phosphate buffered saline (PBS) or the two doses of AAV expressing dual guided TevCas9. Expanded and normal sized repeats from a representative agarose gel of the PCRs products amplified from genomic DNA is shown on the left. Shown on the right is a summary of the quantitation of normal-sized C9ORF72 PCR products relative to a housekeeping gene (n = 3). FIG. IIS shows the results of reverse transcriptase quantitative PCR (RT-qPCR) of the C9ORF72 transcripts in the cerebellum of humanized mice injected with PBS or the two doses of AAV expressing dual guided TevCas9. FIG. 11T shows the results of Western Blot of C9ORF72 protein in the cerebellum of humanized mice injected with PBS or the two doses of AAV expressing dual guided TevCas9.

[0068] FIG. 12A shows a diagram depicting the structure of the self-inactivating Dualase vector. The construct comprises nucleotide sequences encoding a promoter, human codon- optimized TevSaCas9, poly adenylation signal (“poly A”) and guide RNA sequence (“gRNA”). The self-inactivating target site may be in the region between the promoter and TevSaCas9 site (denoted as “Promoter”) or end of the TevSaCas9 coding sequence and beginning of the PolyA sequence (denoted “PolyA”). FIG. 12B shows a gel of the results of a T7E1 editing assay in HEK293 cells transfected with plasmid DNA with TevSaCas9 targeting the beta-2-microglobulin (B2M) gene harvested at 24-, 48-, and 72-hours post-transfection. Lanes marked “None” do not contain and self-inactivating sequence in the vector, lanes marked “Promoter” contain the B2M1 TevSaCas9 target site between the promoter sequence and TevSaCas9 sequence, lanes marked “PolyA” contain the B2M1 TevSaCas9 target site between the end of TevSaCas9 and the PolyA signal sequence. Levels of editing as determined by the amount of digested product relative to substrate is comparable over time between the constructs. FIG. 12C shows a Western blot for hemagglutinin (a-HA) encoded at the 3’-end of TevSaCas9 (“Dualase”) from the same treated cells in FIG. 12B. Lanes marked “None” do not contain and self-inactivating sequence in the vector, lanes marked “Promoter” contain the B2M1 TevSaCas9 target site between the promoter sequence and TevSaCas9 sequence, lanes marked “PolyA” contain the B2M1 TevSaCas9 target site between the end of TevSaCas9 and the PolyA signal sequence. Beta-actin (a- Act) was blotted on the same membrane as a loading control. FIG. 12D shows the production of a PolyA self-inactivating AAV2 virus targeting the B2M gene with a titer measured by quantitative reverse transcription PCR (RT-qPCR) of a 2.4 x 1012viral genomes (vg) per mL and a western blot for Western blot for hemagglutinin (a-HA) encoded at the 3 ’-end of TevSaCas9 (“Dualase”) for cell transduced with the virus. Beta-actin (a- Act) was blotted on the same membrane as a loading control. FIG 12E shows the results of a Western Blot for saCas9 and GAPDH from cell lipofected transfected with plasmid DNA versions of the vectors used to produce the selfinactivating AAV in FIG12D (“Self-inactivating”), as well as a vector that does not contain the self-inactivating sequences (“non-self-inactivating”) and a plasmid expressing GFP (“pAAV- GFP”) as controls over 14 days.

[0069] FIGs. 13A-C show schematics of the AA VS1 target site before (FIG. 13A), after cleavage with Dualase and guide RNA targeting AA VS J (FIG. 13B), and in relation with gRNA- repair template for the AAVS1 site for precise repair. (FIG. 13C).

[0070] FIG. 14 shows a Western blot of immunoprecipitated HA-tagged Dualase or SaCas9 from cells treated with Dualase and gRNA-RT or saCas9 and gRNA-RT. The presence of coimmunoprecipitated Rad52 or PolQ (polymerase theta; Pol 0) was determined using antibodies specific for Rad52 (a Rad52) or PolQ (a PolQ). Rad52 co-precipitates with Dualase- and Cas9- treated cells but PolQ only co-precipitates with Dualase treated cells.

[0071] FIG. 15A shows pictures of gels depicting the editing efficiency by restriction enzyme (RE) in HEK293 cells treated with purified TevCas9 mutant proteins and gRNA-RT targeting AAVS1. The “Tev[WT]-dCas9” lane contains purified TevCas9 protein containing the inactivating D10A+H557A mutation but an active I-TevI domain. The “Tev[R27A]-dCas9” lane contains purified TevCas9 protein containing the inactivating D10A+H557A mutation and an I- TevI domain inactivated with the R27A mutation. Control lanes of saCas9[WT], reagent only, cell only and cells lipofected with Tev-Cas9 3-in-l mRNA control are also shown. FIG. 15B shows pictures of gels depicting the editing efficiency by restriction enzyme (RE) in HEK293 cells treated with mRNA versions of the SaCas9 (WT = wild type; D10A+H557A = inactive; D10A = nickase; H557A = nickase) without the I-TevI domain (“No Tev”) and with the I-TevI domain containing the R27A, V117F, K135R and N140S mutations.

[0072] FIG. 16A shows a schematic of inserting eGFP in-frame into the CFTR coding sequence using a rep-gRNA. eGFP is expressed from the endogenous gene promoter and is separated from the truncated endogenous protein by a T2A cleavable peptide sequence. FIG. 16B shows micrographs of cells lipofected with saCas9 or Dualase mRNA and rep-gRNA encoding eGFP. Phase contrast and GFP shown including cell only and rep-gRNA only controls. FIG. 16C shows a picture of a gel with the insertion site amplified using PCR depicting the larger inserted sequence as Edited and no insertion as Unedited. FIG. 16D shows a schematic of inserting eGFP using a guide RNA targeting the CFTR F508 site and rep-gRNA encoding GFP targeting the CFTR G542 site “bridging rep-gRNA”. The bridging rep-gRNA has 14 bases of homology to the upstream CFTR F508 site. The ~28 kilobase (kb) region between the F508 and G542 sites is removed and replaced with the GFP sequence encoded by the bridging rep-gRNA. FIG. 16E shows micrographs of cells lipofected with Dualase (“TevSaCas9”) or SaCas9 and an equimolar mixture of the upstream guide RNA and bridging rep-gRNA. FIG. 16F shows a picture of a gel with the unedited target site indicated by “~28kb region” and the inserted sequence by “Sequence removed and replaced.” Also shown is a summary of the fraction of deepsequencing reads across the 5’- and 3 ’-junctions of the inserted sequences in which the repaired junction is correct (“Exact repair”) or there are detected insertions or deletions (“Indel”).

[0073] FIGs. 17A-17E show a schematic RNA-templated DNA Repair (“Rep-editing”) design and predicted TevSaCas9 protein engagement with its target site . FIG. 17A shows a schematic of rep-editing outlining a TevSaCas9 target site with the individual DNA targeting components, the domain structure, and the rep-gRNA (also referred to as “gRNA-RT”). The I-TevI linker zinc finger is indicated by yellow dot. Cleavage by the I-TevI domain leaves a 2-nt, 3’ overhang that is complementary to the 3’ end of the rep-gRNA, while cleavage by SaCas9 generates a blunt DNA end. FIG. 17B, shows the AAVS1 target site, expected repair product, and structure and interactions of the rep-gRNA with the target site. FIG. 17C shows identification and distribution of Tev CNNNG nuclease motifs upstream of SaCas9 binding sites in the human genome. FIG. 17D Schematic and predicted processing of the all-in-one construct with individual components indicated (not to scale). MALAT, RNA stability element from the MALAT1 non-coding RNA; HH, hammerhead ribozyme; T2A, self-splicing peptide linker; IRES, internal ribosome entry site. FIG. 17E shows on the left, in vitro processing of the all-in-one mRNA transcript by HEK293 cell extracts, incubated for the indicated time and resolved on an 1% agarose gel. Right, eGFP activity in HEK293 cells transfected with the all-in-one construct.

[0074] FIGs. 18A-18H. Rep-editing at the AAVS1 safe harbor site FIG. 18A shows a representative agarose gel of Bglll digestions of AAVS1 target sites PCR amplified from treated HEK293 cells. AAV; adeno-associated virus 2; pDNA, plasmid DNA; RNP, ribonucleoprotein particle of TevSaCas9 and rep-gRNA; mRNA, all-in-one construct. FIG. 18B shows a summary of editing outcomes at the AA VS J site by analysis method. FIG. 18C shows a summary of editing outcomes by delivery method. Barplots are the mean of all replicates with whiskers representing the standard deviation from the mean. Dots are individual replicates. FIG. 18D shows a plot of apparent nucleotide substitutions from deep sequencing of AAVS1 PCR amplicons from edited cells (blue triangles, 6 replicates) or mock transfected cells (orange circles, 4 replicates) showing fidelity of the editing over the editing window. Points are mean values with whiskers representing the standard deviation from the mean. The region targeted for editing and repair is bounded by dashed vertical lines. FIG. 18E shows an example reads from deep sequencing of the AAVS1 site in TevSaCas9 / rep-gRNA editing cells, with the reference (wild-type) sequence shown on the top line and repair products shown below, with the number ofreads for each sequence indicated on the right. Differences relative to the wild-type sequence are colored by nucleotide. FIG. 18F shows a schematic of reg-gRNA designed to delete 13 -bp and create a Hindlll site in AAVS1. FIG. 18G shows a plot of proportion of deep sequencing reads with lengths differences relative to unedited AA VS1 target site length from edited or mock treated cells. FIG. 18H shows an alignment of the sequencing reads with gaps indicated by dashes (-) and the number of reads indicated on the right with >48% of reads showing the intended 13-bp deletion.

[0075] FIG. 19A shows a schematic of rep-gRNAs used to test base pairing between the 3’ end of rep-gRNA and I-TevI cleavage overhang. All rep-gRNAs targeted the AA VS1 safe harbor site. FIG. 19B shows the impact on editing efficiency of rep-gRNAs with different length 3’ and 5’ overlaps. Shown is a barplot of the ratio repaired to unrepaired sites as judged by quantitative- PCR of the AA VS1 target site in treated cells. Barplots are the mean of 2 biological replicates with error bars representing the standard deviation from the mean. The dark green bars are cells treated with TevSaCas9 / rep-gRNA while light green bars are cells treated with protected rep- gRNA where a single-stranded DNA oligonucleotide complementary to the overhang was codelivered with the rep-gRNA. FIG. 19C shows the results of deep sequencing of the TevCas9 / rep-gRNA treated cells with various overhang lengths with the nucleotide differences to the unmodified (WT) sequence colored. FIG. 19D shows the impact of the terminal two nucleotides of the rep-gRNA on editing. Shown is a representative gel of Bglll digests of AA VS1 target site amplicons from treated HEK293 cells. FIG. 19E shows the impact of mismatches between the crRNA portion of the rep-gRNA on repair at the AA VS1 site with a rep-gRNA with an exact match to the I-TevI overhang (CC) or a wobble base pair (UC). Nucleotide mismatches in the crRNA relative to the target site are indicated by position above the gel image.

[0076] FIG. 20A shows a schematic of methodology used to identify TevSaCas9 off-target sites. FIG. 20B shows a plot of distribution of CNNNG motifs upstream of AA VS1 off-target sites with the indicated mismatches to the gRNA portion. CNNNG that can support repair using the AAVS1 rep-gRNA 3’-GG end are highlighted. FIG. 20C shows the alignment of off-target sites to the on-target AAVS1 sites. Identical nucleotides are colored, and CNNNG motifs in the upstream region are underlined. FIG. 20D shows percent editing as determined by CRISPRaltRations analysis. Points represent individual experiments, ns = not significant as calculated by paired t-tests of treated. FIG. 20E shows percent editing at on- and off-target sitesas determined by CRISPECTOR analysis for HEK293 treated with TevSaCas9 / AAVS1 rep- gRNA by AAV transduction (yellow) or mRNA lipofection (blue). The plots are separated into indels mapped near the predicted SaCas9 cleavage site and Tev cleavage site for each target site. Bars are the mean of three replicates with whiskers representing 95% confidence interval from the CRISPECTOR calculated editing rate.

[0077] FIG. 21A shows identification of repair proteins that are involved in rep-editing through use of small molecule inhibitors. Shown is an agarose gel of editing at the AA VS J target site in treated HEK293 cells. On the right, HEK293 cells were treated with the B02, SCR7, D- 103 or ART558 inhibitors at the same time as transduction with an AAV2 encapsidating TevSaCas9 and the rep-gRNA. Increasing concentration of inhibitor is indicated by a blue triangle. AAVS1 target site was PCR amplified and digested with Bglll; digestion with Bglll indicates a successful repair event. On the left, editing as determined by Bglll digestion of amplified AAVS1 target sites in HEK293 cells transduced with AAV2-TevSaCas9-rep-gRNAand different co-delivered repair sequences as indicated, along with the indicated small molecule inhibitors. FIG. 21B Replicated co-immunoprecipitation of extracts from HEK293 cells transduced with AAV2-TevSaCas9-rep-gRNA orAAV2-SaCas9-rep-gRNA with an anti-HA antibody, followed by Western blot with an anti-PolO or anti-Rad52antibody. Sizes (in kd) are indicated on the left of the gel image. FIG. 21C Model for rep-editing. Cleavage by TevSaCas9- rep-gRNA recruits Rad52 that can bind to RNA, or dsRNA, or mediate RNA:DNA strand exchange at the I-TevI cleavage site. Formation of a hybrid RNA:DNA structure with a 3’-OH recruits Pol0. Resolution of the repair intermediate by a fill-in gap repair process involving DNA ligase and an unidentified DNA polymerase.

[0078] FIG. 22A shows schematics of rep-gRNA design for targeting three mutations in the CFTR gene: a 3nt deletion DF508, G>A transition mutation G542X and G>C transversion mutation W1282X. The expected edited product is shown below, with silent substitutions indicated by a hash symbol (#) and corrective edits indicated by a x-marked circle. FIG 22B shows a representative gel of SspI (DF508 or G542X) or Hindlll (W1282X) restriction digestion analysis of PCR amplicons of 16HBEge cells. TevSaCas9 / rep-gRNA treated cells are indicated by a (+) and transfection reagent only treated cells with a (-).

[0079] FIG. 23A shows an alignment of prevalent reads from deep sequencing of PCR products amplified from total 16HBE C 7 ? G542X cells transfected with TevSaCas9 / rep-gRNA(cells were not enriched or selected prior to analysis). FIG. 23B shows a schematic of formulating an all-in-one messenger RNA (mRNA) expressing TevCas9 and a conditionally cleavable repair template guide RNA (rep-gRNA) in a lipid nanoparticle for intratracheal delivery to a humanized CFTRG542Xmouse. The TevCas9 mRNA also includes a cleavable GFP tag to allow for quantifying uptake into lung cell cells. FIG. 23C shows the results of fluorescent- activated cell sorting (FACS) of harvested lung tissue of the humanized CFTRG542Xmice treated with the TevCas9 all-in-one mRNA and a GFP expressing mRNA control. Approximately 31 % of the cells indicated in the boxed area were counted as GFP positive for the TevCas9 treated mouse and approximately 25% of the cells indicated in the boxed area in the GFP only control mouse. FIG. 23D shows the results of a PCR that detects the repaired sequence from the harvested lung tissue in the TevCas9 treated and GFP control mice. The PCR is calibrated with a lung epithelial cell sample with known repair (“corrected cell control”). A repaired product is only detected in the lung tissue of the TevCas9 all-in-one mRNA treated mice and not in the GFP control mice.

[0080] FIG. 24A shows a schematic of a rep-gRNA design for targeting the human SERPINA1 gene at amino acid position E342K and an alignment of prevalent reads from deep sequencing of PCR products amplified in GM 11423 patient liver fibroblasts transfected with TevSaCas9 / rep- gRNA (cells were not enriched or selected prior to analysis. FIG. 24B shows the dosing and sampling schedule of humanized SERPINA1 E342K mice treated with lipid nanoparticle encapsulated all-in-one TevSaCas9 / re-gRNA to correct the mutation. FIG. 24C shows the results of an enzyme-linked immunosorbent assay (ELISA) of human alpha- 1 -antitrypsin (Al AT) from pre- and post-dosing serum samples from the treated and naive mice at the indicated times. FIG. 24D shows the results of serum biochemistry analysis for aspartate transaminase (AST) and alanine transaminase (ALT) from pre- and post-dosing serum samples from the treated and naive mice at the indicated times. FIG. 24E show the results of reversetranscriptase quantitative PCR (RT-qPCR) directed against the Cas9 domain of TevCas9 to confirm expression of TevSaCas9 in the liver samples of naive and treated mice.

[0081] FIG. 25A shows a schematic of removing and replacing 74 nucleotides between amino acids R553 and G542 of human CFTR using a guide RNA and bridging rep-gRNA strategy. FIG. 25B shows a picture of a gel of PCR amplicons of the CFTR target site from SaCas9 + rep-gRNA and TevSaCas9 + rep-gRNA treated 16HBEge cells digested with the Mfel and SspI and restriction enzymes. The presence of digested products indicates repair.

[0082] FIG. 26A-C shows exemplary combinations and orientations of conditionally cleavable nucleotide sequences of the disclosure. FIG. 26A shows schematics of the exemplary constructs that are compatible as both DNA and RNA constructs using human transfer RNAs (tRNAs) or transfer RNAs with a trans-acting ribozyme encoded in the anticodon loop (tRNA’) The cleavage sites after the DNA versions of the encoded constructs are translated to mRNA or in vitro transcribed RNA versions of the constructs are introduced into the cell, the sites of cleaving of the RNA are indicated by the solid triangles (A). The sites of cleaving of transacting elements are indicated with lines ending with arrows. Messenger RNA versions of constructs have a 5’-UTR and DNA versions of the construct have a promoter sequence. Table 10 lists exemplary human transfer tRNAs that undergo tRNA maturation when expressed or transfected into cells. Some exemplary versions contain a MALAT stabilizing sequence to stabilize the I-TevI and Cas9 coding sequence after cleavage of the mRNA.. FIG. 26B and C show schematic the exemplary constructs that are compatible as DNA constructs only, such as with adeno-associated viral vectors. Ribozymes can only be encoded as DNA since they will self-cleave upon transcription to RNA. Ribozymes cleave on one side only and the orientation of the ribozymes is indicated by the direction of the arrow. Transfer RNAs or trans-acting transfer RNAs may be combined with ribozymes for cutting ofcleaving guide RNAs. FIG. 26B depicts exemplary constructs without MALAT stabilizing sequences and FIG. 26C depicts exemplarys schematics of constructs with MALAT stabilizing sequences. In addition to hammer head (HH) ribozymes and hepatitis delta virus (HDV) ribozymes, Table 9 lists other exemplary ribozymes with activity in human cells.

[0083] FIG. 27 shows the development of a new AAV construct with conditionally cleavable elements. FIG. 27A shows a schematic of the original DNA construct and the conditionally cleavable features that were re-designed in a second DNA construct. Also shown is the mRNA that results from transcription of the construct to liberate the two guide RNAs targeting DMPK CAG repeat sequences. FIG. 27B shows a schematic of the experimental workflow to test AAV2 encapsidating the original (“AAV6”) and re-designed construct (“AAV67”) in DMPK CAG repeat fibroblast cells. Shown are the durations of transduction with the construct before harvesting cells to analyze the genomic DNA and the polymerase chain reaction (PCR) ofgenomic DNA used to analyze the presence or absence of CAG repeat sequences. FIG. 27C shows an agarose gel of the PCR products of genomic DNA extracted from the cells transduced with 5,000, 10,000 or 25,000 multiplicities of infection (MOI) of the AAV2’s the indicated times. Lanes marked with ‘L’ contain a DNA ladder, lanes marked with ‘E” are empty and lanes marked with C contain the PCR products from genomic DNA of untreated cells. FIG. 27D shows the results of quantifying the ratio of the PCRs products above 1 ,000 base pairs (“High molecular weight / MW”) to the PCR products below 1,000 base pairs (“Low molecular weight / LW”) by densitometry of the agarose gel.

[0084] FIGs. 28A and B shows an alignment of prevalent reads from deep sequencing of PCR products amplified from HEK293 cells transfected with the Tev[Vl 17F+K135R+N140S]- saCas9[D10E] variant and an AAVSl-target repair template guide RNA (cells were not enriched or selected prior to analysis). FIG. 28A shows the summary of the proportion of unmodified, inserted / deleted (“NHEJ”), accurately repaired (“HDR”), imperfectly repaired (“imperfect HDR”) and ambiguous reads. FIG. 28B shows an alignment of the most prevalent sequencing reads from the transfected cells with number of reads (#Reads) and percent of reads indicated. The expected repair product is indicated with an Asterix (*).

[0085] FIGs. 29A - C shows in cell editing using a chimeric nuclease comprising LTevI and erCasl2a at the B2M gene. FIG. 29A shows a schematic of the chimeric nuclease features highlighting the LTevI nuclease domain, linker domain and erCasl2a nuclease domain (“Tev- erCasl2a”). FIG. 29B shows the DNA target site in the B2M gene highlighting the erCasl2 guide RNA binding site (“B2M6 crRNA”), erCasl2 protospacer adjacent motif (“PAM”), the DNA spacer sequence between the erCasl2a site and the LTevI site (“B2M6 Tev Spacer”) and the LTevI target site (“B2M6 LTevI site”). Also shown are the LTevI and erCasl2a sense and anti-sense cut sites. FIG. 29C shows pictures of a gel depicting the editing efficiency by T7 endonuclease I (T7E1) in HEK293 using plasmid DNA encoding Tev-erCasl2a, erCasl2a, TevSaCas9 and saCas9 targeting the B2M gene. Shown is the PCR amplicon from the B2M target site (“Sub”) and T7E1 digested product (“Edited”). The percent editing is indicated below the picture of the gel.DETAILED DESCRIPTION

[0086] Provided herein are, inter alia, compositions and methods for chimeric nucleases and chimeric nuclease systems and nucleic acids encoding chimeric nucleases and chimeric nuclease systems.

[0087] The disclosure is based, in part, on the discovery, that the chimeric nucleases and nuclease systems of the disclosure can be expressed from a single nucleic acid using a single promoter in a cell. The chimeric nuclease systems of the disclosure and the nucleic acids encoding the chimeric nuclease systems can be used to edit the genome of a cell. The nucleic acid encoding the chimeric nuclease systems of the disclosure can be transcribed in the cell into a single mRNA comprising the components of the chimeric nuclease system. The mRNA can be further processed in the cell into its individual components, for example a Cas9 nuclease, a guide RNA and a donor polynucleotide. The chimeric nuclease systems of the disclosure can comprise one or more chimeric nucleases and one or more guide polynucleotides. Additionally, the chimeric nuclease systems can comprise a donor polynucleotide. The nucleic acid encoding the chimeric nuclease system of the disclosure can comprise a single promoter sequence, a sequence encoding the chimeric nuclease, and a sequence encoding the guide polynucleotide.

[0088] Additionally, the nucleic acid can comprise nucleic acid sequences that aid in processing of the single mRNA into smaller fragments and / or RNA stabilizing sequences that obviate the need for a stabilizing Poly A. The transcribed mRNA sequence can comprise a nucleic acid sequence encoding for a chimeric nuclease, one or more guide RNAs, and one or more donor polynucleotides, additional sequences such an RNA stabilizing sequences, tRNAs, ribozymes, and ribozyme cut sites.

[0089] The disclosure is also based, in part, on the discovery that chimeric nucleases and chimeric nuclease systems efficiently and precisely replace sequences in the genome of a cell using RNA-templated DNA repair in mammalian cells in the absence of an exogenous reverse transcriptase.

[0090] The advantage of the nucleic acids of the disclosure is that they are small compared to a multipromoter nucleic acid construct and can be packaged into a viral genome, for example an AAV genome. Additional advantages of the nucleic acid designs provided herein are that the nucleic acid can be designed to ensure precise 5’ and 3’ ends of the nucleic acid components after transcription and further processing. Another advantage of the chimeric nucleases andchimeric nuclease systems and nucleic acids of the disclosure is that the system can be used for RNA-mediated repair without delivery of a exogenous reverse transcriptase to the cell. Due to the small size of the nucleic acids encoding the chimeric nucleases and chimeric nuclease systems of the disclosure, the nucleic acids can be packaged into viral vectors for efficient cellular delivery of these all-in-one editing systems.

[0091] Before the embodiments of the disclosure are described, it is to be understood that such embodiments are provided by way of example only, and that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the invention. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention.

[0092] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Various scientific dictionaries that include the terms included herein are well known and available to those in the art. Although any methods and materials similar or equivalent to those described herein find use in the practice or testing of the disclosure, some preferred methods and materials are described. Accordingly, the terms defined immediately below are more fully described by reference to the specification as a whole.Definitions

[0093] All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0094] The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated cases, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0095] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0096] In this application, the use of “or” means “and / or” unless stated otherwise. The terms “and / or” and “any combination thereof’ and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof’ can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C”. The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

[0097] Furthermore, the use of the term “including” as well as other forms, such as “include”, “includes” and “included”, is not limiting.

[0098] Reference in the specification to “some embodiments”, “an embodiment”, “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures.

[0099] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0100] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. In another example, the amount “about 10” includes 10 and any amounts from 9 to 11. In yet another example, the term “about” in relation to a reference numerical value canalso include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. Alternatively, particularly with respect to biological systems or processes, the term “about” can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0101] The term “at least” followed by a number is used herein to denote the start of a range beginning with that number (which may be a range having an upper limit or no upper limit, depending on the variable being defined). For example, “at least 1” means 1 or more than 1.

[0102] The term “at most” followed by a number is used herein to denote the end of a range ending with that number (which may be a range having 1 or 0 as its lower limit, or a range having no lower limit, depending upon the variable being defined). For example, “at most 4” means 4 or less than 4, and “at most 40%” means 40% or less than 40%. When, in this specification, a range is given as “(a first number) to (a second number)” or “(a first number)-(a second number)” this means a range whose lower limit is the first number and whose upper limit is the second number. For example, 25 to 100 mm means a range whose lower limit is 25 mm, and whose upper limit is 100 mm.

[0103] As may be used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid oligomer,” “oligonucleotide,” “nucleic acid sequence,” “nucleic acid fragment” and “polynucleotide” are used interchangeably and are intended to include, but are not limited to, a polymeric form of nucleotides covalently linked together that may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs, derivatives or modifications thereof. Different polynucleotides may have different three-dimensional structures, and may perform various functions, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, and a primer.Polynucleotides useful in the methods of the disclosure may comprise natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or a combination of such sequences.

[0104] The term “identical” or percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence. Methods of alignment of sequences for comparison are well known in the art. Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity is determined by dividing the number of matches in the alignment by the length of the reference sequence, followed by multiplying the resulting value by 100. For example, a peptide sequence that has 1166 matches when aligned with a test sequence having 1554 amino acids is 75.0 percent identical to the test sequence (1166 -1554 * 100=75.0). As the terms are used herein, gaps in the alignment do not decrease the percent sequence identity. Unless otherwise specified, optimal alignment of sequences for comparison is conducted by the global alignment algorithm of Needleman and Wunsch, Mol. Biol. 48:443 (1970) as implemented by EMBOSS Needle (on the World Wide Web at ebi.ac.uk / Tools / psa / emboss_needle / ) (Madeira et al. Nucleic Acids Res. 50(Wl):W276-W279 (2022)). In embodiments, other alignment methods may be used, including without limitation those described in Devereux, et al., Nucleic Acids Res. 12:387-95 (1984) ; Altschul et al., J. Mol. Biol. 215:403-10 (1990) (BLAST); Carrillo and Lipman Siam J. Appl. Math. 48(5) (1988); Computational Molecular Biology (Lesk, AM, ed., 1989); Biocomputing Informatics and Genome Projects, (Smith, DW, ed., 1993); Computer Analysis of Sequence Data, Part I, (Griffin and Griffin, eds., 1994); Sequence Analysis in Molecular Biology (von Heijne, 2012); Sequence Analysis Primer (Gribskov and Devereux, J., eds. 1993). Sequence identity is calculated using the implementation of the Needleman-Wunsch algorithm provided by the National Library of Medicine (on the World Wide Web at blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=GlobalAln)

[0105] For example, sequence identity can be determined by standard methods that are commonly used to compare the similarity of two polypeptide or two polynucleotide sequences. Using a computer program such as EMBOSS Needle or BLAST, two polypeptide or two polynucleotide sequences are aligned for optimal matching of their respective residues (either along the full length of one or both sequences, or along a pre-determined portion of one or both sequences). The programs provide a default opening penalty and a default gap penalty, and ascoring matrix such as PAM 250 (a standard scoring matrix; see Dayhoff et al., in Atlas of Protein Sequence and Structure, vol. 5, supp. 3 (1978)) that can be used in conjunction with the computer program.

[0106] By "binding" is meant attaching by a covalent bond or a non-covalent bond. Non- covalent bonds include those formed by van der Waals forces, hydrogen bonds, ionic bonds, entrapment or physical encapsulation, absorption, adsorption, and / or other intermolecular forces. Binding can be effectuated by any useful means, such as by enzymatic binding (e.g., enzymatic ligation) or by chemical binding e.g., chemical ligation).

[0107] The term “expression,” as used herein, generally refers to the process by which a nucleic acid sequence or a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcript) or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0108] As used herein, “operably linked,” “operable linkage,” “operatively linked”, or grammatical equivalents thereof generally refer to juxtaposition of genetic elements, e.g., a promoter, an enhancer, a polyadenylation sequence, etc., wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a regulatory element, which may comprise promoter or enhancer sequences, is operatively linked to a coding region if the regulatory element helps initiate transcription of the coding sequence. There may be intervening residues between the regulatory element and coding region so long as this functional relationship is maintained.

[0109] A “vector” as used herein, generally refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide and which may be used to mediate delivery of the polynucleotide to a cell. Examples of vectors include plasmids, viral vectors, liposomes, and other gene delivery vehicles. The vector generally comprises genetic elements, e.g., regulatory elements, operatively linked to a gene to facilitate expression of the gene in a target.

[0110] As used herein, “an expression cassette” and “a nucleic acid cassette” are used interchangeably generally to refer to a combination of nucleic acid sequences or elements that are expressed together or are operably linked for expression. In some cases, an expressioncassette refers to the combination of regulatory elements and a gene or genes to which they are operably linked for expression.

[0111] As used herein, an “engineered” object generally indicates that the object has been modified by human intervention. According to non-limiting examples: a nucleic acid may be modified by changing its sequence to a sequence that does not occur in nature; a nucleic acid may be modified by ligating it to a nucleic acid that it does not associate with in nature such that the ligated product possesses a function not present in the original nucleic acid; an engineered nucleic acid may synthesized in vitro with a sequence that does not exist in nature; a protein may be modified by changing its amino acid sequence to a sequence that does not exist in nature; an engineered protein may acquire a new function or property. An “engineered” system comprises at least one engineered component.

[0112] Included in the current disclosure are variants of any of the endonucleases described herein with one or more conservative amino acid substitutions. Such conservative substitutions can be made in the amino acid sequence of a polypeptide without disrupting the three- dimensional structure or function of the polypeptide. Conservative substitutions can be accomplished by substituting amino acids with similar hydrophobicity, polarity, and R chain length for one another. Additionally, or alternatively, by comparing aligned sequences of homologous proteins from different species, conservative substitutions can be identified by locating amino acid residues that have been mutated between species (e.g., non-conserved residues) without altering the basic functions of the encoded proteins. Such conservatively substituted variants may include variants with at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, 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% identity to any one of the endonuclease protein sequences described herein. In some embodiments, such conservatively substituted variants are functional variants. Such functional variants can encompass sequences with substitutions such that the activity of one or more critical active site residues or guide RNA binding residues of the endonuclease are not disrupted.

[0113] Conservative substitution tables providing functionally similar amino acids are available from a variety of references (see, for e.g., Creighton, Proteins: Structures and Molecular Properties (W H Freeman & Co.; 2nd edition (December 1993)). The following eight groups each contain amino acids that are conservative substitutions for one another:1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);7) Serine (S), Threonine (T); and8) Cysteine (C), Methionine (M)

[0114] An amino acid or nucleotide base "position" is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-end). Due to deletions, insertions, truncations, fusions, and the like that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N- terminus will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where a variant has a deletion relative to an aligned reference sequence, there will be no amino acid in the variant that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to a numbered amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0115] The terms "numbered with reference to" or "corresponding to," when used in the context of the numbering of a given amino acid or polynucleotide sequence, refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence.

[0116] The terms “target site” or “target sites”, as used herein, refer to, with respect to the present invention in particular, a position in a gene that when targeted with a nuclease will bebound and / or cleaved by the nuclease. A target site can encompass a multiplicity of nucleotides, such as the cleavage site of an I-TevI or CRISPR / Cas nuclease, or the DNA binding site of an I- TevI nuclease or CRISPR / Cas guide RNA. A target site can be in a gene or intergenic region in any of various cell types and organisms.

[0117] The terms “target” or “targets” or “to target” or “targeting”, as used herein, refer to, with respect to the inventions of the instant application, aiming or directing a nuclease to a particular, selected DNA sequence using, for example, a selected or engineered DNA binding domain or guide RNA.

[0118] The term “viral vector”, as used herein, refers to tools commonly used to deliver genetic material into cells. This process can be performed inside a living organism (in vivo) or in cell culture (in vitro). Examples of viral vectors include AAV vectors, lentiviral vectors, and adenoviral vectors.

[0119] The term "simultaneously", as used herein, refers to the administration of CRISPR / Cas9 complex with multiple gRNA at the same time or substantially the same time. It will be understood that certain procedures can be executed in steps that occur sequentially but are spaced apart by small intervals. For example, electroporation of cells followed by administration of a donor DNA within about an hour.

[0120] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub combination was individually and explicitly disclosed herein.Chimeric Nucleases and Chimeric Nuclease Systems

[0121] Provided herein are, inter alia, chimeric nucleases and chimeric nuclease systems comprising two or more nucleases and a guide RNA. The chimeric nucleases and chimericnuclease systems can be used to introduce modifications in the genome of a cell, for example for insertions, deletions, or mutations.

[0122] In some embodiments, the chimeric nuclease system comprises a CRISPR Cas nuclease domain, a guide RNA, and a GIY-YIG nuclease domain. In some embodiments, the chimeric nuclease comprises a CRISPR Cas nuclease domain and a GIY -YIG nuclease domain. In some embodiments, the chimeric nuclease comprises a fusion protein of a CRISPR Cas nuclease domain and a GIY-YIG nuclease domain. In some embodiments, the CRISPR Cas nuclease domain is located N-terminal or C-terminal (e.g., N-terminal) of the GIY-YIG nuclease domain. In some embodiments, the chimeric nuclease comprises a linker between the CRISPR Cas nuclease domain and a GIY-YIG nuclease domain.CRISPR Cas Nucleases

[0123] CRISPR Cas nucleases are programmable RNA-directed nucleases that have been described to function as an adaptive immune system in microbes. Nuclease targeting of a particular target nucleic acid sequence generally requires both (i) complementary hybridization between the first 6-8 nucleic acids of the target (the target seed) and the crRNA guide; and (ii) the presence of a protospacer-adjacent motif (PAM) sequence within a defined vicinity of the target seed. CRISPR Cas systems are commonly organized into 2 classes, 5 types and 16 subtypes based on shared functional characteristics and evolutionary similarity. Structure, nomenclature and classification of CRISPR systems are reviewed in Makarova st al, Evolution and classification of the CRISPR Cas systems. Nature Reviews Microbiology. 2011 June; 9(6): 467-477.

[0124] Class I CRISPR Cas systems have large, multisubunit effector complexes, and comprise Types I, III, and IV. Type I CRISPR systems comprise a multi-protein complex called Cascade (CRISPR-associated complex for antiviral defense) comprised of subunits CasA, B, C, D and E and a crRNA. The Cascade-crRNA complex recognizes the target nucleic acid through hybridization of the target nucleic acid with crRNA. The bound nucleoprotein complex recruits the Cas3 helicase / nuclease to facilitate cleavage of target nucleic acid. Type III CRISPR systems include the RAMP superfamily of endoribonucleases (e.g., Cas6) that cleave the pre- crRNA array with the help of one or more CRISPR polymerase-like proteins. Type IV CRISPR- Cas systems possess an effector complex that consists of a highly reduced large subunit nuclease (csfl), two genes for RAMP proteins of the Cas5 (csf3) and Cas7 (csf2) groups, and, in somecases, a gene for a predicted small subunit; such systems are commonly found on endogenous plasmids.

[0125] Class 2 CRISPR Cas systems generally have single-polypeptide multidomain nuclease effectors, and comprise Types II, V, and VI. Type II CRISPR Cas systems typically comprise a Cas9 nuclease, a crRNA and a trans-activating CRISPR RNA (tracrRNA). The tracrRNA hybridizes to the crRNA repeat. The tracrRNA / crRNA complex can associate with the nuclease, e.g., a Cas9. The crRNA-tracrRNA-Cas9 complex recognizes the target nucleic acid through hybridization of the target nucleic acid with crRNA. Hybridization of the crRNA to the target nucleic acid activates the Cas9 nuclease for target nucleic acid cleavage. Type V CRISPR systems comprise a different set of Cas-like genes, including Cas 12 nucleases such as Cas 12a (Cpfl), Casl2b, Casl2c, Casl2d, Casl2e, Casl2g, Casl2h, Casl2i, Casl2j, Casl2k, and Casl4. Type VI CRISPR Cas systems are RNA-guided RNA endonucleases.

[0126] In some embodiments, the chimeric nuclease comprises a Cas9 CRISPR Cas nuclease. Cas9 is a programmable Class 2 Type II CRISPR Cas nuclease that forms a complex with a cognate crRNA and a tracrRNA. In some embodiments, the chimeric nuclease system comprises a Cas9 nuclease, a crRNA and a tracrRNA. In some embodiments, the chimeric nuclease comprises a nuclear localization signal (NLS). In some embodiments, the chimeric nuclease comprises two C-terminal nucleoplasmin NLSs separated by a human influenza hemagglutinin (HA) sequence, or two SV40 NLSs separated by a HA sequence or a HA sequence followed by two SV40 NLSs.

[0127] In some embodiments, the Cas9 nuclease comprises a conservative amino acid substitution. In some embodiments, the Cas9 nuclease comprises a mutation in the catalytic domain. In some embodiments, the Cas9 nuclease comprises a mutation that results in nickase activity (nCas9). In some embodiments, the Cas9 nuclease comprises a mutation that results in a catalytically dead Cas9 (dCas9, deactivated Cas9). In some embodiments, the Cas nuclease is a Cas9 domain.

[0128] In some embodiments, the Cas9 nuclease can be from or is derived from: Staphylococcus aureus, Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromo genes, Streptomyces viridochromogenes, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillusdelbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus desulforudis, Clostridium botulinum, Clostridium difficile, Fine goldia magna, Natranaerobius thermophilus, Pelotomaculum the rmopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, or Acaryochloris marina.

[0129] In some embodiments, the Cas9 nuclease is selected from a Cas9 nuclease from F. novicida, T. denticola, Campylobacter jejuni, Alicyclobacillus acidoterrestris, Prevotella and Francisella Acidaminococcus sp. BV3L6, Eubacterium rectale, SpCasl2fl (497 aa), AsCasl2fl (422 aa), S. lugdunensis (Siu) , S. hyicus (Shy), S. microti (Smi), S. pasteuri (Spa), Seawater microbial communities, Freshwater microbial communities from Lake Mendota, Planctomycetes, Agricultural soil microbial communities from Utah to study Nitrogen management - Steer compost 2015, Thermophilic microbial communities from the Joint Bioenergy Institute, California, USA of rice / straw / compost enrichment - eDNA_2, Eggerthella_sp._YY7918, Finegoldia_magna_ATCC_29328, Lactobacillus_rhamnosus_LOCK900, Nitratifractor salsuginis, Streptococcus_gordonii_str._Challis_substr._CHl, Tissierellia bacterium KA00581, Turicibacter sp., or a engineered SpyCas9.

[0130] In some embodiments, the Cas9 nuclease is derived from Staphylococcus aureus, Streptococcus pyogenes, Neisseria meningitidis, Campylobacter jejuni, Streptococcus pasteurianus, Clostridium cellulolyticum, or Geobacillus thermodenitrificans Tl.

[0131] In some embodiments, the Cas9 domain is derived from Staphylocuccus aureus (SaCas9). In some embodiments, the Cas9 domain is derived from Streptococcus pyogenes (spCas9). In some embodiments, the Cas9 domain is derived from Neisseria meningitidis (NmCas9). In some embodiments, the Cas9 domain is derived from Campylobacter jejuni (CjCas9). In some embodiments, the Cas9 domain is derived from Streptococcus pasteurianus (SpCas9), In some embodiments, the Cas9 domain is derived from Clostridiumcellulolyticum(CcCas9). In some embodiments, the Cas9 domain is derived from Geobacillus thermodenitrificans T1 (GtCas9).

[0132] Exemplary Cas9 nucleases and cognate PAM sequences are shown in Table A.Table A

[0133] In some embodiments, the Staphylococcus aureus Cas9 domain comprises a mutation or amino acid substitution corresponding to a position selected from any one of DIO, H557, N580, H840, DI 135, R1335, T1337, T267, L325, V327, D333, A336, 1341, E345, D348, K352, S360, T368, N369, N371, S372, E373, K386, N393, H408, N410, 1414, A415, T438, Y467, N471, D485, M489, E506, R409, T510, N515, Y518, A539, F550, N551, S596, T602, A611, 1617, T620, R650, G654, N667, R685, K695, 1706, K722, A723, K724, M731, F732, K735, S739, P741, E742, E746, Q747, 1754, T755, H757, K760, H761, P778, E781, 1783, N784, D785, T786L, L787, Y788, K792, D794, T798, L799, V801, N803, L804, N805, G806, D813, K814, L818, 1819, S822, E824, L841, G847, D848, Y857, V875, 1876, N884, A888, L890, D894, D895, P897, V903, G920, F924, N929, E936, N937, V941, N942, S943, C945, E947, K951, L952, S956, N957, Q958, A959, N974, G975, V983, N984, N985, D986, 1991, V993, M995, 1996, T999, Y1000, R1001, E1002, L1004, E1005, N1006, M1007, D1009, K1010, R1011, P1012, P1013, 11015, 11016, A1020, S1021, Q1024, K1027, E1039, H1045, 10148, K1050 or a combination thereof.

[0134] In some embodiments, the Staphylococcus aureus Cas9 domain comprises a mutation or substitution corresponding to any one or more of D10A, D10E, H557A, N580A, H840A, D1135E, R1335Q, T1337R, T267A, L325F, V327I, D333G, A336S, I341L, E345D, D348N, K352E, S360A, T368A, N369E, N371E, S372P, E373K, K386T, N393R, H408N, N410S, I414M, A415T, T438S, Y467F, N471K, D485E, M489F, E506K, R409K, T510E, N515K, Y518F, A539P, F550Y, N551H, S596A, T602I, A611S, I617V, T620K, R650K,G654E, N667D, R685K, K695Q, I706V, K722T, A723T, K724N, M731T, F732V, K735Q, S739N, P741L, E742G, E746D, Q747D, I754D, T755I, H757R, K760Q, H761S, P778I, E781K, I783V, N784D, D785E, T786L, L787V, Y788H, K792E, D794T, T798R, L799I, V801I, N803S, L804I, N805K, G806N, D813G, K814E, L818I, I819F, S822P, E824G, L841T, G847S, D848N, Y857H, V875I, I876V, N884K, A888V, L890R, D894G, D895H, P897L, V903I, G920D, F924L, N929Y, E936D, N937G, V941I, N942D, S943L, C945A, E947K, K951R, L952Q, S956N, N957E, Q958K, A959S, N974D, G975K, V983A, N984S, N985D, D986G, 199 IV, V993L, M995F, I996V, T999N, Y1000K, R1001E, E1002D, L1004I, E1005K, N1006M, M1007N, D1009L,K1010S, R1011T, P1012S, P1O13F, I1O15L, I1016R, A1020G, S1021K, Q1024K, K1027S, E1039K, H1045K, I0148M, K1050M or a combination thereof.

[0135] In some embodiments, the Cas9 comprises a mutation at a residue corresponding to amino acid position 10 of SEQ ID NO: 36 or SEQ ID NO: 86. In some embodiments, the Cas9 comprises a mutation at a residue corresponding to amino acid position 557 of SEQ ID NO: 36. In some embodiments, the Cas9 comprises a mutation at a residue corresponding to amino acid position 580 of SEQ ID NO: 36. In some embodiments, the Cas9 comprises a mutation at a residue corresponding to amino acid position 650 of SEQ ID NO: 36. In some embodiments, the Cas9 comprises a mutation at a residue corresponding to amino acid position 840 of SEQ ID NO: 86. In some embodiments, the Cas9 comprises a mutation at a residue corresponding to amino acid position 1135 of SEQ ID NO: 86. In some embodiments, the Cas9 comprises a mutation at a residue corresponding to amino acid position 1335 of SEQ ID NO: 86. In some embodiments, the Cas9 comprises a mutation at a residue corresponding to amino acid position 1337 of SEQ ID NO: 86.

[0136] In some embodiments, the Cas9 comprises a mutation at a residue corresponding to D10 of SEQ ID NO: 36 or SEQ ID NO: 86. In some embodiments, the Cas9 comprises a D10E mutation in SEQ ID NO: 36 or SEQ ID NO: 86. In some embodiments, the Cas9 comprises a D10A mutation in SEQ ID NO: 36 or SEQ ID NO: 86. In some embodiments, the Cas9 comprises a H557A mutation in SEQ ID NO: 36. In some embodiments, the Cas9 comprises a N580A mutation in SEQ ID NO: 36. . In some embodiments, the Cas9 comprises a R650K mutation in SEQ ID NO: 36. In some embodiments, the Cas9 comprises a H840A mutation in SEQ ID NO: 86. In some embodiments, the Cas9 comprises a DI 135E mutation in SEQ ID NO: 86. In some embodiments, the Cas9 comprises a R1335Q mutation of SEQ ID NO:86. In some embodiments, the Cas9 comprises a T1337R mutation in SEQ ID NO:86.

[0137] In some embodiments, the Cas9 comprises a D10E mutation and a H557A mutation in SEQ ID NO: 36. In some embodiments, the Cas9 comprises a D10A mutation and a H557A mutation in SEQ ID NO: 36. In some embodiments, the Cas9 comprises a D10E mutation and a N580A mutation in SEQ ID NO: 36. In some embodiments, the Cas9 comprises a D10A mutation and a N580A mutation in SEQ ID NO: 36. In some embodiments, the Cas9 comprises a D10E mutation and a H840A mutation in SEQ ID NO: 86. In some embodiments, the Cas9 comprises a D10A mutation and a H840A mutation in SEQ ID NO: 86. In some embodiments,the Cas9 comprises a D10E mutation and a DI 135E mutation in SEQ ID NO:86. In some embodiments, the Cas9 comprises a D10A mutation and a DI 135E mutation in SEQ ID NO: 86. In some embodiments, the Cas9 comprises a D10E mutation and a R1335Q mutation in SEQ ID NO: 86. In some embodiments, the Cas9 comprises a D10A mutation and a R1335Q mutation in SEQ ID NO: 86. In some embodiments, the Cas9 comprises a D10E mutation and a T1337R mutation in SEQ ID NO: 86. In some embodiments, the Cas9 comprises a D10A mutation and a T1337R mutation in SEQ ID NO: 86. In some embodiments, the Cas9 comprises a D10E, DI 135E, R1335Q, and a T1337R mutation in SEQ ID NO: 86. In some embodiments, the Cas9 comprises a D10E, H840A, DI 135E, R1335Q, and a T1337R mutation in SEQ ID NO: 86.

[0138] In some embodiments, the Cas9 nuclease is a Staphylococcus aureus (SaCas9) nuclease. In some embodiments, the SaCas9 comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 36. In some embodiments, the SaCas9 comprises an amino acid sequence that is 90% identical to SEQ ID NO: 36. In some embodiments, the SaCas9 comprises an amino acid sequence that is 91% identical to SEQ ID NO: 36. In some embodiments, the SaCas9 comprises an amino acid sequence that is 92% identical to SEQ ID NO: 36. In some embodiments, the SaCas9 comprises an amino acid sequence that is 93% identical to SEQ ID NO: 36. In some embodiments, the SaCas9 comprises an amino acid sequence that is 94% identical to SEQ ID NO: 36. In some embodiments, the SaCas9 comprises an amino acid sequence that is 95% identical to SEQ ID NO: 36. In some embodiments, the SaCas9 comprises an amino acid sequence that is 96% identical to SEQ ID NO: 36. In some embodiments, the SaCas9 comprises an amino acid sequence that is 97% identical to SEQ ID NO: 36. In some embodiments, the SaCas9 comprises an amino acid sequence that is 98% identical to SEQ ID NO: 36. In some embodiments, the SaCas9 comprises an amino acid sequence that is 99% identical to SEQ ID NO: 36. In some embodiments, the SaCas9 comprises an amino acid sequence that is 100% identical to SEQ ID NO: 36.

[0139] In some embodiments, the SaCas9 comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 37. In some embodiments, the SaCas9 comprises an amino acid sequence that is 90% identical to SEQ ID NO: 37. In some embodiments, the SaCas9 comprises an amino acid sequence that is 91% identical to SEQ ID NO: 37. In some embodiments, the SaCas9 comprises an amino acidsequence that is 92% identical to SEQ ID NO: 37. In some embodiments, the SaCas9 comprises an amino acid sequence that is 93% identical to SEQ ID NO: 37. In some embodiments, the SaCas9 comprises an amino acid sequence that is 94% identical to SEQ ID NO: 37. In some embodiments, the SaCas9 comprises an amino acid sequence that is 95% identical to SEQ ID NO: 37. In some embodiments, the SaCas9 comprises an amino acid sequence that is 96% identical to SEQ ID NO: 37. In some embodiments, the SaCas9 comprises an amino acid sequence that is 97% identical to SEQ ID NO: 37. In some embodiments, the SaCas9 comprises an amino acid sequence that is 98% identical to SEQ ID NO: 37. In some embodiments, the SaCas9 comprises an amino acid sequence that is 99% identical to SEQ ID NO: 37. In some embodiments, the SaCas9 comprises an amino acid sequence that is 100% identical to SEQ ID NO: 37.

[0140] In some embodiments, the SaCas9 comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 38.

[0141] In some embodiments, the SaCas9 comprises an amino acid sequence that is 90% identical to SEQ ID NO: 38. In some embodiments, the SaCas9 comprises an amino acid sequence that is 91% identical to SEQ ID NO: 38. In some embodiments, the SaCas9 comprises an amino acid sequence that is 92% identical to SEQ ID NO: 38. In some embodiments, the SaCas9 comprises an amino acid sequence that is 93% identical to SEQ ID NO: 38. In some embodiments, the SaCas9 comprises an amino acid sequence that is 94% identical to SEQ ID NO: 38. In some embodiments, the SaCas9 comprises an amino acid sequence that is 95% identical to SEQ ID NO: 38. In some embodiments, the SaCas9 comprises an amino acid sequence that is 96% identical to SEQ ID NO: 38. In some embodiments, the SaCas9 comprises an amino acid sequence that is 97% identical to SEQ ID NO: 38. In some embodiments, the SaCas9 comprises an amino acid sequence that is 98% identical to SEQ ID NO: 38. In some embodiments, the SaCas9 comprises an amino acid sequence that is 99% identical to SEQ ID NO: 38. In some embodiments, the SaCas9 comprises an amino acid sequence that is 100% identical to SEQ ID NO: 38.

[0142] In some embodiments, the SaCas9 comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 39. In some embodiments, the SaCas9 comprises an amino acid sequence that is 90% identical to SEQ ID NO: 39. In some embodiments, the SaCas9 comprises an amino acid sequence that is91% identical to SEQ ID NO: 39. In some embodiments, the SaCas9 comprises an amino acid sequence that is 92% identical to SEQ ID NO: 39. In some embodiments, the SaCas9 comprises an amino acid sequence that is 93% identical to SEQ ID NO: 39. In some embodiments, the SaCas9 comprises an amino acid sequence that is 94% identical to SEQ ID NO: 39. In some embodiments, the SaCas9 comprises an amino acid sequence that is 95% identical to SEQ ID NO: 39. In some embodiments, the SaCas9 comprises an amino acid sequence that is 96% identical to SEQ ID NO: 39. In some embodiments, the SaCas9 comprises an amino acid sequence that is 97% identical to SEQ ID NO: 39. In some embodiments, the SaCas9 comprises an amino acid sequence that is 98% identical to SEQ ID NO: 39. In some embodiments, the SaCas9 comprises an amino acid sequence that is 99% identical to SEQ ID NO: 39. In some embodiments, the SaCas9 comprises an amino acid sequence that is 100% identical to SEQ ID NO: 39.

[0143] In some embodiments, the SaCas9 comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 40. In some embodiments, the SaCas9 comprises an amino acid sequence that is 90% identical to SEQ ID NO: 40. In some embodiments, the SaCas9 comprises an amino acid sequence that is 91% identical to SEQ ID NO: 40. In some embodiments, the SaCas9 comprises an amino acid sequence that is 92% identical to SEQ ID NO: 40. In some embodiments, the SaCas9 comprises an amino acid sequence that is 93% identical to SEQ ID NO: 40. In some embodiments, the SaCas9 comprises an amino acid sequence that is 94% identical to SEQ ID NO: 40. In some embodiments, the SaCas9 comprises an amino acid sequence that is 95% identical to SEQ ID NO: 40. In some embodiments, the SaCas9 comprises an amino acid sequence that is 96% identical to SEQ ID NO: 40. In some embodiments, the SaCas9 comprises an amino acid sequence that is 97% identical to SEQ ID NO: 40. In some embodiments, the SaCas9 comprises an amino acid sequence that is 98% identical to SEQ ID NO: 40. In some embodiments, the SaCas9 comprises an amino acid sequence that is 99% identical to SEQ ID NO: 40. In some embodiments, the SaCas9 comprises an amino acid sequence that is 100% identical to SEQ ID NO: 40.

[0144] In some embodiments, the SaCas9 comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 46. In some embodiments, the SaCas9 comprises an amino acid sequence that is 90% identical toSEQ ID NO: 46. In some embodiments, the SaCas9 comprises an amino acid sequence that is 91% identical to SEQ ID NO: 46. In some embodiments, the SaCas9 comprises an amino acid sequence that is 92% identical to SEQ ID NO: 46. In some embodiments, the SaCas9 comprises an amino acid sequence that is 93% identical to SEQ ID NO: 46. In some embodiments, the SaCas9 comprises an amino acid sequence that is 94% identical to SEQ ID NO: 46. In some embodiments, the SaCas9 comprises an amino acid sequence that is 95% identical to SEQ ID NO: 46. In some embodiments, the SaCas9 comprises an amino acid sequence that is 96% identical to SEQ ID NO: 46. In some embodiments, the SaCas9 comprises an amino acid sequence that is 97% identical to SEQ ID NO: 46. In some embodiments, the SaCas9 comprises an amino acid sequence that is 98% identical to SEQ ID NO: 46. In some embodiments, the SaCas9 comprises an amino acid sequence that is 99% identical to SEQ ID NO: 46. In some embodiments, the SaCas9 comprises an amino acid sequence that is 100% identical to SEQ ID NO: 46.

[0145] In some embodiments, the SaCas9 comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 47. In some embodiments, the SaCas9 comprises an amino acid sequence that is 90% identical to SEQ ID NO: 47. In some embodiments, the SaCas9 comprises an amino acid sequence that is 91% identical to SEQ ID NO: 47. In some embodiments, the SaCas9 comprises an amino acid sequence that is 92% identical to SEQ ID NO: 47. In some embodiments, the SaCas9 comprises an amino acid sequence that is 93% identical to SEQ ID NO: 47. In some embodiments, the SaCas9 comprises an amino acid sequence that is 94% identical to SEQ ID NO: 47. In some embodiments, the SaCas9 comprises an amino acid sequence that is 95% identical to SEQ ID NO: 47. In some embodiments, the SaCas9 comprises an amino acid sequence that is 96% identical to SEQ ID NO: 47. In some embodiments, the SaCas9 comprises an amino acid sequence that is 97% identical to SEQ ID NO: 47. In some embodiments, the SaCas9 comprises an amino acid sequence that is 98% identical to SEQ ID NO: 47. In some embodiments, the SaCas9 comprises an amino acid sequence that is 99% identical to SEQ ID NO: 47. In some embodiments, the SaCas9 comprises an amino acid sequence that is 100% identical to SEQ ID NO: 47.

[0146] In some embodiments, the SaCas9 comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 48.In some embodiments, the SaCas9 comprises an amino acid sequence that is 90% identical to SEQ ID NO: 48. In some embodiments, the SaCas9 comprises an amino acid sequence that is 91% identical to SEQ ID NO: 48. In some embodiments, the SaCas9 comprises an amino acid sequence that is 92% identical to SEQ ID NO: 48. In some embodiments, the SaCas9 comprises an amino acid sequence that is 93% identical to SEQ ID NO: 48. In some embodiments, the SaCas9 comprises an amino acid sequence that is 94% identical to SEQ ID NO: 48. In some embodiments, the SaCas9 comprises an amino acid sequence that is 95% identical to SEQ ID NO: 48. In some embodiments, the SaCas9 comprises an amino acid sequence that is 96% identical to SEQ ID NO: 48. In some embodiments, the SaCas9 comprises an amino acid sequence that is 97% identical to SEQ ID NO: 48. In some embodiments, the SaCas9 comprises an amino acid sequence that is 98% identical to SEQ ID NO: 48. In some embodiments, the SaCas9 comprises an amino acid sequence that is 99% identical to SEQ ID NO: 48. In some embodiments, the SaCas9 comprises an amino acid sequence that is 100% identical to SEQ ID NO: 48.

[0147] In some embodiments, the SaCas9 comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 49. In some embodiments, the SaCas9 comprises an amino acid sequence that is 90% identical to SEQ ID NO: 49. In some embodiments, the SaCas9 comprises an amino acid sequence that is 91% identical to SEQ ID NO: 49. In some embodiments, the SaCas9 comprises an amino acid sequence that is 92% identical to SEQ ID NO: 49. In some embodiments, the SaCas9 comprises an amino acid sequence that is 93% identical to SEQ ID NO: 49. In some embodiments, the SaCas9 comprises an amino acid sequence that is 94% identical to SEQ ID NO: 49. In some embodiments, the SaCas9 comprises an amino acid sequence that is 95% identical to SEQ ID NO: 49. In some embodiments, the SaCas9 comprises an amino acid sequence that is 96% identical to SEQ ID NO: 49. In some embodiments, the SaCas9 comprises an amino acid sequence that is 97% identical to SEQ ID NO: 49. In some embodiments, the SaCas9 comprises an amino acid sequence that is 98% identical to SEQ ID NO: 49. In some embodiments, the SaCas9 comprises an amino acid sequence that is 99% identical to SEQ ID NO: 49. In some embodiments, the SaCas9 comprises an amino acid sequence that is 100% identical to SEQ ID NO: 49.

[0148] In some embodiments, the SaCas9 comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 50. In some embodiments, the SaCas9 comprises an amino acid sequence that is 90% identical to SEQ ID NO: 50. In some embodiments, the SaCas9 comprises an amino acid sequence that is 91% identical to SEQ ID NO: 50. In some embodiments, the SaCas9 comprises an amino acid sequence that is 92% identical to SEQ ID NO: 50. In some embodiments, the SaCas9 comprises an amino acid sequence that is 93% identical to SEQ ID NO: 50. In some embodiments, the SaCas9 comprises an amino acid sequence that is 94% identical to SEQ ID NO: 50. In some embodiments, the SaCas9 comprises an amino acid sequence that is 95% identical to SEQ ID NO: 50. In some embodiments, the SaCas9 comprises an amino acid sequence that is 96% identical to SEQ ID NO: 50. In some embodiments, the SaCas9 comprises an amino acid sequence that is 97% identical to SEQ ID NO: 50. In some embodiments, the SaCas9 comprises an amino acid sequence that is 98% identical to SEQ ID NO: 50. In some embodiments, the SaCas9 comprises an amino acid sequence that is 99% identical to SEQ ID NO: 50. In some embodiments, the SaCas9 comprises an amino acid sequence that is 100% identical to SEQ ID NO: 50.

[0149] In some embodiments, the SaCas9 comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to the SaCas9 amino acid sequence encoded by SEQ ID NO: 15. In some embodiments, the SaCas9 comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to the SaCas9 amino acid sequence encoded by SEQ ID NO: 16. In some embodiments, the SaCas9 comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to the SaCas9 amino acid sequence encoded by SEQ ID NO: 17. In some embodiments, the SaCas9 comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to the SaCas9 amino acid sequence encoded by SEQ ID NO: 18. In some embodiments, the SaCas9 comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to the SaCas9 amino acid sequence encoded by SEQ ID NO: 19.

[0150] In some embodiments, the Cas9 nuclease is a Streptococcus pyogenes (SpCas9) nuclease. In some embodiments, the Streptococcus pyogenes (SpCas9) nuclease comprises theamino acid sequence :MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFG NLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSD AILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGY AGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGEL HAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPA FLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLL KIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTG WGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKN SRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSD YDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLIT QRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYG DYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKK YGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO 86)

[0151] In some embodiments, the Cas9 nuclease is a Streptococcus pyogenes (SpCas9) nuclease. In some embodiments, the SpCas9 comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 86. In some embodiments, the SpCas9 comprises an amino acid sequence that is 90% identical to SEQ ID NO: 86. In some embodiments, the SpCas9 comprises an amino acid sequence that is 91% identical to SEQ ID NO: 86. In some embodiments, the SpCas9 comprisesan amino acid sequence that is 92% identical to SEQ ID NO: 86. In some embodiments, the SpCas9 comprises an amino acid sequence that is 93% identical to SEQ ID NO: 86. In some embodiments, the SpCas9 comprises an amino acid sequence that is 94% identical to SEQ ID NO: 86. In some embodiments, the SpCas9 comprises an amino acid sequence that is 95% identical to SEQ ID NO: 86. In some embodiments, the SpCas9 comprises an amino acid sequence that is 96% identical to SEQ ID NO: 86. In some embodiments, the SpCas9 comprises an amino acid sequence that is 97% identical to SEQ ID NO: 86. In some embodiments, the SpCas9 comprises an amino acid sequence that is 98% identical to SEQ ID NO: 86. In some embodiments, the SpCas9 comprises an amino acid sequence that is 99% identical to SEQ ID NO: 86. In some embodiments, the SpCas9 comprises an amino acid sequence that is 100% identical to SEQ ID NO: 86.

[0152] ] In some embodiments, the Staphylococcus pyogenes Cas9 domain comprises a mutation corresponding to any one of positions DIO, S29, F32, D39, R40, H41, S42, 148, C80, S87, KI 12, H113, K132, K141, D147, L158, E171, P176, 1186, V189, Q190, Q194, N199, 1201, N202, A203, S204, R205, A210, Q228, L229, G231, S245, T249, S254, D261, T270, N295, T300, D304, V308, N309, 1312, T333, A337, E345, F352, Q354, S355, K356, G366, A367, E396, L398, 1414, D428, F429, D435, K468, S469, E470, T472, E480, A486, S490, F498, K500, N501, N504, K528, V530, E532, G533, A538, T555, K570, F575, D605, E611, R629, E634, T638, R655, R664, R671, K705, E706, Q709, K710, S714, G7115, G717, H721, H723, A725, N726, V743, L747, V748, K772, K775, N776, 1788, G792, K797, Y799, T804, N808, L811, R820, N831, R832, V842, L847, N869, E874, N881, Q885, N888, T893, L911, Y945, D946, L949, E952, A1023, Y1036, G1067, G1077, R1078, N1093, R1114, Ni l 15, DI 117, Al 121, D1125, P1128, K1129, V1146, SI 154, SI 159, L1164, S1172, N1177, P1178, 11179, D1180, K1211, M1213, G1218, N1234, E1243, K1244, E1253, E1260, K1263, H1264, E1271, Q1272, E1275, V1290, L1291, S1292, A1293, N1295, H1297, R1298, D1299, K1300, R1303, E1307, N1308, 11309, 11310, H1311, L1312, L1315, T1316, N1317, Y1326, D1328, V1342, A1345, 11360, S1363, or a combination thereof of SEQ ID NO: 86.

[0153] In some embodiments, the Streptococcus pyogenes Cas9 domain comprises a mutation corresponding to any one of D10E, D10A, S29T, F32M, D39N, R40K, H41Q, S42T, I48L, C80R, S87A, K112D, H113N, K132N, K141E, D147E, L158V, E171Q, P176S, I186K, V189L, Q190H, Q194E, N199R, I201L, N202E, A203E, S204I, R205K, A210G, Q228A, L229F,G231N, S245A, T249M, S254A, D261N, T270S, N295K, T300I, D304G, V308A, N309D, 1312V, T333A, A337V, E345K, F352S, Q354K, S355T, K356T, G366K, A367T, E396D, L398F, 1414V, D428A, F429Y, D435E, K468Q, S469R, E470N, T472A, E480D, A486T, S490L, F498V, K500E, N501H, N504T, K528R, V530I, E532D, G533E, A538E, T555A, K570Q, F575C, D605E, E611D, R629K, E634K, T638K, R655H, R664K, R671K, K705V, E706D, Q709K, K710A, S714F, G7115E, G717K, H721K, H723Q, A725S, N726A, V743I, L747I, V748I, K772Q, K775R, N776R, I788M, G792R, K797E, Y799H, T804A, N808D, L811R, R820K, N831D, R832H, V842I, L847I, N869D, E874A, N881S, Q885R, N888K, T893S, L911A, Y945H, D946G, L949P, E952A, A1023G, Y1036R, G1067E, G1077E, R1078K, N1093T, R1114G, N1115E, D1117A, A1121P, D1125G, P1128T, K1129T, VI 1461, S1154T, S1159P, LI 164V, S1172N, N1177D, P1178S, Il 179V, D1180S, K1211R, M1213L, G1218T, N1234H, E1243D, K1244T, E1253K, E1260D, K1263Q, H1264Y, E1271D, Q1272W, E1275H, V1290L, L1291R, S1292A, A1293T, N1295E, H1297N, R1298T, D1299H, K1300L, R1303S, E1307D, N1308S, I1309M, I1310L, H1311N, L1312A, L1315F, T1316S, N1317R, Y1326F, D1328N, V1342I, A1345S, I1360L, S1363N, or a combination thereof of SEQ ID NO: 86.

[0154] In some embodiments, the Cas9 domain is a Neisseria meningitidis Cas9 nuclease. In some embodiments, the Neisseria meningitidis (NmCas9) nuclease comprises the amino acid sequence : MAAFKPNPINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRVFERAEVPKTGDSLAM ARRLARSVRRLTRRRAHRLLRARRLLKREGVLQAADFDENGLIKSLPNTPWQLRAAAL DRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVANNAHALQTGDFRT PAELALNKFEKESGHIRNQRGDYSHTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGIE TLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSER PLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEMKA YHAISRALEKEGLKDKKSPLNLSSELQDEIGTAFSLFKTDEDITGRLKDRVQPEILEALLK HISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTEEKIYLPPIPADKIRN PVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRKEIEKRQEENRKDREKA AAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLVRLNEKGYVEIDHALPFS RTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVETSRFPRSKKQ RILLQKFDEDGFKECNLNDTRYVNRFLCQFVADHILLTGKGKRRVFASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAFDGKTIDKETGKVL HQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTPEKLRTLLAEKLSSRPEAVHEYV TPLFVSRAPNRKMSGAHKDTLRSAKRFVKHNEKISVKRVWLTEIKLADLENMVNYKNG REIELYEALKARLEAYGGNAKQAFDPKDNPFYKKGGQLVKAVRVEKTQESGVLLNKK NAYTIADNGDMVRVDVFCKVDKKGKNQYFIVPIYAWQVAENILPDIDCKGYRIDDSYTF CFSLHKYDLIAFQKDEKSKVEFAYYINCDSSNGRFYLAWHDKGSKEQQFRISTQNLVLIQ KYQVNELGKEIRPCRLKKRPPVR (SEQ ID NO: 148).

[0155] In some embodiments, the Cas9 domain is a Neisseria meningitidis Cas9 nuclease. In some embodiments, the Neisseria meningitidis Cas9 domain comprises an amino acid sequence as set forth in SEQ ID NO: 148. Other Neisseria meningitidis Cas9 can be found at www.uniprot.org / uniprot / with accession numbers C9X1G5, A1IQ68, E0NB23, A9M1K5, or C6S593.

[0156] In some embodiments, the Neisseria meningitidis Cas9 domain comprises a mutation corresponding to any one of positions 19, D16, D30, E31, A94, 1103, P124, N164, 1213, G229, T241, S376, E393, G454, K471, G490, D660, C665, K764, T770, P803, A841, H842, K843, D844, L846, R847, K854, H855, N856, K858, K862, W865, E868, 1869, A872, D873, N876, Y880, G883, 1886, E887, E890, R895, A898, Y899, G900, G901, N902, A903, K904, Q905, D908, N912, K917, G919, L921, V927, K929, T930, E932, S933, L936, L937, N938, K939, K940, Y943, T944, G949, D950, C958, K965, N966, Q967, F969, A975, E980, N981, 1986, D987, C988, K989, G990, Y991, R992, 1993, D994, Y997, T998, C1000, S1002, H1004, K1005, Y1006, A1010, F1011, Q1012, K1013, D1014, E1015, K1018, V1019, E1020, F1021, A1022, Y1024, 11025, N1026, C1027, D1028, S1029, S1030, N1031, R1033, F1034, Y1035, L1036, A1037, W1038, K1041, G1042, K1044, E1045, Q1046, Q1047, F1048, R1049, 11050, S 1051, T1052, Q1053, N1054, L1055, V1056, L1057, 11058, Y1061, V1063, N1064, or a combination thereof or SEQ ID NO: 148.

[0157] In some embodiments, the Neisseria meningitidis Cas9 domain comprises a mutation corresponding to any one of I9M, D16E, D30E, E31K, A94D, I103V, P124C, N164D, I213N, G229D, T241A, S376T, E393K, G454C, K471E, G490C, D660E, C665R, K764E, T770A, P803S, A841Q, H842G, K843H, D844E, L846V, R847K, K854R, H855L, N856D, K858G, K862L, W865P, E868Q, I869L, A872K, D873G, N876K, Y880R, G883E, I886P, E887K, E890E, R895Q, A898T, Y899H, G900K, G901D, N902D, A903P, K904T, Q905K, D908A,N912E, K917Y, G919T, L921Q, V927I, K929Q, T930V, E932K, S933T, L936W, L937V, N938R, K939N, K940H, Y943N, T944G, G949A, D950T, C958E, K965G, N966G, Q967K, F969Y, A975S, E980K, N981G, I986R, D987A, C988V, K989V, G990A, Y991F, R992K, I993D, D994E, Y997F, T998E, C1000R, S1002I, H1004Y, K1005A, Y1006N, A1010K, F1O11L, Q1012T, K1013A, D1014K, E1015K, K1018N, V1019E, E1020F, F1021L, A1022G, Y1024F, II025V, N1026S, C1027L, D1028N, S1029R, S1O3OA, N1031T, R1033A, F1034I, Y1035D, L1O36I, A1037R, W1038T, K1041T, G1042D, K1044T, E1045K, Q1046G, Q1047E, F1048Q, R1049S, I1O5OV, S1O51G, T1052V, Q1053K, N1054T, L1055A, V1056L, L1057S, IIO58F, Y1061N, V1O63I, N1064D, or a combination thereof of SEQ ID NO: 148.

[0158] In some embodiments, the RNA-guided nuclease Neisseria meningitidis Cas9 domain comprises an amino acid sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 148. In some embodiments, the RNA-guided nuclease Neisseria meningitidis Cas9 domain comprises an amino acid sequence having between 85-90%, 90-95%, 95-97%, 97-98%, or 98-99% sequence identity to SEQ ID NO: 148.

[0159] In some embodiments, the RNA-guide nuclease Cas9 domain is an RNA-guided nuclease Campylobacter jejuni Cas9 domain. In some embodiments, the Campylobacter jejuni (CjCas9) nuclease comprises the amino acid sequence :MARILAFDIGISSIGWAFSENDELKDCGVRIFTKAENPKTGESLALPRRLARSARKRLARR KARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLISPYELRFRALNELLSKQDFAR VILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLANYQSVGEYLYKEYFQKFKENSK EFTNVRNKKESYERCIAQSFLKDGLKLIFKKQREFGFSFSKKFEEEVLSVAFYKRALKDF SHLVGNCSFFTDEKRAPKNSPLAFMFVALTRIINLLNNLKNTEGILYTKDDLNTLLNEVL KNGTLTYKQTKKLLGLSDDYEFKREKGTYFIEFKKYKEFIKALGEHNLSQDDLNEIAKDI TLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFKALKLITPLMLEGKKYDEACN ELNLKVAINEDKKDFLPAFNETYYKDEVTNPVVLRAIKEYRKVLNALLKKYGKVHKINI ELAREVGKNHSQRAKIEKEQNENYKAKKDAELECEKLGLKINSKNILKLRLFKEQKEFC AYSGEKIKISDLQDEKMLEIDHIYPYSRSFDDSYMNKVLVFTKQNQEKLNKTPFEAFGN DSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQKDFKDRNLNDTRYIARLVLNYTKDY LDFLPLSDDENTKLNDTQKGSKVHVEAKSGMLTSALRHTWGFSTKDRNNHLHHAIDAV IIAYANNSIVKAFSDFKKEQESNSAELYAKKISELDYKNKRKFFEPFSGFRQKVLDKIDEI FVSKPERKKPSGALHEETFRKEEEFYQSYGGKEGVLKALELGKIRKVNGKIVKNGDMFRVDIFKHKKTNKFYAVPIYTMDFALKVLPNKAVARSKKGEIKDWILMDENYEFCFSLYK DSLILIQTKDMQEPEFVYYNAFTSSTVSLIVSKHDNKFETLSKNQKILFKNANEKEVIAKS IGIQNLKVFEKYIVSALGEVTKAEFRQREDFKK (SEQ ID NO: 149)

[0160] In some embodiments, the Cas9 domain is an Campylobacter jejuni Cas9 nuclease. In some embodiments, the Campylobacter jejuni Cas9 domain comprises an amino acid sequence as set forth in SEQ ID NO: 149. Other Campylobacter jejuni Cas9 can be found at www.uniprot.org / uniprot / with accession numbers Q0P897, A7H5P1, A0A2U0QR81, A0A5Y4VLH1, or A0A381CRM8. In some embodiments, the RNA-guided nuclease Campylobacter jejuni Cas9 domain comprises a mutation corresponding to any one of positions L5, A6, D8, 19, S12, S13, F18, S19, L24, K25, 131, T40, E42, L50, L58, A59, R61, L58, L65, H67A N74, K77, L98, 199, P101, N110, LI 13, Al 19, A126, R128, 1134, K 140, A144, K147, Q151 , L156, V184, S190, F199, D202, G203, R212, F214, K221, E223, Y232, A235, V243, S247, D251, P256, L261, T269, N276, N277, L285, T287, L291, K300, T305, Q308, L312, G314, Y335, K336, 1339, H345, D351, N353, E354, 1362, K370, D383E, S384, K391, 1396, L403, T405, K413, N419, L421, D430, K432, A437, L453, K457, V462, A465, K472, N477, A492, E495, L525, K526, L527, K531, E532, E542, Q550, E556, H559, Y561, S564, M572, V577, Q581, N587, N596, K600, Q602, K603, Q616, K617, N623, Y624, K633, D634, Y642, N649, D656, L660, D662, K667, V677, E680, K682, L686, H692, T693, V712, 1714, V722, K723, S736, L739, K742, L747, N751, F756, R763, Q764, E772, K777, A786, E790, F792, Q800, S801, G804, L812, E813, V833, 1835, T841, Y845, A855, L856, A863, V864, D879, E883, D900, Q902, K927, F928, V971, T972, or a combination thereof of SEQ ID NO: 149.

[0161] In some embodiments, the RNA-guided nuclease Campylobacter jejuni Cas9 domain comprises a mutation corresponding to any one of L5I, A6G, D8N, D8E, I9L, S12A, S13N, F18L, S19R, L24I, K251, 13 IV, T40N, E42N, L50E, L58V, A59K, R61K, L58V, L65M, H67A, N74K, K77N, L98T, I99Q, P101I, N110S, LI 131, A119S, A126V, R128H, I134S, K140N, A144T, K147E, Q151K, L156M, V184I, S190D, F199L, D202Q, G203E, R212K, F214L, K221K, E223K, Y232F, A235P, V243I, S247I, D251N, P256A, L261S, T269G, N276K, N277S, L285V, T287E, L291I, K300D, T305S, Q308K, L312I, G314N, Y335L, K336N, I339K, H345T, D351I, N353D, E354S, I362T, K370E, D383E, S384K, K391N, I396L, L403Q, T405I, K413R, N419E, L421C, D430E, K432S, A437L, L453I, K457C, V462L, A465D, K472S, N477H, A492K, E495I, L525Q, K526I, L527V, K531E, E532D, E542L, Q550D, E556V, H559Y,Y561R, S564N, M572S, V577T, Q581L, N587G, N596E, K600L, Q602A, K603E, Q616R, K617F, N623F, Y624F, K633T, D634E, Y642W, N649S, D656S, L660I, D662E, K667A, V677Q, E680V, K682S, L686I, H692N, T693F, V712I, 1714V, V722I, K723F, S736K, L739F, K742N, L747S, N751L, F756L, R763K, Q764E, E772N, K777H, A786T, E790L, F792P, Q800N, S801T, G804D, L812V, E813K, V833S, I835L, T841K, Y845H, A855S, L856T, A863T, V864P, D879N, E883N, D900G, Q902K, K927N, F928Y, V971L, T972S, or a combination thereof of SEQ ID NO: 149.

[0162] In some embodiments, the Campylobacter jejuni Cas9 domain comprises an amino acid sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 149. In some embodiments, the Campylobacter jejuni Cas9 domain comprises an amino acid sequence having between 85-90%, 90-95%, 95-97%, 97-98%, or 98-99% sequence identity to SEQ ID NO: 149.

[0163] In some embodiments, the Cas9 domain is an Streptococcus pasteurianus Cas9 nuclease. In some embodiments, the Streptococcus pasteurianus (SpCas9) nuclease comprises the amino acid sequence :MTKKNYSIGLDIGTNSVGWAVITDDYKVPAKKMKVLGNTDKKYIKKNLLGALLFDSGE TAEATRLKRTARRRYTRRKNRLRYLQEIFAEEMTKVDESFFYRLDESFLTTDEKDFERHP IFGNKADEIKYHQEFPTIYHLRKHLADSSEKADLRLVYLALAHMIKFRGHFLIEGELNAE NTDVQKIFADFVGVYDRTFDDSHLSEITVDAASILTEKISKSRRLENLIKYYPTEKKNTLF GNLIALALGLQPNFKMNFKLSEDAKLQFSKDSYNEDLEELLGKIGDDYADLFTSAKNLY DAILLSGILTVDDNSTKAPLSASMIKRYAEHHEDLEKLKEFIKANKSELYHDIFKDETKN GYAGYIENGVKQDEFYKYLKNTLSKIAGSDYFLDKIEREDFLRKQRTFDNGSIPHQIHLQ EMHAILRRQGDYYPFLKENQDRIEKILTFRIPYYVGPLARKDSRFSWAEYHSDEKITPWN FDKVIDKEKSAEKFITRMTLNDLYLPEEKVLPKHSHVYETYAVYNELTKIKYVNEQGKD SFFDSNMKQEIFDHVFKENRKVTKEKLLNYLNKEFPEYRIKDLIGLDKENKSFNASLGTY HDLKKILDKAFLDDKVNEEVIEDIIKTLTLFEDKDMIHERLQKYSDIFTADQLKKLERRH YTGWGRLSYKLINGIRNKENNKTILDYLIDDGSANRNFMQLINDDTLPFKQIIQKSQVVG DVDDIEAVVHDLPGSPAIKKGILQSVKIVDELVKVMGDNPDNIVIEMARENQTTNRGRS QSQQRLKKLQNSLKELGSNILNEEKPSYIEDKVENSHLQNDQLFLYYIQNGKDMYTGDE LDIDHLSDYDIDHIIPQAFIKDDSIDNRVLTSSAKNRGKSDDVPSLDIVRARKAEWVRLY KSGLISKRKFDNLTKAERGGLTEADKAGFIKRQLVETRQITKHVAQILDARFNTESDENDKVIRDVKVITLKSNLVSQFRKDFEFYKVREINDYHHAHDAYLNAVVGTALLKKYPKLAS EFVYGEYKKYDVHKLIAKSSDDHSEMGKATAKYFFYSNLMNFFKRVIRYSNGKVIVRP VVEYSKDTEDIAWDKKSNFRTICKVLSYPQVNIVKKVETQTGGFSKESILPKGDSDKLIP RKTKKAYWDTKKYGGFDSPTVAYSVFVVADVEKGKAKKLKTVKELVGISIMERSFFEE NPVEFLENKGYHNIREDKLIKLPKYSLFEFEGGKRRLLASASELQKGNEMVIPGHLVKLL YHAQRINSFNSTKYLDYVSAHKKEFEKVLSCVEDFANLYVDVEKNLSKIRAVADSMDN FSIEEISNSFINLLTLTALGAPADFNFLGEKIPRKRYTSTKECLNATLIHQSITGLYETRIDLS KIGEE (SEQ ID NO: 150).

[0164] In some embodiments, the Cas9 domain is a Streptococcus pasteurianus Cas9 nuclease. In some embodiments, the Streptococcus pasteurianus Cas9 domain comprises an amino acid sequence as set forth in SEQ ID NO: 150. Other Streptococcus pasteurianus Cas9 can be found at www.uniprot.org / uniprot / with accession number F5X275.

[0165] In some embodiments, the Streptococcus pasteurianus Cas9 domain comprises a mutation corresponding to any one of positions Dl l, E85, A88, T92, E96, Y100, T109, DUO, DI 13, El 15, R116, D125, 1127, K128, E132, S147, 1185, A187, K228, Y229, T232, M255, S271, N273, A294, A327, E355, K357, N379, T380, S382, A385, D439, R440, S464, H469, Y519, 1528, N569, 1581, A607, K632, D633, H635, E636, A647, D648, T703, P705, K712, S713, A724, V750, D882, S951, D977, E979, S1014, H1027, 11030, E1081, D1082, D1086, K1088, S1089, N1090, R1092, T1093, 11094, C1095, Al 138, Y1139, DI 141, T1142, Fl 158, A1168, E1190, E1198, H1202, 11204, R1205, 11210, K1224, S1232, M1240, V1241, 11242, P1243, G1424, K1248, Q1254, N1257, S1258, T1262, K1263, Y1264, D1266, A1270, K1277, D1284, L1288, V1302, N1316, T1346, 11374, or a combination thereof of SEQ ID NO: 150.

[0166] In some embodiments, the Streptococcus pasteurianus Cas9 domain comprises a mutation corresponding to any one of DI IE, DI 1 A, E85D, A88T, T92A, E96D, Y 100Q, T109D, DI ION, D113N, E115D, R116S, D125E, I127D, K128A, E132K, S147T, I185L, A187T, K228N, Y229N, T232K, M255T, S271T, N273E, A294S, A327V, E355K, K357Q, N379G, T380I, S382T, A385N, D439E, R440E, S464A, H469R, Y519F, I528V, N569D, I581V, A607S, K632R, D633E, H635Q, E636Q, A647K, D648Q, T703A, P705S, K712E, S713A, A724T, V750I, D882G, S951R, D977E, E979K, S1014P, H1027R, I1030V, E1081G, D1082E, D1086N, K1088R, S1089T, N1090D, R1092E, T1093K, I1094V, C1095R, Al 138V, Y1139L, D1141E, T1142P, F1158L, A1168T, E1190K, E1198K, H1202Q, I1204V, R1205Q, I1210M, K1224R,S1232T, M1240I, V1241M, I1242L, P1243S, G1424A, K1248A, Q1254H, N1257G, S1258N, T1262A, K1263E, Y1264H, D1266K, A1270E, K1277E, D1284N, L1288V, V1302A, N1316D, T1346N, I1374L,or a combination thereof of SEQ ID NO: 150.

[0167] In some embodiments, the Streptococcus pasteurianus Cas9 domain comprises an amino acid sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 150. In some embodiments, the Streptococcus pasteurianus Cas9 domain comprises an amino acid sequence having between 85-90%, 90-95%, 95-97%, 97-98%, or 98- 99% sequence identity to SEQ ID NO: 150

[0168] In some embodiments, the Cas9 domain is a Clostridium cellulolyticum Cas9 nuclease. In some embodiments, the Clostridium cellulolyticum (CcCas9) nuclease comprises the amino acid sequence :MKYTLGLDVGIASVGWAVIDKDNNKIIDLGVRCFDKAEESKTGESLATARRIARGMRRR ISRRSQRLRLVKKLFVQYEIIKDSSEFNRIFDTSRDGWKDPWELRYNALSRILKPYELVQV LTHITKRRGFKSNRKEDLSTTKEGVVITSIKNNSEMLRTKNYRTIGEMIFMETPENSNKR NKVDEYIHTIAREDLLNEIKYIFSIQRKLGSPFVTEKLEHDFLNIWEFQRPFASGDSILSKV GKCTLLKEELRAPTSCYTSEYFGLLQSINNLVLVEDNNTLTLNNDQRAKIIEYAHFKNEI KYSEIRKLLDIEPEILFKAHNLTHKNPSGNNESKKFYEMKSYHKLKSTLPTDIWGKLHSN KESLDNLFYCLTVYKNDNEIKDYLQANNLDYLIEYIAKLPTFNKFKHLSLVAMKRIIPFM EKGYKYSDACNMAELDFTGSSKLEKCNKLTVEPIIENVTNPVVIRALTQARKVINAIIQK YGLPYMVNIELAREAGMTRQDRDNLKKEHENNRKAREKISDLIRQNGRVASGLDILKW RLWEDQGGRCAYSGKPIPVCDLLNDSLTQIDHIYPYSRSMDDSYMNKVLVLTDENQNK RSYTPYEVWGSTEKWEDFEARIYSMHLPQSKEKRLLNRNFITKDLDSFISRNLNDTRYIS RFLKNYIESYLQFSNDSPKSCVVCVNGQCTAQLRSRWGLNKNREESDLHHALDAAVIA CADRKIIKEITNYYNERENHNYKVKYPLPWHSFRQDLMETLAGVFISRAPRRKITGPAHD ETIRSPKHFNKGLTSVKIPLTTVTLEKLETMVKNTKGGISDKAVYNVLKNRLIEHNNKPL KAFAEKIYKPLKNGTNGAIIRSIRVETPSYTGVFRNEGKGISDNSLMVRVDVFKKKDKYY LVPIYVAHMIKKELPSKAIVPLKPESQWELIDSTHEFLFSLYQNDYLVIKTKKGITEGYYR SCHRGTGSLSLMPHFANNKNVKIDIGVRTAISIEKYNVDILGNKSIVKGEPRRGMEKYNSFKSN (SEQ ID NO: 151)

[0169] In some embodiments, the Cas9 domain is an Clostridium cellulolyticum Cas9 domain. In some embodiments, the Clostridium cellulolyticum Cas9 domain comprises an amino acidsequence as set forth in SEQ ID NO: 151. Other Clostridium cellulolyticum Cas9 can be found at www.uniprot.org / uniprot / with accession number B8I085.

[0170] In some embodiments, the Clostridium cellulolyticum Cas9 domain comprises a mutation corresponding to any one of positions T4, DIO, V9, D20, K21, 127, C33, K36, A47, A49, S64, Q65, E102, L103, T122, 1124, K131, D137, R163, G166, 1169, F170, V183, D184, 1187, E193, K200, K208, L209, D221, N224, E227, F228, S234, V242, K244, L252, T256, C258, S261, V413, M415, K416, R417, K424, Y426, K427, S429, D430, A468, T470, A472, A478, Q481, K482, L485, A497, L535, W540, R541, E544, G554, P556, 1570, Y574, M580, Y584, M585, T592, D593, V606, W607, 1647, N650, S693, L697, E702, S704, A713, V714, 1715, D776, L847, G850, G853, A854, R860, 1900, H904, M905, 1906, E921, Q923, S929, T930, H931, Q939, N994, 1997, N1000, K1001, S1002, 11003, K1005, P1008, or a combination thereof of SEQ ID NO: 151.

[0171] In some embodiments, the Clostridium cellulolyticum Cas9 domain comprises a mutation corresponding to any one of T4S, D10E, V9I, D20N, K21E, I27E, C33I, K36V, A47S, A49P, S64R, Q65H, E102L, L103V, T122V, I124F, K131Q, D137E, R163Q, G166S, I169L, F170L, V183G, D184G, I187T, E193S, K200Q, K208A, L209Y, D221K, N224Q, E227S, F228S, S234T, V242I, K244N, L252K, T256K, C258T, S261F, V413K, M415L, K416R, R417N, K424Q, Y426I, K427P, S429H, D430Q, A468S, T470S, A472V, A478G, Q481K, K482R, L485S, A497M, L535H, W540Y, R541K, E544Q, G554F, P556S, I570V, Y574I, M580F, Y584N, M585N, T592A, D593A, V606W, W607F, I647R, N650H, S693K, L697F, E702Q, S704N, A713V, V7141, 1715V, D776E, L847A, G850P, G853A, A854P, R860K, I900V, H904D, M905V, I906L, E921Y, Q923E, S929D, T930E, H931Y, Q939P, N994Q, I997P, N1000R, K1001M, S1002N, I1003K, K1005H, P1008K, or a combination thereof of SEQ ID NO: 151.

[0172] In some embodiments, the Clostridium cellulolyticum Cas9 domain comprises an amino acid sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 151. In some embodiments, the Clostridium cellulolyticum Cas9 domain comprises an amino acid sequence having between 85-90%, 90-95%, 95-97%, 97-98%, or 98-99% sequence identity to SEQ ID NO: 151.

[0173] In some embodiments, the Cas9 domain is an Geobacillus thermodenitrificans T1 Cas9 nuclease. In some embodiments, the Geobacillus thermodenitrificans T1 (GtCas9) nucleasecomprises the amino acid sequence :MKYKIGLDIGITSIGWAVINLDIPRIEDLGVRIFDRAENPKTGESLALPRRLARSARRRLRR RKHRLERIRRLFVREGILTKEELNKLFEKKHEIDVWQLRVEALDRKLNNDELARILLHLA KRRGFRSNRKSERTNKENSTMLKHIEENQSILSSYRTVAEMVVKDPKFSLHKRNKEDNY TNTVARDDLEREIKLIFAKQREYGNIVCTEAFEHEYISIWASQRPFASKDDIEKKVGFCTF EPKEKRAPKATYTFQSFTVWEHINKLRLVSPGGIRALTDDERRLIYKQAFHKNKITFHDV RTLLNLPDDTRFKGLLYDRNTTLKENEKVRFLELGAYHKIRKAIDSVYGKGAAKSFRPID FDTFGYALTMFKDDTDIRSYLRNEYEQNGKRMENLADKVYDEELIEELLNLSFSKFGHL SLKALRNILPYMEQGEVYSTACERAGYTFTGPKKKQKTVLLPNIPPIANPVVMRALTQA RKVVNAIIKKYGSPVSIHIELARELSQSFDERRKMQKEQEGNRKKNETAIRQLVEYGLTL NPTGLDIVKFKLWSEQNGKCAYSLQPIEIERLLEPGYTEVDHVIPYSRSLDDSYTNKVLV LTKENREKGNRTPAEYLGLGSERWQQFETFVLTNKQFSKKKRDRLLRLHYDENEENEF KNRNLNDTRYISRFLANFIREHLKFADSDDKQKVYTVNGRITAHLRSRWNFNKNREESN LHHAVDAAIVACTTPSDIARVTAFYQRREQNKELSKKTDPQFPQPWPHFADELQARLSK NPKESIKALNLGNYDNEKLESLQPVFVSRMPKRSITGAAHQETLRRYIGIDERSGKIQTV VKKKLSEIQLDKTGHFPMYGKESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKKNGEL GPIIRTIKIIDTTNQVIPLNDGKTVAYNSNIVRVDVFEKDGKYYCVPIYTIDMMKGILPNK AIEPNKPYSEWKEMTEDYTFRFSLYPNDLIRIEFPREKTIKTAVGEEIKIKDLFAYYQTIDS SNGGLSLVSHDNNFSLRSIGSRTLKRFEKYQVDVLGNIYKVRGEKRVGVASSSHSKAGE TIRPL (SEQ ID NO: 152)

[0174] In some embodiments, the Geobacillus thermodenitrificans T1 Cas9 domain comprises an amino acid sequence as set forth in SEQ ID NO: 152. Other Geobacillus thermodenitrificans T1 Cas9 can be found at www.uniprot.org / uniprot / with accession number A0A1W6VMQ3.

[0175] In some embodiments, the Geobacillus thermodenitrificans T1 Cas9 domain comprises a mutation corresponding to any one of positions K2, D8, 114, D35, K41, F74, V75, K91, 1117, R128, T136, Q151, S152, S156, A161, V164, S 171, E178, D179, V185, R192, K195, A199, Y204, 1207, V208, A212, H215, S219, F227, T260, V261, V271, G274, 1276, A278, L279, D282, 1287, K289, H293, F299, V302, N307, R313, L317, L318, V331, G337, K341, S348, A354, A355, K356, R359, M372, T377, R380, E395, D399, E404, S416, T441, R445, N464, E504, S508, M515, Q516, E520, G521, V534, L545, K559, T578, K603, T612, L619, S621, N656, N660, L673, D685, 1699, N708, N717, R737, V738, S752, D756, Q771, N777, N792,E793, 1811, 1824, K839, Q845, K848, T849, L895, 1902, T908, V929, 1943, 1946, M948, F990, T995, V1000, Q1014, D1017, S1019, N1020, G1021, S1024, N1030, N1031, R1035, S1036, 11037, V1067, S1071, A1075, 11079, or a combination thereof of SEQ ID NO: 152.

[0176] In some embodiments, the Geobacillus thermodenitrificans T1 Cas9 domain comprises a mutation corresponding to any one of positions D8, D179, D282, D399, D685, D756, D1071. In some embodiments, the RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain comprises a mutation corresponding to any one of K2R, D8E, D8A, I14V, D35E, K41Q, F74V, V75I, K91E, Il 17V, R128K, T136S, Q151R, S152A, S156G, A161G, V164I, S171A, E178G, D179E, V185I, R192H, K195R, A199S, Y204F, I207M, V208S, A212K, H215N, S219T, F227V, T260I, V261A, V271I, G274S, I276A, A278G, L279P, D282E, I287L, K289E, H293Q, F299Y, V302I, N307R, R313Y, L317I, L318V, V331I, G337D, K341Q, S348K, A354K, A355S, K356S, R359L, M372L, T377A, R380H, E395P, D399N, E404N, S416T, T441S, R445K, N464T, E504D, S508T, M515T, Q516K, E520D, G521E, V534M, L545H, K559R, T578V, K603R, T612I, L619V, S621T, N656M, N660S, L673F, D685E, I699V, N708E, N717D, R737K, V738I, S752A, D756E, Q771R, N777H, N792D, E793Q, 181 IV, I824V, K839T, Q845K, K848A, T849S, L895P, I902V, T908K, V929V, I943V, I946M, M948I, F990L, T995I, V1000G, Q1014K, D1017H, S1019G, N1020T, G1021A, S1024E, N1030C, N1031S, R1035S, S1036G, I1037V, V1067L, S1071A, A1075T, I1079V, or combination thereof of SEQ ID NO: 152.

[0177] In some embodiments, the RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain comprises a mutation corresponding to any one of positions D8E, D179E, D282E, D399N, D685E, D756E, D1071H of SEQ ID NO: 152. In some embodiments, the RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain comprises an amino acid sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 152. In some embodiments, the RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain comprises an amino acid sequence having between 85-90%, 90-95%, 95-97%, 97-98%, or 98- 99% sequence identity to SEQ ID NO: 152.

[0178] In some embodiments, the CRISPR Cas nuclease is a Deltaproteobacteria CasX a Acidaminococcus Cas 12 or a Eubacterium rectale Cas 12a..In some embodiments, the CRISPR Cas nuclease comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identicalto, Acidaminococcus Casl2,Deltaproteobacteria CasX, or Eubacterium rectale Cas 12a.

[0179] In some embodiments, the Cas domain is CasX domain. In some embodiments, the CasX domain comprises the amino acid sequence :QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISN TSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPAPKNIDQRKLIPVKDG NERLTSSGFACSQCCQPLYVYKLEQVNDKGKPHTNYFGRCNVSEHERLILLSPHKPEAN DELVTYSLGKFGQRALDFYSIHVTRESNHPVKPLEQIGGNSCASGPVGKALSDACMGAV ASFLTKYQDIILEHQKVIKKNEKRLANLKDIASANGLAFPKITLPPQPHTKEGIEAYNNVV AQIVIWVNLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDWWDMVCNVKKLI NEKKEDGKVFWQNLAGYKRQEALLPYLSSEEDRKKGKKFARYQFGDLLLHLEKKHGE DWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKE ADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNL YLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKR QGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIK PMNLIGIDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAAKEVEQ RRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTF MAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEK LKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWT KGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQ TNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV (SEQ ID NO: 153).

[0180] In some embodiments, the CasX domain is derived from Planctomycetes bacterium. In some embodiments, the CasX domain is from Deltaproteobacteria. In some embodiments, the CasX domain comprises an amino acid sequence at least about 85%, 90%, 95%, 97%, 98%, or 99% identical, or is identical to SEQ ID NO: 153. In some embodiments, the CasX domain comprises a mutation corresponding to any one of positions Rl l, R12, V14, K15, S17, N18, A22, G23, T25, P38, K41, E42, N46, L47, N53, 154, P57, T61, S62, R63, A64, E75, H82, Q89, P104, N106, 1113, N199, S124, S125, C133, Y137, N145, D146, H151, S161, R165, N177, L180, R202, N205, G215, C219, V236, T241, L248, 1254, S269, 1290, E291, V297, Q299, 1314, E318, Q323, L333, E359, D360, K362, Q366, N367, L368, A369, G370, Y371, H404, H409,G410, E411, Y417, V428, E429, S432, K433, L437, S443, A451, 1464, A470, 1502, L503, 1531, G537, L540, N553, 1559, S563, V571, N579, H589, S607, L608, L620, R623, R624, L644, S646, M652, 1657, R679, L684, N686, H689, S696, T702, T737, L742, Y744, Q748, M751, 1753, A771, R777, P792, S818, R823, V824, E826, K827, A832, T833, M836, 1839, G841, V846, N860, V862, D864, V867, V877, S883, S889, G890, S894, K908, N913, F916, T918, R936, Q938, Y940, K942, S963, R966, K967, K968, or any combination thereof of SEQ ID NO: 153.

[0181] In some embodiments, the CasX domain comprises a mutation or substitution corresponding to any one or more of R11K, R12K, V14S, K15A, S17N, N18A, A22V, G23S, T25S, P38D, K41K, E42K, N46K, L47R, N53V, I54M, P57V, T61N, S62A, R63A, A64N, E75K, H82Q, Q89K, P104S, N106K, I113K, N199K, S124T, S125A, C133G, Y137F, N145S, D146E, H151Y, S161A, R165K, N177S, L180A, R202K, N205T, G215A, C219Y, V236I, T241S, L248I, I254V, S269G, I290V, E291D, V297I, Q299R, I314L, E318D, Q323L, L333V, E359D, D360M, K362R, Q366S, N367G, L368V, A369T, G370A, Y371E, H404Y, H409Y, G410A, E411G, Y417F, V428I, E429A, S432T, K433S, L437R, S443A, A451V, I464L, A470M, I502V, L503V, 153 IL, G537K, L540I, N553S, I559L, S563G, V571L, N579Q, H589T, S607L, L608I, L620I, R623K, R624K, L644V, S646P, M652V, 1657 V, R679E, L684S, N686G, H689D, S696G, T702A, T737S, L742F, Y744H, Q748H, M751V, I753V, A771T, R777K, P792T, S818T, R823G, V824M, E826V, K827R, A832S, T833D, M836A, I839L, G841N, V846A, N860T, V862E, D864E, V867A, V877G, S883K, S889R, G890D, S894F, K908Q, N913D, F916H, T918V, R936N, Q938N, Y940F, K942S, S963A, R966K, K967R, K968R, or a combination thereof of SEQ ID NO: 153.

[0182] In some embodiments, the Casl2 domain is from Acidaminococcus sp. BV3L6. In some embodiments, the Casl2 domain comprises the amino acid sequence : TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAIN KRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFS AEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPF YNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFK QILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKK LETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNE VDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKE KNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPK CSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKG YREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEK EIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELF YRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARAL LPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGI DRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDL KQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCL VLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVW KTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNE TQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLEN DDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADA NGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRN (SEQ ID NO: 154).

[0183] In some embodiments, the Casl2 domain comprises an amino acid sequence at least about 85%, 90%, 95%, 97%, 98%, or 99% identical, or is identical to SEQ ID NO: 154. In some embodiments, the Casl2 domain comprises a mutation corresponding to any one of positions Tl, Q2, E4, G5, N8, L9, K28, H29, 130, Q31, E32, Q33, F35, 136, E37, E38, A41, N43, D44, H45, E48, 152, R55, T59, Y60, A61, D62, Q63, C64, Q66, L67, Q69, L70, N74, S76, A77, D80, S81, Y82, E85, E88, T90, R91, N92, A93, 195, E97, A99, T100, Y101, N103, A104, H106, D107, 1110, R112, T113, DI 14, R159, S169, S185, A187, 1192, D195, K201, T212, R218, N223, 1228, S233, 1236, E237, V239, F242, Q249, Y257, V279, 1284, F305, N313, S324, 1329, S331, T337, L338, L345, E349, S357, 1358, N386, 1393, L396, 1400, S403, V408, Q409, G427, K428, Q436, L442, S468, Q469, S472, L473, L479, E487, S488, A497, L510, A516, K522, Q535, M536, S541, V545, K549, N550, G552, V557, N559, S586, Y596, A601, 1604, A613, S628, E637, A657, K660, G663, Q665, C673, L683, L697, A711, L717, Q723, A733, E735, Y740, K751, K756, G766, 1778, R793, L844, 1858, S865, 1874, H898, 1903, 1916, L931, K941, N945, V951, S958, V959, D965, 1938, H984, A1009, C1024, G1037, T1049, G1055, T1056, Y1068, L1075, V1083, K1085, L1097, H1104, D1106, Dl l l l, L1122, A1134, V1138, D1147, V1160, Pl 161,R1171, R1173, Y1176, N1205, D1207, S1220, V1221, A1230, N1237, L1243, M1259, Q1274, G1291, Q1295, A1299, or L1304 of SEQ ID NO: 154.

[0184] In some embodiments, the Casl2 domain comprises a mutation or substitution corresponding to any one or more of T1S, Q2N, E4S, G5E, N8H, L9K, K28E, H29N, I30L, Q31T, E32A, Q33Y, F35M, I36V, E37N, E38D, A41L, N43S, D44E, H45N, E48K, 15 IN, R55K, T59Y, Y60F, A61I, D62E, Q63E, C64T, Q66K, L67H, Q69A, L70I, N74P, S76Y, A77K, D80T, S81A, Y82F, E85D, E88L, T90N, R91N, N92T, A93N, I95R, E97I, A99D, T100N, Y101C, N103K, A104S, H106A, D107G, I110E, R112K, T113V, D114P, R159K, S169V, S185A, A187S, I192L, D195E, K201I, T212K, R218N, N223T, I228T, S233G, I236L, E237D, V239I, F242V, Q249C, Y257F, V279T, I284V, F305Y, N313S, S324N, I329L, S331A, T337E, L338K, L345I, E349Q, S357L, I358A, N386D, I393V, L396A, I400L, S403N, V408I, Q409E, G427D, K428D, Q436A, L442I, S468V, Q469L, S472A, L473V, L479T, E487D, S488D, A497V, L510I, A516V, K522Q, Q535S, M536N, S541D, V545E, K549Q, N550Q, G552C, V557E, N559E, S586N, Y596Q, A601S, I604L, A613D, S628N, E637T, A657D, K660R, G663N, Q665K, C673H, L683V, L697V, A711G, L717F, Q723E, A733L, E735D, Y740F, K751E, K756A, G766A, I778V, R793P, L844F, I858V, S865T, I874L, H898N, I903V, I916A, L931F, K941N, N945Q, V951I, S958T, V959A, D965E, I938V, H984Q, A1009S, C1024Y, G1037S, T1049E, G1055R, T1056N, Y1068F, L1075A, V1083R, K1085G, L1097I, H1104K, DI 106N, DI 11 IN, LI 122K, Al 134D, VI 1381, DI 147A, VI 160E, Pl 161F, R1171Q, R1173E, Y1176L, N1205T, D1207N, S1220L, V1221T, A1230E, N1237S, L1243I, M1259K, Q1274L, G1291A, Q1295N, A1299N, or L1304K of SEQ ID NO: 154.

[0185] Additional Cas9 nuclease amino acid sequences are listed in Table 1. Table 1 Exemplary Cas9 nuclease amino acid sequences.

[0186] In some embodiments, the chimeric nuclease comprises a Cas nuclease or nuclease domain selected from Table 1. In some embodiments, the chimeric nuclease comprises a Cas nuclease or nuclease domain selected from SEQ ID NOs: 161-191. In some embodiments, the Cas nuclease or nuclease domain comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NOs: 161- 191.

[0187] In some embodiments, the chimeric nuclease further comprises additional protein fusions. In some embodiments, the chimeric nuclease comprises a self-cleaving peptide, for example a T2A peptide.GIY-YIG Nucleases

[0188] In some embodiments, the chimeric nuclease comprises a GIY-YIG nuclease. In some embodiments, the chimeric nuclease comprises a GIY-YIG nuclease catalytic domain.

[0189] GIY -YIG nucleases are a family of homing endonucleases that cut DNA. GIY -YIG nucleases are folded into two structural and functional domains, an N-terminal catalytic domain and a C-terminal DNA-binding domain, separated by a flexible linker. In some embodiments, the chimeric nuclease comprises the N-terminal catalytic domain of the GIY-YIG nuclease. In someembodiments, the chimeric nuclease comprises the N-terminal catalytic domain and the flexible linker of the GIY-YIG nuclease. In some embodiments, the chimeric nuclease comprises the N- terminal catalytic domain and the flexible linker of the GIY -YIG nuclease but lacks the C- terminal DNA-binding domain of the GIY -YIG nuclease.

[0190] In some embodiments, the GIY-YIG nuclease is an I-TevI nuclease. I-TevI is a sitespecific, sequence-tolerant homing endonuclease encoded by the td intron of bacteriophage T4 (Uniprot ID A0A7S9XH31, SEQ ID NO: 155).). In some embodiments, the chimeric nuclease comprises the catalytic domain of an I-TevI nuclease.

[0191] In some embodiments, the I-TevI is a modified I-TevI. In some embodiments, the I- TevI is a catalytic domain of I-TevI. In some embodiments, the I-TevI comprises a conservative amino acid substitution. In some embodiments, the I-TevI comprises a mutation in the catalytic domain. In some embodiments, the I-TevI is a nickase.

[0192] In some embodiments, the I-TevI nuclease comprises the amino acid sequence of MKSGIYQIKNTLNNKVYVGSAKDFEKRWKRHFKDLEKGCHSSIKLQRSFNKHGNVFEC SIEEEIPYEKDEIIERENFWIKEENSKINGYNIADATFGDTCSTHPEKEEIIKKRSETVKAKM EKEGPDGRKAEYS KPGS KNGRWNPETHKFCKCGVRIQTS A YTCS KCRNRSGENNSFFN HKHSDITKSKISEKMKGKKPSNIKKISCDGVIFDCAADAARHFKISSGEVTYRVKSDKWN WFYINA (SEQ ID NO: 155).

[0193] In some embodiments, the I-TevI nuclease comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 155.

[0194] In some embodiments, the I-TevI nuclease comprises the amino acid sequence of MKSGIYQIKNTLNNKVYVGSAKDFEKRWKRHFKDLEKGCHSSIKLQRSFNKHGNVFEC SILEEIPYEKDLIIERENFWIKELNSKINGYNIADATFGDTCSTHPLKEEIIKKRSETVKAKM LKLGPDGRKALYSKPGSKNGRWNPETHKFCKCGVRIQTSAYTCSKCRN (SEQ ID NO: 741).

[0195] In some embodiments, the I-TevI nuclease comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 741.

[0196] In some embodiments, the I-TevI nuclease comprises the amino acid sequence of MGKSGIYQIKNTLNNKVYVGSAKDFEKRWKRHFKDLEKGCHSSIKLQRSFNKHGNVFECSILEEIPYEKDLIIERENFWIKELNSKINGYNIADATFGDTCSTHPLKEEIIKKRSETVKAK MLKLGPDGRKALYSKPGSKNGRWNPETHKFCKCGVRIQTSAYTCSKCRNRSGENNSFF NHKHSDITKSKISEKMKGKKPSNIKKISCDGVIFDCAADAARHFKISSGLVTYRVKSDKW NWFYINA (SEQ ID NO: 156).

[0197] In some embodiments, the I-TevI nuclease comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 156.

[0198] In some embodiments, the I-TevI nuclease comprises the amino acid sequence of KSGIYQIKNTLNNKVYVGSAKDFEKRWKRHFKDLEKGCHSSIKLQRSFNKHGNVFECSI LEEIPYEKDLIIERENFWIKELNSKINGYNIADATFGDTCSTHPLKEEIIKKRSETVKAKMLKLGPDGRKALYSKPGSKNGRWNPETHKFCKCGVRIQTSAYTCSKCRNRSGENNSFFNH KHSDITKSKISEKMKGKKPSNIKKISCDGVIFDCAADAARHFKISSGLVTYRVKSDKWN WFYINA (SEQ ID NO: 157).

[0199] In some embodiments, the I-TevI nuclease comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 157.

[0200] In some embodiments, the I-TevI nuclease catalytic domain comprises the amino acid sequence of MKSGIYQIKNTLNNKVYVGSAKDFEKRWKRHFKDLEKGCHSSIKLQRSFNKHGNVFEC SILEEIPYEKDLIIERENFWIKELNSKINGYNIA (SEQ ID NO: 24).

[0201] In some embodiments, the I-TevI nuclease catalytic domain comprises the amino acid sequence of MGKSGIYQIKNTLNNKVYVGSAKDFEKRWKRHFKDLEKGCHSSIKLQRSFNKHGNVFE CSILEEIPYEKDLIIERENFWIKELNSKINGYNIA (SEQ ID NO: 158).

[0202] In some embodiments, the I-TevI nuclease catalytic domain comprises the amino acid sequence of KSGIYQIKNTLNNKVYVGSAKDFEKRWKRHFKDLEKGCHSSIKLQRSFNKHGNVFECSI LEEIPYEKDLIIERENFWIKELNSKINGYNIA (SEQ ID NO: 159).

[0203] In some embodiments, the I-TevI nuclease catalytic domain comprises the amino acid sequence of MGKSGIYQIKNTLNNKVYVGSAKDFERRWKRHFKDLEKGCHSSIKLQRSFNKHGNVFECSILEEIPYEKDLIIERENFWIKELNSKINGYNIADASFGDTCSTHPLKEEIIKKRSETVKAK MLKLGPDGRKALYSKPGSKNGRWNPETHKFCKCGVRIRTSAYTCSKCRN (SEQ ID NO: 272).

[0204] In some embodiments, the I-TevI nuclease comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 272.

[0205] In some embodiments, the I-TevI nuclease catalytic domain comprises the amino acid sequence of MGKSGIYQIKNTLNNKVYVGSAKDFEKRWKRHFKDLEKGCHSSIKLQRSFNKHGNVFE CSILEEIPYEKDLIIERENFWIKELNSKINGYNIADATFGDTCSTHPLKEEIIKKRSETFKAK MLKLGPDGRKALYSRPGSKSGRWNPETHKFCKCGVRIQTSAYTCSKCRN (SEQ ID NO: 740).

[0206] In some embodiments, the I-TevI nuclease comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 740.

[0207] In some embodiments, the I-TevI sequence comprises a mutation in an amino acid position corresponding to K26 of SEQ ID NO: 155. In some embodiments, the I-TevI sequence comprises a mutation in an amino acid position corresponding to T95 of SEQ ID NO: 155. In some embodiments, the I-TevI sequence comprises a mutation in a amino acid position corresponding to Q158 of SEQ ID NO: 155. In some embodiments, the I-TevI sequence comprises a mutation in an amino acid position corresponding to VI 17 of SEQ ID NO: 155. In some embodiments, the I-TevI sequence comprises a mutation in a amino acid position corresponding to KI 35 of SEQ ID NO: 155. In some embodiments, the I-TevI sequence comprises a mutation in an amino acid position corresponding to N140 of SEQ ID NO: 155.

[0208] In some embodiments, the I-TevI sequence comprises mutations in amino acid positions corresponding to K26 and T95 of SEQ ID NO: 155. In some embodiments, the I-TevI sequence comprises mutations in amino acid positions corresponding to K26 and Q158 of SEQ ID NO: 155. In some embodiments, the I-TevI sequence comprises mutations in amino acid positions corresponding to T95 and Q158 of SEQ ID NO: 155. In some embodiments, the I-TevI sequence comprises mutations in amino acid positions corresponding to K26, T95, and Q158 of SEQ ID NO: 155.

[0209] In some embodiments, the I-TevI sequence comprises mutations in amino acid positions corresponding to V117, K135, and N140 of SEQ ID NO: 155.

[0210] In some embodiments, the I-TevI sequence comprises a K26R mutation in an amino acid position corresponding to of SEQ ID NO: 155. In some embodiments, the I-TevI sequence comprises a T95S mutation in an amino acid position corresponding to of SEQ ID NO: 155. In some embodiments, the I-TevI sequence comprises a Q158R mutation in an amino acid position corresponding to of SEQ ID NO: 155. In some embodiments, the I-TevI sequence comprises a V 117F mutation in an amino acid position corresponding to of SEQ ID NO: 155. In some embodiments, the I-TevI sequence comprises a K135R mutation in a amino acid position corresponding to of SEQ ID NO: 155. In some embodiments, the I-TevI sequence comprises a N140S mutation in an amino acid position corresponding to of SEQ ID NO: 155.

[0211] In some embodiments, the I-TevI sequence comprises K26R and T95S mutations in amino acid positions corresponding to of SEQ ID NO: 155. In some embodiments, the I-TevI sequence comprises to K26R and Q158R mutations in amino acid positions corresponding of SEQ ID NO: 155. In some embodiments, the I-TevI sequence comprises T95S and Q158R mutations in amino acid positions corresponding to of SEQ ID NO: 155. In some embodiments, the I-TevI sequence comprises K26R, T95S, and Q158R mutations in amino acid positions corresponding to of SEQ ID NO: 155.

[0212] In some embodiments, the I-TevI sequence comprises to VI 17F, K135R, and N140S mutations in amino acid positions corresponding of SEQ ID NO: 155.

[0213] In some embodiments, the I-TevI sequence comprises mutations in amino acid positions corresponding to R27, V117, K135, and N140 of SEQ ID NO: 155.

[0214] In some embodiments, the I-TevI nickase domain comprises the R27A, VI 17F, K135R, and N140S mutations. In some embodiments, the I-TevI comprises a mutation that results in a catalytically dead I-TevI. In some embodiments, the I-TevI comprises a mutation that results in a catalytically dead I-TevI at a residue corresponding to amino acid position R27A of SEQ ID NO: 24. In some embodiments, the I-TevI comprises a mutation that results in a catalytically dead I-TevI at a residue corresponding to amino acid position R28A of SEQ ID NO: 25.

[0215] In some embodiments, the I-TevI comprises a mutation at a residue corresponding to amino acid position 26 of SEQ ID NO: 24. In some embodiments, the I-TevI comprises a K26R mutation of SEQ ID NO: 24. In some embodiments, the I-TevI comprises a mutation at aresidue corresponding to amino acid position 27 of SEQ ID NO: 25. In some embodiments, the I-TevI comprises a K27R mutation of SEQ ID NO: 25.

[0216] In certain embodiments, the modified I-TevI nuclease domain comprises a substitution selected from any one or more of T1 IV, V16I, N14G, E25D, K26R, E36S, K37N, G38N, C39V, S41H, L45F, F49Y, I60V, and E81I corresponding to amino acid positions in SEQ ID NO: 155.

[0217] In some embodiments, the I-TevI nuclease comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 156. In some embodiments, the I-TevI nuclease comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 157. In some embodiments, the I-TevI nuclease comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 158. In some embodiments, the I-TevI nuclease comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 159. In some embodiments, the I-TevI nuclease comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 24. In some embodiments, the I-TevI comprises an amino acid sequence that is 90% identical to SEQ ID NO: 24. In some embodiments, the I-TevI comprises an amino acid sequence that is 91% identical to SEQ ID NO: 24. In some embodiments, the I- TevI comprises an amino acid sequence that is 92% identical to SEQ ID NO: 24. In some embodiments, the I-TevI comprises an amino acid sequence that is 93% identical to SEQ ID NO: 24. In some embodiments, the I-TevI comprises an amino acid sequence that is 94% identical to SEQ ID NO: 24. In some embodiments, the I-TevI comprises an amino acid sequence that is 95% identical to SEQ ID NO: 24. In some embodiments, the I-TevI comprises an amino acid sequence that is 96% identical to SEQ ID NO: 24. In some embodiments, the I-TevI comprises an amino acid sequence that is 97% identical to SEQ ID NO: 24. In some embodiments, the I- TevI comprises an amino acid sequence that is 98% identical to SEQ ID NO: 24. In some embodiments, the I-TevI comprises an amino acid sequence that is 99% identical to SEQ ID NO: 24. In some embodiments, the I-TevI comprises an amino acid sequence that is 100% identical to SEQ ID NO: 24.

[0218] In some embodiments, the I-TevI nuclease comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQID NO: 25. In some embodiments, the I-TevI comprises an amino acid sequence that is 90% identical to SEQ ID NO: 25. In some embodiments, the I-TevI comprises an amino acid sequence that is 91% identical to SEQ ID NO: 25. In some embodiments, the I-TevI comprises an amino acid sequence that is 92% identical to SEQ ID NO: 25. In some embodiments, the I- TevI comprises an amino acid sequence that is 93% identical to SEQ ID NO: 25. In some embodiments, the I-TevI comprises an amino acid sequence that is 94% identical to SEQ ID NO: 25. In some embodiments, the I-TevI comprises an amino acid sequence that is 95% identical to SEQ ID NO: 25. In some embodiments, the I-TevI comprises an amino acid sequence that is 96% identical to SEQ ID NO: 25. In some embodiments, the I-TevI comprises an amino acid sequence that is 97% identical to SEQ ID NO: 25. In some embodiments, the I-TevI comprises an amino acid sequence that is 98% identical to SEQ ID NO: 25. In some embodiments, the I- TevI comprises an amino acid sequence that is 99% identical to SEQ ID NO: 25. In some embodiments, the I-TevI comprises an amino acid sequence that is 100% identical to SEQ ID NO: 25.

[0219] In some embodiments, the GIY-YIG nuclease is an I-Bmol nuclease. I-Bmol is encoded by a group I intron that interrupts the thymidylate synthase (TS) gene (thy A) of Bacillus mojavensis s87-18. In some embodiments, the I-Bmol nuclease comprises the amino acid sequence of MKSGVYKITNKNTGKFYIGSSEDCESRLKVHFRNLKNNRHINRYLNNSFNKHGEQVFIG EVIHILPIEEAIAKEQWYIDNFYEEMYNISKSAYHGGDLTSYHPDKRNIILKRADSLKKVY LKMTSEEKAKRWQCVQGENNPMFGRKHTETTKLKISNHNKLYYSTHKNPFKGKKHSEE SKTKLSEYASQRVGEKNPFYGKTHSDEFKTYMSKKFKGRKPKNSRPVIIDGTEYESATE ASRQLNVVPATILHRIKSKNEKYSGYFYK (SEQ ID NO: 737).

[0220] In some embodiments, the I-Bmol nuclease comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 737.

[0221] In some embodiments, the GIY-YIG nuclease is an Eco29kl nuclease. An exemplary Eco29kl nuclease is Eco29kl restriction endonuclease from Ectothiorhodospira magna.

[0222] In some embodiments, the Eco29kl nuclease comprises the amino acid sequence of MTDDKVIPFNPLDKRHLGESVGQAMLRQPVVPMAKLSRFRGAGIYAIYYTGNFEAYQGI AACNRDDRFAAPIYVGKAVPKGARKGSGSLDTSPGAVLFSRLAQHGKSIQEVKNLDINDFYCRYLIVDDIWIPLGESLLIAKFNPLWNSVLDGFGNHDPGKGRHAGLRPRWDVVHPGR AWAGRCQAREETAEKILREAVNFLASNPPPGDW (SEQ ID NO: 738).

[0223] In some embodiments, the Eco29kl nuclease comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 738.

[0224] In some embodiments, the Eco29kl nuclease is a Eco29kl restriction endonuclease from Escherichia coli.

[0225] In some embodiments, the Eco29kl nuclease comprises the amino acid sequence of

[0226] MTGKVIPFNPLDKQNLGASVAEALLSKDAHPLEELTSFQGAGIYAIYYTGDHPA YRQLAELNRDGQFRLPIYVGKAVPAGARMGLTNPDKVGNVLFRRLKEHAESIRAAENL SIEDFYCRFLVVDDIWIPLGESLVISRFKPIWNSSIDGFGNHDPGKHRYTGLRPRWDFMHP GRGWAQNLRERDETVDELIRDSIQYLQNLPPCLAQKFIEAEGD (SEQ ID NO: 739).

[0227] In some embodiments, the Eco29kl nuclease comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 739.Linkers

[0228] In some embodiments, the chimeric nuclease comprises a linker domain. In some embodiments, the linker domain is located between the CRISPR Cas nuclease and the GIY-YIG nuclease. In some embodiments, the linker domain is located between the CRISPR Cas nuclease domain and the GIY-YIG nuclease domain.

[0229] In some embodiments, the linker comprises the I-TevI linker (amino acids 93 - 150 of I-TevI (SEQ ID NO: 155). The linker may alternatively or further comprise a flexible amino acid linker comprising from 10 to 100 amino acids. Such linkers can be unstructured or comprise a Gly-Ser linker.

[0230] In certain embodiments, the linker comprises an amino acid substitution selected from any one or more of a position corresponding to residue T95S, S101Y, Al 19D, K120N, K135N, K135R, P126S, D127K, N140S, T147I, Q158R, A161V, or S165G of SEQ ID NO: 155. In certain embodiments, the linker comprises a substitution selected from any one or more of position corresponding to residue T95S, VI 17F, K135R, N140S, or Q158R of SEQ ID NO: 155.

[0231] In some embodiments, the linker comprises a mutation selected from a mutation corresponding to any one of T95S, S101Y, A119D, K120N, K135N, K135R, P126S, D127K, N140S, T147I, Q158R, A161V, V117F, S165G of SEQ ID NO: 155, or a combination thereof.

[0232] In some embodiments, the linker domain comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 26. In some embodiments, the linker domain comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 27. In some embodiments, the linker domain comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 28. In some embodiments, the linker domain comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 29. In some embodiments, the linker domain comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 30. In some embodiments, the linker domain comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 31. In some embodiments, the linker domain comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 32. In some embodiments, the linker domain comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 33. In some embodiments, the linker domain comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 34. In some embodiments, the linker domain comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 35.

[0233] In some embodiments, the linker additionally comprises a hinge sequence. In some embodiments, the hinge sequence comprises the amino acid sequence GGSGGTGGSG.

[0234] In some embodiments, the I-TevI domain and linker sequence comprises SEQ ID NO: 225. In some embodiments, the I-TevI domain and linker sequence comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NO: 225.

[0235] In some embodiments, the I-TevI is I-TevI [R27A] and the Cas9 is SaCas9 [D10A+H557A].

[0236] In some embodiments, the I-TevI is I-TevI WT and the Cas9 is CasX. In some embodiments, the chimeric nuclease comprises the amino acid sequence ofMGKSGIYQIKNTLNNKVYVGSAKDFEKRWKRHFKDLEKGCHSSIKLQRSFNKHGNVFE CSILEEIPYEKDLIIERENFWIKELNSKINGYNIADATFGDTCSTHPLKEEIIKKRSETVKAK MLKLGPDGRKALYSKPGSKNGRWNPETHKFCKCGVRIQTSAYTCSKCRNGGSGGTGGS QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISN TSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPAPKNIDQRKLIPVKDG NERLTSSGFACSQCCQPLYVYKLEQVNDKGKPHTNYFGRCNVSEHERLILLSPHKPEAN DELVTYSLGKFGQRALDFYSIHVTRESNHPVKPLEQIGGNSCASGPVGKALSDACMGAV ASFLTKYQDIILEHQKVIKKNEKRLANLKDIASANGLAFPKITLPPQPHTKEGIEAYNNVV AQIVIWVNLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDWWDMVCNVKKLI NEKKEDGKVFWQNLAGYKRQEALLPYLSSEEDRKKGKKFARYQFGDLLLHLEKKHGE DWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKE ADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNL YLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKR QGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIK PMNLIGIDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAAKEVEQ RRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTF MAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEK LKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWT KGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQ TNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV (SEQ ID NO: 160).

[0237] In some embodiments, the chimeric nuclease comprises an amino acid sequence selected form Table 2.Table 2 Exemplary chimeric nuclease amino acid sequences

[0238] In some embodiments, the chimeric nuclease comprises a chimeric nuclease sequence selected from Table 2. In some embodiments, the chimeric nuclease comprises is selected from SEQ ID NOs: 192-223. In some embodiments, the chimeric nuclease comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identical to SEQ ID NOs: 192-223. In some embodiments, the chimeric nuclease has cleavage activity against an I-TevI recognition site in double-stranded DNA. In some embodiments, the chimeric nuclease has cleavage activity against an I-TevI recognition site in double-stranded DNA and cleavage activity against a Cas9 recognition site in double-stranded DNA. In some embodiments, the chimeric nuclease has binding activity against an I-TevI recognition site in double-stranded DNA. In some embodiments, the chimeric nuclease has cleavage activity against an I-TevI recognition site in double-stranded DNA and binding activity against a Cas9 recognition site in double-stranded DNA. In some embodiments, the chimeric nuclease has nickase activity against an I-TevI recognition site in double-stranded DNA and a nickase activity against a Cas9 recognition site in double-stranded DNA.Guide Polynucleotides

[0239] In some embodiments, the chimeric nuclease or the chimeric nuclease system further comprises a guide polynucleotide. In some embodiments, the guide polynucleotide is a guide RNA. In certain embodiments, the guide RNA targets a genomic target site in a cell. In certain embodiments, the guide RNA targets a disease-causing mutation in a mammalian cell. In certain embodiments, the mammalian cell is a human cell. In certain embodiments, the guide RNA targets a bacterial or a vital sequence.

[0240] In some embodiments, the guide RNA is a single guide RNA (sgRNA) (e.g., a fusion of a crRNA and a tracrRNA) or a dual guide RNA (crRNA and tracrRNA).

[0241] A single guide RNA (sgRNA) can comprise, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single guide linker, a minimum tracrRNA sequence, a 3' tracrRNA sequence and / or an optional tracrRNA extension sequence. A scaffold sequence can comprise all the elements without the spacer sequence. The optional tracrRNA extension can comprise elements that contribute additional functionality (e.g., stability) to the guide RNA. The single guide linker can link the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension can comprise one or more hairpins. In particular embodiments, the disclosure provides for an sgRNA comprising a spacer sequence and a tracrRNA sequence.In some embodiments, the crRNA and / or tracrRNA is from Staphylococcus aureus, Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromo genes, Streptomyces viridochromogenes, Str ep to sporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus desulforudis, Clostridium botulinum, Clostridium difficile, Fine goldia magna, Natranaerobius thermophilus, Pelotomaculum the rmopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, or Acaryochloris marina Cas9 system.

[0242] In some embodiments, a variant of a guide RNA scaffold sequence may be used. In some embodiments, the scaffold sequence to follow the guide sequence at its 3’ end is SEQ ID NO: 75.

[0243] In some embodiments, the guide RNA comprises the nucleic acid sequence set forth in SEQ ID NO: 53. In some embodiments, the guide RNA comprises the nucleic acid sequence 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 53. In some embodiments, theguide RNA comprises the nucleic acid sequence 95% identical to SEQ ID NO: 53. In some embodiments, the guide RNA comprises the nucleic acid sequence 96% identical to SEQ ID NO: 53. In some embodiments, the guide RNA comprises the nucleic acid sequence 97% identical to SEQ ID NO: 53. In some embodiments, the guide RNA comprises the nucleic acid sequence 98% identical to SEQ ID NO: 53. In some embodiments, the guide RNA comprises the nucleic acid sequence 99% identical to SEQ ID NO: 53. In some embodiments, the guide RNA comprises the nucleic acid sequence 100% identical to SEQ ID NO: 53.

[0244] In some embodiments, the guide RNA comprises the nucleic acid sequence set forth in SEQ ID NO: 76. In some embodiments, the guide RNA comprises the nucleic acid sequence 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 76. In some embodiments, the guide RNA comprises the nucleic acid sequence 95% identical to SEQ ID NO: 76. In some embodiments, the guide RNA comprises the nucleic acid sequence 96% identical to SEQ ID NO:76. In some embodiments, the guide RNA comprises the nucleic acid sequence 97% identical to SEQ ID NO: 76. In some embodiments, the guide RNA comprises the nucleic acid sequence 98% identical to SEQ ID NO: 76. In some embodiments, the guide RNA comprises the nucleic acid sequence 99% identical to SEQ ID NO: 76. In some embodiments, the guide RNA comprises the nucleic acid sequence 100% identical to SEQ ID NO: 76.

[0245] In some embodiments, the guide RNA comprises the nucleic acid sequence set forth in SEQ ID NO: 77. In some embodiments, the guide RNA comprises the nucleic acid sequence 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 77. In some embodiments, the guide RNA comprises the nucleic acid sequence 95% identical to SEQ ID NO: 77. In some embodiments, the guide RNA comprises the nucleic acid sequence 96% identical to SEQ ID NO:77. In some embodiments, the guide RNA comprises the nucleic acid sequence 97% identical to SEQ ID NO: 77. In some embodiments, the guide RNA comprises the nucleic acid sequence 98% identical to SEQ ID NO: 77. In some embodiments, the guide RNA comprises the nucleic acid sequence 99% identical to SEQ ID NO: 77. In some embodiments, the guide RNA comprises the nucleic acid sequence 100% identical to SEQ ID NO: 77.

[0246] In some embodiments, the guide RNA comprises the nucleic acid sequence set forth in SEQ ID NO: 78. In some embodiments, the guide RNA comprises the nucleic acid sequence 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 78. In some embodiments, the guide RNA comprises the nucleic acid sequence 95% identical to SEQ ID NO: 78. In someembodiments, the guide RNA comprises the nucleic acid sequence 96% identical to SEQ ID NO:78. In some embodiments, the guide RNA comprises the nucleic acid sequence 97% identical to SEQ ID NO: 78. In some embodiments, the guide RNA comprises the nucleic acid sequence 98% identical to SEQ ID NO: 78. In some embodiments, the guide RNA comprises the nucleic acid sequence 99% identical to SEQ ID NO: 78. In some embodiments, the guide RNA comprises the nucleic acid sequence 100% identical to SEQ ID NO: 78.

[0247] In some embodiments, the guide RNA comprises the nucleic acid sequence set forth in SEQ ID NO: 79. In some embodiments, the guide RNA comprises the nucleic acid sequence 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 79. In some embodiments, the guide RNA comprises the nucleic acid sequence 95% identical to SEQ ID NO: 79. In some embodiments, the guide RNA comprises the nucleic acid sequence 96% identical to SEQ ID NO:79. In some embodiments, the guide RNA comprises the nucleic acid sequence 97% identical to SEQ ID NO: 79. In some embodiments, the guide RNA comprises the nucleic acid sequence 98% identical to SEQ ID NO: 79. In some embodiments, the guide RNA comprises the nucleic acid sequence 99% identical to SEQ ID NO: 79. In some embodiments, the guide RNA comprises the nucleic acid sequence 100% identical to SEQ ID NO: 79.

[0248] In some embodiments, the guide RNA comprises the nucleic acid sequence set forth in SEQ ID NOs: 76-85. In some embodiments, the guide RNA comprises the nucleic acid sequence set forth in SEQ ID NO: 234-235.

[0249] In some embodiments, the guide RNA is modified. In certain embodiments, the guide RNA comprises one or more of: a non-natural internucleoside linkage, a nucleic acid mimetic, a modified sugar moiety, and a modified nucleobase. In some embodiments, the guide RNA comprises modified nucleotides. In certain embodiments, the modified nucleotide comprises one or more of: a 5-methylcytosine; a 5 -hydroxymethyl cytosine; a xanthine; a hypoxanthine; a 2- aminoadenine; a 6-methyl derivative of adenine; a 6-methyl derivative of guanine; a 2-propyl derivative of adenine; a 2-propyl derivative of guanine; a 2-thiouracil; a 2-thio thy mine; a 2- thiocytosine; a 5-halouracil; a 5-halocytosine; a 5-propynyl uracil; a 5-propynyl cytosine; a 6-azo uracil; a 6-azo cytosine; a 6-azo thymine; a pseudouracil; a 4-thiouracil; an 8-halo; an 8-amino; an 8-thiol; an 8-thioalkyl; an 8-hydroxyl; a 5-halo; a 5-bromo; a 5 -trifluoromethyl; a 5- substituted uracil; a 5-substituted cytosine; a 7-methylguanine; a 7-methyladenine; a 2-F- adenine; a 2-amino-adenine; an 8-azaguanine; an 8-azaadenine; a 7-deazaguanine; a 7-deazaadenine; a 3 -deazaguanine; a 3-deazaadenine; a tricyclic pyrimidine; a phenoxazine cytidine; a phenothiazine cytidine; a substituted phenoxazine cytidine; a carbazole cytidine; a pyridloinclole cytidine; a 7-cleaza-adenine; a 7-dleazaguanosine; a 2- aminopyridine; a 2- pyridone; a 5-substituted pyrimidine; a 6-azapyrimidine; an N-2, N-6 or 0-6 substituted purine; a 2-aminopropyladenine; a 5-propynyluracil; or a 5-propynylcytosine.

[0250] In certain embodiments, the non-natural internucleoside linkage comprises one or more of: a phosphorothioate, a phosphoramidate, a non-phosphodiester, a heteroatom, a chiral phosphorothioate, a phosphorodithioate, a phosphotriester, an aminoalkylphosphotriester, a 3'- alkylene phosphonates, a 5'-alkylene phosphonate, a chiral phosphonate, a phosphinate, a 3'- amino phosphoramidate, an aminoalkylphosphoramidate, a phosphorodiamidate, a thionophosphoramidate, a thionoalkylphosphonate, a thionoalkylphosphotriester, a selenophosphate, and a boranophosphate. In certain embodiments, the nucleic acid mimetic comprises one or more of a peptide nucleic acid (PNA), morpholino nucleic acid, cyclohexenyl nucleic acid (CeNAs), or a locked nucleic acid (LNA). In certain embodiments, the modified sugar moiety comprises one or more of 2'-0-(2-methoxyethyl), 2'-dimethylaminooxyethoxy, 2'- dimethylaminoethoxyethoxy, 2'-0-methyl, or 2'-fluoro.

[0251] In some embodiments, the chimeric nuclease targets a site in a gene. In some embodiments, the chimeric nuclease targets a site in the intron or the exon of a gene. In some embodiments the gene is B2M or AA VS1. In some embodiments the gene is CFTR. In some embodiments the gene is SERPINA 1 (coding for Alpha- 1 -antitrypsin). In some embodiments the gene is DMPK. In some embodiments the gene is C9ORF72. In some embodiments, the guide RNA comprises a spacer that targets a target site in B2M or AAVS1. In some embodiments, the guide RNA comprises a spacer that targets a target site in CFTR. In some embodiments, the guide RNA comprises a spacer that targets a target site in SERPINA 1. . In some embodiments, the guide RNA comprises a spacer that targets a target site in DMPK. . In some embodiments, the guide RNA comprises a spacer that targets a target site in C9ORF72.

[0252] In some embodiments, the gene is C9ORF72 or DMPK. In some embodiments, the spacer comprises a nucleic acid sequence set forth in SEQ ID NOs: 20 to 23.

[0253] In some embodiments, the spacer comprises a nucleic acid sequence set forth in SEQ ID NO: 41 or SEQ ID NO: 51. In some embodiments, the spacer comprises a nucleic acid sequence set forth in SEQ ID NO: 76 to SEQ ID NO: 85.

[0254] It is to be understood, that in the sequence if the nucleic acid sequence is a DNA a T is a Thymine, if the nucleic acid sequence is an RNA, T is a Uracil.CFTR

[0255] The cystic fibrosis transmembrane conductance regulator ( CFTR) gene encodes a member of the ATP -binding cassette (ABC) transporter superfamily. The encoded protein functions as a chloride channel, making it unique among members of this protein family, and controls ion and water secretion and absorption in epithelial tissues. Channel activation is mediated by cycles of regulatory domain phosphorylation, ATP-binding by the nucleotide- binding domains, and ATP hydrolysis. Mutations in this gene cause cystic fibrosis (CF), the most common lethal genetic disorder in populations of Northern European descent. Mutations in the CFTR gene (GRCh38.pl4 GCF_000001405.40, NM_000492.4, NC_000007.14 Reference GRCh38.pl4 ) can lead to suboptimal ion transport and fluid retention, causing the prominent clinical manifestations of abnormal thickening of the mucus in lungs and pancreatic insufficiency. The most frequently occurring mutation in cystic fibrosis, DeltaF508, results in impaired folding and trafficking of the encoded protein. The CFTR protein, is found across a wide range of organs including pancreas, kidney, liver, lungs, gastrointestinal tracts, and reproductive tracts, making CF a multiorgan disease. In the lung, dysfunctional CFTR can hinder mucociliary clearance, rendering the organ susceptible to bacterial infections and inflammation, ultimately leading to airway occlusion, respiratory failure, and premature death. CF remains the most common and lethal genetic disease among the Caucasian population with 70,000-100,000 sufferers estimated worldwide, highlighting a real need for the development of better treatments.

[0256] Mutations in the CFTR include, but are not limited to c.!521_1523del (p.Phe508del), C.1624OT (p.Gly542Ter) or c.3846G>A (p.Trp!282Ter).In some embodiments, the guide polynucleotides target sites in the intronic or exonic regions of the CFTR gene.

[0257] In some embodiments, the target region for the chimeric nuclease is the CFTR F508 - R553X region on Chr7: 117,559,464-117,587,836 relative to the hg38 genome. In some embodiments, the chimeric nuclease cuts directly adjacent to 5’, 3’, and / or inside of the region of Chr7: 117,559,464-117,587,836.SERPINA1

[0258] The Serpin Family A Member 1 protein encoded by the SERPINA1 gene (GRCh38.pl4 (GCF_000001405.40), NG_008290.1 RefSeqGene) is a serine protease inhibitor belonging to the serpin superfamily whose targets include elastase, plasmin, thrombin, trypsin, chymotrypsin, and plasminogen activator. SERPINA1 protein is produced in the liver, the bone marrow, by lymphocytic and monocytic cells in lymphoid tissue, and by the Paneth cells of the gut. Defects in this gene are associated with chronic obstructive pulmonary disease, emphysema, and chronic liver disease and plays a role in Alpha 1 -antitrypsin deficiency. Several transcript variants encoding the same protein have been found for this gene. The SERPINA1 E342K mutation is involved in Alpha 1 -antitrypsin deficiency.

[0259] In some embodiments, the guide polynucleotides target sites in the intronic or exonic regions of the SERPINA1 gene.DMPK

[0260] DM1 Protein Kinase (DMPK) is a serine-threonine kinase that is closely related to other kinases that interact with members of the Rho family of small GTPases. Substrates for this enzyme include myogenin, the beta-subunit of the L-type calcium channels, and phospholemman. The DMPK gene (NG_009784.1 RefSeqGene, GRCh38.pl4 (GCF_000001405.40)) is located on 19ql3.32. The 3' untranslated region of this gene contains 5-38 copies of a (CTG)n * (CAG)n trinucleotide repeat. Expansion of this unstable motif to 40- 5,000 copies causes myotonic dystrophy type I, which increases in severity with increasing repeat element copy number. Repeat expansion is associated with condensation of local chromatin structure that disrupts the expression of genes in this region. Several alternatively spliced transcript variants of this gene have been described. Removal of the excess (CTG)n * (CAG)n trinucleotide repeat sequences can stabilize the 3' untranslated region of DMPK in a cell. In some embodiments, one or more guide polynucleotide target (CAG)n sites in the 3' untranslated region of the DMPK gene for excision.

[0261] In some embodiments, the spacer of the guide polynucleotide comprises a nucleic acid sequence set forth in SEQ ID NOs: 22 to 23. In some embodiments, the spacer of the guide polynucleotide comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 136-147.

[0262] In some embodiments, the chimeric nuclease system targets the intron region of DMPK set forth in SEQ ID NO: 4.

[0263] In some embodiments, the chimeric nuclease system is a dual-guided chimeric nuclease. In some embodiments, the chimeric nuclease system is a dual-guided Tev-dCas9. Exemplary excision sequences in DMPK that can be excised with dual-guided nucleases of the disclosure are listed in Table 3A. Exemplary regions for excision are listed in Table 3B. In some embodiments, the excised sequence comprises SEQ ID NO: 236. In some embodiments, the excised sequence comprises any one of SEQ ID NOs: 237 to 247.Table 3A Sequences in the 3' untranslated region of Dmpk that can be targeted for be excision with a dual-guided Tev-dCas9*[CAG]40+ stands for 40 or more CAG repeatsTable 3B Range of chromosomal locations that can be cut with dual Tev-dCas9 in Dmpk

[0264] Table 4A shows an exemplary dual-guided chimeric nuclease targeting the nucleic acid sequence of SEQ ID NO: 236 in the DPMK gene for excision.Table 4A Exemplary nucleotide sequences of combinations of nuclease + guide RNA + conditionally cleavable elements targeting DMPK.

[0265] Table 4B shows an exemplary dual-guided chimeric nuclease targeting the nucleic acid sequence of SEQ ID NO: 240 in the DPMK gene for excision.Table 4B Exemplary nucleotide sequences of combinations of nuclease + guide RNA + conditionally cleavable elements targeting DMPK.Table 5 Exemplary amino acid sequences of a chimeric nuclease and nucleic acid sequences of two guide RNAs after transcription, processing, and translation.C9ORF72

[0266] C9orf72-SMCR8 Complex Subunit (C9ORF72) plays an important role in the regulation of endosomal trafficking, and has been shown to interact with Rab proteins that are involved in autophagy and endocytic transport. Expansion of a GGGGCC repeat from 2-22 copies to 700-1600 copies in the intronic sequence between alternate 5' exons in transcripts from this gene is associated with 9p-linked ALS (amyotrophic lateral sclerosis) and FTD (frontotemporal dementia). Studies suggest that hexanucleotide expansions could result in the selective stabilization of repeat-containing pre-mRNA, and the accumulation of insoluble dipeptide repeat protein aggregates that could be pathogenic in FTD-ALS patients (PMID: 23393093). Alternative splicing results in multiple transcript variants encoding different isoforms.

[0267] In some embodiments, the gene is C9ORF72. In some embodiments, the spacer comprises a nucleic acid sequence set forth in SEQ ID NOs: 20 or 21.

[0268] In some embodiments, the spacer comprises a nucleic acid sequence set forth any one of SEQ ID NOs: 132-135.

[0269] In some embodiments, the chimeric nuclease targets the intron region of C9ORF72 set forth in SEQ ID NOs: 2 or 3.1n some embodiments, the removed C9orf72 G4C2 repeat comprises a [G4C2]24+ repeat.Table 6. Range of chromosomal locations that can be cut with dual Tev-dCas9 in C9orf72Table 7 Exemplary nucleotide sequences of combinations of nuclease + guide RNA + conditionally cleavable elements targeting C9orf72

[0270] In some embodiments, the gene is C9ORF72. In some embodiments, the spacer comprises a nucleic acid sequence set forth in SEQ ID NOs: 20 to 23.

[0271] In some embodiments, the chimeric nuclease system is a dual-guided chimeric nuclease system. Dual-guided nuclease system comprise two chimeric nucleases that bind and cleave at two sites in a genome of a cell, preferably on the same chromosome. In some embodiments, the two chimeric nucleases are the same chimeric nuclease with two different guide polynucleotidesthat can bind up- or downstream of each other on a DNA sequence. In some embodiments, the two chimeric nucleases are different chimeric nuclease with two different guide polynucleotides.

[0272] In some embodiments, the chimeric nuclease system comprises a first chimeric nuclease and a first guide RNA and a second chimeric nuclease and a second guide RNA. In some embodiments, the first chimeric nuclease and the first guide RNA target a first target site, and the second chimeric nuclease and the second guide RNA target a second target site. In some embodiments, the first chimeric nuclease comprises an active I-TevI nuclease and an inactive dCas nuclease. In some embodiments, the second chimeric nuclease comprises an active I-TevI nuclease and an inactive dCas nuclease. In some embodiments, the first chimeric nuclease comprises an active I-TevI nuclease and an inactive dCas nuclease and the second chimeric nuclease comprises an active I-TevI nuclease and an inactive dCas nuclease.

[0273] In some embodiments, the first and the second guide RNA target different target sites in the genome of a cell. In some embodiments, the distance between the first target site and the second target site is 100 bases. In some embodiments, the distance between the first target site and the second target site is 200 bases. In some embodiments, the distance between the first target site and the second target site is 300 bases. In some embodiments, the distance between the first target site and the second target site is 400 bases. In some embodiments, the distance between the first target site and the second target site is 500 bases. In some embodiments, the distance between the first target site and the second target site is 600 bases. In some embodiments, the distance between the first target site and the second target site is 700 bases. In some embodiments, the distance between the first target site and the second target site is 800 bases. In some embodiments, the distance between the first target site and the second target site is 900 bases. In some embodiments, the distance between the first target site and the second target site is 1000 bases. In some embodiments, the distance between the first target site and the second target site is 2000 bases. In some embodiments, the distance between the first target site and the second target site is 3000 bases. In some embodiments, the distance between the first target site and the second target site is 4000 bases. In some embodiments, the distance between the first target site and the second target site is 5000 bases. In some embodiments, the distance between the first target site and the second target site is 6000 bases. In some embodiments, the distance between the first target site and the second target site is 7000 bases. In some embodiments, the distance between the first target site and the second target site is 8000 bases. Insome embodiments, the distance between the first target site and the second target site is 9000 bases. In some embodiments, the distance between the first target site and the second target site is 10000 bases. In some embodiments, the distance between the first target site and the second target site is 11000 bases. In some embodiments, the distance between the first target site and the second target site is 12000 bases. In some embodiments, the distance between the first target site and the second target site is 13000 bases. In some embodiments, the distance between the first target site and the second target site is 14000 bases. In some embodiments, the distance between the first target site and the second target site is 15000 bases. In some embodiments, the distance between the first target site and the second target site is 20000 bases. In some embodiments, the distance between the first target site and the second target site is 25000 bases. In some embodiments, the distance between the first target site and the second target site is 28000 bases. In some embodiments, the distance between the first target site and the second target site is 30000 bases. In some embodiments, the distance between the first target site and the second target site is more than 30000 bases. In some embodiments, the distance between the first target site and the second target site is between 100 bases and 1000 bases. In some embodiments, the distance between the first target site and the second target site is between 100 bases and 10000 bases. In some embodiments, the distance between the first target site and the second target site is between 100 bases and 20000 bases. In some embodiments, the distance between the first target site and the second target site is between 100 bases and 30000 bases. In some embodiments, the distance between the first target site and the second target site is between 1000 bases and 10000 bases. In some embodiments, the distance between the first target site and the second target site is between 1000 bases and 20000 bases. In some embodiments, the distance between the first target site and the second target site is between 1000 bases and 30000 bases. In some embodiments, the distance between the first target site and the second target site is between 5000 bases and 30000 bases.

[0274] In some embodiments, the first guide RNA and the second guide target the same strand of genomic DNA. In some embodiments, the first guide RNA and the second guide target the opposite strand of genomic DNA. In some embodiments, the first guide RNA is targets a first chimeric nuclease to a first I-TevI target site and cleaves atthe first I-TevI target site in a genome of a cell and the second guide RNA is targets a second chimeric nuclease to a second I-TevItarget site in the genome of the cell and cleaves at the second I-TevI target site, wherein the cleavage creates a nucleotide overhang at the second I-TevI target site.

[0275] In some embodiments, the first guide RNA is targets a first chimeric nuclease to a Cas9 target site and cleaves at the Cas9 target site in a genome of a cell and the second guide RNA is targets a second chimeric nuclease to a I-TevI target site in the genome of the cell and cleaves at the I-TevI target site, wherein the cleavage creates a nucleotide overhang at the I-TevI target site.

[0276] In some embodiments, the second guide RNA targets a region 5’ to the donor polynucleotide target site. In some embodiments, the 3’ end of the donor polynucleotide is complementary to the overhang created by the cleavage of second I-TevI at the second I-TevI target site.

[0277] In some embodiments, the guide RNA further comprises a donor polynucleotide. In some embodiments, the donor polynucleotide is attached to the 3’ end of the guide RNA. In some embodiments, the donor polynucleotide comprises a repair template. In some embodiments, the repair template comprises a 2 nucleotide overhang compared with the target site. In some embodiments, the repair template is 36 bases long.

[0278] In some embodiments, the guide RNA further comprises one or more synthetic tRNA sequences encoding trans-acting ribozyme sequences. In some embodiments, the tRNA is located 5’ of the guide RNA. In some embodiments, the tRNA is located at the 5’ end of the guide RNA. In some embodiments, the ribozyme is capable of cleaving the tRNA from the guide RNA.Nucleic Acids

[0279] Provided herein are, inter alia, nucleic acids encoding the chimeric nucleases and the chimeric nuclease systems described herein.

[0280] In one aspect, the nucleic acid comprises a polynucleotide encoding a chimeric nuclease comprising a GIY-YIG nuclease domain and an RNA-guided nuclease domain; a polynucleotide encoding a guide RNA (gRNA), and a polynucleotide encoding a tRNA. In some embodiments, the nucleic acid comprises a polynucleotide encoding a chimeric nuclease comprising an I-TevI nuclease domain and an RNA-guided nuclease domain; a polynucleotide encoding a guide RNA (gRNA), and a polynucleotide encoding a tRNA.

[0281] Exemplary designs for nucleic acids encoding the chimeric nucleases and the chimeric nuclease systems described herein are shown in FIG. 1, FIG. 2, FIG. 26A, FIG. 26B, FIG. 26C, and FIG. 27A.

[0282] In another aspect, the nucleic acid comprises a polynucleotide encoding a guide RNA and a polynucleotide encoding a tRNA.

[0283] In some embodiments, the nucleic acid further comprises one or more additional guide RNAs.

[0284] In some embodiments, the nucleic acid is a DNA or RNA. In some embodiments, the DNA is circular plasmid DNA, linear double-strand DNA, single strand DNA, or chimeric RNA and DNA.

[0285] In some embodiments, the RNA is mRNA. In some embodiments, the mRNA comprises nucleic acid mimetics selected from the group of peptide nucleic acid (PNA), morpholino nucleic acid, cyclohexenyl nucleic acid (CeNAs), and locked nucleic acid (LNA). In some embodiments, the mRNA comprises modified sugar moieties, optionally wherein the modified sugar moiety is selected from the group of N1 -methylpseudouridine, 9-Methyladenine, 2'-O-(2-methoxyethyl), 2'-dimethylaminooxyethoxy, 2'-dimethylaminoethoxyethoxy, 2'-O- methyl, and 2'-fluoro. In some embodiments, the mRNA comprises a modified nucleobase, optionally wherein the modified nucleobase is selected from the group of a 5-methylcytosine; a 5 -hydroxymethyl cytosine; a xanthine; a hypoxanthine; a 2-aminoadenine; a 6-methyl derivative of adenine; a 6-methyl derivative of guanine; a 2-propyl derivative of adenine; a 2-propyl derivative of guanine; a 2-thiouracil; a 2-thiothymine; a 2-thiocytosine; a 5-halouracil; a 5- halocytosine; a 5-propynyl uracil; a 5-propynyl cytosine; a 6-azo uracil; a 6-azo cytosine; a 6-azo thymine; a pseudouracil; a 4-thiouracil; an 8-halo; an 8-amino; an 8-thiol; an 8-thioalkyl; an 8- hydroxyl; a 5-halo; a 5-bromo; a 5 -trifluoromethyl; a 5-substituted uracil; a 5-substituted cytosine; a 7-methylguanine; a 7-methyladenine; a 2-F-adenine; a 2-amino-adenine; an 8- azaguanine; an 8-azaadenine; a 7-deazaguanine; a 7-deazaadenine; a 3-deazaguanine; a 3- deazaadenine; a tricyclic pyrimidine; a phenoxazine cytidine; a phenothiazine cytidine; a substituted phenoxazine cytidine; a carbazole cytidine; a pyridoindole cytidine; a 7-deaza- adenine; a 7-deazaguanosine; a 2-aminopyridine; a 2-pyridone; a 5-substituted pyrimidine; a 6- azapyrimidine; an N-2, N-6 or O-6 substituted purine; a 2-aminopropyladenine; a 5- propynyluracil; or a 5-propynylcytosine. In some embodiments, the mRNA comprises a non-naturally occurring or a non-natural internucleoside linkage selected from the group of a phosphorothioate, a phosphoramidate, a non-phosphodiester, a heteroatom, a chiral phosphorothioate, a phosphorodithioate, a phosphotriester, an aminoalkylphosphotriester, a 3'- alkylene phosphonates, a 5'-alkylene phosphonate, a chiral phosphonate, a phosphinate, a 3'- amino phosphoramidate, an aminoalkylphosphoramidate, a phosphorodiamidate, a thionophosphoramidate, a thionoalkylphosphonate, a thionoalkylphosphotriester, a selenophosphate, or a boranophosphate.

[0286] In some embodiments, the nucleic acid is about 5kb in length. In some embodiments, the nucleic acid is less than 5kb in length. In some embodiments, the nucleic acid can be packaged into an AAV.

[0287] In some embodiments, the nucleic acid is transcribed into a single mRNA encoding the chimeric nuclease, one or more guide RNA, and any additional donor polynucleotides. In some embodiments, the single mRNA is processed by a ribozyme or the cellular mRNA processing machinery into individual components at one or more cut sites or transacting cut sites.

[0288] In some embodiments, the cut site is a ribozyme cut site. In some embodiments, the cut site is a tRNA cut site. In some embodiments, the guide RNA sequence is directly adjacent to a 5’ cut site. In some embodiments, the guide RNA sequence is directly adjacent to a 3’ cut site.

[0289] In some embodiments, the guide RNA sequence is directly adjacent to a 5’ cut site and 3’ cut site. The cut sites in the mRNA can increase the efficiency and amount of available guide RNA sequences in the cell after transcription.

[0290] In some embodiments, the nucleic acid comprises from 5’ to 3’ end a promoter (when the nucleic acid is a DNA) or untranslated region (UTR) (when the nucleic acid is an RNA) sequence, a chimeric nuclease sequence, a cut site, a tRNAl sequence, a cut site, a gRNAl sequence, a cut site, a tRNA2 sequence, a cut site, a gRNA2 sequence, a cut site, a tRNA3 sequence, a cut site, a poly A sequence.

[0291] In some embodiments, the nucleic acid comprises from 5’ to 3’ end a promoter / un translated region (UTR) sequence, a chimeric nuclease sequence, a cut site, a tRNAl sequence, a cut site, a gRNAl sequence, a cut site, a tRNA2 sequence, a cut site, a gRNA2 sequence, a transacting target site / cut site, and a poly A sequence.

[0292] In some embodiments, the nucleic acid comprises from 5’ to 3’ end a promoter / untranslated region (UTR) sequence, a chimeric nuclease sequence, a transacting targetsite / cut site, a gRNAl sequence, a tRNAl sequence, a cut site, a gRNA2 sequence, a cut site, a tRNA2 sequence, a cut site, and a poly A sequence.

[0293] In some embodiments, the nucleic acid comprises from 5’ to 3’ end a promoter or UTR sequence, a chimeric nuclease sequence, a MALAT sequence, a cut site, a tRNAl sequence, a cut site, a gRNAl sequence, a cut site, a tRNA2 sequence, a cut site, a gRNA2 sequence, a cut site, a tRNA3 sequence, a cut site, a poly A sequence.

[0294] In some embodiments, the nucleic acid comprises from 5’ to 3’ end a promoter / UTR sequence, a chimeric nuclease sequence, a MALAT sequence, a cut site, a tRNAl sequence, a cut site, a gRNAl sequence, a cut site, a tRNA2 sequence, a cut site, a gRNA2 sequence, a transacting target site / cut site, and a poly A sequence.

[0295] In some embodiments, the nucleic acid comprises from 5’ to 3’ end a promoter / untranslated region (UTR) sequence, a chimeric nuclease sequence, a MALAT sequence, a transacting target site / cut site, a gRNAl sequence, a tRNAl sequence, a cut site, a gRNA2 sequence, a cut site, a tRNA2 sequence, a cut site, and a poly A sequence.

[0296] In some embodiments, the nucleic acid comprises from 5’ to 3’ end a promoter sequence, a chimeric nuclease sequence, a Ribol sequence, a cut site, a gRNAl sequence, a cut site, a tRNAl sequence, a cut site, a gRNA2 sequence, a cut site, a tRNA2 sequence, a cut site, and a poly A sequence.

[0297] In some embodiments, the nucleic acid comprises from 5’ to 3’ end a promoter sequence, a chimeric nuclease sequence, a Ribol sequence, a cut site, a gRNAl sequence, a cut site, a tRNAl sequence, a cut site, a gRNA2 sequence, a cut site, a Ribo2 sequence, and a poly A sequence.

[0298] In some embodiments, the nucleic acid comprises from 5’ to 3’ end a promoter sequence, a chimeric nuclease sequence, a Ribol sequence, a cut site, a gRNAl sequence, a cut site, a tRNAl sequence, a cut site, a gRNA2 sequence, a transacting target site / cut site, and a poly A sequence.

[0299] In some embodiments, the nucleic acid comprises from 5’ to 3’ end a promoter sequence, a chimeric nuclease sequence, a transacting target site / cut site, a gRNAl sequence, a cut site, a tRNAl sequence, a cut site, a gRNA2 sequence, a Ribo2 sequence, and a poly A sequence.

[0300] In some embodiments, the nucleic acid comprises from 5’ to 3’ end a promoter sequence, a chimeric nuclease sequence, a cut site a tRNAl sequence, a cut site, a gRNAl sequence, a transacting target site / cut site, a Ribol sequence, a cut site, a gRNA2 sequence, a Ribo2 sequence, and a poly A sequence.

[0301] In some embodiments, the nucleic acid comprises from 5’ to 3’ end a promoter sequence, a chimeric nuclease sequence, a cut site, a tRNAl sequence, a cut site, a gRNAl sequence, a cut site, a Ribol sequence, a transacting target site / cut site, a gRNA2 sequence, a Ribo2 sequence, and a poly A sequence.

[0302] In some embodiments, the nucleic acid comprises from 5’ to 3’ end a promoter sequence, a chimeric nuclease sequence, a transacting target site / cut site, a Ribol sequence, a cut site, a gRNAl sequence, a cut site, a tRNAl sequence, a cut site, a gRNA2 sequence, a cut site, a tRNA2 sequence, a cut site, and a poly A sequence.

[0303] In some embodiments, the nucleic acid comprises from 5’ to 3’ end a promoter sequence, a chimeric nuclease sequence, a MALAT sequence, a Ribol sequence, a cut site, a gRNAl sequence, a tRNAl sequence, a cut site, a gRNA2 sequence, a cut site, a tRNA2 sequence, a cut site, and a poly A sequence.

[0304] In some embodiments, the nucleic acid comprises from 5’ to 3’ end a promoter sequence, a chimeric nuclease sequence, a MALAT sequence, a Ribol sequence, a cut site, a gRNAl sequence, a tRNAl sequence, a cut site, a gRNA2 sequence, a cut site, a Ribo2 sequence, and a poly A sequence.

[0305] In some embodiments, the nucleic acid comprises from 5’ to 3’ end a promoter sequence, a chimeric nuclease sequence, a MALAT sequence, a Ribol sequence, a cut site, a gRNAl sequence, a tRNAl sequence, a cut site, a gRNA2 sequence, a transacting target site / cut site, and a poly A sequence.

[0306] In some embodiments, the nucleic acid comprises from 5’ to 3’ end a promoter sequence, a chimeric nuclease sequence, a MALAT sequence, a transacting target site / cut, a gRNAl sequence, a tRNAl sequence, a cut site, a gRNA2 sequence, a cut site, a Ribo2 sequence, and a poly A sequence.

[0307] In some embodiments, the nucleic acid comprises from 5’ to 3’ end a promoter sequence, a chimeric nuclease sequence, a MALAT sequence, a tRNAl sequence, a cut site, agRNAl sequence, a transacting target site / cut, a Ribol sequence, a cut site, a gRNA2 sequence, a cut site, a Ribo2 sequence, and a poly A sequence.

[0308] In some embodiments, the nucleic acid comprises from 5’ to 3’ end a promoter sequence, a chimeric nuclease sequence, a MALAT sequence, a tRNAl sequence, a cut site, a gRNAl sequence, a cut site, a Ribol sequence, a transacting target site / cut site, a gRNA2 sequence, a Ribo2 sequence, and a poly A sequence.

[0309] In some embodiments, the nucleic acid comprises from 5’ to 3’ end a promoter sequence, a chimeric nuclease sequence, a MALAT sequence, a transacting target site / cut site, a Ribol sequence, a gRNAl sequence, a cut site, a tRNAl sequence, a cut site, a gRNA2 sequence, a cut site, a tRNA2 sequence, a cut site, and a poly A sequence.

[0310] In some embodiments, the transacting target site / cut site is a cut site for a ribozyme or a tRNA.

[0311] In some embodiments, the nucleic acid additionally comprises one or more donor nucleotide sequences.

[0312] In some embodiments, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NOs: 15-19. In some embodiments, the nucleic acid comprises a nucleic acid sequence as set forth in SEQ ID NOs: 113, 114, 115, 116, 117, or 118.

[0313] In some embodiments, the nucleic acid further comprises a self-inactivating nucleic acid sequence. In some embodiments, the self-inactivating nucleic acid sequence comprises a nuclease binding and cleavage sites between the promoter and start codon of the nuclease, between the nuclease and the poly adenylation sequence, or the 5’ untranslated region.Donor Polynucleotides

[0314] In some embodiments, the nucleic acid further comprises a nucleic acid sequence encoding for a donor polynucleotide. In some embodiments, the nucleic acid comprises two or more donor polynucleotides. In some embodiments, the donor polynucleotide is a single stranded nucleic acid. In some embodiments, the donor polynucleotide is a double stranded nucleic acid. In some embodiments, the donor polynucleotide is a partially double stranded nucleic acid. In some embodiments, the donor polynucleotide is an RNA. In some embodiments, the first strand of the double stranded donor polynucleotide is DNA and the second strand is RNA. In some embodiments, the donor polynucleotide is a cis acting repair template encoding the sense strand of the repair sequence. In some embodiments, the donor polynucleotide is a cisacting repair template encoding the complement or reverse complement of a repair sequence. In some embodiments, the donor polynucleotide is a trans acting repair template of the repair sequence. In some embodiments, the donor polynucleotide comprises a cis-acting single-strand RNA polynucleotide annealed to a complementary single-strand DNA polynucleotide.

[0315] In some embodiments, the repair template is 20 bases long. In some embodiments, the repair template is 25 bases long. In some embodiments, the repair template is 30 bases long. In some embodiments, the repair template is 35 bases long. In some embodiments, the repair template is 40 bases long. In some embodiments, the repair template is 45 bases long. In some embodiments, the repair template is 50 bases long. In some embodiments, the repair template is 55 bases long. In some embodiments, the repair template is 60 bases long. In some embodiments, the repair template is 65 bases long. In some embodiments, the repair template is 70 bases long. In some embodiments, the repair template is 75 bases long. In some embodiments, the repair template is 80 bases long. In some embodiments, the repair template is 85 bases long. In some embodiments, the repair template is 90 bases long. In some embodiments, the repair template is 95 bases long. In some embodiments, the repair template is 100 bases long. In some embodiments, the repair template is 150 bases long. In some embodiments, the repair template is 200 bases long. In some embodiments, the repair template is 250 bases long. In some embodiments, the repair template is 300 bases long. In some embodiments, the repair template is 350 bases long. In some embodiments, the repair template is 400 bases long. In some embodiments, the repair template is 450 bases long. In some embodiments, the repair template is 500 bases long. In some embodiments, the repair template is 600 bases long. In some embodiments, the repair template is 700 bases long. In some embodiments, the repair template is 800 bases long. In some embodiments, the repair template is 900 bases long. In some embodiments, the repair template is 1000 bases long. In some embodiments, the repair template is at least 900 bases long. In some embodiments, the repair template is between 20 and 100 bases long. In some embodiments, the repair template is between 20 and 200 bases long. In some embodiments, the repair template is between 20 and 300 bases long. In some embodiments, the repair template is between 20 and 400 bases long. In some embodiments, the repair template is between 20 and 500 bases long. In some embodiments, the repair template is between 20 and 600 bases long. In some embodiments, the repair template is between 20 and 700 bases long. In some embodiments, the repair template is between 20 and 800 bases long. In some embodiments, therepair template is between 20 and 900 bases long. In some embodiments, the repair template is between 20 and 1000 bases long.

[0316] In some embodiments, the donor polynucleotide is attached to the guide RNA. In some embodiments, the donor polynucleotide is attached to the guide RNA through the 5’ end of the donor polynucleotide and the 3’ end of the guide RNA. In some embodiments, the donor polynucleotide is attached to the guide RNA through a linker. In some embodiments, the linker is attached to the 5’ end of the donor polynucleotide and the 3’ end of the guide RNA.

[0317] In some embodiments, the linker is one nucleotide long. In some embodiments, the linker is 2 nucleotides long. In some embodiments, the linker is 3 nucleotides long. In some embodiments, the linker is 4 nucleotides long. In some embodiments, the linker is 5 nucleotides long. In some embodiments, the linker is 6 nucleotides long. In some embodiments, the linker is 7 nucleotides long. In some embodiments, the linker is 8 nucleotides long. In some embodiments, the linker is 9 nucleotides long. In some embodiments, the is 10 nucleotides long. In some embodiments, the linker is 11 nucleotides long. In some embodiments, the linker is 12 nucleotides long. In some embodiments, the linker is 13 nucleotides long. In some embodiments, the linker is 14 nucleotides long. In some embodiments, the linker is 15 nucleotides long. In some embodiments, the linker is 16 nucleotides long. In some embodiments, the linker is 17 nucleotides long. In some embodiments, the linker is 18 nucleotides long. In some embodiments, the linker is 19 nucleotides long. In some embodiments, the linker is 20 nucleotides long. In some embodiments, the linker is 25 nucleotides long. In some embodiments, the linker is 30 nucleotides long.

[0318] In some embodiments, the one or more donor polynucleotides are separated by one or more ribozyme cleavage sites.

[0319] In some embodiments, the target site for the donor polynucleotide is the I-TevI cleavage site. In some embodiments, the target site for the donor polynucleotide is the Cas cleavage site.

[0320] In some embodiments, the one or more donor polynucleotides comprise a 2 nucleotide overhang compared to corresponding bases 3’ adjacent to the target site. In some embodiments, the one or more donor polynucleotides comprise a 3’ nucleotide overhang compared to the target site. In some embodiments, the one or more donor polynucleotides comprise a 4-nucleotide overhang compared to the target site. In some embodiments, the one or more donorpolynucleotides comprise a 5-nucleotide overhang compared to the target site. In some embodiments, the one or more donor polynucleotides comprise a 6-nucleotide overhang compared to the target site. In some embodiments, the one or more donor polynucleotides comprise a 7-nucleotide overhang compared to the target site. In some embodiments, the one or more donor polynucleotides comprise a 8-nucleotide overhang compared to the target site. In some embodiments, the one or more donor polynucleotides comprise a 9-nucleotide overhang compared to the target site. In some embodiments, the one or more donor polynucleotides comprise a 10-nucleotide overhang compared to the target site. In some embodiments, the one or more donor polynucleotides comprise a 11 -nucleotide overhang compared to the target site. In some embodiments, the one or more donor polynucleotides comprise a 12-nucleotide overhang compared to the target site. In some embodiments, the one or more donor polynucleotides comprise a 13-nucleotide overhang compared to the target site. In some embodiments, the one or more donor polynucleotides comprise a 14-nucleotide overhang compared to the target site. In some embodiments, the one or more donor polynucleotides comprise a 15-nucleotide overhang compared to the target site. In some embodiments, the one or more donor polynucleotides comprise a 16-nucleotide overhang compared to the target site. In some embodiments, the one or more donor polynucleotides comprise a 17-nucleotide overhang compared to the target site. In some embodiments, the one or more donor polynucleotides comprise a 18-nucleotide overhang compared to the target site. In some embodiments, the one or more donor polynucleotides comprise a 19-nucleotide overhang compared to the target site. In some embodiments, the one or more donor polynucleotides comprise a 20-nucleotide overhang compared to the target site. In some embodiments, the one or more donor polynucleotides comprise a 2 to 16-nucleotide overhang compared to the target site. In some embodiments, the one or more donor polynucleotides comprise a 2 to 18-nucleotide overhang compared to the target site. In some embodiments, the one or more donor polynucleotides comprise a 2 to 20- nucleotide overhang compared to the target site. In some embodiments, the one or more donor polynucleotides comprise a 4 to 16-nucleotide overhang compared to the target site. In some embodiments, the one or more donor polynucleotides comprise a 4 to 18-nucleotide overhang compared to the target site. In some embodiments, the one or more donor polynucleotides comprise a 4 to 20-nucleotide overhang compared to the target site.

[0321] In some embodiments, the second guide RNA targets a region 5’ to the donor polynucleotide target site. In some embodiments, the 3’ end of the donor polynucleotide is complementary to the overhang created by the cleavage of the second I-TevI at the second I-TevI target site.

[0322] In some embodiments, the one or more donor polynucleotides are at least 30 bases long. In some embodiments, the one or more donor polynucleotides are at least between 30 and 1500 bases long. In some embodiments, the one or more donor polynucleotides are at least between 30 and 100 bases long. In some embodiments, the one or more donor polynucleotides are at least between 40 and 90 bases long. In some embodiments, the one or more donor polynucleotides are at least between 50 and 80 bases long. In some embodiments, the one or more donor polynucleotides are at least between 60 and 70 bases long. In some embodiments, the one or more donor polynucleotides are at least between 100 and 1000 bases long. In some embodiments, the one or more donor polynucleotides are at least between 100 and 1500 bases long. In some embodiments, the one or more donor polynucleotides are at least between 200 and 800 bases long. In some embodiments, the one or more donor polynucleotides are at least between 300 and 700 bases long. In some embodiments, the one or more donor polynucleotides are at least between 400 and 600 bases long. In some embodiments, the one or more donor polynucleotides are at least 500 bases long.

[0323] In some embodiments, the donor polynucleotide can be inserted in the AA VS J safe harbor site. The nucleic acid sequence for the AAVS1 integration site is set forth in SEQ ID NO: 1.

[0324] In some embodiments, the donor polynucleotide comprises a donor polynucleotide sequence selected from Table 8.

[0325] In some embodiments, the donor polynucleotide comprises one of the nucleic acid sequences set for in SEQ ID NOs: 12-14.

[0326] In some embodiments, the donor polynucleotide comprises one of the nucleic acid sequences set for in SEQ ID NOs: 42-45.

[0327] In some embodiments, the donor polynucleotide comprises the B2M inactivating sequence SEQ ID NO: 52.

[0328] In some embodiments, the donor polynucleotide can be inserted in the CFTR gene. In some embodiments, the donor polynucleotide can be used for the repairing the CFTR gene. Insome embodiments, the donor polynucleotide comprises a nucleic acid sequence SEQ ID NO: 67 to 69. In some embodiments, the donor polynucleotide comprises a nucleic acid sequence 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 67 to 69, or 71-74.

[0329] In some embodiments, the donor polynucleotide can be inserted in the SERPINA1 gene. In some embodiments, the donor polynucleotide can be used for the repairing the SERPINA1 gene. In some embodiments, the donor polynucleotide comprises a SERPINA1 sequence SEQ ID NO: 70. In some embodiments, the donor polynucleotide comprises a nucleic acid sequence 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 70 or 75. In some embodiments, the donor polynucleotide comprises a nucleic acid sequence 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 226 to 229. In some embodiments, the donor polynucleotide comprises a SERPINA1 nucleic acid sequence selected from SEQ ID NOs: 119-122. In some embodiments, the donor polynucleotide comprises a nucleic acid sequence 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NO: 119-122.

[0330] In some embodiments, the guide sequence with donor polynucleotide comprises a SERPINA1 nucleic acid sequence selected from SEQ ID NOs: 123-126. In some embodiments, the donor polynucleotide comprises a nucleic acid sequence 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 123-126.

[0331] Table 8 shows exemplary sequences for the target site and donor polynucleotide. All sequences are paired with an exemplary scaffold sequence GTTTTAGTACTCTGGAAACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCT CGTCAACTTGTTGGCGAGAT (SEQ ID NO: 87).

[0332] In some embodiments, the guide polynucleotide and donor polynucleotide comprise a nucleic acid sequence 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOs: 88-100. In some embodiments, the guide polynucleotide and donor polynucleotide comprise a nucleic acid sequence 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOs: 101-103 or 105-108. In some embodiments, the guide polynucleotide and donor polynucleotide comprise a nucleic acid sequence 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOs: 104 or 109. In some embodiments, the guide polynucleotide and donor polynucleotide comprise a nucleic acid sequence 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOs: 230 - 233.

[0333] TABLE 8: Exemplary Donor Polynucleotide Sequences and Target Sites0334] In some embodiments, the donor polynucleotide encodes a protein or a fragment thereof. In some embodiments, the protein is a fluorescent protein. In some embodiments, the fluorescent protein is a GFP, an eGFP, a RFP, a YFP, a BFP, or a CFP.

[0335] In some embodiments, the donor polynucleotide further comprises a coding sequence for a self-cleaving peptide. Exemplary self-cleaving peptides include T2A, P2A, E2A, and F2A self-cleaving peptides. In some embodiments, the T2A self-cleaving peptide comprises a sequence of EGRGSLLTCGDVEENPGP. In some embodiments, the P2A self-cleaving peptide comprises a sequence of ATNFSLLKQAGDVEENPGP. In some embodiments, the E2A selfcleaving peptide comprises a sequence of QCTNY ALLKLAGDVESNPGP. In some embodiments, the F2A self-cleaving peptide comprises a sequence of VKQTLNFDLLKLAGDVESNPGP. In some embodiments, the T2A self-cleaving peptide comprises a sequence of EGRGSLLTCGDVEENPGP. Any of the foregoing can also include anN terminal GSG linker. For example, a T2A self-cleaving peptide can also comprise a sequence of GSGEGRGSLLTCGDVEENPGP.

[0336] In some embodiments, the donor polynucleotide further comprises a trans-acting double-stranded RNA polynucleotide with 3’- or 5 ’-overhanging nucleotide. In some embodiments, the donor polynucleotide further comprises a cis-acting single-strand RNA polynucleotide with sequence similarity to the target strand of the nuclease. In some embodiments, the donor polynucleotide further comprises a cis-acting single-strand RNA polynucleotide with sequence similarity to the non-target strand of the nuclease. In some embodiments, the donor polynucleotide further comprises one or more binding sites for a genome modifying factor optionally a binding site for a site-specific recombinase, such as serine recombinases or LoxP target sites. In some embodiments, the donor polynucleotide further comprises a repair template for a protein coding sequence. In some embodiments, the donor polynucleotide further comprises one or more exons with splice acceptor and donor sequences. In some embodiments, the donor polynucleotide further comprises one or more selectable sequences selected from the group of NeoR, BsdR, HygR, PuroR, and BleoR genes. In some embodiments, the donor polynucleotide further comprises one or more drug-inducible regulatory sequences for controlled gene expression. In some embodiments, the donor polynucleotide comprises a combination of the above.

[0337] In some embodiments, the donor polynucleotide comprises a self-complementing single-strand RNA repair template separated by ribozyme cleavage sites that when transcribed acts as a trans-acting double strand nucleic acid to repair target sequences through the NHEJ pathway.Promoters

[0338] In another aspect, provided herein are certain expression control sequences useful for expressing the recombinant nucleic acids provided herein. Non-limiting exemplary embodiments of the recombinant nucleic acids of the disclosure can include one or more of the following features.

[0339] In some embodiments, any of the recombinant nucleic acids provided herein can be operably linked, e.g., placed under the control of, other structural elements (e.g., promotersequences) required for expression of such recombinant nucleic acids in host cells, in subjects, or in ex-vivo cell-free expression systems.

[0340] As used herein, the terms “promoter” and “promoter sequence” are used interchangeably to refer to a DNA sequence that promotes the expression of a protein coding open reading frame or a nucleotide sequence encoding ana functional RNA (e.g., a guide polynucleotide or a donor polynucleotide). an shRNA). Those skilled in the art understand that different promoters direct gene expression in different tissues or cell types, at different stages of development, or in response to different environmental or physiological conditions.

[0341] In some embodiments, the nucleic acid further comprises one or more promoters.

[0342] In some embodiments, the nucleic acid further comprises one promoter driving the expression of the chimeric nuclease and the guide RNA together. In some embodiments, the promoter is operably linked to the nucleic acid encoding the chimeric nuclease. In some embodiments, the promoter is operably linked to the nucleic acid encoding the guide RNA. In some embodiments, the promoter is operably linked to the nucleic acid sequence encoding the whole operon of the disclosure.guide RNA.

[0343] In some embodiments, the promoter is a CMV promoter, SV40 promoter, minimal cytomegalovirus (CMV) promoter, or a human elongation factor- 1 alpha (EFla) promoter. In some embodiments, the promoter is a mini CMV promoter. In some embodiments, the promoter is an MND promoter. In some embodiments, the promoter is a U6 promoter. In some embodiments, the promoter is a T7 promoter.

[0344] In some embodiments, the promoter is a tissue or cell specific promoter. In some embodiments, the promoter is a muscle-specific synthetic promoter SPc5-12 neuronal-specific promoter hSYNl, aldhlLl promoter, cTNT promoter, alpha-MHC promoter, SPc5-12 promoter, MUC2 promoter, Ksp-cadherin promoter, Albumin promoter, HAS promoter, insulin promoter, rhodopsin promoter, rNSE promoter, or a Cone-opsin promoter. In some embodiments, the promoter is a T7 promoter.

[0345] In some embodiments, the promoter is a CMV promoter set forth in SEQ ID NO: 6.

[0346] In some embodiments, the donor polynucleotide is inserted into the genome in frame with an endogenous coding sequence. In some embodiments, the donor polynucleotide is inserted into the genome in frame for expression from an endogenous promoter.Ribozymes and Transfer RN As

[0347] In some embodiments, the nucleic acid further comprises a nucleic acid sequence encoding one or more self-cleaving ribozymes or endogenous RNA processing sequences, for example tRNAs. Self-cleaving ribozymes are catalytic RNA molecules that cleave their own phosphodiester backbone. Introduction of self-cleaving ribozymes can ensure clean 5’ or 3’ ends of a guide RNA, depending on placement of the ribozyme in an mRNA. In some embodiments, the ribozyme is placed 5’ end of the guide RNA. In some embodiments, the ribozyme is placed directly adjacent to the 5’ end of the guide RNA.

[0348] In some embodiments, the ribozyme is a hammerhead ribozyme or a hepatitis delta virus (HDV) ribozyme. In some embodiments, the ribozyme is a hammerhead ribozyme encoded in a nucleic acid sequence according to SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 111. In some embodiments, the ribozyme is a HDV ribozyme encoded in a nucleic acid sequence according to SEQ ID NO: 10 or SEQ ID NO: 112.

[0349] Exemplary Ribozyme sequences are listed in Table 9. In some embodiments, the ribozyme encoded in a nucleic acid sequence according to any one of SEQ ID NOs:289 to 301.Table 9. Exemplary ribozyme sequences for conditionally cleavable elements

[0350] In some embodiments, the ribozyme sequence is located inside the tRNA sequence and is trans-acting. In some embodiments the tRNA sequence comprising a ribozyme sequence is set forth in SEQ ID NO: 5. It is to be understood that the DNA sequence is transcribed into an RNA sequence and the ribozyme is active when present as an RNA. In some embodiments, the tRNA is a glycine arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine tRNAIn some embodiments, one or more tRNAs separate one or more guide RNAs. tRNAs are conditionally cleaved inside cells when expressed as RNA through the endogenous tRNA maturation process resulting in cleavage of an RNA into two or more parts. In some embodiments, one or more tRNAs separate an RNA stability sequence and one or more tRNAs. In some embodiments, the tRNA is encoded in a nucleic acid sequence according to SEQ ID NO: 127 or SEQ ID NO: 128. In some embodiments, the tRNA is encoded in a nucleic acid sequence according to Table 10. In some embodiments, the tRNA is encoded in a nucleic acid sequence according to any one of SEQ ID NO: 302 to SEQ ID NO: 733.Table 10. Exemplary tRNA sequences for conditionally cleavable elementsRNA Stabilizing Sequences

[0001] In some embodiments, the nucleic acid further comprises RNA stabilizing polynucleotide.

[0002] In some embodiments, the nucleic acid comprises a polyadenylation signal. In some embodiments, the polyadenylation signal comprises a simian virus 40 (SV40), a globin, P- globin, a human growth hormone (hGH), a bovine growth hormone (BGH), a herpes simplex virus type 1, a thymidine kinase (HSV TK), or a synthetic polyadenylation (Synt poly A) poly adenylation signal.

[0003] In some embodiments, the RNA stabilizing polynucleotide is a PolyA signal. The poly(A) tail acts as the binding site for poly(A)-binding protein. Poly (A) -binding protein promotes export from the nucleus and translation, and inhibits degradation. In some embodiments, the polyadenylation signal is set forth in SEQ ID NO: 11. Generally, PolyA signals are located at the 3’ end of an mRNA transcript. A PolyA signal typically cannot be located at the 5 ’ end or inside of an mRNA transcript. Ribozyme cleavage of the single mRNA transcribed from a nucleic acid described herein, exposes a free 3’ end on an mRNA. To protect the 3’ end from degradation, a second RNA stabilizing polynucleotide other than a Poly A can be introduced.

[0004] In some embodiments, the RNA stabilizing polynucleotide is a metastasis-associated lung adenocarcinoma transcript 1 (MALAT) 3' sequence, or a 3’-end of the multiple endocrine neoplasia beta transcript (MEN P). In some embodiments, the MALAT sequence is a nucleic acid sequence as set forth in SEQ ID NO: 7.

[0005] In some embodiments, the RNA stabilizing polynucleotide a triple helix RNA structure or a 3 ’-end of a RNA transcript lacking a canonical polyadenylation signal.

[0006] In some embodiments, the RNA stabilizing polynucleotide is located at the 3’ end of the polynucleotide encoding the chimeric nuclease. In some embodiments, the RNA stabilizing polynucleotide is located 5’ of a ribozyme cleavage site. In some embodiments, the RNA stabilizing polynucleotide protects the mRNA encoding for the polypeptide portion of the chimeric nuclease from degradation.

[0007] In some embodiments, the nucleic acid further comprises a 5’UTR and / or a 3’UTR sequence.

[0008] In some embodiments, a composition comprising a chimeric nuclease polypeptide comprising an I-TevI domain and a RNA-guided nuclease domain and a nucleic acid of the disclosure is provided.

[0009] In some embodiments, a composition comprising a chimeric nuclease nucleic acid encoding a chimeric nuclease comprising an I-TevI domain and a RNA-guided nuclease domain and a nucleic acid of the disclosure is provided. In some embodiments, the chimeric nuclease nucleic acid is an mRNA.Additional Elements

[0010] In another aspect, the nucleic acids provided herein can further include additional regulatory elements. In some embodiments, the additional regulatory element can be a WPRE sequence, a polyA sequence, and / or a viral replication and packaging control sequence, an long terminal repeat (LTR) sequence.

[0011] In some embodiments, the recombinant nucleic acid includes a poly adenylation (PolyA) signal. In some embodiments, the polyadenylation signal includes a simian virus 40 (SV40), a globin, P-globin, a human growth hormone (hGH), a bovine growth hormone (BGH), a herpes simplex virus type 1, a thymidine kinase (HSV TK), or a synthetic polyadenylation (Synt poly A) polyadenylation signal.

[0012] In some embodiments, the viral replication and packaging control sequence is a long terminal repeat (LTR) sequence. In some embodiments, the viral replication and packaging control sequence is an inverted terminal repeat (ITR) sequence.Vectors

[0013] In some embodiments, the nucleic acids of the disclosure can be incorporated into an expression vector or a vector used for virus production. The vector can be a plasmid, phage, or cosmid, into which another DNA segment can be inserted so as to bring about the replication of the inserted segment. In some embodiments, the expression vector can be an integrating vector. Accordingly, also provided herein are vectors, plasmids, or viruses containing one or more of the nucleic acids encoding any of the nucleic acids disclosed herein. The nucleic acid described above can be contained within a vector that is capable of directing their expression in, for example, a cell that has been transduced with the vector. Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available or readily prepared by a skilled artisan. Additional vectors can also be found, for example, in Ausubel, F.M., et al.,Current Protocols in Molecular Biology, (Current Protocol, 1994) and Sambrook et al., “Molecular Cloning: A Laboratory Manual,” 2nd ED. (1989).Viral Vectors

[0014] In some embodiments, the nucleic acid of the disclosure can be packaged in a viral particle or viral vector. Methods for generating viral vectors from various virus types are known in the art. Exemplary types of viral particles that may be recombinantly engineered as delivery vehicles include retroviruses, lentivirus (e.g., HIV and its derivatives and SIV), adeno-associated virus, adenovirus, MMLV retrovirus, MSCV retrovirus, baculovirus, vesicular stomatitis virus, herpes simplex virus, and vaccinia virus. Examples include adeno-associated virus (AAV) particles used for gene therapy. In some embodiments, the virus can be an AAV. In some embodiments, the AAV is a AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV7, AAV8, AAV9, AAV10, AAV-DJ, AAV2.5T, or AAVmyo. In some embodiments, the virus can be a lentivirus.Pharmaceutical Compositions

[0015] In another aspect, provided herein are compositions and pharmaceutical compositions suitable for administration to a human subject including any of the chimeric nucleases and chimeric nuclease systems, nucleic acids, vectors or viral vectors disclosed and described herein. The chimeric nucleases and chimeric nuclease systems, nucleic acids, vectors, or viral vectors of the disclosure can be formulated as compositions, including pharmaceutical compositions. Such compositions generally include one or more chimeric nucleases and chimeric nuclease systems, nucleic acids, vectors, or viral vectors as disclosed and described herein, and a pharmaceutically acceptable excipient, e.g., a carrier. In some embodiments, the compositions of the disclosure are formulated for the treatment, or management of a health condition. In some embodiments, the health condition is a congenital disease. For example, the compositions of the disclosure can be formulated as a therapeutic composition, or a pharmaceutical composition including a pharmaceutically acceptable excipient, or a mixture thereof. In some embodiments, the compositions of the present disclosure are formulated for use as a therapy for cystic fibrosis. In some embodiments, the compositions of the present disclosure are formulated for use as a therapy for alpha- 1 -antitrypsin deficiency. In some embodiments, the compositions of the present disclosure are formulated for use as a therapy for myotonic dystrophy type 1. . In someembodiments, the compositions of the present disclosure are formulated for use as a therapy for amyotrophic lateral sclerosis or frontotemporal dementia.

[0016] Accordingly, in one aspect, provided herein are pharmaceutical compositions including a pharmaceutically acceptable excipient and a nucleic acid, vector, or viral vector of the disclosure.

[0017] In some embodiments, a pharmaceutical composition comprising a chimeric nuclease, a chimeric nuclease system, a nucleic acid, a vector, a viral vector, an AAV of the disclosure, a composition of the disclosure, or an LNP composition of the disclosure; and an excipient is provided.

[0018] In some embodiments, the compositions described herein, e.g., chimeric nucleases and chimeric nuclease systems, nucleic acids, vectors, or viral vectors, and / or pharmaceutical compositions are incorporated into therapeutic compositions for use in methods of preventing or treating a subject who has, who is suspected of having, or who may be at high risk for developing cystic fibrosis. In some embodiments, the compositions described herein, e.g., chimeric nucleases and chimeric nuclease systems, nucleic acids, vectors, or viral vectors, and / or pharmaceutical compositions are incorporated into therapeutic compositions for use in methods of preventing or treating a subject who has, who is suspected of having, or who may be at high risk for developing alpha- 1 -antitrypsin deficiency. In some embodiments, the compositions described herein, e.g., chimeric nucleases and chimeric nuclease systems, nucleic acids, vectors, or viral vectors, and / or pharmaceutical compositions are incorporated into therapeutic compositions for use in methods of preventing or treating a subject who has, who is suspected of having, or who may be at high risk for developing myotonic dystrophy type 1. In some embodiments, the compositions described herein, e.g., chimeric nucleases and chimeric nuclease systems, nucleic acids, vectors, or viral vectors, and / or pharmaceutical compositions are incorporated into therapeutic compositions for use in methods of preventing or treating a subject who has, who is suspected of having, or who may be at high risk for developing amyotrophic lateral sclerosis or frontotemporal dementia.

[0019] In these cases, the composition should be sterile, formulated to facilitate administration to a human subject, stable under the conditions of manufacture and storage such that is resistant to contamination by microorganisms such as bacteria and fungi.

[0020] In some embodiments, the composition is formulated for one or more of intramuscular administration, intranodal administration, intravenous administration, intratracheal administration, intraperitoneal administration, or intra-cranial administration.Cellular Delivery

[0021] The chimeric nucleases and chimeric nuclease systems, nucleic acids, vectors or viral vectors disclosed and described herein can be delivered to a cell for genome editing.

[0022] In some embodiments, the delivery is a non-viral delivery or a viral delivery. In some embodiments, the nuclease can be delivered as a ribonucleoprotein complex, a DNA encoding the nuclease system, or as messenger RNA encoding the nuclease system, or a combination thereof. For example, the polypeptide portion of the nuclease can be delivered to a cell as a protein, and the guide RNA / donor can be delivered as an RNA.

[0023] In some embodiments, the disclosure provides for a vector comprising the nucleic acids described herein. In some embodiments, the chimeric nuclease systems and nucleic acids encoding for chimeric nuclease systems can delivered to the cell using lipofection or polymer- based transfection. In some embodiments, the nucleic acid is comprised in a lipid nanoparticle (LNP). In some embodiments, the chimeric nuclease system is comprised in a lipid nanoparticle.

[0024] In some embodiments, the nucleic acid encoding for a chimeric nuclease system can be delivered to a cell using viral delivery. In some embodiments, the nucleic acid is packaged into a viral vector. Viral vectors can be used to transduce mammalian cells with the nucleic acid of the disclosure. In some embodiments, the virus is a lentivirus, adeno-associated virus (AAV), adenovirus, retrovirus, or modified Herpes Simplex Virus (HSV). In some embodiments, the AAV is a AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV7, AAV8, AAV9, AAV10, AAV-DJ, AAV2.5T or AAVmyo.

[0025] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell (such as T-cells), hematopoietic stems cells, mesenchymal stem cells, or induced pluripotent stem cells (iPSCs). Cell types that can be modified ex vivo by the methods and nuclease described herein include immune cells such as T ceil or NK-cells, or pluripotent cells such as mesenchymal stem cells, hematopoietic stems cell, or cell otherwise induced to pluripotency using techniques known in the art. In some embodiments, the cell is a muscle cell. In some embodiments, the cell is a brain cell.Methods of Using Chimeric Nuclease Systems

[0026] In one aspect of the disclosure, methods for editing the genome of a cell with the chimeric nuclease systems and nucleic acids encoding for chimeric nuclease systems described herein are provided.

[0027] In some embodiments, the method comprises contacting a cell with the nucleic acid of the disclosure. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the method comprises contacting a cell with a virus comprising a nucleic acid of the disclosure. In some embodiments, the method comprises contacting a cell with a vector comprising a nucleic acid of the disclosure. In some embodiments, the method comprises contacting a cell with an LNP comprising a nucleic acid of the disclosure.

[0028] In some embodiments, a method of removing precise lengths of DNA from the genome of a cell is provided, the method comprising, providing to the cell a chimeric nuclease that cleaves at two sites to remove the precise length of DNA and leaves two different DNA ends. The two DNA ends provide the necessary target site for accurate repair through error-free NHEJ or RNA-dependent PolQ- mediated repair enabling repair in all cell cycle phases including G1 / G0.

[0029] In some embodiments, a method of editing the genome of a cell at a chimeric nuclease target site is provided, the method comprising providing to the cell a chimeric nuclease with a repair polynucleotide with a 2 base overhang corresponding to the bases 3’ adjacent to the I-TevI target site in the genome of the cell. In some embodiments, a method of editing the genome of a cell at a target site is provided, the method comprising providing to the cell a chimeric nuclease with a repair polynucleotide with a 2-18 base overhang corresponding to the bases 3’ adjacent to the I-TevI target site in the genome of the cell. In some embodiments, a method of editing the genome of a cell at a target site is provided, the method comprising providing to the cell a chimeric nuclease with a repair polynucleotide with a 14 base overhang corresponding to the bases 3’ adjacent to the I-TevI target site in the genome of the cell. In some embodiments, the repair polynucleotide comprises a donor polynucleotide. In some embodiments, the repair polynucleotide comprises a guide polynucleotide.

[0030] In some embodiments, a method of delivering messenger RNA encoding a chimeric nuclease comprising an I-TevI domain and an RNA-guided nuclease domain to a cell is providedthe method comprising contacting the cell with a polynucleotide encoding one or more guide RNAs and polynucleotide donor.

[0031] In some embodiments, a method genetically modifying the genome of a cell is provided, the method comprising contacting the cell with a nucleic acid of the disclosure, a vector of the disclosure, a viral vector of the disclosure, or a AAV of the disclosure, the composition of the disclosure, or the LNP composition of the disclosure.

[0032] In some embodiments, the modification comprises an insertion, deletion, substitution, or mutation of the genome of a cell.

[0033] In some embodiments, the insertion of the donor polynucleotide into the genome of the cell results in removal of sequences between a I-TevI target site and a Cas9 target site.

[0034] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0035] In some embodiments, a method of inserting or replacing a sequence at a chimeric nuclease target site in a genome in a cell is provided, the method comprising: contacting the cell with a nucleic acid comprising a chimeric nuclease comprising a Cas9 domain and a I-TevI domain; and a nucleic acid comprising a guide polynucleotide and a donor polynucleotide; wherein the guide polynucleotide and the chimeric nuclease form a complex, and the complex binds and cleaves the genomic DNA at a Cas9 target site and a I-TevI target site; wherein the 3’ end of the donor polynucleotide comprises at least 2 bases complementarity over the 5 ’ end of the I-TevI target site; and wherein the donor polynucleotide is incorporated into the chimeric nuclease target site at a position 5’ to the Cas9 target site.

[0036] In some embodiments, a cellular polymerase is targeted to the chimeric nuclease target site. In some embodiments, the cellular polymerase is polymerase theta.

[0037] In some embodiments, the 3’ end of the guide polynucleotide and the 5’ end of the donor polynucleotide are connected.

[0038] In some embodiments, a method of editing the genome of a cell at a target site is provided, the method comprising providing to the cell a chimeric nuclease with a repair polynucleotide that is a cis-acting single strand nucleic acid encoding the sense strand of the repair sequence or an antisense strand of the repair sequence and is repaired through an RNA- dependent Rad52-mediated repair pathway.

[0039] In some embodiments, a method of editing the genome of a cell at a target site is provided, the method comprising providing to the cell a purified protein chimeric nuclease complexed with one or more synthetic guide RNA sequences separated by one or more synthetic tRNA sequences encoding trans-acting ribozyme sequences, further comprising donor DNA sequences separated by one or more ribozyme cleavage sites.

[0040] In some embodiments, a method of editing the genome of a cell at a target site to create large, predictable deletions in target sites through error-free NHEJ is provided, the method comprising providing to the cell a dual-guided chimeric nuclease system comprising a catalytically inactive Cas9 domain a catalytically active I-TevI domain, and two guide RNAs targeting two different target sites.

[0041] In some embodiments, a method of replacing at least a portion of a CFTR gene in the genome of a cell is provided, the method comprising contacting a nucleic acid of the disclosure, a vector of the disclosure, a viral vector of the disclosure, or a AAV of the disclosure, the composition of the disclosure, or the LNP composition of the disclosure to the cell. In some embodiments, the guide RNA and donor polynucleotide target mutations in the CFTR gene. In some embodiments, the chimeric nuclease with the guide RNA and donor polynucleotide target and replace the CFTR c.l521_1523del (p.Phe508del), C.1624G>T (p.Gly542Ter), C.1652G>A (p.Gly551Asp), c.!657C>T (p.Arg553Ter), or c.3846G>A (p.Trp!282Ter) mutations.

[0042] In some embodiments, a method of replacing at least a portion of a SERPINA1 gene in the genome of a cell is provided, the method comprising contacting a nucleic acid of the disclosure, a vector of the disclosure, a viral vector of the disclosure, or a AAV of the disclosure, the composition of the disclosure, or the LNP composition of the disclosure to the cell. In some embodiments, the guide RNA and donor polynucleotide target mutations in the SERPINA1 gene. In some embodiments, the guide RNA and donor polynucleotide target and replace the SERPINA1 c.1096 A (p.Glu342Lys) mutation.

[0043] In some embodiments, a method of excising at least a portion of a DMPK gene in the genome of a cell is provided, the method comprising contacting a nucleic acid of the disclosure, a vector of the disclosure, a viral vector of the disclosure, or a AAV of the disclosure, the composition of the disclosure, or the LNP composition of the disclosure to the cell. In some embodiments, a dual-guided chimeric nuclease excises and deletes (CTG)n * (CAG)n trinucleotide repeats in the 3' untranslated region of the DMPK gene.

[0044] In some embodiments, a method of excising at least a portion of a C9ORF72 gene in the genome of a cell is provided, the method comprising contacting a nucleic acid of the disclosure, a vector of the disclosure, a viral vector of the disclosure, or a AAV of the disclosure, the composition of the disclosure, or the LNP composition of the disclosure to the cell. In some embodiments, a dual-guided chimeric nuclease excises and deletes GGGGCC repeats in the intronic sequence between alternate 5' exons of the C9ORF72 gene. In some embodiments, the chimeric nuclease, guide RNA, and donor polynucleotide target and remove large hexanucleotide GGGGCC repeats between Exon la and Exon lb of the C9ORF72 gene.

[0045] In some embodiments, a method of removing at least a portion of a DMPK gene in the genome in a cell is provided, the method comprising contacting the nucleic acid of the disclosure, a vector of the disclosure, a viral vector of the disclosure, or a AAV of the disclosure, the composition of the disclosure, or LNP composition of the disclosure to a cell. In some embodiments, two guide RNAs of the disclosure target two different mutations in the DMPK gene. In some embodiments, the chimeric nuclease, guide RNA, and donor polynucleotide target and remove large triplet CAG repeat in the 3’ untranslated region of the DMPK gene.

[0046] In some embodiments, a method of tunable editing the genome of a cell at a target site is provided, the method comprising providing to the cell a nucleic acid expressing a chimeric nuclease system described herein, the nucleic acid further comprising a self-inactivating sequence cleaved by the chimeric nuclease. In some embodiments, the genome target site is B2M and the targeting sequence is SEQ ID NO: 51 or SEQ ID NO: 130. In some embodiments, the self-inactivating sequence is SEQ ID NO: 52 or SEQ ID NO: 131.

[0047] In some embodiments, a method of inserting a sequence into a target site of the genome of a cell at the target site is provided, the method comprising providing to the cell a nucleic acid comprising a nuclease system described herein and a donor polynucleotide.

[0048] In some embodiments, a method of inserting a polypeptide sequence into a target site of the genome of a cell at the target site is provided, the method comprising providing to the cell a nucleic acid comprising a nuclease system described herein and a donor polynucleotide.

[0049] In some embodiments, a method of removing large repeated sequences of DNA that cause disease from the genome of a cell is provided, the method comprising, providing a chimeric nuclease that cleaves at two sites to remove the large repeated sequences of DNA.

[0050] In some embodiments, a method of inserting a donor polynucleotide in frame with an endogenous promoter is provided, the method comprising providing to the cell a nucleic acid comprising a nuclease system described herein and a donor polynucleotide encoding for a polypeptide and T2A cleavable peptide sequence.

[0051] In some embodiments, a method of replacing at least a portion of a DMPK gene in the genome in a cell, the method comprising contacting the nucleic acid of the disclosure, a vector of the disclosure, a viral vector of the disclosure, or a AAV of the disclosure, the composition of the disclosure, or LNP composition of the disclosure to the cell. In some embodiments, one or more guide RNAs target mutations in the DMPK gene. In some embodiments, one or more guide RNAs target CAG triplet polynucleotide sequences in the 3 ’ untranslated region of the DMPK gene.

[0052] In some embodiments, the excision of deletion is 30 base pairs in length. In some embodiments, the deletion is 35 base pairs in length. In some embodiments, the deletion is 40 base pairs in length. In some embodiments, the deletion is 45 base pairs in length. In some embodiments, the deletion is 50 base pairs in length. In some embodiments, the deletion is 60 base pairs in length. In some embodiments, the deletion is 70 base pairs in length. In some embodiments, the deletion is 80 base pairs in length. In some embodiments, the deletion is 90 base pairs in length. In some embodiments, the deletion is 150 base pairs in length. In some embodiments, the deletion is 30 base pairs in length. In some embodiments, the deletion is 200 base pairs in length. In some embodiments, the deletion is 300 base pairs in length. In some embodiments, the deletion is 400 base pairs in length. In some embodiments, the deletion is 500 base pairs in length. In some embodiments, the deletion is 600 base pairs in length. In some embodiments, the deletion is 700 base pairs in length. In some embodiments, the deletion is 800 base pairs in length. In some embodiments, the deletion is 900 base pairs in length. In some embodiments, the deletion is 1000 base pairs in length. In some embodiments, the deletion is 1500 base pairs in length. In some embodiments, the deletion is 2000 base pairs in length. In some embodiments, the deletion is 2500 base pairs in length. In some embodiments, the deletion is 3000 base pairs in length. In some embodiments, the deletion is 4000 base pairs in length. In some embodiments, the deletion is 5000 base pairs in length. In some embodiments, the deletion is 6000 base pairs in length. In some embodiments, the deletion is 7000 base pairs in length. In some embodiments, the deletion is 8000 base pairs in length. In some embodiments, the deletionis 9000 base pairs in length. In some embodiments, the deletion is 10000 base pairs in length. In some embodiments, the deletion is more than 10000 base pairs in length.Methods of Treatment

[0053] In another aspect, provided herein are methods of using the chimeric nucleases and chimeric nuclease systems, nucleic acids, vectors or viral vectors disclosed and described herein for the treatment of human subjects in need thereof.

[0054] Such the chimeric nucleases and chimeric nuclease systems, nucleic acids, vectors or viral vectors disclosed and described herein are suitable for use as gene therapy for the treatment of a subject in need thereof. In some embodiments, the chimeric nucleases and chimeric nuclease systems, nucleic acids, vectors or viral vectors disclosed and described herein include therapeutic agents for use in methods of treating a subject who has, is suspected of having, or may be at high risk for developing one or more health conditions or diseases treatable with such gene therapies. Exemplary health conditions or diseases can include, without limitation, congenital diseases, for example cystic fibrosis, alpha- 1 -antitrypsin deficiency, myotonic dystrophy type 1, amyotrophic lateral sclerosis or frontotemporal dementia.Cystic Fibrosis

[0055] Cystic fibrosis (CF) is an autosomal-recessive disease resulting from mutations in the CFTR gene, which encodes an epithelial anion channel. The CFTR protein, cystic fibrosis transmembrane conductance regulator, is found across a wide range of organs including pancreas, kidney, liver, lungs, gastrointestinal tracts, and reproductive tracts, making CF a multiorgan disease. Mutations in CFTR gene (GRCh38.pl4 GCF_000001405.40, NM_000492.4) can lead to suboptimal ion transport and fluid retention, causing the prominent clinical manifestations of abnormal thickening of the mucus in lungs and pancreatic insufficiency. In the lung, dysfunctional CFTR can hinder mucociliary clearance, rendering the organ susceptible to bacterial infections and inflammation, ultimately leading to airway occlusion, respiratory failure, and premature death. CF remains the most common and lethal genetic disease among the Caucasian population with 70,000-100,000 sufferers estimated worldwide, highlighting a real need for the development of better treatments.

[0056] Mutations in CFTR include, but are not limited to c.l521_1523del (p.Phe508del), C.1624G>T (p.Gly542Ter) or c.3846G>A (p.Trp!282Ter).

[0057] In some embodiments, a method of treating cystic fibrosis in a patient in need thereof is provided, the method comprising, administering a nucleic acid of the disclosure, a vector of the disclosure, a viral vector of the disclosure, or an AAV of the disclosure, the composition of the disclosure, or the LNP composition of the disclosure.

[0058] In some embodiments, the guide RNA and donor polynucleotide target mutations in the CFTR gene.

[0059] In some embodiments, the guide RNA and donor polynucleotide target and replace the CFTR c.!521_1523del (p.Phe508del), C.1624G>T (p.Gly542Ter), c.!652G>A (p.Gly551Asp), c.!657C>T (p.Arg553Ter) or c.3846G>A (p.Trp!282Ter) mutations.Alpha 1 -antitrypsin deficiency

[0060] Alpha 1 -antitrypsin deficiency is a genetic disorder that affects the lungs and sometimes the liver. The affected gene in Alpha- 1 -antitrypsin deficiency is SERPINA1 , on chromosome 14 (GRCh38.pl4 (GCF_000001405.40) (coding for Alpha- 1 -antitrypsin). The protein encoded by this gene is a serine protease inhibitor belonging to the serpin superfamily whose targets include elastase, plasmin, thrombin, trypsin, chymotrypsin, and plasminogen activator. This protein is produced in the liver, the bone marrow, by lymphocytic and monocytic cells in lymphoid tissue, and by the Paneth cells of the gut. Defects in this gene are associated with chronic obstructive pulmonary disease, emphysema, and chronic liver disease.

[0061] In some embodiments, a method of treating alpha- 1 -antitrypsin deficiency in a patient in need thereof is provided, the method comprising, administering a nucleic acid of the disclosure, a vector of the disclosure, a viral vector of the disclosure, or a AAV of the disclosure, the composition of the disclosure, or the LNP composition of the disclosure to the patient.

[0062] In some embodiments, the guide RNA and donor polynucleotide target mutations in the SERPINA1 gene. In some embodiments, the guide RNA and donor polynucleotide target and replace the SERPINA1 c.!096G>A (p.Glu342Lys) mutation.Myotonic Dystrophy Type 1

[0063] Myotonic dystrophy type 1 (DM1) is a multisystem disorder that affects skeletal and smooth muscle as well as the eye, heart, endocrine system, and central nervous system. The clinical findings, which span a continuum from mild to severe, have been categorized into three somewhat overlapping phenotypes: mild, classic, and congenital. DM1 is caused by expansion of a CTG trinucleotide repeat in the noncoding region of DMPK. The diagnosis of DM1 issuspected in individuals with characteristic muscle weakness and is confirmed by molecular genetic testing of DMPK. CTG repeat length exceeding 34 repeats is abnormal. Molecular genetic testing can detect pathogenic variants in nearly 100% of affected individuals.

[0064] In some embodiments, a method of treating myotonic dystrophy type 1 in a patient in need thereof, the method comprising, administering a nucleic acid of the disclosure, a vector of the disclosure, a viral vector of the disclosure, or a AAV of the disclosure, the composition of the disclosure, or LNP composition of the disclosure to the patient.Amyotrophic Lateral Sclerosis

[0065] Amyotrophic lateral sclerosis (ALS) is a neurological disorder that affects motor neurons, the nerve cells in the brain and spinal cord that control voluntary muscle movement and breathing. C9orf72 frontotemporal dementia and / or amyotrophic lateral sclerosis (C9orf72- FTD / ALS) is characterized most often by frontotemporal dementia (FTD) and upper and lower motor neuron disease (MND). C9orf72-FTD / ALS shows heterozygous abnormal G4C2 (GGGGCC, C4C2) hexanucleotide repeat expansion in C9orf72 that can be identified by molecular genetic testing.

[0066] In some embodiments, a method of treating amyotrophic lateral sclerosis or frontotemporal dementia in a patient in need thereof, the method comprising, administering a nucleic acid of the disclosure, a vector of the disclosure, a viral vector of the disclosure, or a AAV of the disclosure, the composition of the disclosure, or LNP composition of the disclosure to the patient.KITSAlso provided herein are various kits for the practice of a method described herein as well as written instructions for making and using the same. In particular, some embodiments relate to kits for methods of treating disease in a subject in need thereof. For example, provided herein, in some embodiments, are kits that include one or more nucleic acids, and / or pharmaceutical compositions as provided and described herein, as well as written instructions for using the same. In some embodiments, the kits of the disclosure further include one or more means useful for the administration of the engineered immune cells, and / or pharmaceutical compositions to a subject. For example, in some embodiments, the kits of the disclosure further include one or more bags,syringes (including pre-filled syringes) used to administer any one of the provided engineered immune cells, and / or pharmaceutical compositions to a subject.

[0067] In some embodiments, a kit can further include instructions for using the components of the kit to practice the methods disclosed herein. For example, the kit can include a package insert including information concerning the pharmaceutical compositions and dosage forms in the kit. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information regarding a combination of the disclosure may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer / distributor information and intellectual property information.

[0068] The instructions for practicing the methods are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate, such as paper or plastic, etc. The instructions can be present in the kit as a package insert, in the labeling of the container of the kit or components thereof (e.g., associated with the packaging or subpackaging), etc. The instructions can be present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD-ROM, diskette, flash drive, etc. In some instances, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the internet), can be provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.EXAMPLES

[0069] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.EXAMPLE 1. Design of an all in one chimeric nuclease cassette

[0070] This example describes the design and synthesis of an all-in-one chimeric nuclease cassette.

[0071] Briefly, a chimeric nuclease cassette is designed comprising a promoter, a chimeric nuclease, an mRNA stabilizing sequence, a gRNA, a modified tRNA comprising a ribozyme sequence, a repair template, and a poly A tail. An exemplary nucleic acid expression cassette is shown in FIG. 1, FIG. 2, FIG. 26A, FIG. 26B, and FIG 26C. The Minimal CMV promoter drives the transcription of 3-in-l construct containing Dualase (chimeric nuclease), long noncoding RNA MALAT-1, synthetic transfer RNA containing sequence-specific ribozyme (tRNA’), gRNA fused with repair template (RT), and synthetic poly A signal sequence (synt[A]). Upon transcription, RNA maturation at 3’ end of MALAT and both ends of tRNA’ result in the separation of Dualase-MALAT RNA, tRNA, and gRNA. U-rich repeat on MALAT will help protect Dualase RNA from degradation while tRNA’ will recognize and cleave at the region between RTs as well as between RT and synt[A] sequence. After translation, Dualase can form a complex with gRNA (RNP) and cleave target site. The presence of in-place repair template fused to 3’ end of gRNA (cis-sense and antisense) or abundant free-floating repair template (trans-antisense and trans-sense) serve as a bridge between two cleavage sites and local reference template for cell repair machinery.

[0072] Another exemplary chimeric nuclease cassette is designed comprising a promoter, a chimeric nuclease, a self-cleaving peptide, a protein tag, an mRNA stabilizing sequence, a gRNA, a modified tRNA comprising a ribozyme sequence, a repair template, and a poly A tail. An exemplary nucleic acid expression cassette is shown in FIG. 2. The all-in-one mRNA cassette encoding necessary elements for efficient accurate target site disruption or repair. T7 promoter drives the transcription of 3-in-l construct containing Dualase, long non-coding RNA MALAT-1, synthetic transfer RNA containing sequence-specific ribozyme (tRNA’), gRNA fused with repair template (RT), and synthetic poly A signal sequence (synt[A]). Conditions are established where tRNA, MALAT maturation as well as ribozyme cleavage activities are inactive in storage buffer. Upon delivery, RNA maturation at 3 ’ end of MALAT and both ends of tRNA’ result in the separation of Dualase-MALAT RNA, tRNA, and gRNA. U-rich repeat on MALAT helps protect Dualase RNA from degradation while tRNA’ will recognize and cleave at the region between RTs as well as between RT and synt[A] sequence. After translation, Dualaseforms a complex with gRNA (RNP) and cleave target site. The presence of in-place repair template fused to 3 ’ end of gRNA (cis-sense and antisense) or abundant free-floating repair template (trans-antisense and trans-sense) serve as a bridge between two cleavage sites and local reference template for cell repair machinery.EXAMPLE 2. Delivery of Chimeric Nuclease

[0073] This example describes the delivery of a chimeric nuclease system to a cell.

[0074] Briefly, the chimeric nuclease is expressed in E. coli and purified. RNA comprising a gRNA fused to a repair template is produced. The purified chimeric nuclease and the guide RNA / repair template are assembled into a ribonucleoprotein (RNP) complex and co-delivered to a cell, for example with an LNP or by electroporation.

[0075] FIG. 3 shows a diagram of a dual-cleaving nuclease ribonucleoprotein (RNP) complex with in all-in-one guide RNA repair cassette. Dualase and 2-in- 1 gRNA-RT are incubated to form ribonucleoprotein complex (RNP) and delivered into target cells where they interact and cleave the target site after nuclei translocation.EXAMPLE 3. Gene Editing using a Chimeric Nuclease

[0076] This example describes the genome editing in a cell using a chimeric nuclease targeting the AA VS1 locus in a cell.

[0077] Briefly, HEK293 were treated with cells AAV-Dualase-AAVS7. The genomic DNA was collected, purified and sequenced. The data was analyzed with Tracking of Indels by DE composition (TIDE). The results show a 35bp deletion with AAV-Dualase-AA VS1. Amplicons from samples treated with AAV-Dualase- A V.S7 were Sanger-sequenced and analyzed using TIDE approach. Portion of deletion, insertion at defined length as well as unmodified reads were characterized as bar graph. Dotted line P <0.01 (B) Chromatograms of sequenced samples treated with AAV-Dualase-AA VS1 or reagents only in comparison with reference sequence (FIG. 4A and B).EXAMPLE 4. Gene Editing using an all-in-one Chimeric Nuclease Cassette

[0078] This example describes the genome editing in a cell using an all-in-one chimeric nuclease targeting the AAVS1 locus in a cell.

[0079] Briefly, Dualase together with guide RNA and a donor nucleic acid were co-expressed in HEK293 cells from one nucleotide sequence operably linked to a promoter sequence, one ormore self-cleaving guide RNA sequences and one or more self-cleaving repair template sequences to co-deliver exogenous donor nucleic acid. The genomic DNA was collected, purified and sequenced. Next generation sequencing (NGS) showed high and accurate target site repair. NGS data was analyzed using two bioinformatic tools (FIG. 5A) CRIS.Py and Geneious alignment platforms, as well as (FIG. 5B) CRISPRESSO2. “Indels” represent repair events resulting in an insertion or deletion. “Precise repair” represents sequencing reads with complete alignment to the repaired sequence. “Repair + SNP” represents sequence alignments with repair and another nucleotide change. And “Other” represents other sequence modifications in the sample. Analysis of individual sequences using CRIS.Py and Geneious identified -10% of reads with repair + SNP or Other sequences which likely result from sequencing errors since the cell only sample contained the same number of reads. Total and aligned reads were used to identify editing outcome (p <0.001). An alignment of the sequencing reads of the cis-antisense RNA repair template guide RNA shows the accuracy of repair at the I-TevI and Cas9 site with no other changes than the expected repair outcome above the limits of detection of sequencing indicated by the dotted line (FIG. 5C). The results show that the gene editor inserts RNA sequences with high efficiency and accuracy in the target site (FIGs. 5A, 5B and C)EXAMPLE 5. Gene Editing using an all-in-one Chimeric Nuclease Cassette and DNA repair inhibitors

[0080] This example describes the genome editing in a cell using an all-in-one AAV chimeric nuclease targeting the AA VS J locus in a cell together with NHEJ inhibitors.

[0081] Briefly, HEK293 cells were treated with AAV expressing Dualase and gRNA-RT targeting AAVS1 (all-in-one) as well as a control of AAV expressing Dualase targeting the AAVS1 co-delivered with the indicated repair template (co-delivery) by lipofection in the presence of increasing doses of inhibitors blocking the (FIG. 6A) non-homologous end-joining (NHEJ), (FIG. 6B) homologous-directed repair (HDR), or (FIG. 6C) Rad52-dependent pathways. NHEJ, Rad51 and Rad52 inhibitors with cis-sense and cis-antisense repair template Samples were analyzed for editing efficiency by restriction enzyme (RE) digestion efficiency using a unique RE at the inserted repair template site. The results show, that repair using the cis- sense and cis-antisense all-in-one constructs was only inhibited by the Rad52 inhibitor. (FIG. 6A-C) Control inhibition of co-delivered repair templates indicate the inhibitor was functioningas expected. NHEJ (DNA) = duplex DNA without homology, HDR (DNA) = duplex DNA with homology arms and NHEJ (RNA) = single-strand RNA without homology.EXAMPLE 6. Gene Editing using an all-in-one Chimeric Nuclease Cassette and Repair Template

[0082] This example describes the directional insertion of a repair template with Dualase ribonucleoprotein (RNP) complex formed with an AAVS1 -targeting guide RNA combined with RNA repair templates by directional polymerase chain reaction (PCR).

[0083] Briefly, HEK293 cells were lipofected with Dualase together with guide RNA and repair template delivered separately (co-delivery) or Dualase with guide RNA and RNA repair template fused (“All-in-one”), lipofection reagent only (’’Reagent only”) or cells only (“Mock”). Shown for each reaction are the undigested PCR amplicon of the AA VS J site (“Sub”) and digested PCR amplicon of the AA VS J site (“Digested”). The genomic DNA was extracted and purified. Samples were analyzed for editing efficiency by restriction enzyme (RE) digestion efficiency using a unique RE at the inserted repair template site. The results show, that a PCR product is present in the correct orientation “Right oriented RT” only if the repair template is inserted correctly and a PCR product is present in the incorrect orientation (“Wrong oriented RT”) if the repair template inserted incorrectly. Dualase RNP complex with the all-in-one repair template accurately inserted as evidenced by the correctly oriented PCR products (FIG. 7A). With co-delivered repair template, the repair template inserts in both the correct and incorrect orientation (FIG. 7B).EXAMPLE 7. Gene Editing using an all-in-one Chimeric Nuclease Cassette and cis-sense or cis-antisense Repair Template

[0084] This example describes the analysis of directional insertion of repair template with AAVS1 -targeting Dualase all-in-one mRNA with cis-sense or cis-antisense repair templates by directional polymerase chain reaction (PCR).

[0085] Briefly, HEK293 cells were lipofected with Dualase (Tev[VKN]-SaCas9[WT] or Tev[VKN]-SaCas9[D10E]), SaCas9[WT] or lipofection reagent only. The genomic DNA was extracted and purified. Samples were analyzed for editing efficiency by restriction enzyme (RE) digestion (Bglll digest) efficiency using a unique RE at the inserted repair template site. Results are shown for each reaction are the undigested PCR amplicon of the AAVS1 site (“Full length”), digested PCR amplicon of the AA VS J site (“Bglll digest”), PCR product if the repair templateinserted only in the correct orientation (“Right oriented RT”) and PCR product if the repair template inserted only in the incorrect orientation (“Wrong oriented RT”) (FIG. 8). Control cells treated with Dualase mRNA targeting the AA VS J site with co-delivered repair template (codelivery) is also shown. Cis-sense or cis-antisense sense repair template accurately inserted with the all-in-one Dualase mRNA as evidenced by the correctly oriented PCR products. No insertion was detected with the cis-sense or anti-sense repair templates with saCas9[WT] only as evidenced by no Bglll digestion nor directional PCR products. Co-delivered Dualase mRNA and repair template was inserted in both the correct and incorrect orientation.EXAMPLE 8. Gene Editing using an all-in-one Chimeric Nuclease Cassette and NHEJ and Rad52 inhibitors

[0086] This example describes the analysis of the editing efficiency in cell with messenger RNA (mRNA) Dualase, NHEJ and Rad52 inhibitors, and trans dsRNA repair template.

[0087] Briefly, HEK293 cells were treated with AAV expressing Dualase and gRNA-RT (also referred to as repair template guide RNA or “rep-gRNA") targeting AAVS1 (all-in-one) as well as a control of AAV expressing Dualase targeting the AA VS J co-delivered with the indicated repair template (co-delivery) by lipofection in the presence of increasing doses of inhibitors blocking the non-homologous end-joining (NHEJ inhibitor) or Rad52 pathway (“Rad52 inhibitor”).Samples were analyzed for editing efficiency by looking for restriction enzyme (RE) digestion efficiency using a unique RE at the inserted repair template insert site. Repair using the trans-RT all-in-one constructs was inhibited by both the NHEJ and Rad52 inhibitor demonstrating both these pathways can be used with self-complementing trans repair template (FIG. 9). Control inhibition of co-delivered repair templates indicate the inhibitor was functioning as expected. NHEJ (DNA) = duplex DNA without homology, HDR (DNA) = duplex DNA with homology arms & NHEJ (RNA) = single-strand RNA without homology.EXAMPLE 9. Gene Editing using a dual guide Chimeric Nuclease for Deletion

[0088] This example describes the precise removal of large repeat sequences using a dualguide TevCas9 nuclease.

[0089] FIG. 10A shows a schematic of precise removal of large repeat sequences using a dual-guide TevCas9 nuclease in which the guide RNAs target opposite strands of the duplex DNA to orient two I-TevI domains head-to-head. A TevCas9 nuclease (1) containing the inactivating D10A+H557A mutation but an active I-TevI domain (2) is targeted to the 5 ’-end ofa repeated sequence (3) using a synthetic guide RNA. A second TevCas9 nuclease (4) containing the inactivating D10A+H557A mutation but an active I-TevI domain is targeted to 3’- end of the repeated sequence but the on the opposing strand such that the I-TevI domains of both nucleases are pointed into the repeat sequence. Upon binding and cleavage (5) by the I-TevI nuclease domain, two complementary 3’ 2-nucleotide overhangs remain (6 and 7). Through the non-homologous end joining pathway, the cell can repair these complementary overhangs (8) and the large repeated sequence is removed (9) leaving a defined number of repeats in the genomic DNA (10).

[0090] FIG. 10B shows a schematic of precise removal of large repeat sequences using a dual-guide TevCas9 nuclease in which the guide RNAs target the same strands of the duplex DNA to orient two I-TevI domains in tandem. A TevCas9 nuclease (1) containing the inactivating D10A+H557A mutation but an active I-TevI domain (2) is targeted to the 5 ’-end of a repeated sequence (3) using a synthetic guide RNA. A second TevCas9 nuclease (4) containing the inactivating D10A+H557A mutation but an active I-TevI domain is targeted downstream of the 3 ’-end of the repeated sequence on the same strand such that the I-TevI domains of both nucleases are pointed in the same direction. Upon binding and cleavage (5) by the I-TevI nuclease domain, two complementary 3’ 2-nucleotide overhangs remain (6 and 7). Through the non-homologous end joining pathway, the cell can repair these complementary overhangs (8) and the large repeated sequence is removed (9) leaving a defined number of repeats in the genomic DNA (10).EXAMPLE 10. Gene Editing using a dual guide Chimeric Nuclease for Deletion of CAG triplet or GGGGCC hexanucleotide repeats in cells and humanized mice

[0091] This example describes the precise removal of CAG triplet repeat expansion in DMPK 3’-UTR or GGGGCC hexanucleotide repeat between Exon la and lb in C9ORF72 using an all- in-one AAV encoding TevCas9 together with dual guides targeting the 5’- and 3 ’-ends of the repeat expansion in vivo.

[0092] Briefly, DMPKMUTCAG repeat cells were transduced with all-in-one AAV TevCas9 and SaCas9 dual guides (SEQ ID NO: 117). (FIG. 11A) shows a diagram of the orientation of the dual-guided Tev[KTQ]-Cas9[D10A+H557A] on the DMPK CAG repeat target site. (FIG. 11B) shows a diagram of the sequences in the all-in-one TevCas9 AAV. Shown are the hammerhead ribozyme (HHribo), single guide RNAs (sgRNAs), glycine tRNA (Gly tRNA), hepatitis delta virus ribozy,e (HDVribo) and poly adenylation sequence (Poly A). (FIG. 11C) shows the in vitro activity of dual guided TevCas9 on DMPKMUTCAG repeat DNA sequences demonstrating I-TevI domain activity at this target. (FIG. 11D) shows an alignment of Sanger sequencing reads of amplicons generated from cells transduced with an all-in-one AAV encoding TevCas9 together with dual guides together with the expected cleavage demonstrating removal on repeated CAG sequence. (FIG. HE) shows the workflow of the experiment to quantify repeat collapse using repeat-primed PCR in DMPKMUTCAG repeat cells transduced with all-in-one AAV TevCas9 and SaCas9 dual guides. Transduced cells were harvested and genomic DNA extracted for PCR-amplification using a primer outside and inside the repeat expansion. The resulting PCR products were analyzed on an Agilent Bioanalyzer and peaks corresponding to the repeat were quantified relative to DMPKMUTCAG repeat cells only. More than 65% of the repeats were collapsed in the TevCas9 treated cells relative whereas -30% of the repeat was collapsed in the SaCas9 treated cells. (FIG. HF) shows transduced cells were subjected to quantitative RT-PCR to quantify the levels of DMPKMUTand DMPKWTtranscripts after each treatment using primers specific for each transcript. TevCas9 treatment resulted in a statistically significant increase in DMPKWTtranscript and decrease in DMPKMUTtranscript relative to untreated cells (p < 0.01). Neither TevCas9 containing a single targeting guide RNA nor SaCas9 dual guide treatments resulted in statistically significant transcript level changes relative to cells only. (FIG. 11G) shows transduced cells were subjected to RT-PCR to quantify the levels of mis-spliced CLCN 1 Exon 6 transcripts after each treatment using primers specific for the misspliced sequence. TevCas9 single guide or TevCas9 dual guide resulted in a statistically significant decrease in mis-spliced CLCN 1 Exon 6.

[0093] For C9ORF72 GGGGCC repeat removal (FIG. 11H), FIG. HI shows the in vitro activity of dual guided TevCas9 on C9ORF72MUTGGGGCC repeat DNA sequences demonstrating I-TevI domain activity at this target. Patient motor neuron progenitor cells containing a large GGGGCC repeat expansion were matured into motor neurons (FIG. HJ) and then transduced with all-in-one AAV encoding TevCas9 or saCas9 and dual guides targeting the C9ORF72 repeat region. The maturation was confirmed by Western blot by the expression of motor-neuron specific marker (ISL1). FIG. HK shows the results of repeat-primed PCR quantifying the removal of the larger repetitive sequence in TevCas9- or Cas9-transduced cellswith approximately 50% removal in TevCas9-treated cells and less than 5% removal in Cas9- treated cells. FIG. 11L shows sequencing the collapsed repeat in TevCas9-treated cells shows 4 of 9 sequences that had precise repeat collapses as seen in the sequences with asterix (*). FIG. 11M shows C9ORF72 guide RNAs targeting Tev[VKN]-saCas9[D10A+H557A] to are specific for the C9ORF72 target site since not off-targets in the genome are detected at levels that exceed the reported limits of detection of the deep sequencing assay. FIG. UN shows the requirement for TevSaCas9 since active SaCas9 targeted by the same C9ORF72 targeting guide RNAs has a detectable off-target in Chromosome 11. FIG. 11O demonstrates that C9ORF72 protein expression can be recovered with in patient derived matured motor neurons with transduction of AAV expressing Tev[VKN]-saCas9[D10A+H557A] and the dual C9ORF72 guides. FIG. IIP shows that the build-up of poly-GR dipeptides expressed from the C9ORF72 repeat sequence can be reduced by transducing patient derived motor neurons with AAV expressing TevfVKN]- saCas9[D10A+H557A] and the dual C9ORF72 guides (SEQ ID NO: 115). Toxic build-up of dipeptides contributes to the degeneration of the motor neurons in disease. FIG. 11Q shows the expression of TevCas9 by RT-qPCR in the cerebellum of humanized C9ORF72 repeat expansion mice that were intracranially injected with two doses of Tev[VKN]-saCas9[D10A+H557A] and the dual C9ORF72 guides demonstrating transduction of brain tissue. FIG. HR shows the dosedependent recovery of normal sized C9orf72 repeat sequences in humanized mice that were intracranially injected with two doses of Tev[VKN]-saCas9[D10A+H557A] and the dual C9ORF72 guides. FIG. IIS shows the recovery of C9ORF72 transcripts by RT-qPCR in cerebellum tissue in mice humanized C9ORF72 repeat expansion mice that were intracranially injected with two doses of Tev[VKN]-saCas9[D10A+H557A] and the dual C9ORF72 guides. FIG. 11T shows the recovery of C9ORF72 protein expression by Western Blot in cerebellum tissue in mice humanized C9ORF72 repeat expansion mice that were intracranially injected with two doses of Tev[VKN]-saCas9[D10A+H557A] and the dual C9ORF72 guides.EXAMPLE 11. Gene Editing using a self-inactivating Chimeric NucleaseThis example describes the gene editing in the B2M gene with a self-inactivating TevSaCas9.

[0094] Briefly, HEK293 cells transfected with plasmid DNA with TevSaCas9 targeting the beta-2-microglobulin (B2M) gene harvested at 24-, 48-, and 72-hours post-transfection. FIG. 12A depicts the structure of the self-inactivating vector. The construct comprises nucleotide sequences encoding a promoter, human codon-optimized TevSaCas9, poly adenylation signal(“poly A”) and guide RNA sequence (“gRNA”). The self-inactivating target site may be in the region between the promoter and TevSaCas9 site (denoted as “Promoter”) or end of the TevSaCas9 coding sequence and beginning of the PolyA sequence (denoted “PolyA”). The results of T7E1 editing assay in HEK293 cells transfected with plasmid DNA with TevSaCas9 targeting the beta-2-microglobulin (B2M) gene harvested at 24-, 48-, and 72-hours posttransfection are shown in FIG. 12B. Lanes marked “None” do not contain and self-inactivating sequence in the vector, lanes marked “Promoter” contain the B2M1 TevSaCas9 target site between the promoter sequence and TevSaCas9 sequence, lanes marked “PolyA” contain the B2M1 TevSaCas9 target site between the end of TevSaCas9 and the PolyA signal sequence. Levels of editing as determined by the amount of digested product relative to substrate is comparable over time between the constructs. FIG. 12C shows the results of a Western Blot for hemagglutinin (a-HA) encoded at the 3’-end of TevSaCas9 (“Dualase”) from the same treated cells in B. Lanes marked “None” do not contain and self-inactivating sequence in the vector, lanes marked “Promoter” contain the B2M1 TevSaCas9 target site between the promoter sequence and TevSaCas9 sequence, lanes marked “PolyA” contain the B2M1 TevSaCas9 target site between the end of TevSaCas9 and the PolyA signal sequence. The presence of a band in the a-HA blot indicate the TevSaCas9 protein is expressed. The levels of TevSaCas9 expression are stable over time in the vector containing no inactivating sequence, decrease over 72 hours in the vector containing the promoter inactivating sequence and are reduced to undetectable levels in 72 hours in the vector contain the polyA inactivating sequence. FIG 12D shows the production and titer of an AAV2 capsid encapsulating the self-inactivating TevCas9 targeting B2M as well as a picture of an SDS-polyacrylamide gel showing the successful assembly of the capsid with the VP1, VP2 and VP3 capsid structural proteins. As seen in the western blot for an HA-tag on the TevCas9 protein, after 72 hours, the levels of TevCas9 protein are greatly reduced when compared to the beta-actin housekeeping gene indicating inactivation as occurred. FIG 12E shows the results of a Western Blot for saCas9 and GAPDH from cell lipofected transfected with plasmid DNA versions of the vectors used to produce the self-inactivating AAV in FIGI 2D (“Self-inactivating”), as well as a vector that does not contain the self-inactivating sequences (“non-self-inactivating”) and a plasmid expressing GFP (“pAAV-GFP”) as controls. Transfected cells were harvested at 48 hours, (48h), 72 hours (72h), 7 days (7d) and 14 days (14d) and lysed to recover total protein the presence of expressed Cas9 was detected with the Cas9 antibody andthe housekeeping gene GAPDH was used to assess consistency of the loaded sample. SaCas9- sized bands are only detected in the non-self-inactivating control samples at 48h and 72h and not in the self-inactivating samples, demonstrating the presence of the self-inactivating target sites in the expression vectors limit expression of TevSaCas9.EXAMPLE 12. Gene Editing using TevCas9 and rep-gRNA to recruit endogenous polymerase theta (PolO or PolO)

[0095] This example described the use of the TevCas9 gene editor and rep-gRNA in cells to recruit endogenous polymerases.

[0096] Briefly, HEK293 cells were lipofected with the TevCas9 or Cas9 mRNA and AAVS1- targeting rep-gRNA (FIG 13A-C). Cells were lysed and the extract was precipitated using magnetic beads conjugated to an anti-HA antibody which specifically recognizes a C-terminal HA-tag on TevCas9 or Cas9. After washing the magnetic beads, the precipitated proteins are resolved on a polyacrylamide gel and Western blotted using various antibodies. FIG. 14 shows the Western blots using a Rad52-specific antibody and polymerase theta- specific (Pol Q or Pol 0) antibody showing polymerase theta co-precipitates only with TevCas9.EXAMPLE 13. Gene Editing using an inactivated or nickase Chimeric Nuclease

[0097] This example describes the gene editing of the AA VS J site with TevCas9 using a nuclease inactivated or a nickase gene editor.

[0098] Briefly, HEK293 cells were lipofected with purified TevCas9 and Cas9 proteins and 2- in-1 guide RNAs targeting the AAVS1 site. Samples were analyzed for editing efficiency by looking for restriction enzyme (RE) digestion efficiency using a unique RE at the inserted repair template insert site. Digestion was observed using purified TevCas9 containing the inactivating D10A+H557A mutations in the Cas9 domain (“Tev[WT]-dCas9”) and TevCas9 containing the inactivating D10A+H557A mutations in the Cas9 together with the inactivating R27A mutation in the I-TevI domain (“Tev[R27A]-dCas9”; FIG. 15A). No digestion is seen in Cas9 only or reagent only treated cells or with untreated cells (“Cell only”). Digestion is seen in a control of cells lipofected with Tev-Cas9 3-in-l mRNA. Digestion was also seen with the mRNA version of TevCas9 which contains the mutations R27A+V117F+K135R+N140S and the Cas9 domain with either the D10A or the H557A nickase mutations (FIG. 15B).EXAMPLE 14. In frame insertion of a GFP into the genome of a cell

[0099] This example describes the in frame insertion of GFP into the genome of a mammalian cell with TevCas9.

[0100] Briefly, a rep-gRNA was designed comprising a T2A sequence and an eGFP coding sequence for insertion into the G542 site of the CFTR gene (FIG. 16A) to cover G542X, R553X and G551D mutations. Cells were lipofected with the TevCas9 and the rep-RNA encoding eGFP or saCas9 with a rep-RNA encoding eGFP. The results show that GFP is expressed from the endogenous CFTR promoter in the TevCas9 condition only and not in the SaCas9, rep-gRNA or reagent only conditions (FIG. 16B). The eGFP is separated from the truncated endogenous protein by the self-cleaving T2A peptide. FIG. 16C shows that the inserted sequence is detected as a larger band when amplifying the CFTR target site region by PCR. The results of the PCR of the target region shows that the ratio of edited to unedited is -50%. Another approach uses a combination of a guide RNA upstream (5’) of a rep-gRNA to remove a large -28 kilobase sequence and replace it with a 921 base pair (bp) GFP coding sequence. The rep-gRNA encoding GFP spans the region between CFTR F508 and G542 to cut out a ~28kb region between Exon 11 and Exon 12 and replace with GFP. FIG. 16D shows a schematic of a guide RNA (sgRNA) targeting the F508 region of CFTR and a rep-gRNA targeting the G542 region of CFTR. Lung epithelial cells were lipofected with TevCas9 or Cas9 mRNA combined with an equimolar mixture of the guide RNA and rep-gRNA. The results show that GFP is expressed from the endogenous CFTR promoter only in the TevCas9 condition and not in the Cas9 condition (FIG. 16E). FIG. 16F shows a picture of a gel of PCR amplicons of the target site demonstrating that the -28 kilobase (kb) region between the F508 and G542 sites is removed and replaced as evidenced by the -1500 base pair product in the TevCas9 lane.EXAMPLE 15. A modified gRNA enables targeted DSB repair in human cells

[0101] This example describes the determination of targeted DSB repair in human cells by a modified gRNA and TevSaCas9 nuclease.

[0102] To test directional, RNA-templated repair using the TevSaCas9 nuclease, a modified gRNA was created by fusing an RNA repair sequence to the 3’ end of standard SaCas9 single guide RNA (sgRNA) composed of the crRNA portion that is the complement of the target site and the tracrRNA portion to create a rep-gRNA (repair templated gRNA) (FIGs. 17A and 17B).The putative repair scenario where the 3’ end of the rep-gRNA was the complement of the 2-nt 3’ overhang generated by the I-TevI nuclease domain (hereafter, Tev) that would form a priming site for repair (FIG. 17A), possibly through reverse transcription of the RNA repair template. The location of the targeted edit can be at any position between the Tev and SaCas9 cleavage sites up to the scaffold region. First, a rep-gRNA was designed where the crRNA portion targeted a site in the AA VS J safe harbor locus (FIG. 17B). The AA VS J rep-gRNA contained a repair sequence of 37 nucleotides in length that was dissimilar to the AA VS J sequence and included a diagnostic Bglll site. Crucially, the 3’ end of the rep-gRNA possessed two complementary nucleotides (5’-CC-3’) to the 2-nt 3’ overhang left by Tev cleavage at the appropriately positioned S’-CA'fGGj.G-S’ site (the 2-nt 3’ overhang product is 5’-GG-3’) (FIG. 17B). To confirm activity of TevSaCas9 and SaCas9 at the AA VS1 site, a sgRNA targeted to this site was used and demonstrated robust activity in vitro and in HEK293 cells where the primary editing product was a 35-bp deletion corresponding to the distance between the Tev and SaCas9 cleavage sites.

[0103] To determine the number of putative genomic targets for cleavage of CNNNG motifs by Tev within the TevSaCas9 construct, target sites were predicted where the CNNNG motif was spaced 5-31 bp from the gRNA binding site consistent with spacing preferences for the native I- TevI nuclease and other Tev-based chimeric nucleases. It is estimated that ~75% of SaCas9 sites in the human genome, defined by the presence of an NNGRRT PAM site and 21 -nt gRNA binding site, possessed appropriately spaced CNNNG motif that could support Tev cleavage (FIG. 17C). To deliver TevSaCas9 and the rep-gRNA to cells, an all-in-one format construct was created, where a single RNA polymerase II transcript of ~4.5 kb contains the TevSaCas9 coding sequence and rep-gRNA and that would fit within a single AAV viral vector (FIG. 17D). This setup would avoid the requirement for a separate RNA polymerase III promoter commonly used to express the gRNA. Since this setup removes a poly(A) tail from the TevSaCas9 coding sequence, a MALAT1 sequence was included at the 3’-end of TevSaCas9 for stability. To stimulate post-transcriptional processing of the all-in-one mRNA, an alanine tRNA sequence (ala tRNA) was inserted between the TevSaCas9-eGFP coding region and rep-gRNA, as well as a trans-acting hammerhead (HH) ribozyme sequence in the hairpin of the alanine tRNA to act on a HH cleavage site located between the 3 ’end of the rep-gRNA and the poly(A) signal. To confirm correct processing, in vitro transcribed TevSaCas9-eGFP / rep-gRNA all-in-one mRNA wasincubated with a HEK293 cell extract and processing into the two predicted RNA products was observed (TevSaCas9-eGFP and rep-gRNA) on a 1% agarose gel (FIG. 17E). HEK293 cells were transfected with the TevSaCas9-eGFP / rep-gRNA all-in-one mRNA and observed robust eGFP activity demonstrating the MALAT sequencing can provide RNA stability (FIG. 17F).EXAMPLE 16. Rep-editing in human cell lines

[0104] This example describes the determination of targeted DSB repair in human cells by a modified gRNA and TevSaCas9 nuclease.

[0105] The TevSaCas9 / rep-gRNA or SaCas9 / rep-gRNA all-in-one construct targeting the AAVS1 safe harbor site was introduced into HEK293 cells by four different methods; AAV2 transduction, lipid nanoparticles containing mRNA, plasmid DNA (pDNA) transfection, or purified RNPs (FIG. 18A). Robust editing at the AAVS1 site was with TevSaCas9 / rep-gRNA observed with all four delivery modalities by Bglll digests (56 + / - 12%) and by deep sequencing (52 + / - 14%)) of target sites PCR amplified from genomic DNA (cells were not enriched prior to DNA isolation) but not with SaCas9 / rep-gRNA (FIGs. 18A and 18B). AAV2 delivery resulted in the highest editing rates (67 + / - 10%)) whereas the editing rate for RNP transfections was lowest (43 + / - 11% across all methods of analysis) (FIG. 18C), likely reflecting lower RNP transfection versus AAV2 transduction efficiency. Using PCR primers that can distinguish correctly from incorrectly oriented editing events, it was shown that TevSaCas9 / rep-gRNA editing generated correctly orientated repair products. Analysis of deep sequencing reads from 6 independent TevSaCas9-rep-gRNA editing events confirmed both the directional insertion and fidelity of the editing, with 42 + / - 6% of reads corresponding to the expected repair product with no other templated nucleotide changes (FIG. 18D and FIG. 18E). Less than 0.1% of reads corresponding to incorrect repair products, while 1.6 + / - 1.1% of reads possessed indels. No reads were identified where repair extended beyond the RNA repair template region to include sequence in the stem loop structure in the scaffold region of the rep-gRNA, indicating that editing and repair is specified by only the RNA repair template portion of the rep-gRNA.Moreover, apparent nucleotide substitutions in sequencing reads for edited cells were indistinguishable from those in mock treated cells (FIG. 18D).

[0106] A rep-gRNA was designed to introduce a 13 -bp deletion ...

Claims

CLAIMSWhat is claimed is:

1. A nucleic acid comprising(i) a polynucleotide encoding a chimeric nuclease comprising an I-TevI domain and a RNA-guided nuclease domain;(ii) a polynucleotide encoding a first guide RNA (gRNA); and(iii) a polynucleotide encoding a tRNA; wherein the polynucleotides in (ii) - (iii) are in sequential order.

2. A nucleic acid comprising(i) a polynucleotide encoding a chimeric nuclease comprising a GIY -YIG nuclease domain and a RNA-guided nuclease domain;(ii) a polynucleotide encoding a first guide RNA (gRNA); and(iii) a polynucleotide encoding a tRNA; wherein the polynucleotides in (ii) - (iii) are in sequential order.

3. The nucleic acid of claim 1 or claim 2, further comprising(iv) an RNA stabilizing polynucleotide located downstream of (i).

4. A nucleic acid comprising(i) a polynucleotide encoding a first guide RNA (gRNA); and(ii) a polynucleotide encoding a tRNA; wherein the polynucleotides in (i) - (ii) are in sequential order.

5. The nucleic acid of any one of claims 1 to 4, further comprising two or more donor polynucleotides positioned in tandem; and a ribozyme polynucleotide; wherein the donor polynucleotides are in sequential order and the donor polynucleotide located upstream comprises a ribozyme cleavage site sequence at the 3’ end.

6. The nucleic acid of any one of claims 1 to 5, wherein the nucleic acid is DNA or RNA.

7. The nucleic acid of claim 6, wherein the DNA is circular plasmid DNA, linear doublestrand DNA, single strand DNA, or chimeric RNA and DNA.

8. The nucleic acid of claim 6, wherein the RNA is mRNA.

9. The nucleic acid of claim 8, wherein the mRNA comprises nucleic acid mimetics selected from the group of peptide nucleic acid (PNA), morpholino nucleic acid, cyclohexenyl nucleic acid (CeNAs), and locked nucleic acid (LNA).

10. The nucleic acid of claim 8 or claim 9, wherein the mRNA comprises modified sugar moieties, optionally wherein the modified sugar moiety is selected from the group of Nl- methylpseudouridine, 9-Methyladenine, 2'-O-(2-methoxyethyl), 2'- dimethylaminooxyethoxy, 2'-dimethylaminoethoxyethoxy, 2'-O-methyl, and 2'-fluoro.

11. The nucleic acid of any one of claims 8 to 10, wherein the mRNA comprises a modified nucleobase, optionally wherein the modified nucleobase is selected from the group of a 5- methylcytosine; a 5 -hydroxymethyl cytosine; a xanthine; a hypoxanthine; a 2- aminoadenine; a 6-methyl derivative of adenine; a 6-methyl derivative of guanine; a 2- propyl derivative of adenine; a 2-propyl derivative of guanine; a 2-thiouracil; a 2- thiothymine; a 2-thiocytosine; a 5-halouracil; a 5-halocytosine; a 5-propynyl uracil; a 5- propynyl cytosine; a 6-azo uracil; a 6-azo cytosine; a 6-azo thymine; a pseudouracil; a 4- thiouracil; an 8-halo; an 8-amino; an 8-thiol; an 8-thioalkyl; an 8-hydroxyl; a 5-halo; a 5- bromo; a 5 -trifluoromethyl; a 5-substituted uracil; a 5-substituted cytosine; a 7- methylguanine; a 7-methyladenine; a 2-F-adenine; a 2-amino-adenine; an 8-azaguanine; an 8-azaadenine; a 7-deazaguanine; a 7-deazaadenine; a 3-deazaguanine; a 3- deazaadenine; a tricyclic pyrimidine; a phenoxazine cytidine; a pheno thiazine cytidine; a substituted phenoxazine cytidine; a carbazole cytidine; a pyridoindole cytidine; a 7- deaza-adenine; a 7-deazaguanosine; a 2-aminopyridine; a 2-pyridone; a 5-substituted pyrimidine; a 6-azapyrimidine; an N-2, N-6 or 0-6 substituted purine; a 2- aminopropyladenine; a 5-propynyluracil; and a 5-propynylcytosine.

12. The nucleic acid of any one of claims 8 to 11, wherein the mRNA comprises a non- naturally occurring or a non-natural internucleoside linkage selected from the group of a phosphoro thioate, a phosphoramidate, a non-phosphodiester, a heteroatom, a chiral phosphoro thioate, a phosphorodithioate, a phosphotriester, an aminoalkylphosphotriester, a 3'-alkylene phosphonates, a 5'-alkylene phosphonate, a chiral phosphonate, a phosphinate, a 3'-amino phosphoramidate, an aminoalky Iphosphoramidate, a phosphorodiamidate, a thionophosphoramidate, a thionoalkylphosphonate, a thionoalkylphosphotriester, a selenophosphate, and a boranophosphate.

13. The nucleic acid of any one of claims 1 to 12 wherein the RNA-guided nuclease is selected from the group of Staphylococcus aureus Cas9 (“saCas9”), Streptococcus pyogenes Cas9, Acidaminococcus Casl2, Deltaproteobacteria CasX, and Eubacterium rectale Casl2a.

14. The nucleic acid of claim 13, wherein the Cas is a deactivated Cas (dCas).

15. The nucleic acid of claim 13, wherein the Cas is a nickase (nCas) or a dCas.

16. The nucleic acid of any one of claims 1 to 15, wherein the I-TevI is a nickase and the I- TevI is a I-TevI nickase domain.

17. The nucleic acid of claim 16, wherein the I-TevI nickase domain comprises mutations at amino acid residues R27A, V117F, K135R, and N140S at amino acid residues corresponding to amino acid residues in SEQ ID NO: 155.

18. The nucleic acid of any one of claims 1 to 15, wherein the I-TevI is deactivated.

19. The nucleic acid of claim 18, wherein the I-TevI deactivating mutation is the R27A mutation at an amino acid residue corresponding to amino acid residues in SEQ ID NO: 155..

20. The nucleic acid of any one of claims 5 to 19, wherein the ribozyme polynucleotide is located within the tRNA polynucleotide.

21. The nucleic acid of any one of claims 1 to 20, further comprising a polynucleotide encoding a second gRNA, optionally wherein the second gRNA polynucleotide is located 3 ’ of the tRNA polynucleotide.

22. The nucleic acid of any one of claims 3 or 5 to 21, wherein the order of the polynucleotides is chimeric nuclease, RNA stabilizing polynucleotide, guide RNA, tRNA, and guide RNA.

23. The nucleic acid of any one of claims 5 to 21, wherein the order of the polynucleotides is chimeric nuclease, RNA stabilizing polynucleotide, guide RNA, tRNA / ribozyme, guide RNA, donor polynucleotide 1, and donor polynucleotide 2.

24. The nucleic acid of any one of claims 3 or 5 to 23, wherein the RNA stabilizing polynucleotide comprises a metastasis-associated lung adenocarcinoma transcript 1 (MALAT) 3' sequence, or a 3 ’-end of the multiple endocrine neoplasia beta transcript (MEN P).

25. The nucleic acid of any one of claims 3 or 5 to 23, wherein the RNA stabilizing sequence comprises a triple helix RNA structure or a 3 ’-end of a RNA transcript lacking a canonical poly adenylation signal.

26. The nucleic acid of any one of claims 5 to 25, wherein the donor polynucleotide is single stranded or double stranded.

27. The nucleic acid of any one of claims 5 to 25, wherein the donor polynucleotide is DNA or RNA.

28. The nucleic acid of any one of claims 5 to 25, wherein one strand of the double stranded donor polynucleotide is DNA and one strand is RNA.

29. The nucleic acid of any one of claims 1 to 25, wherein the donor polynucleotide comprises a cis-acting single-strand RNA polynucleotide annealed to a complementary single-strand DNA polynucleotide.

30. The nucleic acid of any one of claims 1 to 28, wherein the single or double stranded donor polynucleotide comprises a 2 to 18 nucleotide overhang at the 3’ end.

31. The nucleic acid of any one of claims 5 to 29, wherein the single or double stranded donor polynucleotide comprises a 14 nucleotide overhang at the 3’ end.

32. The nucleic acid of any one of claims 26 to 31 , wherein the overhang at the 3 ’ end is a single-stranded RNA polynucleotide.

33. The nucleic acid of any one of claims 1 to 32, additionally comprising a second guide RNA capable of targeting a region 5’ to the donor polynucleotide target site.

34. The nucleic acid of claim 33, wherein the first guide RNA is capable of targeting a first chimeric nuclease to a first I-TevI or Cas9 target site and cleaving at the first I-TevI or Cas9 target site in a genome of a cell and the second guide RNA is capable of targeting a second chimeric nuclease to a second I-TevI target site in the genome of the cell and cleaving at the second I-TevI target site, wherein the cleavage creates a nucleotide overhang at the second I-TevI target site.

35. The nucleic acid of claim 34, wherein the 3’ end of the donor polynucleotide is complementary to the overhang created by the cleavage of second I-TevI at the second I- TevI target site.

36. The nucleic acid of any one of claims 1 to 35, wherein the guide RNA and the donor polynucleotide target mutations in the CFTR gene.

37. The nucleic acid of any one of claims 1 to 35, wherein the guide RNA and donor polynucleotide target and replace the CFTR c.!521_1523del (p.Phe508del), c.!624G>T (p.Gly542Ter), c.!652G>A (p.Gly551Asp), C.1657OT (p.Arg553Ter) or c.3846G>A (p.Trp!282Ter) mutations.

38. The nucleic acid of any one of claims 1 to 35, wherein the guide RNA and the donor polynucleotide target mutations in the SERPINA1 gene.

39. The nucleic acid of any one of claims 1 to 35, wherein the guide RNA and donor polynucleotide target and replace the SERPINA1 c.!096G>A (p.Glu342Lys) mutation.

40. The nucleic acid of any one of claims 1 to 39, further comprising a promoter.

41. The nucleic acid of claim 40, wherein the promoter is selected from the group of CMV promoter, SV40 promoter, minimal cytomegalovirus (CMV) promoter, and an human elongation factor- 1 alpha (EFla) promoter.

42. The nucleic acid of claim 41, wherein the promoter is selected from the group of musclespecific synthetic promoter SPc5-12 neuronal-specific promoter hSYNl, aldhlLl, cTNT, alpha-MHC, SPc5-12, MUC2, Ksp-cadherin, Albumin, HAS, insulin, rhodopsin, rNSE, and Cone-opsin promoters.

43. The nucleic acid of any one of claims 1 to 42, wherein the tRNA comprises a glycine arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine tRNA.

44. The nucleic acid of any one of claims 1 to 42, wherein the ribozyme comprises a hammerhead ribozyme or a hepatitis delta virus (HDV) ribozyme.

45. The nucleic acid of any one of claims 1 to 42, wherein the donor polynucleotide comprises a trans-acting double-stranded RNA polynucleotide with 3’- or 5 ’-overhanging nucleotides; a cis-acting single-strand RNA polynucleotide with sequence similarity to the target strand of the nuclease; a cis-acting single-strand RNA polynucleotide with sequence similarity to the non-target strand of the nuclease;one or more binding sites for a genome modifying factor optionally a binding site for a site-specific recombinase, such as serine recombinases or LoxP target sites; a repair template for a protein coding sequence; one or more exons with splice acceptor and donor sequences; one or more selectable sequences selected from the group of NeoR, BsdR, HygR, PuroR, and BleoR genes; one or more drug-inducible regulatory sequences for controlled gene expression; and / or a 2 nucleotide overhang at the 3’ end.

46. The nucleic acid of any one of claims 1 to 45, further comprising a poly adenylation signal.

47. The nucleic acid of claim 46, wherein the polyadenylation signal comprises a simian virus 40 (SV40), a globin, P-globin, human growth hormone (hGH), bovine growth hormone (BGH), herpes simplex virus type 1 thymidine kinase (HSV TK), or synthetic polyadenylation (Synt poly A) polyadenylation signal.

48. The nucleic acid of any one of claims 1 to 47, further comprising a self-inactivating sequence.

49. The nucleic acid of any one of claims 1 to 48, wherein the nucleic acid is about 5kb in length.

50. The nucleic acid of any one of claims 1 to 48, wherein the nucleic acid is less than 5kb in length.

51. The nucleic acid of any one of claims 1 to 50, wherein the nucleic acid is packaged in a virus.

52. The nucleic acid of claim 51, wherein the virus is a lentivirus, adeno-associated virus (AAV), adenovirus, retrovirus, or modified Herpes Simplex Virus (HSV).

53. The nucleic acid of claim 52, wherein the AAV is a AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV7, AAV8, AAV9, AAV10, AAV-DJ, AAV2.5T or AAVmyo.

54. A vector comprising a nucleic acid of any one of claims 1 to 53.

55. A viral vector comprising a nucleic acid of any one of claims 1 to 53.

56. An AAV virus comprising a nucleic acid of any one of claims 1 to 53.

57. A cell comprising a nucleic acid of any one of claims 1 to 53, a vector of claim 54, a viral vector of claim 55, or a AAV of claim 56.

58. A composition comprising a chimeric nuclease polypeptide comprising an I-TevI domain and a RNA-guided nuclease domain and a nucleic acid of any one of claims 4 to 53.

59. A composition comprising a chimeric nuclease nucleic acid encoding a chimeric nuclease comprising an I-TevI domain and a RNA-guided nuclease domain and a nucleic acid of any one of claims 4 to 53.

60. The composition of claim 59, wherein the chimeric nuclease nucleic acid is an mRNA.

61. An LNP composition comprising a nucleic acid of any one of claims 1 to 50 or the composition of any one of claims 58 to 60.

62. A pharmaceutical composition comprising a nucleic acid of any one of claims 1 to 53, a vector of claim 54, a viral vector of claim 55, or a AAV of claim 56, the composition of claim 58, or the LNP composition of claim 61; and an excipient.

63. A method of delivering messenger RNA encoding a chimeric nuclease comprising an I- TevI domain and an RNA-guided nuclease domain to a cell the method comprising contacting the cell with a polynucleotide encoding one or more guide RNAs and polynucleotide donor.

64. A method of genetically modifying the genome of a cell, the method comprising contacting the cell with a nucleic acid of any one of claims 1 to 53, a vector of claim 54, a viral vector of claim 55, or a AAV of claim 56, the composition of claim 58, or the LNP composition of claim 61.

65. The method of claim 64, wherein the modification comprises an insertion, deletion, substitution, or mutation of the genome.

66. The method of any one of claims 63 to 65, wherein the insertion of the donor polynucleotide into the genome of the cell results in removal of sequences between a I- TevI target site and a Cas9 target site.

67. The method of claim 64 or 65, wherein the cell is a mammalian cell.

68. The method of any one of claims 64 to 65, wherein the cell is a human cell.

69. A method of inserting or replacing a sequence at a chimeric nuclease target site in a genome in a cell, the method comprising:contacting the cell with a nucleic acid comprising one or more nucleic acid sequences encoding a chimeric nuclease comprising a Cas9 domain and a I-TevI domain; and a nucleic acid comprising a guide polynucleotide and a donor polynucleotide; wherein the guide polynucleotide and the chimeric nuclease form a complex, and the complex binds and cleaves the genomic DNA at a Cas9 target site and a I- TevI target site; wherein the 3 ’ end of the donor polynucleotide comprises at least 2 bases of complementarity over the 5’ end of the I-TevI target site after I-TevI cleavage; and wherein the donor polynucleotide is incorporated into the chimeric nuclease target site at a position 5’ to the Cas9 target site.

70. The method of claim 69, wherein the 3’ end of the guide polynucleotide and the 5’ end of the donor polynucleotide are connected.

71. The method of claim 69, wherein a cellular polymerase is targeted to the chimeric nuclease target site.

72. The method of claim 71, wherein the cellular polymerase is polymerase theta.

73. A method of replacing at least a portion of a CFTR gene in the genome in a cell, the method comprising contacting the nucleic acid of any one of claims 1 to 53, a vector of claim 54, a viral vector of claim 55, or a AAV of claim 56, the composition of claim 58, or the LNP composition of claim 61 to the cell.

74. The method of claim 73, wherein the guide RNA and donor polynucleotide target mutations in the CFTR gene.

75. The method of any one of claims 73 to 74 , wherein the guide RNA and donor polynucleotide target and replace the CFTR c.!521_1523del (p.Phe508del), c.!624G>T (p.Gly542Ter), c.!652G>A (p.Gly551Asp), C.1657OT (p.Arg553Ter), or c.3846G>A (p.Trp!282Ter) mutations.

76. A method of treating cystic fibrosis in a patient in need thereof, the method comprising, administering a nucleic acid of any one of claims 1 to 53, a vector of claim 54, a viralvector of claim 55, or a AAV of claim 56, the composition of claim 58, or the LNP composition of claim 61 to the patient.

77. The method of claim 76, wherein the guide RNA and donor polynucleotide target mutations in the CFTR gene.

78. The method of claim 76, wherein the guide RNA and donor polynucleotide target and replace the CFTR c.!521_1523del (p.Phe508del), C.1624G>T (p.Gly542Ter), c.!652G>A (p.Gly551Asp), C.1657OT (p.Arg553Ter) or c.3846G>A (p.Trpl282Ter) mutations.

79. A method of replacing at least a portion of a SERPINA1 gene in the genome in a cell, the method comprising contacting the nucleic acid of any one of claims 1 to 53, a vector of claim 54, a viral vector of claim 55, or a AAV of claim 56, the composition of claim 58, or the LNP composition of claim 61 to the cell.

80. The method of claim 79, wherein the guide RNA and donor polynucleotide target mutations in the SERPINA1 gene.

81. The method of claim 79, wherein the guide RNA and donor polynucleotide target and replace the SERPINA1 c.!096G>A (p.Glu342Lys) mutation.

82. A method of treating alpha- 1 -antitrypsin deficiency in a patient in need thereof, the method comprising, administering a nucleic acid of any one of claims of claims 1 to 53, a vector of claim 54, a viral vector of claim 55, or a AAV of claim 56, the composition of claim 58, or the LNP composition of claim 61 to the patient.

83. The method of claim 82, wherein the guide RNA and donor polynucleotide target mutations in the SERPINA1 gene.

84. The method of claim 82, wherein the guide RNA and donor polynucleotide target and replace the SERPINA1 c.!096G>A (p.Glu342Lys) mutation.

85. A method of replacing at least a portion of a DMPK gene in the genome in a cell, the method comprising contacting the nucleic acid of any one of claims 1 to 53, a vector of claim 54, a viral vector of claim 55, or a AAV of claim 56, the composition of claim 58, or the LNP composition of claim 61 to the cell.

86. The method of claim 85, wherein the one or more guide RNAs target mutations in the DMPK gene.

87. The method of claim 85, wherein the one or more guide RNAs target CAG triplet polynucleotide sequences in the 3 ’ untranslated region of the DMPK gene.

88. A method of treating myotonic dystrophy type 1 in a patient in need thereof, the method comprising, administering a nucleic acid of any one of claims 1 to 53, a vector of claim 54, a viral vector of claim 55, or a AAV of claim 56, the composition of claim 58, or the LNP composition of claim 61 to the patient.

89. A method of replacing at least a portion of a C9ORF72 gene in the genome in a cell, the method comprising contacting the nucleic acid of any one of claims 1 to 53, a vector of claim 54, a viral vector of claim 55, or a AAV of claim 56, the composition of claim 58, or the LNP composition of claim 61 to the cell.

90. The method of claim 89, wherein the one or more guide RNAs target mutations in the C9ORF72 gene.

91. The method of claim 89, wherein the one or more guide RNAs target GGGGCC hexanucleotide repeat sequences between Exon la and Exon lb of the C9ORF72 gene.

92. A method of treating amyotrophic lateral sclerosis or frontotemporal dementia in a patient in need thereof, the method comprising, administering a nucleic acid of any one of claims 1 to 53, a vector of claim 54, a viral vector of claim 55, or a AAV of claim 56, the composition of claim 58, or the LNP composition of claim 61 to the patient.

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