Patent Publication Number: US-2023151353-A1

Title: Direct replacement genome editing

Description:
CROSS-REFERENCE 
     This application claims the benefit of U.S. Provisional Application Ser. No. 63/278,886 filed on Nov. 12, 2021, and U.S. Provisional Application Ser. No. 63/341,200 filed on May 12, 2022, the entireties of which are hereby incorporated by reference. 
    
    
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on Nov. 7, 2022, is named “Replace Therapeutic 62942-701201” and is 703,885 bytes in size.” 
     BACKGROUND 
     Improved gene editing methods are needed for modifying nucleic acids. 
     SUMMARY 
     Disclosed herein, in some aspects, are systems or compositions comprising: a DNA-binding protein coupled to a DNA ligase. The DNA-binding protein may include an endonuclease. The endonuclease may include an RNA-guided endonuclease. In some aspects, the coupling is covalent. Some aspects include a fusion protein comprising the DNA-binding protein (e.g. endonuclease such as an RNA-guided endonuclease) and the DNA ligase. Some aspects include a composition comprising: a cell containing a DNA-binding protein (e.g. endonuclease such as an RNA-guided endonuclease) and a DNA ligase, both of which are heterologous to the cell. In some aspects, the DNA-binding protein is amino (N)-terminal relative to the DNA ligase within the fusion protein. In some aspects, the DNA-binding protein is carboxy (C)-terminal relative to the DNA ligase within the fusion protein. In some aspects, the connection comprises a linker comprising 1-100 amino acids. In some aspects, the coupling is non-covalent. In some aspects, the composition comprises a first polypeptide comprising at least part of the DNA-binding protein, and a second polypeptide comprising at least part of the DNA ligase, wherein the first and second polypeptides are non-covalently coupled. In some aspects, the first polypeptide comprises a first heterodimerization domain that binds a second heterodimerization domain, and wherein the second polypeptide comprises the second heterodimerization domain. In some aspects, the heterodimer domains comprise a leucine zipper, PDZ domain, streptavidin, streptavidin binding protein, foldon domain, hydrophobic moiety, or a functional binding fragment thereof. In some aspects, the first polypeptide comprises a first intein that binds a second intein, and wherein the second polypeptide comprises the second intein. In some aspects, the ligase comprises a hairpin binding motif, and wherein the DNA-binding protein and the DNA ligase are coupled with a nucleic acid comprising a scaffold that binds to the DNA-binding protein and a hairpin that binds to the hairpin binding motif. In some aspects, the hairpin binding motif comprises an MS2 coat protein (MCP) peptide, and wherein the hairpin comprises an MS2 hairpin. In some aspects, the DNA-binding protein and the DNA ligase are coupled with a heterobifunctional molecule comprising an endonuclease binding domain and a DNA ligase binding domain. In some aspects, the heterobifunctional molecule comprises a small molecule. In some aspects, the DNA-binding protein comprises a class II CRISPR/Cas endonuclease. In some aspects, the DNA-binding protein comprises a Cas9 endonuclease. In some aspects, the DNA-binding protein comprises a nickase. In some aspects, the DNA-binding protein comprises an amino acid sequence at least 80% identical to the amino acid sequence of any one of SEQ ID NOS: 1-13, or a functional fragment thereof. In some aspects, the DNA ligase ligates DNA strands base paired to a DNA splint. In some aspects, the DNA ligase ligates DNA strands base paired to an RNA splint. In some aspects, the DNA ligase comprises an amino acid sequence at least 80% identical to the amino acid sequence of any one of SEQ ID NOS: 55-96, or a functional fragment thereof. In some aspects, the DNA-binding protein or the DNA ligase comprises a nuclear localization signal, chromatin modifying domain, cell penetrating peptide, or tag polypeptide. Some aspects include a guide RNA and an integrating nucleic acid. Some aspects include one or more nucleic acids encoding the composition. Some aspects include a cell comprising the composition, or comprising the one or more nucleic acids. 
     Disclosed herein, in some aspects, are editing methods, comprising: contacting a target nucleic acid in a cell with an endonuclease at a predetermined locus of the target nucleic acid, thereby introducing a nick at the predetermined locus of the target nucleic acid; introducing a pre-synthesized integrating nucleic acid to the cell; and ligating a 5′ end of the pre-synthesized integrating nucleic acid to a 3′ end of the nick at the predetermined locus of the target nucleic acid. In some aspects, the endonuclease comprises a class II CRISPR/Cas endonuclease. In some aspects, the endonuclease comprises Cas9 nickase. Some aspects include contacting the endonuclease and the predetermined locus of the target nucleic acid with a guide nucleic acid. In some aspects, said ligating is performed by a ligase coupled to the endonuclease. In some aspects, the pre-synthesized integrating nucleic acid comprises a mutation in relation to the target nucleic acid. In some aspects, the nick comprises a single phosphodiester strand break in the otherwise double stranded target nucleic acid. In some aspects, the nick comprises a non-sticky, non-blunt end of a strand of the target nucleic acid. In some aspects, the target nucleic acid comprises a chromosome of the cell. In some aspects, the cell is eukaryotic. 
     Disclosed herein, in some aspects, are editing systems, comprising: a ligase; an endonuclease that introduces a nick at a predetermined locus of a target nucleic acid; and a pre-synthesized integrating nucleic acid comprising a 5′ end that is ligated by the ligase to a 3′ end of the nick at the predetermined locus of the target nucleic acid. In some aspects, the endonuclease comprises a class II CRISPR/Cas endonuclease. In some aspects, the endonuclease comprises Cas9 nickase. Some aspects include a guide nucleic acid that brings the endonuclease into proximity with the predetermined locus of the target nucleic acid. In some aspects, the ligase is coupled to the endonuclease. In some aspects, the pre-synthesized integrating nucleic acid comprises a mutation in relation to the target nucleic acid. In some aspects, the nick comprises a single phosphodiester strand break in the otherwise double stranded target nucleic acid. In some aspects, the nick comprises a non-sticky, non-blunt end of a strand of the target nucleic acid. In some aspects, the target nucleic acid comprises a chromosome of a cell. In some aspects, the cell is eukaryotic. 
     Disclosed herein, in some aspects, are systems of nucleic acids comprising: a guide nucleic acid comprising: (a) a spacer complementary to a region of a genomic locus of a genomic strand, (b) a scaffold for complexing with a DNA-binding protein, (c) an optional donor binding site that is at least partially complementary to an integrating nucleic acid, and (d) a flap binding site that is at least partially identical or complementary to a genomic flap at or adjacent to the genomic locus; and an integrating nucleic acid comprising a 5′ end to be ligated to a 3′ terminus of the genomic strand generated by a DNA-binding protein. The DNA-binding protein may include an endonuclease. The endonuclease may include an RNA-guided endonuclease. Disclosed herein, in some aspects, are systems of nucleic acids comprising: a guide nucleic acid comprising: (a) a spacer complementary to a region of a genomic locus of a genomic strand, (b) a scaffold for complexing with a DNA-binding protein, and (c) an optional donor binding site that is at least partially complementary to a splinting nucleic acid; an integrating nucleic acid comprising a 5′ end to be ligated to a 3′ terminus of the genomic strand generated by a DNA-binding protein; and a splinting nucleic acid comprising a flap binding site that is at least partially identical or complementary to a genomic flap at or adjacent to the genomic locus, and comprising an optional guide binding site that is at least partially complementary to a guide nucleic acid. In some aspects, the genomic strand is in a cell. In some aspects, the splinting nucleic acid further comprises a donor binding site that is at least partially identical or complementary to a portion of the integrating nucleic acid. In some aspects, the guide nucleic acid comprises a sequence of linking nucleic acids between the scaffold and the donor binding site. In some aspects, the guide nucleic acid or the integrating nucleic acid comprises a modified internucleoside linkage. In some aspects, the modified internucleoside linkage comprises a phosphorothioate linkage. In some aspects, the modified internucleoside linkage is between any of the 4 terminal nucleosides at a 5′ end or at a 3′ end of the guide nucleic acid or the integrating nucleic acid. In some aspects, the guide nucleic acid or the integrating nucleic acid comprises a modified nucleoside. In some aspects, the modified nucleoside comprises a locked nucleic acid (LNA), a 2′ fluoro, a 2′ O-alkyl, or a combination thereof. In some aspects, the modified nucleoside is any of the 3 terminal nucleosides at a 5′ end or at a 3′ end of the guide nucleic acid or the integrating nucleic acid. The modified nucleoside may include an LNA, a 2′fluoro, a 2′ O-alkyl, a methylated cytosine, an inverted thymidine, or a combination thereof. 
     Disclosed herein, in some aspects, are compositions, comprising: a DNA-binding protein connected to a DNA ligase. The DNA-binding protein may include an endonuclease. The endonuclease may include an RNA-guided endonuclease. In some aspects, the connection between the DNA-binding protein and the DNA ligase is covalent. Some aspects include a fusion protein comprising the DNA-binding protein upstream of the DNA ligase. Some aspects include a fusion protein comprising the DNA-binding protein downstream of the DNA ligase. In some aspects, the connection comprises a linker comprising 1-100 amino acids. In some aspects, the composition comprises a first polypeptide comprising at least part of the DNA-binding protein, and a second polypeptide comprising at least part of the DNA ligase, wherein the first and second polypeptides are bound together covalently or non-covalently. In some aspects, the first polypeptide comprises a first heterodimerization domain that binds a second heterodimerization domain, and wherein the second polypeptide comprises the second heterodimerization domain. In some aspects, the heterodimer domains comprise a leucine zipper, PDZ domain, streptavidin, streptavidin binding protein, foldon domain, hydrophobic moiety, or a functional binding fragment thereof. In some aspects, the first polypeptide comprises a first intein that binds a second intein, and wherein the second polypeptide comprises the second intein. In some aspects, the DNA-binding protein and the DNA ligase are bound together by a small molecule. In some aspects, the DNA-binding protein comprises a class II CRISPR/Cas endonuclease. In some aspects, the DNA-binding protein comprises a Cas9 endonuclease. In some aspects, the DNA-binding protein comprises a nickase. In some aspects, the DNA-binding protein comprises an amino acid sequence at least 80% identical to the amino acid sequence of any one of SEQ ID NOS: 1-13, or a functional fragment thereof. In some aspects, the DNA ligase ligates DNA strands base paired to a DNA splint. In some aspects, the DNA ligase ligates DNA strands base paired to an RNA splint. In some aspects, the DNA ligase comprises an amino acid sequence at least 80% identical to the amino acid sequence of any one of SEQ ID NOS: 55-96, or a functional fragment thereof. In some aspects, the DNA-binding protein or the DNA ligase comprises a nuclear localization signal, chromatin modifying domain, cell penetrating peptide, or tag polypeptide. Some aspects include a guide RNA and an integrating nucleic acid. Some aspects relate to a cell comprising the composition. Some aspects include a nucleic acid encoding the composition. Some aspects include one or more nucleic acids encoding the first or second polypeptides. Some aspects include an editing method (e.g. nucleic acid) which uses the composition. Some aspects include a method of treatment using the composition. Some aspects include administering the composition to a subject. 
     Disclosed herein, in some aspects, are fusion proteins, comprising: a DNA-binding protein fused to a DNA ligase. The DNA-binding protein may include an endonuclease. The endonuclease may include an RNA-guided endonuclease. Disclosed herein, in some aspects, are protein complexes, comprising: a DNA-binding protein bound to a DNA ligase. In some aspects, the endonuclease and the DNA ligase are bound together through heterodimerization domains. In some aspects, the heterodimerization domains comprise leucine zippers, PDZ domains, streptavidin and streptavidin binding protein, foldon domains, hydrophobic polypeptides, an antibody that binds the Cas nickase, or an antibody that binds the DNA ligase, or one or more binding fragments thereof. Disclosed herein, in some aspects, are cells comprising the fusion protein or the protein complex. Disclosed herein, in some aspects, are cells comprising a heterologous DNA-binding protein and a DNA ligase that was introduced into the cell. Some aspects include a nuclease that is different from the DNA-binding protein. Disclosed herein, in some aspects, are guide nucleic acids, comprising: a spacer at least partially reverse complementary to a first region of a target nucleic acid; a scaffold configured to bind to an endonuclease; and a flap binding site at least partially reverse complementary to a nucleic acid flap, and an integrating nucleic acid binding site. Disclosed herein, in some aspects, are integrating nucleic acids, comprising: a single or double-stranded DNA region to be inserted into a target nucleic acid, wherein the single or double-stranded DNA region is flanked by at least one additional single-stranded region comprising a guide binding site. Disclosed herein, in some aspects, are editing systems, comprising a DNA-binding protein, the guide nucleic acid, and the integrating nucleic acid. Disclosed herein, in some aspects, are editing methods, comprising: contacting a target nucleic acid with the editing system and a DNA ligase. 
     Disclosed herein, in some aspects, are systems comprising: at least one DNA-binding protein; at least one guide nucleic acid comprising: a spacer at least partially complementary to a genomic locus in a cell; a scaffold for complexing with the at least one DNA-binding protein; and an optional donor binding site that is at least partially complementary to an integrating nucleic acid; and at least one DNA ligase; and the integrating nucleic acid, comprising a flap binding site at least partially reverse complementary to a nucleic acid flap and optionally comprising a guide binding site that is at least partially complementary to the at least one guide nucleic acid, wherein the at least one DNA-binding protein cleaves or nicks at least one strand of the genomic locus, and wherein the at least one DNA ligase ligates an end of the integrating nucleic acid to the genomic flap site, thereby replacing a region of the genomic locus with the integrating nucleic acid in the cell. The DNA-binding protein may include an endonuclease. The endonuclease may include an RNA-guided endonuclease. In some aspects, the integrating nucleic acid comprises a single-stranded DNA. In some aspects, the integrating nucleic acid comprises a double-stranded DNA. 
     Disclosed herein, in some aspects, are systems comprising: at least one DNA-binding protein comprising a first DNA-binding protein and an optional second DNA-binding protein; at least one guide nucleic acid comprising a first guide nucleic acid and a second guide nucleic acid, the first guide nucleic acid comprising: a first spacer complementary to a first region of a genomic locus in a cell; a first scaffold for complexing with the first DNA-binding protein; and an optional first donor binding site that at least partially complementary to an integrating nucleic acid; and a first flap binding site that is at least partially identical or complementary to a first genomic flap at or adjacent to the genomic locus; and the second guide nucleic acid comprising: a second spacer complementary to a second region of the genomic locus in the cell; a second scaffold for complexing with the first or second DNA-binding protein; an optional second donor binding site that at least partially complementary to the integrating nucleic acid; and a second flap binding site that is at least partially identical or complementary to a second genomic flap at or adjacent to the genomic locus; at least one DNA ligase comprising a first DNA ligase and an optional second DNA ligase; and at least one integrating nucleic acid comprising a first strand and a second strand: wherein the first strand comprises an optional first guide binding site that is at least partially complementary to the first guide nucleic acid; and wherein the second strand comprises an optional second guide binding site that is at least partially complementary to the second guide nucleic acid, wherein the first DNA-binding protein and/or the second DNA-binding protein each cleaves or nicks at least one strand of the genomic locus in the cell; and wherein the first DNA ligase ligates an end of the first strand of the integrating nucleic acid to the first genomic flap; and the first or second DNA ligase ligates an end of the second strand of the integrating nucleic acid to the second genomic flap, thereby replacing a region of the genomic locus with the integrating nucleic acid in the cell. In some aspects, the integrating nucleic acid comprises a double-stranded DNA duplex region. The DNA-binding protein may include an endonuclease. The endonuclease may include an RNA-guided endonuclease. In some aspects, the integrating nucleic acid comprises a 5′ overhang optionally comprising the first guide binding site. In some aspects, the integrating nucleic acid comprises a 5′ overhang optionally comprising the second guide binding site. 
     Disclosed herein, in some aspects, are systems comprising: at least one DNA-binding protein; at least one guide nucleic acid comprising: a spacer complementary to a genomic locus in a cell; a scaffold for complexing with the at least one DNA-binding protein; and an optional donor binding site that is at least partially complementary to an integrating nucleic acid; at least one DNA ligase; and the integrating nucleic acid that: comprises an optional guide binding site that is at least partially complementary to the at least one guide nucleic acid; and comprises a flap binding site that is at least partially identical or complementary to a genomic flap at or adjacent to the genomic locus, wherein the at least one DNA-binding protein cleaves or nicks at least one strand of the genomic locus; and wherein the at least one DNA ligase ligates an end of the integrating nucleic acid to the genomic flap, thereby replacing a region of the genomic locus with the integrating nucleic acid in the cell. The DNA-binding protein may include an endonuclease. The endonuclease may include an RNA-guided endonuclease. In some aspects, the integrating nucleic acid comprises a DNA comprising a 3′ overhang. In some aspects, the 3′ overhang comprises the guide binding site. In some aspects, the 3′ overhang comprises the flap binding site. In some aspects, the at least one DNA ligase ligates a strand of the integrating nucleic acid to the genomic nucleic acid sequence. 
     Disclosed herein, in some aspects, are systems comprising: at least one DNA-binding protein comprising a first DNA-binding protein and an optional second DNA-binding protein; at least one guide nucleic acid comprising a first guide nucleic acid and a second guide nucleic acid, the first guide nucleic acid comprising: a first spacer complementary to a first region of a genomic locus in a cell; a first scaffold for complexing with the first DNA-binding protein; and an optional first donor binding site that at least partially complementary to an integrating nucleic acid; and the second guide nucleic acid comprising: a second spacer complementary to a second region of the genomic locus in the cell; a second scaffold for complexing with the first or second DNA-binding protein; and an optional second donor binding site that at least partially complementary to the integrating nucleic acid; and at least one DNA ligase comprising a first DNA ligase and an optional second DNA ligase; and the integrating nucleic acid comprising a first strand and a second strand: wherein the first strand comprises an optional first guide binding site that is at least partially complementary to the first guide nucleic acid; wherein the second strand comprises an optional second guide binding site that is at least partially complementary to the second guide nucleic acid; wherein the first strand comprises a first flap binding site that is at least partially identical or complementary to a first genomic flap at or adjacent to the genomic locus; and wherein the second strand comprises a second flap binding site that is at least partially identical or complementary to a second genomic flap at or adjacent to the genomic locus; wherein the first DNA-binding protein and/or the second DNA-binding protein each cleaves or nicks at least one strand of the genomic locus in the cell; and wherein the first DNA ligase ligates an end of the first strand of the integrating nucleic acid to the first genomic flap; and the first or second DNA ligase ligates an end of the second strand of the integrating nucleic acid to the second genomic flap, thereby replacing a region of the genomic locus with the integrating nucleic acid in the cell. The DNA-binding protein may include an endonuclease. The endonuclease may include an RNA-guided endonuclease. In some aspects, the integrating nucleic acid comprises a double-stranded DNA duplex region. In some aspects, the double-stranded DNA comprises a 3′ overhang optionally comprising the first guide binding site, and comprising the first flap binding site. In some aspects, the double stranded DNA comprises a 3′ overhang optionally comprising the second guide binding site, and comprising the second flap binding site. 
     The DNA-binding protein may include an endonuclease. The endonuclease may include an RNA-guided endonuclease. In some aspects, the at least one DNA-binding protein comprises a Cas protein or a functional fragment thereof. In some aspects, the Cas protein or the functional fragment thereof comprises nickase activity. In some aspects, the at least one DNA-binding protein comprises a Cas9 nickase or a functional fragment thereof. In some aspects, the at least one DNA ligase ligates nucleic acids bound to DNA. In some aspects, the at least one DNA ligase ligates nucleic acids bound to RNA. In some aspects, the at least one DNA ligase comprises a PBCV-1 DNA ligase. In some aspects, the at least one DNA ligase is operatively coupled to the at least one DNA-binding protein. In some aspects, the at least one DNA ligase is fused to the at least one DNA-binding protein as a fusion polypeptide. In some aspects, the at least one DNA-binding protein and the at least one DNA ligase each comprises a heterodimer domain. In some aspects, the at least one DNA-binding protein and the at least one DNA ligase forms a heterodimer via the heterodimer domain. In some aspects, the at least one DNA-binding protein comprises a linker. In some aspects, the linker connects the Cas protein or a functional fragment thereof to the heterodimer domain. In some aspects, the at least one DNA-binding protein comprises a localization signal sequence. In some aspects, the at least one DNA ligase comprises a localization signal sequence. In some aspects, the localization signal sequence comprises a nuclear localization sequence (NLS). In some aspects, the a least one DNA-binding protein or the at least one DNA ligase are directed to nucleus of the cell by the NLS. In some aspects, the at least one integrating nucleic acid corrects at least one genetic mutation in the at least one genomic locus. In some aspects, the at least one integrating nucleic acid inserts a coding sequence. In some aspects, the coding sequence encodes a full length protein. In some aspects, the at least one integrating nucleic acid inserts a non-coding sequence. In some aspects, the non-coding sequence knocks out an endogenous gene. In some aspects, the non-coding sequence comprises a regulatory element. Some aspects further include a nuclease. In some aspects, the nuclease comprises an exonuclease for digesting the genomic flap. In some aspects, the nuclease comprises a human flap endonuclease 1 (hFEN1), a human exonuclease 5 (hEXO5), a T5 exonuclease, a T7 exonuclease, an exonuclease VIII, a flap endonuclease domain of  E. coli  PolI, a RecJF, a Lambda exonuclease, a Xni (ExoIXI), a SaFEN ( Staphylococcus aureus  FEN), a nuclease BAL-31, or a fragment thereof. In some aspects, the heterologous nuclease comprises an endonuclease for digesting the genomic flap, and the endonuclease is different from the at least one DNA-binding protein. In some aspects, the at least one DNA-binding protein comprises at least one additional functional domain. In some aspects, the at least one additional functional domain comprises a chromatin modifying domain. In some aspects, the at least one additional functional domain comprises a cell penetrating peptide. In some aspects, the at least one guide nucleic acid comprises at least one nucleic acid modification. In some aspects, the at least one nucleic acid modification comprises a modification to a backbone, a sugar, a base, or a combination thereof. In some aspects, the at least one DNA-binding protein is complexed with the at least one guide nucleic acid. In some aspects, the at least one guide nucleic acid is complexed with the integrating nucleic acid. In some aspects, the at least one DNA-binding protein, the at least one guide nucleic acid, the at least one at least one DNA ligase, the integrating nucleic acid, or a combination thereof is encoded by a polynucleotide. In some aspects, the polynucleotide comprises mRNA. In some aspects, the polynucleotide comprises a vector. In some aspects, the vector comprises a viral vector. In some aspects, the at least one DNA-binding protein, the at least one guide nucleic acid, the at least one at least one DNA ligase, the integrating nucleic acid, or a combination thereof is encapsulated by at least one lipid nanoparticle. In some aspects, the cell comprises a bacterial cell, an eukaryotic cell, or a plant cell. In some aspects, the eukaryotic cell comprises a mammalian cell. Some aspects include a composition comprising the system. Some aspects include a cell comprising the system. Some aspects include a cell line comprising the cell. Some aspects include a pharmaceutical composition comprising the system. Some aspects include a pharmaceutical composition comprising the composition. Some aspects include a pharmaceutical composition comprising the cell. Some aspects include a pharmaceutically acceptable: excipient, carrier, or diluent. In some aspects, the pharmaceutical composition is formulated for administering intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, intratumorally, pulmonarily, endotracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, by inhalation, by inhaled nebulized form, by intraluminal-GI route, or a combination thereof to a subject in need thereof. Some aspects include a kit comprising: the system, the composition, or the pharmaceutical composition and a container. In some aspects, include method for modifying a cell comprising contacting a cell with the system. In some aspects, include method for modifying a cell comprising contacting a cell with the composition. In some aspects, include method for modifying a cell comprising contacting a cell with the pharmaceutical composition. In some aspects, the cell is not a dividing cell. In some aspects, the integrating nucleic acid is inserted into the genomic locus of the cell independent of endogenous non-homologous end joining (NHEJ) and independent of endogenous homology-directed repair (HDR). Some aspects include a method for treating a disease or condition in subject in need thereof comprising: contacting the cell or the subject with the system, the composition, or the pharmaceutical composition; replacing a genomic locus in a cell with an integrating nucleic acid, thereby treating the disease or condition in the subject. In some aspects, the cell is not a dividing cell. In some aspects, the integrating nucleic acid is inserted into the genomic locus of the cell independent of endogenous non-homologous end joining (NHEJ) and independent of endogenous homology-directed repair (HDR). 
     Disclosed herein, in some aspects, are guide nucleic acids comprising: a spacer that is at least partially complementary to a genomic locus in a cell; a scaffold for complexing with a DNA-binding protein; and a donor binding site that is at least partially complementary to an integrating nucleic acid. The DNA-binding protein may include an endonuclease. The endonuclease may include an RNA-guided endonuclease. In some aspects, the guide nucleic acid comprises a flap binding site that is at least partially complementary to a genomic sequence of the genomic locus. In some aspects, the guide nucleic acid comprises at least one nucleic acid modification. In some aspects, the at least one nucleic acid modification comprises a modification to a backbone, a sugar, a base, or a combination thereof. In some aspects, the guide nucleic acid comprises RNA sequence. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG.  1 A  illustrates a guide nucleic acid, an endonuclease, a ligase, and a donor strand at a genomic locus. 
         FIG.  1 B  follows sequentially from  FIG.  1 A , and illustrates a donor strand incorporated into one side of a genomic locus, the donor strand having displaced a genomic flap. 
         FIG.  1 C  follows sequentially from  FIG.  1 B , and illustrates a donor strand incorporated into one side of a genomic locus, and a nick appearing where a genomic flap has been removed. 
         FIG.  2 A  illustrates 2 guide nucleic acids, 2 endonucleases, 2 ligases, and a donor strand at a genomic locus. 
         FIG.  2 B  follows sequentially from  FIG.  2 A , and illustrates a donor strand incorporated into a genomic locus, the donor strand having displaced 2 genomic flaps. 
         FIG.  2 C  follows sequentially from  FIG.  2 B , and illustrates a donor strand incorporated into a genomic locus, and 2 nicks appearing where genomic flaps have been removed. 
         FIG.  3 A  illustrates a guide nucleic acid, an endonuclease, a ligase, and a donor strand at a genomic locus. 
         FIG.  3 B  follows sequentially from  FIG.  3 A , and illustrates a donor strand incorporated into one side of a genomic locus, the donor strand having displaced a genomic flap. 
         FIG.  3 C  follows sequentially from  FIG.  3 B , and illustrates a donor strand incorporated into one side of a genomic locus, and a nick appearing where a genomic flap has been removed. 
         FIG.  4 A  illustrates 2 guide nucleic acids, 2 endonucleases, 2 ligases, and a donor strand at a genomic locus. 
         FIG.  4 B  follows sequentially from  FIG.  4 A , and illustrates a donor strand incorporated into a genomic locus, the donor strand having displaced 2 genomic flaps. 
         FIG.  4 C  follows sequentially from  FIG.  4 B , and illustrates a donor strand incorporated into a genomic locus, and 2 nicks appearing where genomic flaps have been removed. 
         FIG.  5 A  illustrates a guide nucleic acid, an endonuclease, a ligase, and a donor strand at a genomic locus. 
         FIG.  5 B  follows sequentially from  FIG.  5 A , and illustrates a donor strand incorporated into a genomic locus, the donor strand having displaced a genomic flap. 
         FIG.  5 C  follows sequentially from  FIG.  5 B , and illustrates a donor strand incorporated into one side of a genomic locus, and a nick appearing where a genomic flap has been removed. 
         FIG.  6 A  illustrates 2 guide nucleic acids, 2 endonucleases, 2 ligases, and a donor strand at a genomic locus. 
         FIG.  6 B  follows sequentially from  FIG.  6 A , and illustrates a donor strand incorporated into a genomic locus, the donor strand having displaced 2 genomic flaps. 
         FIG.  6 C  follows sequentially from  FIG.  6 B , and illustrates a donor strand incorporated into a genomic locus, and 2 nicks appearing where genomic flaps have been removed. 
         FIG.  7    illustrates some examples of fusion protein arrangements. 
         FIG.  8 A  illustrates an exemplary nicking and ligation pattern of an integrating nucleic acid. 
         FIG.  8 B  illustrates a DNA gel showing a pattern associated with 1-Sided Replacer 2 performed in vitro using 30 nt GBS/DBS and thermostable T4 ligase. Using a 30 nt GBS/DBS combination, a donor containing a protospacer adjacent motif (PAM) mutation, and a thermostable T4 ligase (Hi-T4, NEB), we were able to produce a final Replacer product (Lane 3) corresponding to the size of our control product (Lane 1). Replacer products were not detected in the absence of nicking Cas9 (Cas9n) (Lane 2), or in the absence of the bottom donor which serves as the splint (Lanes 4 &amp; 5). 
         FIG.  8 C  illustrates an exemplary nucleic acid gel showing pattern associated with in vitro 1-Sided Replacer 2 using variable length GBS/DBS combinations and T4 ligase. Using regular T4 ligase (NEB), we were to produce a final Replacer product corresponding to the size of the control when using multiple GBS/DBS combinations, including no GBS/DBS, 20 nt GBS/DBS, and 30 nt GBS/DBS. Additionally, in this experiment, recoded dsDNA donors containing PAM mutation were more efficient at producing final Replacer products compared to PAM mutant dsDNA donors that were not recoded. 
         FIG.  9    illustrates measurement of a percentage of cells expressing green fluorescent protein (GFP), indicating gene editing from BFP to GFP by a 1-sided Replacer 2 with nicking Cas9 and DNA ligase. 
         FIG.  10    illustrates sequencing reads merged and aligned to an amplicon of interest and a percentage of total reads that matched an intended edit via a 1-sided replacer 2 with a nicking Cas9 and a T4 DNA ligase. 
         FIG.  11    illustrates sequencing reads merged and aligned to an amplicon of interest and a percentage of total reads that matched an intended edit via a 2-sided replacer 2 with a nicking Cas9 and a T4 DNA ligase. 
         FIG.  12    illustrates measurement of a percentage of cells expressing green fluorescent protein (GFP), indicating gene editing from BFP to GFP via a 1-Sided Replacer 2 with a nicking Cas9 and a T4 DNA Ligase. 
     
    
    
     DETAILED DESCRIPTION 
     Introduction 
     Recent advances in gene editing tools have enabled precision editing of genomes for therapeutic, agricultural, industrial, and research purposes. Some nuclease-based tools such as CRISPR-Cas9 use a guide RNA to target the Cas9 protein to a specific DNA sequence specified by the spacer sequence in the guide RNA. Cas9 nuclease activity then cleaves the DNA resulting in a double-stranded break (DSB). DSBs are typically repaired through endogenous DNA repair mechanisms including non-homologous end joining (NHEJ) or homology-directed repair (HDR). However, NHEJ results in a spectrum of nucleotide insertions and deletions (indels) that hinder its utility for precision editing. HDR efficiency is very low in nondividing cells and may require DNA replication. Even when HDR editing is detectable, DSB-induced indels are often prevalent, meaning that HDR may not be feasible when precision editing is desired. 
     Homology-independent targeted insertion (HITI) utilizes NHEJ DNA repair mechanisms active in nondividing cells for CRISPR-guided transgene integration in nondividing cells such as primary neurons, retinal pigment epithelial cells, and HSPCs. However, due to the generation of DSBs from Cas9, HITI generates high frequencies of indels, resulting in unintended mutations in addition to DSB associated toxicity. 
     Other methods for gene editing have additional limitations. Tools employing fusions of nicking Cas nucleases with nucleotide deaminases (e.g. base editors) can perform certain nucleotide mutations, e.g. cytosine base editors can convert C to T. While some base editors can perform precision editing at high efficiency, they are inherently limited to specific edits determined by the deaminase variant so they are only applicable to specific substitution mutations and further cannot perform precise insertion or deletion edits. Moreover, base editors are generally limited to a small editing window within a subset of the protospacer region and are therefore significantly limited by protospacer adjacent motif (PAM) availability. Finally, base editors can exhibit bystander mutations within the editing region (e.g. if two C&#39;s are present) and have demonstrated DNA and RNA off-target deaminase activity. 
     Existing precision editing technologies have limitations that hamper their practical applicability in a variety of ways. In particular, they may rely on endogenous cellular machinery for editing, for example HDR machinery for nuclease-based editing and mismatch repair for base editing. No system has been reported that is independent of all endogenous factors. Reliance on endogenous factors is problematic because different cell types have different activity levels of these endogenous factors, and in many cases the activity is not sufficient to provide useful levels of editing. An example where this reliance is particularly problematic is nondividing cells, which comprise the majority of cells in adults and therefore are not amenable to many existing precision editing tools. 
     Accordingly, there remains a need for a system or a method for effective gene editing or for modifying gene expression by gene editing. Particularly, there remains a need for the system or method for gene editing or modifying gene expression, where the system or the method do not rely on the endogenous components or mechanism of a cell. There also remains a need for a system or a method for correcting genetic mutations in a cell. In some cases, the correction of genetic mutation can treat a disease or condition in subject in need thereof. As will be seen below, the systems, methods, and compositions disclosed herein may be useful for addressing these needs or limitations. 
     Overview 
     Described herein are self-contained gene editing systems. In some such self-contained systems, every aspect of gene editing may be controlled. Some such systems do not rely on host cell machinery to perform an editing function, or to replace or repair any aspect of a target nucleic acid such as a genomic locus. Some such systems are unaffected by a cell&#39;s nucleotide triphosphate (dNTP) concentration because the editing may be performed without use of a polymerase. For example, an integrating nucleic acid may be delivered and inserted into a genetic locus without transcribing a template. The editing may exclude a need to rely on a cell repair system such as HDR or NHEJ. The editing may be performed without cell cycling. The gene editing may take place in a cell, or may even be performed in vitro. For example, the gene editing may even be performed in a test tube or outside of a cell. 
     Described herein are systems and methods for editing DNA with a donor strand without generating a double-stranded break in the genome using CRISPR-guided DNA ligases and guide nucleic acids targeting the genomic region of interest. DNA ligases are enzymes which chemically join two DNA molecules via a phosphodiester bond. DNA ligases may or may not require hybridization of the DNA molecules to a DNA or RNA backbone or “splint” which is reverse complementary to the DNA sequences that are to be ligated. Targeting of ligases to genomic nicks generated by CRISPR nucleases enables precise replacement of genomic DNA with donor strands optionally recruited by guide nucleic acids into targeted loci. The CRISPR-guided DNA ligases can be composed of DNA ligases that are fused, recruited, or unfused to the RNA-guided endonuclease by utilizing peptide linkers, heterodimerization domains, or two separate peptides, respectively. 
     Some aspects include a cell containing or comprising an RNA-guided endonuclease and a DNA ligase, both of which are introduced into the cell. The endonuclease or ligase may be heterologous to the cell. The endonuclease and ligase may be heterologous to the cell. The ligase may be endogenous to the cell. In some aspects, a cell comprises an RNA-guided endonuclease and a DNA ligase, both of which are heterologous to the cell. The cell may include a composition or system described herein. The cell may be used or included in a system, composition, or method described herein. 
     A system described herein may include a heterologous endonuclease comprising an RNA-guided endonuclease such as nicking Cas9 as well as a heterologous ligase (e.g., a DNA ligase) that can utilize an RNA splint. The guide nucleic acid optionally recruits a donor strand to the site targeted by the endonuclease (e.g., a targeted genomic locus) and also generates a splint across from the donor strand (donor strand) and genomic flap generated by the nicking Cas9, resulting in ligation of the donor strand and the genomic flap by the DNA ligase. In some embodiments, the ligase is or comprises an endogenous ligase. The system can utilize one or more guide nucleic acids that together can comprise the following components, optionally in the following order: 5′ spacer—scaffold—donor binding site (optional)—flap binding site 3′. The donor strand (donor strand) can comprise the following sequence components: 5′ guide binding site—donor strand 3′. The guide binding site of the donor strand is at least partially reverse complementary to the donor binding site of the guide nucleic acid such that the donor hybridizes to the guide and is localized to the target site of the RNA guided endonuclease. The 5′ end of the donor sequence and the 3′ end of the genomic flap generated by nuclease nicking activity are ligated by the DNA ligase, splinted by the donor binding site and a flap binding site of the guide nucleic acid(s). 
       FIG.  1 A- 1 C  illustrate a non-limiting example of a system (1-sided Replacer 1). The example includes a guide nucleic acid comprising: a spacer for targeting a genomic locus; a scaffold for complexing and recruiting an endonuclease described herein; a donor binding site for complexing with a donor strand; and a flap binding site for complexing with a genomic flap of the genomic locus. The guide nucleic acid is shown complexed with an endonuclease (e.g., a Cas9 nickase, nCas9) operatively coupled to a ligase. The guide nucleic acid may direct the endonuclease to a genomic locus that is bound by the spacer of the guide nucleic acid. The guide nucleic acid is also shown as partially complementary to a donor strand (complexing between the donor binding site of the guide nucleic acid and guide binding site of the donor strand). The endonuclease, when directed by the guide nucleic acid, can cleave or nick at least one strand of the genomic locus, and the ligase can ligate one end of the donor strand with the cleaved or nicked end of the genomic locus, thus incorporating the donor strand into the genomic locus. The incorporation of the donor strand into the genomic locus may generate a genomic flap that can be digested and removed by a nuclease. 
       FIG.  2 A- 2 C  illustrate a non-limiting example of a system (2-sided Replacer 1). The guide nucleic acid in the example, similar to the guide nucleic acid of  FIG.  1 A , comprises: a spacer for targeting a genomic locus; a scaffold for complexing and recruiting an endonuclease described herein; a donor binding site for complexing with a donor strand; and a flap binding site for complexing with a genomic flap of the genomic locus. In  FIG.  2 A , a first guide nucleic acid is shown complexed with a first endonuclease operatively coupled with a first ligase and a second guide nucleic acid is complexed with a second endonuclease operatively coupled with a second ligase. The first endonuclease and the second nuclease may each cleave at least one strand of the genomic locus. The two cleaved ends of the genomic locus can then be ligated to the two ends of the donor strand, thereby incorporating the donor strand into the genomic locus. The insertion of the donor strand at the genomic locus may generate two genomic flaps that can be digested and removed by a nuclease. 
       FIG.  3 A- 3 C  illustrate a non-limiting example of a system (1-sided Replacer 2). In the example, a guide nucleic acid comprises: a spacer for targeting a genomic locus; a scaffold for complexing and recruiting an endonuclease described herein; and a donor binding site for complexing with a donor strand. Also shown in  FIG.  3 A  is a donor strand comprising at least one overhang, where the overhang comprises: a flap binding site for complexing with a genomic flap of the genomic locus; and a guide binding site for complexing with the guide nucleic acid (via the donor binding site of the guide nucleic acid). The guide nucleic acid can be complexed with an endonuclease (e.g., nCas9) operatively coupled to a ligase. The guide nucleic acid in the example directs the endonuclease and the ligase to a genomic locus that is bound by the spacer of the guide nucleic acid. The guide nucleic acid in the example is also partially complementary to a donor strand (complexing between the donor binding site of the guide nucleic acid and guide binding site of the donor strand). The endonuclease, when directed by the guide nucleic acid, can cleave at least one strand of the genomic locus, and the ligase can ligate one end of the donor strand with the cleaved end of the genomic locus, thus incorporating the donor strand into the genomic locus. The incorporation of the donor strand into the genomic locus may generate a genomic flap that can be digested and removed by a nuclease. 
       FIG.  4 A- 4 C  illustrates a non-limiting example of a system (2-sided Replacer 2). In the example, where the guide nucleic acid, similar to the guide nucleic acid of  FIG.  3 A , comprises a spacer for targeting a genomic locus; a scaffold for complexing and recruiting an endonuclease described herein; and a donor binding site for complexing with a donor strand. Also shown in  FIG.  4 A  is a donor strand comprising two overhangs, where the overhangs each comprise a flap binding site for complexing with a genomic flap of the genomic locus; and a guide binding site for complexing with a guide nucleic acid (via a donor binding site of the guide nucleic acid). The flap binding site of the donor strand can bring the donor strand in close proximity with the genomic locus after a genomic flap is generated after the endonuclease cleaves at least one strand of the genomic locus. In  FIG.  4 A , a first guide nucleic acid is shown complexed with a first endonuclease operatively coupled with a first ligase and a second guide nucleic acid is complexed with a second endonuclease operatively coupled with a second ligase. In the example, the first endonuclease and the second nuclease each cleave at least one strand of the genomic locus. The two cleaved ends of the genomic locus can then be ligated to the two ends of the donor strand, thereby incorporating the donor strand into the genomic locus. In the example, the insertion of the donor strand at the genomic locus generates two genomic flaps that can be digested and removed by a nuclease. 
     A system described herein (Replacer 3) may include a heterologous endonuclease comprising an RNA-guided endonuclease such as nicking Cas9 as well as a ligase (e.g., a DNA ligase) that can utilize a DNA splint. The guide nucleic acid optionally recruits a donor strand to the site targeted by the endonuclease (e.g., a targeted genomic locus) and also generates a splint across from the donor strand (donor strand) and genomic flap generated by the nicking Cas9, resulting in ligation of the donor strand and the genomic flap by the DNA ligase. At least part of the flap binding site and donor binding site on the guide nucleic acid are DNA such that ligases that utilize DNA splints are able to catalyze the intended reaction. The system can utilize one or more guide nucleic acids that together can comprise the following components, optionally in the following order: 5′ spacer—scaffold—donor binding site (optional)—flap binding site 3′. The donor strand (donor strand) can comprise the following sequence components: 5′ guide binding site—donor strand 3′. The guide binding site of the donor strand is at least partially reverse complementary to the donor binding site of the guide nucleic acid such that the donor hybridizes to the guide and is localized to the target site of the RNA guided endonuclease. The 5′ end of the donor sequence and the 3′ end of the genomic flap generated by nuclease nicking activity are ligated by the DNA ligase, splinted by the donor binding site and a flap binding site of the guide nucleic acid(s). 
       FIG.  5 A- 5 C  illustrate a non-limiting example of a system (1-sided Replacer 3). The example includes a guide nucleic acid comprising: a spacer for targeting a genomic locus; a scaffold for complexing and recruiting an endonuclease described herein; a donor binding site for complexing with a donor strand; and a flap binding site for complexing with a genomic flap of the genomic locus, wherein at least part of the flap binding site and donor binding site are comprised of DNA. The guide nucleic acid is shown complexed with an endonuclease (e.g., a Cas9 nickase, nCas9) operatively coupled to a ligase (e.g., an endogenous ligase or an exogenous ligase). The guide nucleic acid may direct the endonuclease to a genomic locus that is bound by the spacer of the guide nucleic acid. The guide nucleic acid is also shown as partially complementary to a donor strand (complexing between the donor binding site of the guide nucleic acid and guide binding site of the donor strand). The endonuclease, when directed by the guide nucleic acid, can cleave at least one strand of the genomic locus, and the ligase can ligate one end of the donor strand with the cleaved end of the genomic locus, thus incorporating the donor strand into the genomic locus. The incorporation of the donor strand into the genomic locus may generate a genomic flap that can be digested and removed by a nuclease. 
       FIG.  6 A- 6 C  illustrate a non-limiting example of a system (2-sided Replacer 3). The guide nucleic acid in the example, similar to the guide nucleic acid of  FIG.  5 A , comprises: a spacer for targeting a genomic locus; a scaffold for complexing and recruiting an endonuclease described herein; a donor binding site for complexing with a donor strand; and a flap binding site for complexing with a genomic flap of the genomic locus, wherein at least part of the flap binding site and donor binding site are comprised of DNA. In  FIG.  6 A , a first guide nucleic acid is shown complexed with a first endonuclease operatively coupled with a first ligase and a second guide nucleic acid is complexed with a second endonuclease operatively coupled with a second ligase. The first endonuclease and the second nuclease may each cleave at least one strand of the genomic locus. The two cleaved ends of the genomic locus can then be ligated to the two ends of the donor strand, thereby incorporating the donor strand into the genomic locus. The insertion of the donor strand at the genomic locus may generate two genomic flaps that can be digested and removed by a nuclease. 
     Ligation may be performed using a DNA ligase that can utilize an RNA splint such as SplintR ligase—also known as PBCV-1 DNA Ligase—from  Chlorella  virus. In some aspects, the system utilizes two guide nucleic acids targeting the CRISPR-guided ligase to target sites on opposite strands flanking the genomic region of interest. In some aspects, each guide nucleic acid interacts with a corresponding donor strand in the manner described above, resulting in ligation of both donor strands which are reverse complementary with each other in the donor strand regions. 
     A ligase that is fused or recruited to an endonuclease, or supplied in trans, can utilize DNA as a splint, and a donor strand acts as the splint for the genomic flap generated by the endonuclease and another donor strand. In some aspects, the donor strand comprises: 5′ donor strand—flap binding site—guide binding site (optional) 3′. The flap binding site on one donor strand (Donor2) can be reverse complementary to the genomic flap, while the optional guide binding site on Donor2 is reverse complementary to the optional donor binding site of a guide nucleic acid (Guide 1), and the donor strand can be at least partially reverse complementary to a different donor strand (Donor1). The 5′ end of this Donor1 and the 3′ end of the genomic flap can be ligated using the flap binding site and donor strand of the Donor2 as a splint. Such 2-sided approach utilizing dual guide nucleic acids with different spacer sequences can be adopted with Donor2, which provides the splint at the first genomic site and can be ligated on its 5′ end to a 3′ end of a different genomic flap at a nick created using a second Replacer2 guide nucleic acid (Guide2) with a spacer sequence that targets a second site. The donor binding site on the second guide nucleic acid system can optionally recruit Donor1 via hybridization with its optional guide binding site, and the Donor1 acts as the DNA splint for ligation of Donor2 to the 3′ end of the genomic flap at the target site of the second guide nucleic acid. 
     Following ligation, the remaining flaps of native genomic DNA can be excised via exogenously delivered or endogenous flap endonucleases or exonucleases. Examples of exogenous nucleases that can be introduced into the cell include human flap endonuclease 1 (hFEN1), human exonuclease 5 (hEXO5), T5 exonuclease, T7 exonuclease, exonuclease VIII, the flap endonuclease domain of  E. coli  PolI, RecJF, Lambda exonuclease, Xni (ExoIXI) from  Escherichia coli , SaFEN ( Staphylococcus aureus  FEN), nuclease BAL-31, or fragments thereof. The endonucleases or exonucleases can optionally be fused, recruited, or unfused to the RNA-guided endonuclease or DNA ligase by utilizing peptide linkers, heterodimerization domains, or two separate peptides, respectively. 
     In some aspects, the system, composition, or method described herein utilizes additional protein that binds to the cleaved or nicked site. For example, the system, composition, or method described herein can include Ku protein or Gam protein from bacteriophage Mu, where the binding of the Ku protein or Gam protein can increase ligation efficiency of the integration nucleic acid at the cleaved or nicked site. 
     A system or method described herein may use a nicking endonuclease and, therefore, does not generate double stranded breaks. Furthermore, the system described herein addresses the issue of poor editing efficiencies in nondividing cells through a mechanism of action which only depends on the exogenous components delivered to the cells using mRNA, viral vectors, guide nucleic acids, DNA, or peptides, or any other modalities. Therefore, the system does not require the presence of cell cycle-dependent endogenous cell processes or components such as HDR or dNTPS. As such, the system described herein allows efficiency that is not hindered in nondividing cells. Furthermore, the system enables replacement of both strands of a targeted region of the genome, which can increase editing efficiency. 
     A donor strand may contain a high degree of homology with the replaced genomic DNA. These donors may contain mutations to the genomic DNA such as pathogenic mutation correction, disabling of CRISPR protospacer adjacent motif (PAM) sites, disruption of the guide&#39;s spacer sequences, other substitution mutations, or a combination thereof. Additional substitution mutations may be included to increase donor-donor homology versus donor-genome homology to promote hybridization of donor strands and incorporation into the genome. Donor strands may also encode deletions or insertions of nucleotides, or may encode a complex combination of the above which then replaces the target genomic DNA. Optionally, guide and donor strands may be chemically modified using nucleic acid chemistries such as phosphorothioate bonds or 2′-O-methylation. Optionally, guide nucleic acids may include hairpin sequences. Optionally, any combination of guide nucleic acids, donor strands, and proteins can be complexed, using an annealing reaction (gradual reduction in temperature) for example, prior to delivering the editing components to the cell. 
     Protein components (e.g. nicking Cas9, ligase) may be modified using nuclear localization signals, cell penetrating peptides, or chromatin disrupting peptides in order to improve delivery efficiency to genomic targets. 
     The predominant cellular DNA repair pathway for resolving small (&lt;13 nt) mismatches between genomic DNA strands is mismatch repair (MMR). For single stranded donor ligation, the ligated donor strand forms a DNA heteroduplex with the reverse complementary genomic DNA strand. This may also occur with competitive hybridization between ligated donor strand strands and genomic DNA strands. In these cases, MMR activity can excise and revert mismatches in the donor strand using the genomic strand as a template, resulting in reduced editing. Expression of dominant negative versions of MMR proteins has been shown to inhibit the MMR pathway and improve editing outcome in cases where similar DNA heteroduplexes are generated. In some aspects, dominant negative MMR peptides such as MSH2 (G674A) and MLH1 (de1754-756) may be delivered as part of the system described herein to improve genomic editing capability, particularly in cells which overexpress the MMR pathway. In some aspects, these dominant negative MMR peptides can be delivered as a fusion (e.g., fused with any component of the system described herein), recruited, or as separate peptides. 
     Endonucleases 
     Disclosed herein are endonucleases. The endonuclease may be included in a composition, system or method disclosed herein. The endonuclease may be recombinant. The endonuclease may be coupled to a ligase. The endonuclease may be coupled directly or indirectly to the ligase. The coupling may be covalent or non-covalent. The endonuclease may be bound or connected to a ligase. The endonuclease may be recruited to, be part of a fusion protein with, or be used in conjunction with the ligase. The endonuclease may be heterologous. Heterologous may indicate a source from without a cell. Where a heterologous endonuclease is described, a non-heterologous (e.g. endogenous) endonuclease may be used in some instances. The endonuclease may be encoded in a cell. The endonuclease may be delivered to the cell in trans. The endonuclease may catalyze cleavage of a phosphate bond within an integrating nucleic acid. The endonuclease may be guided by a guide nucleic acid to cleave or nick a target nucleic acid for ligation of an integrating nucleic acid at the cleavage or nick site. The endonuclease may include any aspect included in  FIG.  1 A- 6 C . 
     The endonuclease may be non-naturally occurring. The endonuclease may be engineered. The endonuclease may be synthetic. The endonuclease may be pre-synthetized. The endonuclease may be added to a subject or a cell. The endonuclease may be encoded by a nucleic acid. The encoding nucleic acid may be engineered, synthetic, or added to a subject or a cell. 
     At least part of the endonuclease may be included in a first polypeptide. At least part of the endonuclease may be included in a second polypeptide. The endonuclease may be split into two or more polypeptides bound together. The first polypeptide may include an N-terminal portion of the endonuclease. The first polypeptide may include a C-terminal portion of the endonuclease. The second polypeptide may include the N-terminal portion of the endonuclease. The second polypeptide may include the C-terminal portion of the endonuclease. The first or second polypeptide comprising a part of the endonuclease may be fused with at least part, or the whole, of the ligase. 
     Described herein, in some aspects, is a system comprising at least one endonuclease. In some aspects, the endonuclease is a programmable endonuclease, where the endonuclease can be complexed with and directed by a guide nucleic acid described herein to a genomic locus. The endonuclease may bind DNA. In some aspects, the endonuclease is a RNA-guided endonuclease. In some aspects, the endonuclease can introduce a single-stranded break. Examples of RNA-guided endonucleases can include CRISPR/Cas endonucleases (e.g., class 2 CRISPR/Cas endonucleases such as a type II, type V, or type VI CRISPR/Cas endonucleases). A CRISPR/Cas endonuclease is also referred to as a CRISPR/Cas effector polypeptide. A suitable endonuclease is a CRISPR/Cas endonuclease (e.g., a class 2 CRISPR/Cas endonuclease such as a type II, type V, or type VI CRISPR/Cas endonuclease). In some cases, a suitable RNA-guided endonuclease is a class 2 CRISPR/Cas endonuclease. In some cases, a suitable RNA-guided endonuclease is a class 2 type II CRISPR/Cas endonuclease (e.g., a Cas9 protein). In some cases, an endonuclease includes a class 2 type V CRISPR/Cas endonuclease (e.g., a Cpf1 protein, a C2c1 protein, or a C2c3 protein). In some cases, a suitable RNA-guided endonuclease is a class 2 type VI CRISPR/Cas endonuclease (e.g., a C2c2 protein; also referred to as a “Cas13a” protein). Also suitable for use is a CasX protein. Also suitable for use is a CasY protein. In some aspects, the endonuclease can include any one of the Cas described herein complexed with a guide nucleic acid (e.g., a gRNA) as an RNP complex. 
     In some cases, the endonuclease is a Type II CRISPR/Cas endonuclease. In some cases, the endonuclease is a Cas9. Cas9 functions as an RNA-guided endonuclease that uses a dual-guide RNA having a crRNA and trans-activating crRNA (tracrRNA) for target recognition and cleavage by a mechanism involving two nuclease active sites in Cas9 that together generate double-stranded DNA breaks (DSBs), or can individually generate single-stranded DNA breaks (SSBs). The Type II CRISPR endonuclease Cas9 and engineered dual- (dgRNA) or single guide RNA (sgRNA) form a ribonucleoprotein (RNP) complex that can be targeted to a desired DNA sequence. Guided by a dual-RNA complex or a chimeric single-guide RNA, Cas9 generates site-specific DSBs or SSBs within double-stranded DNA (dsDNA) target nucleic acids, which are repaired either by non-homologous end joining (NHEJ) or homology-directed recombination (HDR). The Cas9 can be guided to a target site (e.g., stabilized at a target site) within a target nucleic acid sequence by virtue of its association with the RNA-binding segment of the Cas9 to guide RNA. A Cas9 protein can bind and/or modify (e.g., cleave, nick, methylate, demethylate, etc.) a target nucleic acid and/or a polypeptide associated with target nucleic acid (e.g., methylation or acetylation of a histone tail)(e.g., when the Cas9 protein includes a fusion partner with an activity). In some cases, the Cas9 protein is a naturally-occurring protein (e.g., naturally occurs in bacterial and/or archaeal cells). In other cases, the Cas9 protein is not a naturally-occurring polypeptide (e.g., the Cas9 protein is a variant Cas9 protein, a chimeric protein, and the like). 
     Naturally occurring Cas9 proteins may bind a Cas9 guide RNA, are thereby directed to a specific sequence within a target nucleic acid (a target site), and cleave the target nucleic acid (e.g., cleave dsDNA to generate a double strand break, cleave ssDNA, cleave ssRNA, etc.). A chimeric Cas9 protein may include a fusion protein comprising a Cas9 polypeptide fused to a heterologous protein (referred to as a fusion partner), where the heterologous protein provides an activity (e.g., one that is not provided by the Cas9 protein). The fusion partner can provide an activity, e.g., enzymatic activity (e.g., nuclease activity, activity for DNA and/or RNA methylation, activity for DNA and/or RNA cleavage, activity for histone acetylation, activity for histone methylation, activity for RNA modification, activity for RNA-binding, activity for RNA splicing etc.). In some cases, a portion of the Cas9 protein (e.g., the RuvC domain and/or the HNH domain) exhibits reduced nuclease activity relative to the corresponding portion of a wild type Cas9 protein (e.g., in some cases the Cas9 protein is a nickase). In some cases, the Cas9 protein is enzymatically inactive, or has reduced enzymatic activity relative to a wild-type Cas9 protein (e.g., relative to  Streptococcus pyogenes  Cas9). In some cases, the Cas9 is a Cas9 nickase. The Cas9 nickase can be generated by mutating a Cas9 nuclease domain. Non-limiting example of the Cas9 nickase can include SpCas9, SaCas9, CjCas9, GeoCas9, HpaCas9, and NmeCas9. In some aspects, the endonuclease described herein comprises any one of the Cas9 in Table 1. In some aspects, the endonuclease described herein comprises a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more identical to the polypeptide sequence of any one of the Cas9 in Table 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Non-limiting examples of Cas9 polypeptide sequence 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                 Name 
                 Cas9 polypeptide sequence 
                 NO: 
               
               
                   
               
            
           
           
               
               
               
            
               
                 SpyCas9 
                 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSG 
                 1 
               
               
                   
                 ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK 
                   
               
               
                   
                 HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHF 
                   
               
               
                   
                 LIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLEN 
                   
               
               
                   
                 LIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA 
                   
               
               
                   
                 QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLK 
                   
               
               
                   
                 ALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVK 
                   
               
               
                   
                 LNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI 
                   
               
               
                   
                 PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN 
                   
               
               
                   
                 EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT 
                   
               
               
                   
                 VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDIL 
                   
               
               
                   
                 EDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD 
                   
               
               
                   
                 KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA 
                   
               
               
                   
                 GSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRI 
                   
               
               
                   
                 EEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH 
                   
               
               
                   
                 IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK 
                   
               
               
                   
                 FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE 
                   
               
               
                   
                 VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEF 
                   
               
               
                   
                 VYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE 
                   
               
               
                   
                 TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI 
                   
               
               
                   
                 ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEK 
                   
               
               
                   
                 NPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKY 
                   
               
               
                   
                 VNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLD 
                   
               
               
                   
                 KVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD 
                   
               
               
                   
                 ATLIHQSITGLYETRIDLSQLGGD 
                   
               
               
                   
               
               
                 Nicking 
                 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSG 
                 2 
               
               
                 SpyCas9 
                 ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK 
                   
               
               
                 (H840A) 
                 HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHF 
                   
               
               
                   
                 LIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLEN 
                   
               
               
                   
                 LIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA 
                   
               
               
                   
                 QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLK 
                   
               
               
                   
                 ALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVK 
                   
               
               
                   
                 LNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI 
                   
               
               
                   
                 PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN 
                   
               
               
                   
                 EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT 
                   
               
               
                   
                 VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDIL 
                   
               
               
                   
                 EDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD 
                   
               
               
                   
                 KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA 
                   
               
               
                   
                 GSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRI 
                   
               
               
                   
                 EEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDA 
                   
               
               
                   
                 IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK 
                   
               
               
                   
                 FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE 
                   
               
               
                   
                 VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEF 
                   
               
               
                   
                 VYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE 
                   
               
               
                   
                 TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI 
                   
               
               
                   
                 ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEK 
                   
               
               
                   
                 NPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKY 
                   
               
               
                   
                 VNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLD 
                   
               
               
                   
                 KVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD 
                   
               
               
                   
                 ATLIHQSITGLYETRIDLSQLGGD 
                   
               
               
                   
               
               
                 Nicking 
                 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSG 
                 3 
               
               
                 SpyCas9 
                 ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK 
                   
               
               
                 (H840A) 
                 HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHF 
                   
               
               
                 R221K 
                 LIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRKLEN 
                   
               
               
                 N394K 
                 LIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA 
                   
               
               
                   
                 QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLK 
                   
               
               
                   
                 ALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVK 
                   
               
               
                   
                 LKREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI 
                   
               
               
                   
                 PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN 
                   
               
               
                   
                 EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT 
                   
               
               
                   
                 VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDIL 
                   
               
               
                   
                 EDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD 
                   
               
               
                   
                 KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA 
                   
               
               
                   
                 GSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRI 
                   
               
               
                   
                 EEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDA 
                   
               
               
                   
                 IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK 
                   
               
               
                   
                 FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE 
                   
               
               
                   
                 VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEF 
                   
               
               
                   
                 VYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE 
                   
               
               
                   
                 TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI 
                   
               
               
                   
                 ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEK 
                   
               
               
                   
                 NPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKY 
                   
               
               
                   
                 VNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLD 
                   
               
               
                   
                 KVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD 
                   
               
               
                   
                 ATLIHQSITGLYETRIDLSQLGGD 
                   
               
               
                   
               
               
                 Nicking 
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSG 
                 4 
               
               
                 SpyCas9 
                 ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK 
                   
               
               
                 (D10A) 
                 HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHF 
                   
               
               
                   
                 LIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLEN 
                   
               
               
                   
                 LIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA 
                   
               
               
                   
                 QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLK 
                   
               
               
                   
                 ALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVK 
                   
               
               
                   
                 LNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI 
                   
               
               
                   
                 PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN 
                   
               
               
                   
                 EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT 
                   
               
               
                   
                 VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDIL 
                   
               
               
                   
                 EDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD 
                   
               
               
                   
                 KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA 
                   
               
               
                   
                 GSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRI 
                   
               
               
                   
                 EEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH 
                   
               
               
                   
                 IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK 
                   
               
               
                   
                 FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE 
                   
               
               
                   
                 VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEF 
                   
               
               
                   
                 VYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE 
                   
               
               
                   
                 TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI 
                   
               
               
                   
                 ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEK 
                   
               
               
                   
                 NPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKY 
                   
               
               
                   
                 VNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLD 
                   
               
               
                   
                 KVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD 
                   
               
               
                   
                 ATLIHQSITGLYETRIDLSQLGGD 
                   
               
               
                   
               
               
                 SauCas9 
                 MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRL 
                 5 
               
               
                   
                 KRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHL 
                   
               
               
                   
                 AKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSI 
                   
               
               
                   
                 NRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWK 
                   
               
               
                   
                 DIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEK 
                   
               
               
                   
                 FQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDIT 
                   
               
               
                   
                 ARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGT 
                   
               
               
                   
                 HNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSP 
                   
               
               
                   
                 VVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNER 
                   
               
               
                   
                 IEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIP 
                   
               
               
                   
                 RSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKG 
                   
               
               
                   
                 RISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVK 
                   
               
               
                   
                 VKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVM 
                   
               
               
                   
                 ENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELI 
                   
               
               
                   
                 NDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKL 
                   
               
               
                   
                 KLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDD 
                   
               
               
                   
                 YPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKK 
                   
               
               
                   
                 LKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENM 
                   
               
               
                   
                 NDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG* 
                   
               
               
                   
               
               
                 Nicking 
                 MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRL 
                 6 
               
               
                 SauCas9 
                 KRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHL 
                   
               
               
                 (N580A) 
                 AKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSI 
                   
               
               
                   
                 NRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWK 
                   
               
               
                   
                 DIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEK 
                   
               
               
                   
                 FQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDIT 
                   
               
               
                   
                 ARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGT 
                   
               
               
                   
                 HNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSP 
                   
               
               
                   
                 VVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNER 
                   
               
               
                   
                 IEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIP 
                   
               
               
                   
                 RSVSFDNSFNNKVLVKQEEASKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKG 
                   
               
               
                   
                 RISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVK 
                   
               
               
                   
                 VKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVM 
                   
               
               
                   
                 ENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELI 
                   
               
               
                   
                 NDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKL 
                   
               
               
                   
                 KLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDD 
                   
               
               
                   
                 YPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKK 
                   
               
               
                   
                 LKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENM 
                   
               
               
                   
                 NDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG* 
                   
               
               
                   
               
               
                 KKH- 
                 MGKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARR 
                 7 
               
               
                 SaCas9 
                 LKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLH 
                   
               
               
                   
                 LAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGS 
                   
               
               
                   
                 INRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW 
                   
               
               
                   
                 KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYE 
                   
               
               
                   
                 KFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDI 
                   
               
               
                   
                 TARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTG 
                   
               
               
                   
                 THNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILS 
                   
               
               
                   
                 PVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNE 
                   
               
               
                   
                 RIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHII 
                   
               
               
                   
                 PRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGK 
                   
               
               
                   
                 GRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDV 
                   
               
               
                   
                 KVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKV 
                   
               
               
                   
                 MENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKL 
                   
               
               
                   
                 INDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQK 
                   
               
               
                   
                 LKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITD 
                   
               
               
                   
                 DYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAK 
                   
               
               
                   
                 KLKKISNQAEFIASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLEN 
                   
               
               
                   
                 MNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKK 
                   
               
               
                   
               
               
                 Nicking 
                 MGKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARR 
                 8 
               
               
                 KKH- 
                 LKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLH 
                   
               
               
                 SaCas9 
                 LAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGS 
                   
               
               
                 (N580A) 
                 INRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW 
                   
               
               
                   
                 KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYE 
                   
               
               
                   
                 KFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDI 
                   
               
               
                   
                 TARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTG 
                   
               
               
                   
                 THNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILS 
                   
               
               
                   
                 PVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNE 
                   
               
               
                   
                 RIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHII 
                   
               
               
                   
                 PRSVSFDNSFNNKVLVKQEEASKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGK 
                   
               
               
                   
                 GRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDV 
                   
               
               
                   
                 KVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKV 
                   
               
               
                   
                 MENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKL 
                   
               
               
                   
                 INDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQK 
                   
               
               
                   
                 LKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITD 
                   
               
               
                   
                 DYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAK 
                   
               
               
                   
                 KLKKISNQAEFIASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLEN 
                   
               
               
                   
                 MNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKK 
                   
               
               
                   
               
               
                 CjCas9 
                 MARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLALPRRLARSARK 
                 9 
               
               
                   
                 RLARRKARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLISPYELRFRALNEL 
                   
               
               
                   
                 LSKQDFARVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLANYQSVGEYLYK 
                   
               
               
                   
                 EYFQKFKENSKEFTNVRNKKESYERCIAQSFLKDELKLIFKKQREFGFSFSKKFEE 
                   
               
               
                   
                 EVLSVAFYKRALKDFSHLVGNCSFFTDEKRAPKNSPLAFMFVALTRIINLLNNLKN 
                   
               
               
                   
                 TEGILYTKDDLNALLNEVLKNGTLTYKQTKKLLGLSDDYEFKGEKGTYFIEFKKYK 
                   
               
               
                   
                 EFIKALGEHNLSQDDLNEIAKDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKD 
                   
               
               
                   
                 HLNISFKALKLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLPAFNETYYKDEVT 
                   
               
               
                   
                 NPVVLRAIKEYRKVLNALLKKYGKVHKINIELAREVGKNHSQRAKIEKEQNENYKA 
                   
               
               
                   
                 KKDAELECEKLGLKINSKNILKLRLFKEQKEFCAYSGEKIKISDLQDEKMLEIDHI 
                   
               
               
                   
                 YPYSRSFDDSYMNKVLVFTKQNQEKLNQTPFEAFGNDSAKWQKIEVLAKNLPTKKQ 
                   
               
               
                   
                 KRILDKNYKDKEQKNFKDRNLNDTRYIARLVLNYTKDYLDFLPLSDDENTKLNDTQ 
                   
               
               
                   
                 KGSKVHVEAKSGMLTSALRHTWGFSAKDRNNHLHHAIDAVIIAYANNSIVKAFSDF 
                   
               
               
                   
                 KKEQESNSAELYAKKISELDYKNKRKFFEPFSGFRQKVLDKIDEIFVSKPERKKPS 
                   
               
               
                   
                 GALHEETFRKEEEFYQSYGGKEGVLKALELGKIRKVNGKIVKNGDMFRVDIFKHKK 
                   
               
               
                   
                 TNKFYAVPIYTMDFALKVLPNKAVARSKKGEIKDWILMDENYEFCFSLYKDSLILI 
                   
               
               
                   
                 QTKDMQEPEFVYYNAFTSSTVSLIVSKHDNKFETLSKNQKILFKNANEKEVIAKSI 
                   
               
               
                   
                 GIQNLKVFEKYIVSALGEVTKAEFRQREDFKK 
                   
               
               
                   
               
               
                 GeoCas9 
                 MRYKIGLDIGITSVGWAVMNLDIPRIEDLGVRIFDRAENPQTGESLALPRRLARSA 
                 10 
               
               
                   
                 RRRLRRRKHRLERIRRLVIREGILTKEELDKLFEEKHEIDVWQLRVEALDRKLNND 
                   
               
               
                   
                 ELARVLLHLAKRRGFKSNRKSERSNKENSTMLKHIEENRAILSSYRTVGEMIVKDP 
                   
               
               
                   
                 KFALHKRNKGENYTNTIARDDLEREIRLIFSKQREFGNMSCTEEFENEYITIWASQ 
                   
               
               
                   
                 RPVASKDDIEKKVGFCTFEPKEKRAPKATYTFQSFIAWEHINKLRLISPSGARGLT 
                   
               
               
                   
                 DEERRLLYEQAFQKNKITYHDIRTLLHLPDDTYFKGIVYDRGESRKQNENIRFLEL 
                   
               
               
                   
                 DAYHQIRKAVDKVYGKGKSSSFLPIDFDTFGYALTLFKDDADIHSYLRNEYEQNGK 
                   
               
               
                   
                 RMPNLANKVYDNELIEELLNLSFTKFGHLSLKALRSILPYMEQGEVYSSACERAGY 
                   
               
               
                   
                 TFTGPKKKQKTMLLPNIPPIANPVVMRALTQARKVVNAIIKKYGSPVSIHIELARD 
                   
               
               
                   
                 LSQTFDERRKTKKEQDENRKKNETAIRQLMEYGLTLNPTGHDIVKFKLWSEQNGRC 
                   
               
               
                   
                 AYSLQPIEIERLLEPGYVEVDHVIPYSRSLDDSYTNKVLVLTRENREKGNRIPAEY 
                   
               
               
                   
                 LGVGTERWQQFETFVLTNKQFSKKKRDRLLRLHYDENEETEFKNRNLNDTRYISRF 
                   
               
               
                   
                 FANFIREHLKFAESDDKQKVYTVNGRVTAHLRSRWEFNKNREESDLHHAVDAVIVA 
                   
               
               
                   
                 CTTPSDIAKVTAFYQRREQNKELAKKTEPHFPQPWPHFADELRARLSKHPKESIKA 
                   
               
               
                   
                 LNLGNYDDQKLESLQPVFVSRMPKRSVTGAAHQETLRRYVGIDERSGKIQTVVKTK 
                   
               
               
                   
                 LSEIKLDASGHFPMYGKESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKKNGEPG 
                   
               
               
                   
                 PVIRTVKIIDTKNQVIPLNDGKTVAYNSNIVRVDVFEKDGKYYCVPVYTMDIMKGI 
                   
               
               
                   
                 LPNKAIEPNKPYSEWKEMTEDYTFRFSLYPNDLIRIELPREKTVKTAAGEEINVKD 
                   
               
               
                   
                 VFVYYKTIDSANGGLELISHDHRFSLRGVGSRTLKRFEKYQVDVLGNIYKVRGEKR 
                   
               
               
                   
                 VGLASSAHSKPGKTIRPLQSTRD 
                   
               
               
                   
               
               
                 HpaCas9 
                 MENKNLNYILGLDLGIASVGWAVVEIDEKENPLRLIDVGVRTFERAEVPKTGESLA 
                 11 
               
               
                   
                 LSRRLARSARRLTQRRVARLKKAKRLLKSENILLSTDERLPHQVWQLRVEGLDHKL 
                   
               
               
                   
                 ERQEWAAVLLHLIKHRGYLSQRKNESKSENKELGALLSGVDNNHKLLQQATYRSPA 
                   
               
               
                   
                 ELAVKKFEVEEGHIRNQQGAYTHTFSRLDLLAEMELLFSRQQHFGNPFASEKLLEN 
                   
               
               
                   
                 LTALLMWQKPALSGEAILKMLGKCTFEDEYKAAKNTYSAERFVWITKLNNLRIQEN 
                   
               
               
                   
                 GLERALNDNERLALMEQPYDKNRLFYSQVRSILKLSDEAIFKGLRYSGEDKKAIET 
                   
               
               
                   
                 KAVLMEMKAYHQIRKVLEGNNLKAEWAELKANPTLLDEIGTAFSLYKTDEDISAYL 
                   
               
               
                   
                 AGKLSQPVLNALLENLSFDKFIQLSLKALYKLLPLMQQGLRYDEACREIYGDHYGK 
                   
               
               
                   
                 KTEENHHFLPQIPADEIRNPVVLRTLTQARKVINGVVRLYGSPARIHIETGREVGK 
                   
               
               
                   
                 SYKDRRELEKRQEENRKQRENAIKEFKEYFPHFAGEPKAKDILKMRLYKQQNAKCL 
                   
               
               
                   
                 YSGKPIELHRLLEKGYVEVDHALPFSRTWDDSFNNKVLVLANENQNKGNLTPFEWL 
                   
               
               
                   
                 DGKHNSERWRAFKALVETSAFPYAKKQRILSQKLDEKGFIERNLNDTRYVARFLCN 
                   
               
               
                   
                 FIADNMHLTGEGKRKVFASNGQITALLRSRWGLAKSREDNDRHHALDAVVVACSTV 
                   
               
               
                   
                 AMQQKITRFVRFEAGDVFTGERIDRETGEIIPLHFPTPWQFFKQEVEIRIFSDNPK 
                   
               
               
                   
                 LELENRLPDRPQANHEFVQPLFVSRMPTRKMTGQGHMETVKSAKRLNEGISVIKMP 
                   
               
               
                   
                 LTKLKLKDLELMVNREREKDLYDTLKARLEAFNDDPAKAFAEPFIKKGGAIVKSVR 
                   
               
               
                   
                 VEQIQKSGVLVREGNGVADNASMVRVDVFTKGGKYFLVPIYTWQVAKGILPNKAAT 
                   
               
               
                   
                 QYKDEEDWEVMDNSATFKFSLHPNDLVKLVTKKKTILGYFNGLNRATGNIDIKEHD 
                   
               
               
                   
                 LDKSKGKQGIFEGVGIKLALSFEKYQVDELGKNIRLCKPSKRQPVR 
                   
               
               
                   
               
               
                 SmuCas9 
                 MMMEKFHYVLGLDLGIASVGWAAIEIDKETETSIGLLDCGVRTFERAEVPKTGDSL 
                 12 
               
               
                   
                 AKARREARSTRRLIRRRSHRLLRLKRLLKREIFRQPETFKDLPINAWQLRVKGLDS 
                   
               
               
                   
                 RLNEYEWAAVLLHLVKHRGYLSQRKSEMSETDSKSEMGRLLAGVAENHQLLQQEQY 
                   
               
               
                   
                 RTPAELALKKFVKHFRNKGGDYAHTFNRLDLQAELHLLFQKQRELGNPFTSPELER 
                   
               
               
                   
                 QVDDLLMTQRSALQGDAILKMLGHCGFEPEQFKAAKNTFSAERFIWLTKLNNLRIQ 
                   
               
               
                   
                 DQGKERALTADERTKLLDEPYKKSKLTYAQVRKLLSLPQTAIFKGLRYDLEHDKKA 
                   
               
               
                   
                 ENSTLMEMKSYHNIRQTLEKSGLKTEWQSIATQPEILDAIGTAFSIYKTDEDISHE 
                   
               
               
                   
                 LKTCRLPENVLNELLKNINFDGFIQLSLTALRKILPLMEQGYRYDEACTQIYGNHH 
                   
               
               
                   
                 SGSLQQESKQFLPHIPIDDVRNPVVFRTLTQARKVVNAIIRRYGSPARVHIEMARE 
                   
               
               
                   
                 LGKSKSDRDRIEKQQQKNKKERENAVAKFKEDFPDFVGEPRGKDILKMRLYEQQHG 
                   
               
               
                   
                 KCLYSGHDIDINRLNEKGYVEIDHALPFSRTWDDSQNNKVLVLGSENQNKRNQTPD 
                   
               
               
                   
                 EYLDGANNSQRWLEFQARVQTCHFSYGKKQRIQLAKLDDETEKGFLERNLNDTRYI 
                   
               
               
                   
                 ARFMCQFVQENLYLTGKGKRLVFASNGGMTATLRNLWGLRKVREDNDRHHALDAIV 
                   
               
               
                   
                 VACSTASMQQKITKAFQRHESIEYVDTETGEVKFRIPQPWDFFRQEVMIRVFSDQP 
                   
               
               
                   
                 CEDLVEKLSARPEALHDNVTPLFVSRAPNRKMSGQGHLETIKSAKRLSEENSMVKK 
                   
               
               
                   
                 PLTTLKLKDIPEIVGYPSREPQLYAALKTRLETHDDDPIKAFAKPFYKPNKNGELG 
                   
               
               
                   
                 ALVRSVRVKGVQNTGVMVHDGKGIADNATMVRVDVYTKAGKNYLVPVYVWQVAQGI 
                   
               
               
                   
                 LPNRAVTSGKSEADWDLIDESFEFKFSLSRGDLVEMISNKGRIFGYYNGLDRANGS 
                   
               
               
                   
                 IGIREHDLEKSKGKDGVHRVGVKTATAFNKYHVDPLGKEIHRCSSEPRPTLKIKSK 
                   
               
               
                   
                 K 
                   
               
               
                   
               
               
                 NmeCas9 
                 MAAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRVFERAEVPKTGD 
                 13 
               
               
                   
                 SLAMARRLARSVRRLTRRRAHRLLRTRRLLKREGVLQAANFDENGLIKSLPNTPWQ 
                   
               
               
                   
                 LRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVAGNAHA 
                   
               
               
                   
                 LQTGDFRTPAELALNKFEKESGHIRNQRSDYSHTFSRKDLQAELILLFEKQKEFGN 
                   
               
               
                   
                 PHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWL 
                   
               
               
                   
                 TKLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLR 
                   
               
               
                   
                 YGKDNAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTD 
                   
               
               
                   
                 EDITGRLKDRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEI 
                   
               
               
                   
                 YGDHYGKKNTEEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIE 
                   
               
               
                   
                 TAREVGKSFKDRKEIEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYE 
                   
               
               
                   
                 QQHGKCLYSGKEINLGRLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGN 
                   
               
               
                   
                 QTPYEYFNGKDNSREWQEFKARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRY 
                   
               
               
                   
                 VNRFLCQFVADRMRLTGKGKKRVFASNGQITNLLRGFWGLRKVRAENDRHHALDAV 
                   
               
               
                   
                 VVACSTVAMQQKITRFVRYKEMNAFDGKTIDKETGEVLHQKTHFPQPWEFFAQEVM 
                   
               
               
                   
                 IRVFGKPDGKPEFEEADTLEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSG 
                   
               
               
                   
                 QGHMETVKSAKRLDEGVSVLRVPLTQLKLKDLEKMVNREREPKLYEALKARLEAHK 
                   
               
               
                   
                 DDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQKTGVWVRNHNGIADNATMVRVDV 
                   
               
               
                   
                 FEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQLIDDSFNFKFSLHPNDLVE 
                   
               
               
                   
                 VITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILEGIGVKTALSFQKYQID 
                   
               
               
                   
                 ELGKEIRPCRLKKRPPVR 
               
               
                   
               
            
           
         
       
     
     Some aspects include an endonuclease such as an RNA-guided endonuclease. The RNA-guided endonuclease may comprise a class II CRISPR/Cas endonuclease. The RNA-guided endonuclease may comprise a Cas9 endonuclease. The RNA-guided endonuclease may comprise a nickase. The RNA-guided endonuclease may comprise an amino acid sequence at least 80% identical to the amino acid sequence of any one of SEQ ID NOS: 1-13, or a functional fragment thereof. 
     The endonuclease may introduce a single-strand break in a target nucleic acid. The endonuclease may introduce a single-strand break in a target nucleic acid without cleaving a strand opposite the single strand break. The endonuclease may include a nickase. In some instances, the endonuclease may exclude an endonuclease that introduces a double strand break. The endonuclease may exclude a restriction enzyme. 
     The endonuclease may be included as part of a fusion protein. In some cases, an endonuclease is a fusion protein that is fused to a heterologous polypeptide such as the heterologous ligase described herein. The heterologous polypeptide may include a fusion partner. The fusion protein may include a fusion partner such as a DNA ligase, a nuclear localization signal, chromatin modifying domain, cell penetrating peptide, or tag polypeptide. The fusion protein may include one or more fusion partner. The fusion protein may include a ligase. The fusion protein may include a nuclear localization signal, chromatin modifying domain, cell penetrating peptide, or tag polypeptide. 
     The fusion partner may be connected to the N-terminus of the endonuclease. The fusion partner may be connected to the C-terminus of the endonuclease. The endonuclease may be connected at an N-terminus or a C-terminus to a linker. The fusion partner may be connected by the fusion partner&#39;s N-terminus or C-terminus. The fusion partner may be connected by the fusion partner&#39;s N-terminus to the endonuclease. The fusion partner may be connected by the fusion partner&#39;s C-terminus to the endonuclease. The fusion partner may be connected at an N-terminus or a C-terminus to a linker. 
     In some cases, the endonuclease comprises a linker, where the linker covalently connects the endonuclease to the heterologous polypeptide. The linker may connect the endonuclease to any fusion partner. A linker may also connect any fusion partner to another fusion partner. The linker polypeptide may have any of a variety of amino acid sequences. Proteins can be joined by a spacer peptide, generally of a flexible nature, although other chemical linkages are not excluded. Suitable linkers include polypeptides of between 4 amino acids and 40 amino acids in length, or between 4 amino acids and 25 amino acids in length. These linkers can be produced by using synthetic, linker-encoding oligonucleotides to couple the proteins, or can be encoded by a nucleic acid sequence encoding the fusion protein. Peptide linkers with a degree of flexibility can be used. The linking peptides may have virtually any amino acid sequence, bearing in mind that the preferred linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art. A variety of different linkers are commercially available and are considered suitable for use. Examples of linker polypeptides include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n, (GGSGGS)n, and (GGGS)n, where n is an integer of at least one); glycine-alanine polymers; and alanine-serine polymers. Exemplary linkers can comprise amino acid sequences including, but not limited to, GGSG, GGSGG, GSGSG, GSGGG, GGGSG, GSSSG, and the like. Also suitable is a linker having the sequence (GGGGS)n, where n is an integer of from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). The ordinarily skilled artisan will recognize that design of a peptide conjugated to any desired element can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure. 
     One or more linkers may be included in a fusion protein. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linkers, or a range of linkers defined by any two of the aforementioned integers, may be included in the fusion protein. A linker may connect to an N-terminal end of at least part of the endonuclease. A linker may connect to an N-terminal end of at least part of a fusion partner. A linker may connect to an N-terminal end of at least part of a fusion ligase. A linker may connect to an N-terminal end of a nuclear localization signal. A linker may connect to an N-terminal end of a chromatin modifying domain. A linker may connect to an N-terminal end of a cell penetrating peptide. A linker may connect to an N-terminal end of a tag polypeptide. A linker may connect to a C-terminal end of at least part of the endonuclease. A linker may connect to a C-terminal end of at least part of a fusion partner. A linker may connect to a C-terminal end of at least part of a fusion ligase. A linker may connect to a C-terminal end of a nuclear localization signal. A linker may connect to a C-terminal end of a chromatin modifying domain. A linker may connect to a C-terminal end of a cell penetrating peptide. A linker may connect to a C-terminal end of a tag polypeptide. 
     A linker may comprise a number or range of amino acids or residues. The linker may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 amino acid residues. The linker may, in some aspects, include no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 12, no more than 13, no more than 14, no more than 15, no more than 20, no more than 25, no more than 30, no more than 35, no more than 40, no more than 45, no more than 50, no more than 55, no more than 60, no more than 65, no more than 70, no more than 75, no more than 80, no more than 85, no more than 90, no more than 95, or no more than 100 amino acid residues. A linker may include 1-10 amino acids, 1-25 amino acids, or 1-100 amino acids. 
     Linkers may be included anywhere in a polypeptide chain or protein described herein. For example, a linker may separate an endonuclease from a ligase. A linker may separate an endonuclease from a nuclear localization signal, a chromatin modifying domain, a cell penetrating peptide, or a tag polypeptide. 
     In some cases, the endonuclease comprises a nuclear localization sequence (e.g., one or more nuclear localization signals or NLSs for targeting to the nucleus). In some aspects, the NLS described herein comprises any one of the NLS in Table 2. In some aspects, the NLS described herein comprises a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more identical to the polypeptide sequence of any one of NLS in Table 2. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Non-limiting examples of NLS 
               
               
                 polypeptide sequence 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 SEQ 
               
               
                   
                   
                 NLS polypeptide 
                 ID 
               
               
                   
                 Name 
                 sequence 
                 NO: 
               
               
                   
                   
               
               
                   
                 NLS1 
                 KRTADGSEFESPKKKRKV 
                 14 
               
               
                   
                   
               
               
                   
                 NLS2 
                 SGGSKRTADSQHSTPPKT 
                 15 
               
               
                   
                   
                 KRKVEFEPKKKRKV 
                   
               
               
                   
                   
               
               
                   
                 NLS3 
                 KRPAATKKAGQAKKKK 
                 16 
               
               
                   
                   
               
               
                   
                 NLS4 
                 KKTELQTTNAENKTKKL 
                 17 
               
               
                   
                   
               
               
                   
                 NLS5 
                 KRGINDRNFWRGENGRK 
                 18 
               
               
                   
                   
                 TR 
                   
               
               
                   
                   
               
               
                   
                 NLS6 
                 RKSGKIAAIVVKRPRK 
                 19 
               
               
                   
                   
               
               
                   
                 NLS7 
                 PKKKRKV 
                 20 
               
               
                   
                   
               
               
                   
                 NLS8 
                 MDSLLMNRRKFLYQFK 
                 21 
               
               
                   
                   
                 NVRWAKGRRETYLC 
                   
               
               
                   
                   
               
               
                   
                 SGGSx2-bpNLS- 
                 SGGSSGGSKRTADGSE 
                 22 
               
               
                   
                 SGGSx2 
                 FESPKKKRKVSGGSSG 
                   
               
               
                   
                   
                 GS 
                   
               
               
                   
                   
               
               
                   
                 SGGSx2-XTEN16- 
                 SGGSSGGSSGSETPGT 
                 23 
               
               
                   
                 SGGSx2 
                 SESATPESSGGSSGGS 
                   
               
               
                   
                   
                 S 
                   
               
               
                   
                   
               
               
                   
                 SGGSx10 
                 SGGSSGGSSGGSSGGS 
                 24 
               
               
                   
                   
                 SGGSSGGSSGGSSGGS 
                   
               
               
                   
                   
                 SGGSSGGS 
               
               
                   
                   
               
            
           
         
       
     
     A polynucleotide encoding an NLS polypeptide may be used. An example of such a polynucleotide may be SGGSx2-bpNLS-SGGSx2: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 25) 
               
               
                   
                 TCCGGCGGAAGCTCTGGTGGCAGCAAGCGGAC 
               
               
                   
                   
               
               
                   
                 CGCCGACGGCTCTGAATTCGAGAGCCCTAAGA 
               
               
                   
                   
               
               
                   
                 AGAAAAGAAAGGTGAGCGGAGGCTCTAGCGGC 
               
               
                   
                   
               
               
                   
                 GGAAGC. 
               
            
           
         
       
     
     In some aspects, the endonuclease comprises a dimerization domain. The dimerization domain can be located at the N-terminus or C-terminus of the endonuclease. In some aspects, the dimerization domain allows the endonuclease to form a heterodimer with another polypeptide (e.g., the heterologous ligase). In some aspects, the dimerization domain allows the endonuclease to be functionally coupled with another polypeptide. Non-limiting examples of the dimerization domains can include a leucine zipper, an FKBP, an FRB, a Calcineurin A, a CyP-Fas, a GyrB, a GAI, a GID1, a SNAP tag, a Halo tag, a Bcl-xL, a Fab, a LOV domain, or SpyTag/SpyCatcher. Other example of dimerization domain can include an antibody such as anyone of heavy chain domain 2 (CH2) of IgM (MHD2) or IgE (EHD2), immunoglobulin Fc region, heavy chain domain 3 (CH3) of IgG or IgA, heavy chain domain 4 (CH4) of IgM or IgE, Fab, Fab2, leucine zipper motifs, barnase-barstar dimers, miniantibodies, or ZIP miniantibodies. In some aspects, the dimerization domain described herein comprises any one of the dimerization domain in Table 3. In some aspects, the dimerization domain described herein comprises a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more identical to the polypeptide sequence of any one of dimerization domain in Table 3. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Non-limiting examples of dimerization 
               
               
                 domain sequence 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Dimerization 
                 SEQ 
               
               
                   
                 Name 
                 domain sequence 
                 ID NO: 
               
               
                   
                   
               
               
                   
                 Leucine zipper 
                 LEIEAAFLERENTALETRVAE 
                 26 
               
               
                   
                 EE12RR345L 
                 LRQRVQRLRNRVSQYRTRYGP 
                   
               
               
                   
                   
                 LGGGK 
                   
               
               
                   
                   
               
               
                   
                 Leucine zipper 
                 LEIRAAFLRQRNTALRTEVAE 
                 27 
               
               
                   
                 RR12EE345L 
                 LEQEVQRLENEVSQYETRYGP 
                   
               
               
                   
                   
                 LGGGK 
                   
               
               
                   
                   
               
            
           
         
       
     
     In some aspects, the endonuclease comprises at least one additional domain. In some aspects, the at least one additional domain is a functional domain. For example, the functional domain can comprises a chromatin modifying domain or a cell penetrating peptide. In some aspects, the chromatin modifying domain described herein comprises any one of the chromatin modifying domain in Table 4. In some aspects, the chromatin modifying domain described herein comprises a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more identical to the polypeptide sequence of any one of chromatin modifying domain in Table 4. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Non-limiting examples of chromatin 
               
               
                 modifying domain polypeptide 
               
               
                 sequence 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Chromatin modifying domain 
                 SEQ ID 
               
               
                   
                 Name 
                 polypeptide sequence 
                 NO: 
               
               
                   
                   
               
               
                   
                 H1G 
                 STDHPKYSDMIVAAIQAEKN 
                 28 
               
               
                   
                 (histone 
                 RAGSSRQSIQKYIKSHYKVG 
                   
               
               
                   
                 H1 central 
                 ENADSQIKLSIKRLVTTGVL 
                   
               
               
                   
                 globular 
                 KQTKGVGASGSFRLAKSDEP 
                   
               
               
                   
                 domain) 
                   
                   
               
               
                   
                   
               
               
                   
                 HMGB1 
                 MGKGDPKKPRGKMSSYAFFV 
                 29 
               
               
                   
                   
                 QTCREEHKKKHPDASVNFSE 
                   
               
               
                   
                   
                 FSKKCSERWKTMSAKEKGKF 
                   
               
               
                   
                   
                 EDMAKADKARYEREMKTYIP 
                   
               
               
                   
                   
                 PKGETKKKFKDPNAPKRPPS 
                   
               
               
                   
                   
                 AFFLFCSEYRPKIKGEHPGL 
                   
               
               
                   
                   
                 SIGDVAKKLGEMWNNTAADD 
                   
               
               
                   
                   
                 KQPYEKKAAKLKEKYEKDIA 
                   
               
               
                   
                   
                 AYRAKGKPDAAKKGVVKAEK 
                   
               
               
                   
                   
                 SKKKKEEEEDEEDEEDEEEE 
                   
               
               
                   
                   
                 EDEEDEDEEEDDDDE 
                   
               
               
                   
                   
               
               
                   
                 HMGB2 
                 MGKGDPNKPRGKMSSYAFFV 
                 30 
               
               
                   
                   
                 QTCREEHKKKHPDSSVNFAE 
                   
               
               
                   
                   
                 FSKKCSERWKTMSAKEKSKF 
                   
               
               
                   
                   
                 EDMAKSDKARYDREMKNYVP 
                   
               
               
                   
                   
                 PKGDKKGKKKDPNAPKRPPS 
                   
               
               
                   
                   
                 AFFLFCSEHRPKIKSEHPGL 
                   
               
               
                   
                   
                 SIGDTAKKLGEMWSEQSAKD 
                   
               
               
                   
                   
                 KQPYEQKAAKLKEKYEKDIA 
                   
               
               
                   
                   
                 AYRAKGKSEAGKKGPGRPTG 
                   
               
               
                   
                   
                 SKKKNEPEDEEEEEEEEDED 
                   
               
               
                   
                   
                 EEEEDEDEE 
                   
               
               
                   
                   
               
               
                   
                 HMGB3 
                 MAKGDPKKPKGKMSAYAFFV 
                 31 
               
               
                   
                   
                 QTCREEHKKKNPEVPVNFAE 
                   
               
               
                   
                   
                 FSKKCSERWKTMSGKEKSKF 
                   
               
               
                   
                   
                 DEMAKADKVRYDREMKDYGP 
                   
               
               
                   
                   
                 AKGGKKKKDPNAPKRPPSGF 
                   
               
               
                   
                   
                 FLFCSEFRPKIKSTNPGISI 
                   
               
               
                   
                   
                 GDVAKKLGEMWNNLNDSEKQ 
                   
               
               
                   
                   
                 PYITKAAKLKEKYEKDVADY 
                   
               
               
                   
                   
                 KSKGKFDGAKGPAKVARKKV 
                   
               
               
                   
                   
                 EEEDEEEEEEEEEEEEEEDE 
                   
               
               
                   
                   
               
               
                   
                 HMGN1 
                 MPKRKVSSAEGAAKEEPKRR 
                 32 
               
               
                   
                 (HN1) 
                 SARLSAKPPAKVEAKPKKAA 
                   
               
               
                   
                   
                 AKDKSSDKKVQTKGKRGAKG 
                   
               
               
                   
                   
                 KQAEVANQETKEDLPAENGE 
                   
               
               
                   
                   
                 TKTEESPASDEAGEKEAKSD 
                   
               
               
                   
                   
               
               
                   
                 HMGN2 
                 MPKRKAEGDAKGDKAKVKDE 
                 33 
               
               
                   
                   
                 PQRRSARLSAKPAPPKPEPK 
                   
               
               
                   
                   
                 PKKAPAKKGEKVPKGKKGKA 
                   
               
               
                   
                   
                 DAGKEGNNPAENGDAKTDQA 
                   
               
               
                   
                   
                 QKAEGAGDAK 
               
               
                   
                   
               
            
           
         
       
     
     In some aspects, the cell penetrating peptide described herein comprises any one of the cell penetrating peptide in Table 5. In some aspects, the cell penetrating peptide described herein comprises a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more identical to the polypeptide sequence of any one of cell penetrating peptide in Table 5. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Non-limiting examples of cell penetrating 
               
               
                 peptide polypeptide sequence 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 SEQ 
               
               
                   
                   
                 Cell penetrating 
                 ID 
               
               
                   
                 Name 
                 peptide sequence 
                 NO: 
               
               
                   
                   
               
               
                   
                 Penetratin 
                 RQIKIYFQNRRMKWKK 
                 34 
               
               
                   
                   
               
               
                   
                 TAT 
                 RKKRRQRRR 
                 35 
               
               
                   
                   
               
               
                   
                 R8 
                 RRRRRRRR 
                 36 
               
               
                   
                   
               
               
                   
                 DPV3 
                 RKKRRRESRKKRRRES 
                 37 
               
               
                   
                   
               
               
                   
                 DPV6 
                 GRPRESGKKRKRKRLKP 
                 38 
               
               
                   
                   
               
               
                   
                 R9-TAT 
                 GRRRRRRRRRPPQ 
                 39 
               
               
                   
                   
               
               
                   
                 pVEC 
                 LLIILRRRIRKQAHAHSK 
                 40 
               
               
                   
                   
               
               
                   
                 ARF(19-31) 
                 RVRVFVVWHIPRLT 
                 41 
               
               
                   
                   
               
               
                   
                 MPG 
                 GALFLGFLGAAGSTMGA 
                 42 
               
               
                   
                   
                 WSQPKKKRKV 
                   
               
               
                   
                   
               
               
                   
                 Transportan 
                 GWTLNSAGYLLGKINLK 
                 43 
               
               
                   
                   
                 ALAALAKKIL 
                   
               
               
                   
                   
               
               
                   
                 Bip4 
                 VSALK 
                 44 
               
               
                   
                   
               
               
                   
                 C105Y 
                 CSIPPEVKFNPFVYLI 
                 45 
               
               
                   
                   
               
               
                   
                 Melittin 
                 GIGAVLKVLTTGLPALI 
                 46 
               
               
                   
                   
                 SWIKRKRQQ 
                   
               
               
                   
                   
               
               
                   
                 gH625 
                 HGLASTLTRWAHYNALIRAF 
                 47 
               
               
                   
                   
               
            
           
         
       
     
     In some aspects, the endonuclease comprises a tag, where the tag can be used for increasing expression, identifying, or purifying the endonuclease. In some aspects, the tag described herein comprises any one of the tag sequence in Table 6. In some aspects, the tag described herein comprises a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more identical to the polypeptide sequence of any one of the tag sequence in Table 6. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Non-limiting examples of tag 
               
               
                 polypeptide sequence 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Tag polypeptide 
                 SEQ ID 
               
               
                   
                 Name 
                 sequence 
                 NO: 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 FLAG 
                 DYKDDDDK 
                 48 
               
               
                   
                   
               
               
                   
                 His-Tag 
                 HHHHHH 
                 49 
               
               
                   
                   
               
               
                   
                 CBP 
                 KRRWKKNFIAVSAANRFKK 
                 50 
               
               
                   
                   
                 ISSSGAL 
                   
               
               
                   
                   
               
               
                   
                 MBP 
                 MKIKTGARILALSALTTMMF 
                 51 
               
               
                   
                   
                 SASALAKIEEGKLVIWINGD 
                   
               
               
                   
                   
                 KGYNGLAEVGKKFEKDTGIK 
                   
               
               
                   
                   
                 VTVEHPDKLEEKFPQVAATG 
                   
               
               
                   
                   
                 DGPDIIFWAHDRFGGYAQSG 
                   
               
               
                   
                   
                 LLAEITPDKAFQDKLYPFTW 
                   
               
               
                   
                   
                 DAVRYNGKLIAYPIAVEALS 
                   
               
               
                   
                   
                 LIYNKDLLPNPPKTWEEIPA 
                   
               
               
                   
                   
                 LDKELKAKGKSALMFNLQEP 
                   
               
               
                   
                   
                 YFTWPLIAADGGYAFKYENG 
                   
               
               
                   
                   
                 KYDIKDVGVDNAGAKAGLTF 
                   
               
               
                   
                   
                 LVDLIKNKHMNADTDYSIAE 
                   
               
               
                   
                   
                 AAFNKGETAMTINGPWAWSN 
                   
               
               
                   
                   
                 IDTSKVNYGVTVLPTFKGQP 
                   
               
               
                   
                   
                 SKPFVGVLSAGINAASPNKE 
                   
               
               
                   
                   
                 LAKEFLENYLLTDEGLEAVN 
                   
               
               
                   
                   
                 KDKPLGAVALKSYEEELAKD 
                   
               
               
                   
                   
                 PRIAATMENAQKGEIMPNIP 
                   
               
               
                   
                   
                 QMSAFWYAVRTAVINAASGR 
                   
               
               
                   
                   
                 QTVDEALKDAQTRITK 
                   
               
               
                   
                   
               
               
                   
                 Myc 
                 EQKLISEEDL 
                 52 
               
               
                   
                   
               
               
                   
                 GST 
                 MKLFYKPGACSLASHITLRE 
                 53 
               
               
                   
                   
                 SGKDFTLVSVDLMKKRLENG 
                   
               
               
                   
                   
                 DNYFAVNPKGQVPALLLDDG 
                   
               
               
                   
                   
                 TLLTEGVAIMQYLADSVPDR 
                   
               
               
                   
                   
                 QLLAPVNSISRYKTIEWLNY 
                   
               
               
                   
                   
                 IATELHKGFTPLFRPDTPEE 
                   
               
               
                   
                   
                 YKSTVRAQLEKKLQYVNEAL 
                   
               
               
                   
                   
                 KDEHWICGQRFTIADAYLFT 
                   
               
               
                   
                   
                 VLRWAYAVKLNLEGLEHIAA 
                   
               
               
                   
                   
                 FMQRMAERPEVQDALSAEGL 
                   
               
               
                   
                   
                 K 
                   
               
               
                   
                   
               
               
                   
                 HA 
                 YPYDVPDYA 
                 54 
               
               
                   
                   
               
               
                   
                 HA 
                 YAYDVPDYA 
                 210 
               
               
                   
                   
               
               
                   
                 HA 
                 YDVPDYASL 
                 211 
               
               
                   
                   
               
            
           
         
       
     
     In some embodiments, the endonuclease can be expressed as split construct as one or more exteins fused to one or more inteins. Intein technology may be used to deliver large proteins into a cell by expressing the protein as two or more shorter peptide segments (exteins). Each extein may be expressed as a fusion with an intein peptide (e.g., an Npu C intein or an Npu N intein). An intein may autocatalyze fusion of two or more exteins and may autocatalyze excision of the intein from its corresponding extein. The result may be a protein complex comprising a first extein fused to a second extein and lacking inteins. An intein may be positioned N-terminal of the extein, or an intein may be positioned C-terminal of the extein. An extein may comprise a cysteine residue positioned adjacent to the intein (e.g., at the C-terminal end of an extein with an intein fused to the C-terminal end of the extein). The Cas nickase may be expressed as two or more segments. A first of the Cas nickase segment may comprise an N-terminal portion of the Cas nickase. A first segment of the Cas nickase may comprise a first intein. A second segment of the Cas nickase may comprise a C-terminal portion of the Cas nickase. A second segment of the Cas nickase may comprise a second intein. An intein may be fused to a C-terminus of an N-terminal portion of the Cas nickase. An intein may be fused to an N-terminus of a C-terminal portion of the Cas nickase. A nucleic acid sequence encoding an extein-intein fusion may fit into a delivery vector (e.g., an adeno-associated virus (AAV) vector). 
     DNA Ligases 
     Disclosed herein are ligases. The ligase may be or include a DNA ligase. The ligase may be included in a composition, system or method disclosed herein. The ligase may be recombinant. The ligase may be coupled to the endonuclease. The ligase may be coupled directly or indirectly to the endonuclease. The coupling may be covalent or non-covalent. The ligase may be bound or connected to the endonuclease. The ligase may be recruited to, be part of a fusion protein with, or be used in conjunction with an endonuclease. The ligase may be heterologous. The ligase may be endogenous. Where a heterologous ligase is described, a non-heterologous (e.g. endogenous) ligase may be used in some cases. The ligase may be encoded in a cell. The ligase may be delivered to the cell in trans. The ligase may form a phosphodiester bond by joining two nucleic acid ends together. The ligase may join an end (e.g. 5′ or 3′ end) of a target nucleic acid to an integrating nucleic acid (e.g. a 3′ or 5′ end of the integrating nucleic acid). The ligase ligates an integrating nucleic acid to a cleaved or nicked end of a target nucleic acid where the cleaved or nicked end has been generated by an endonuclease such as an RNA-guided endonuclease. The ligase may include any aspect included in  FIG.  1 A- 6 C . 
     The ligase may be non-naturally occurring. The ligase may be engineered. The ligase may be synthetic. The ligase may be pre-synthetized. The ligase may be added to a subject or a cell. The ligase may be encoded by a nucleic acid. The encoding nucleic acid may be engineered, synthetic, or added to a subject or a cell. 
     At least part of the ligase may be included in a first polypeptide. At least part of the ligase may be included in a second polypeptide. The ligase may be split into two polypeptides bound together. The first polypeptide may include an N-terminal portion of the ligase. The first polypeptide may include a C-terminal portion of the ligase. The second polypeptide may include the N-terminal portion of the ligase. The second polypeptide may include the C-terminal portion of the ligase. The first or second polypeptide comprising a part of the ligase may be fused with at least part, or the whole, of the endonuclease. 
     Examples of DNA ligases are hLIG1, T4 ligase, T7 ligase, and ligases from  Aquifex  aeolicus VFS,  Neisseria meningitidis  serogroup A strain Z2491,  Neisseria meningitidis  serogroup B strain MC58,  Pseudomonas aeruginosa  PA01,  Vibrio cholerae  El Tor N1696, Vaccinia virus, and Emiliania huxleyi virus. 
     The ligase may comprise a ligase that can ligate a substrate comprising DNA. In some aspects, the ligase comprises a ligase that can ligate a substrate comprising a DNA splint. For example, a DNA ligase may ligate a 5′ phosphate to a 3′ hydroxyl of two DNA strands that are hybridized to another DNA strand. The splinting DNA strand may include an RNA portion. For example, a DNA ligase may ligate a 5′ phosphate to a 3′ hydroxyl of two DNA strands that are hybridized across from a DNA portion of an RNA/DNA hybrid strand. In some aspects, the ligase comprises a ligase that can ligate a substrate comprising a DNA/RNA. In some aspects, the ligase comprises a ligase that can ligate a substrate comprising a RNA splint. For example, a DNA ligase may ligate a 5′ phosphate to a 3′ hydroxyl of two DNA strands that are hybridized to an RNA strand. The RNA strand may include a DNA portion. For example, a DNA ligase may ligate a 5′ phosphate to a 3′ hydroxyl of two DNA strands that are hybridized across from an RNA portion of an RNA/DNA hybrid strand. 
     In some aspects, the ligase described herein comprises any one of the ligase in Table 7. In some aspects, the ligase described herein comprises a polypeptide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more identical to the polypeptide sequence of any one of the ligase in Table 7. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Non-limiting examples of ligase 
               
               
                 polypeptide sequence 
               
            
           
           
               
               
               
            
               
                   
                 Ligase polypeptide 
                 SEQ ID 
               
               
                 Name 
                 sequence 
                 NO: 
               
               
                   
               
               
                 splintR 
                 MAITKPLLAATLENIEDVQF 
                 55 
               
               
                 ( chlorella  virus 
                 PCLATPKIDGIRSVKQTQML 
                   
               
               
                 DNA ligase PBCV1) 
                 SRTFKPIRNSVMNRLLTELL 
                   
               
               
                   
                 PEGSDGEISIEGATFQDTTS 
                   
               
               
                   
                 AVMTGHKMYNAKFSYYWFDY 
                   
               
               
                   
                 VTDDPLKKYIDRVEDMKNYI 
                   
               
               
                   
                 TVHPHILEHAQVKIIPLIPV 
                   
               
               
                   
                 EINNITELLQYERDVLSKGF 
                   
               
               
                   
                 EGVMIRKPDGKYKFGRSTLK 
                   
               
               
                   
                 EGILLKMKQFKDAEATIISM 
                   
               
               
                   
                 TALFKNTNTKTKDNFGYSKR 
                   
               
               
                   
                 STHKSGKVEEDVMGSIEVDY 
                   
               
               
                   
                 DGVVFSIGTGFDADQRRDFW 
                   
               
               
                   
                 QNKESYIGKMVKFKYFEMGS 
                   
               
               
                   
                 KDCPRFPVFIGIRHEEDR 
                   
               
               
                   
               
               
                 hLIG1 
                 MQRSIMSFFHPKKEGKAKKP 
                 56 
               
               
                   
                 EKEASNSSRETEPPPKAALK 
                   
               
               
                   
                 EWNGVVSESDSPVKRPGRKA 
                   
               
               
                   
                 ARVLGSEGEEEDEALSPAKG 
                   
               
               
                   
                 QKPALDCSQVSPPRPATSPE 
                   
               
               
                   
                 NNASLSDTSPMDSSPSGIPK 
                   
               
               
                   
                 RRTARKQLPKRTIQEVLEEQ 
                   
               
               
                   
                 SEDEDREAKRKKEEEEEETP 
                   
               
               
                   
                 KESLTEAEVATEKEGEDGDQ 
                   
               
               
                   
                 PTTPPKPLKTSKAETPTESV 
                   
               
               
                   
                 SEPEVATKQELQEEEEQTKP 
                   
               
               
                   
                 PRRAPKTLSSFFTPRKPAVK 
                   
               
               
                   
                 KEVKEEEPGAPGKEGAAEGP 
                   
               
               
                   
                 LDPSGYNPAKNNYHPVEDAC 
                   
               
               
                   
                 WKPGQKVPYLAVARTFEKIE 
                   
               
               
                   
                 EVSARLRMVETLSNLLRSVV 
                   
               
               
                   
                 ALSPPDLLPVLYLSLNHLGP 
                   
               
               
                   
                 PQQGLELGVGDGVLLKAVAQ 
                   
               
               
                   
                 ATGRQLESVRAEAAEKGDVG 
                   
               
               
                   
                 LVAENSRSTQRLMLPPPPLT 
                   
               
               
                   
                 ASGVFSKFRDIARLTGSAST 
                   
               
               
                   
                 AKKIDIIKGLFVACRHSEAR 
                   
               
               
                   
                 FIARSLSGRLRLGLAEQSVL 
                   
               
               
                   
                 AALSQAVSLTPPGQEFPPAM 
                   
               
               
                   
                 VDAGKGKTAEARKTWLEEQG 
                   
               
               
                   
                 MILKQTFCEVPDLDRIIPVL 
                   
               
               
                   
                 LEHGLERLPEHCKLSPGIPL 
                   
               
               
                   
                 KPMLAHPTRGISEVLKRFEE 
                   
               
               
                   
                 AAFTCEYKYDGQRAQIHALE 
                   
               
               
                   
                 GGEVKIFSRNQEDNTGKYPD 
                   
               
               
                   
                 IISRIPKIKLPSVTSFILDT 
                   
               
               
                   
                 EAVAWDREKKQIQPFQVLTT 
                   
               
               
                   
                 RKRKEVDASEIQVQVCLYAF 
                   
               
               
                   
                 DLIYLNGESLVREPLSRRRQ 
                   
               
               
                   
                 LLRENFVETEGEFVFATSLD 
                   
               
               
                   
                 TKDIEQIAEFLEQSVKDSCE 
                   
               
               
                   
                 GLMVKTLDVDATYEIAKRSH 
                   
               
               
                   
                 NWLKLKKDYLDGVGDTLDLV 
                   
               
               
                   
                 VIGAYLGRGKRAGRYGGFLL 
                   
               
               
                   
                 ASYDEDSEELQAICKLGTGF 
                   
               
               
                   
                 SDEELEEHHQSLKALVLPSP 
                   
               
               
                   
                 RPYVRIDGAVIPDHWLDPSA 
                   
               
               
                   
                 VWEVKCADLSLSPIYPAARG 
                   
               
               
                   
                 LVDSDKGISLRFPRFIRVRE 
                   
               
               
                   
                 DKQPEQATTSAQVACLYRKQ 
                   
               
               
                   
                 SQIQNQQGEDSGSDPEDTY 
                   
               
               
                   
               
               
                 hLIG1 
                 TPRKPAVKKEVKEEEPGAPG 
                 57 
               
               
                 (233-919) 
                 KEGAAEGPLDPSGYNPAKNN 
                   
               
               
                   
                 YHPVEDACWKPGQKVPYLAV 
                   
               
               
                   
                 ARTFEKIEEVSARLRMVETL 
                   
               
               
                   
                 SNLLRSVVALSPPDLLPVLY 
                   
               
               
                   
                 LSLNHLGPPQQGLELGVGDG 
                   
               
               
                   
                 VLLKAVAQATGRQLESVRAE 
                   
               
               
                   
                 AAEKGDVGLVAENSRSTQRL 
                   
               
               
                   
                 MLPPPPLTASGVFSKFRDIA 
                   
               
               
                   
                 RLTGSASTAKKIDIIKGLFV 
                   
               
               
                   
                 ACRHSEARFIARSLSGRLRL 
                   
               
               
                   
                 GLAEQSVLAALSQAVSLTPP 
                   
               
               
                   
                 GQEFPPAMVDAGKGKTAEAR 
                   
               
               
                   
                 KTWLEEQGMILKQTFCEVPD 
                   
               
               
                   
                 LDRIIPVLLEHGLERLPEHC 
                   
               
               
                   
                 KLSPGIPLKPMLAHPTRGIS 
                   
               
               
                   
                 EVLKRFEEAAFTCEYKYDGQ 
                   
               
               
                   
                 RAQIHALEGGEVKIFSRNQE 
                   
               
               
                   
                 DNTGKYPDIISRIPKIKLPS 
                   
               
               
                   
                 VTSFILDTEAVAWDREKKQI 
                   
               
               
                   
                 QPFQVLTTRKRKEVDASEIQ 
                   
               
               
                   
                 VQVCLYAFDLIYLNGESLVR 
                   
               
               
                   
                 EPLSRRRQLLRENFVETEGE 
                   
               
               
                   
                 FVFATSLDTKDIEQIAEFLE 
                   
               
               
                   
                 QSVKDSCEGLMVKTLDVDAT 
                   
               
               
                   
                 YEIAKRSHNWLKLKKDYLDG 
                   
               
               
                   
                 VGDTLDLVVIGAYLGRGKRA 
                   
               
               
                   
                 GRYGGFLLASYDEDSEELQA 
                   
               
               
                   
                 ICKLGTGFSDEELEEHHQSL 
                   
               
               
                   
                 KALVLPSPRPYVRIDGAVIP 
                   
               
               
                   
                 DHWLDPSAVWEVKCADLSLS 
                   
               
               
                   
                 PIYPAARGLVDSDKGISLRF 
                   
               
               
                   
                 PRFIRVREDKQPEQATTSAQ 
                   
               
               
                   
                 VACLYRKQSQIQNQQGEDSG 
                   
               
               
                   
                 SDPEDTY 
                   
               
               
                   
               
               
                 hLIG1 
                 PKRRTARKQLPKRTIQEVLE 
                 58 
               
               
                 (119-919) 
                 EQSEDEDREAKRKKEEEEEE 
                   
               
               
                   
                 TPKESLTEAEVATEKEGEDG 
                   
               
               
                   
                 DQPTTPPKPLKTSKAETPTE 
                   
               
               
                   
                 SVSEPEVATKQELQEEEEQT 
                   
               
               
                   
                 KPPRRAPKTLSSFFTPRKPA 
                   
               
               
                   
                 VKKEVKEEEPGAPGKEGAAE 
                   
               
               
                   
                 GPLDPSGYNPAKNNYHPVED 
                   
               
               
                   
                 ACWKPGQKVPYLAVARTFEK 
                   
               
               
                   
                 IEEVSARLRMVETLSNLLRS 
                   
               
               
                   
                 VVALSPPDLLPVLYLSLNHL 
                   
               
               
                   
                 GPPQQGLELGVGDGVLLKAV 
                   
               
               
                   
                 AQATGRQLESVRAEAAEKGD 
                   
               
               
                   
                 VGLVAENSRSTQRLMLPPPP 
                   
               
               
                   
                 LTASGVFSKFRDIARLTGSA 
                   
               
               
                   
                 STAKKIDIIKGLFVACRHSE 
                   
               
               
                   
                 ARFIARSLSGRLRLGLAEQS 
                   
               
               
                   
                 VLAALSQAVSLTPPGQEFPP 
                   
               
               
                   
                 AMVDAGKGKTAEARKTWLEE 
                   
               
               
                   
                 QGMILKQTFCEVPDLDRIIP 
                   
               
               
                   
                 VLLEHGLERLPEHCKLSPGI 
                   
               
               
                   
                 PLKPMLAHPTRGISEVLKRF 
                   
               
               
                   
                 EEAAFTCEYKYDGQRAQIHA 
                   
               
               
                   
                 LEGGEVKIFSRNQEDNTGKY 
                   
               
               
                   
                 PDIISRIPKIKLPSVTSFIL 
                   
               
               
                   
                 DTEAVAWDREKKQIQPFQVL 
                   
               
               
                   
                 TTRKRKEVDASEIQVQVCLY 
                   
               
               
                   
                 AFDLIYLNGESLVREPLSRR 
                   
               
               
                   
                 RQLLRENFVETEGEFVFATS 
                   
               
               
                   
                 LDTKDIEQIAEFLEQSVKDS 
                   
               
               
                   
                 CEGLMVKTLDVDATYEIAKR 
                   
               
               
                   
                 SHNWLKLKKDYLDGVGDTLD 
                   
               
               
                   
                 LVVIGAYLGRGKRAGRYGGF 
                   
               
               
                   
                 LLASYDEDSEELQAICKLGT 
                   
               
               
                   
                 GFSDEELEEHHQSLKALVLP 
                   
               
               
                   
                 SPRPYVRIDGAVIPDHWLDP 
                   
               
               
                   
                 SAVWEVKCADLSLSPIYPAA 
                   
               
               
                   
                 RGLVDSDKGISLRFPRFIRV 
                   
               
               
                   
                 REDKQPEQATTSAQVACLYR 
                   
               
               
                   
                 KQSQIQNQQGEDSGSDPEDT 
                   
               
               
                   
                 Y 
                   
               
               
                   
               
               
                 hLIG3 isoform 1 
                 MSLAFKIFFPQTLRALSRKE 
                 59 
               
               
                   
                 LCLFRKHHWRDVRQFSQWSE 
                   
               
               
                   
                 TDLLHGHPLFLRRKPVLSFQ 
                   
               
               
                   
                 GSHLRSRATYLVFLPGLHVG 
                   
               
               
                   
                 LCSGPCEMAEQRFCVDYAKR 
                   
               
               
                   
                 GTAGCKKCKEKIVKGVCRIG 
                   
               
               
                   
                 KVVPNPFSESGGDMKEWYHI 
                   
               
               
                   
                 KCMFEKLERARATTKKIEDL 
                   
               
               
                   
                 TELEGWEELEDNEKEQITQH 
                   
               
               
                   
                 IADLSSKAAGTPKKKAVVQA 
                   
               
               
                   
                 KLTTTGQVTSPVKGASFVTS 
                   
               
               
                   
                 TNPRKFSGFSAKPNNSGEAP 
                   
               
               
                   
                 SSPTPKRSLSSSKCDPRHKD 
                   
               
               
                   
                 CLLREFRKLCAMVADNPSYN 
                   
               
               
                   
                 TKTQIIQDFLRKGSAGDGFH 
                   
               
               
                   
                 GDVYLTVKLLLPGVIKTVYN 
                   
               
               
                   
                 LNDKQIVKLFSRIFNCNPDD 
                   
               
               
                   
                 MARDLEQGDVSETIRVFFEQ 
                   
               
               
                   
                 SKSFPPAAKSLLTIQEVDEF 
                   
               
               
                   
                 LLRLSKLTKEDEQQQALQDI 
                   
               
               
                   
                 ASRCTANDLKCIIRLIKHDL 
                   
               
               
                   
                 KMNSGAKHVLDALDPNAYEA 
                   
               
               
                   
                 FKASRNLQDVVERVLHNAQE 
                   
               
               
                   
                 VEKEPGQRRALSVQASLMTP 
                   
               
               
                   
                 VQPMLAEACKSVEYAMKKCP 
                   
               
               
                   
                 NGMFSEIKYDGERVQVHKNG 
                   
               
               
                   
                 DHFSYFSRSLKPVLPHKVAH 
                   
               
               
                   
                 FKDYIPQAFPGGHSMILDSE 
                   
               
               
                   
                 VLLIDNKTGKPLPFGTLGVH 
                   
               
               
                   
                 KKAAFQDANVCLFVFDCIYF 
                   
               
               
                   
                 NDVSLMDRPLCERRKFLHDN 
                   
               
               
                   
                 MVEIPNRIMFSEMKRVTKAL 
                   
               
               
                   
                 DLADMITRVIQEGLEGLVLK 
                   
               
               
                   
                 DVKGTYEPGKRHWLKVKKDY 
                   
               
               
                   
                 LNEGAMADTADLVVLGAFYG 
                   
               
               
                   
                 QGSKGGMMSIFLMGCYDPGS 
                   
               
               
                   
                 QKWCTVTKCAGGHDDATLAR 
                   
               
               
                   
                 LQNELDMVKISKDPSKIPSW 
                   
               
               
                   
                 LKVNKIYYPDFIVPDPKKAA 
                   
               
               
                   
                 VWEITGAEFSKSEAHTADGI 
                   
               
               
                   
                 SIRFPRCTRIRDDKDWKSAT 
                   
               
               
                   
                 NLPQLKELYQLSKEKADFTV 
                   
               
               
                   
                 VAGDEGSSTTGGSSEENKGP 
                   
               
               
                   
                 SGSAVSRKAPSKPSASTKKA 
                   
               
               
                   
                 EGKLSNSNSKDGNMQTAKPS 
                   
               
               
                   
                 AMKVGEKLATKSSPVKVGEK 
                   
               
               
                   
                 RKAADETLCQTKVLLDIFTG 
                   
               
               
                   
                 VRLYLPPSTPDFSRLRRYFV 
                   
               
               
                   
                 AFDGDLVQEFDMTSATHVLG 
                   
               
               
                   
                 SRDKNPAAQQVSPEWIWACI 
                   
               
               
                   
                 RKRRLVAPC 
                   
               
               
                   
               
               
                 hLIG3 isoform 2 
                 MSLAFKIFFPQTLRALSRKE 
                 60 
               
               
                   
                 LCLFRKHHWRDVRQFSQWSE 
                   
               
               
                   
                 TDLLHGHPLFLRRKPVLSFQ 
                   
               
               
                   
                 GSHLRSRATYLVFLPGLHVG 
                   
               
               
                   
                 LCSGPCEMAEQRFCVDYAKR 
                   
               
               
                   
                 GTAGCKKCKEKIVKGVCRIG 
                   
               
               
                   
                 KVVPNPFSESGGDMKEWYHI 
                   
               
               
                   
                 KCMFEKLERARATTKKIEDL 
                   
               
               
                   
                 TELEGWEELEDNEKEQITQH 
                   
               
               
                   
                 IADLSSKAAGTPKKKAVVQA 
                   
               
               
                   
                 KLTTTGQVTSPVKGASFVTS 
                   
               
               
                   
                 TNPRKFSGFSAKPNNSGEAP 
                   
               
               
                   
                 SSPTPKRSLSSSKCDPRHKD 
                   
               
               
                   
                 CLLREFRKLCAMVADNPSYN 
                   
               
               
                   
                 TKTQIIQDFLRKGSAGDGFH 
                   
               
               
                   
                 GDVYLTVKLLLPGVIKTVYN 
                   
               
               
                   
                 LNDKQIVKLFSRIFNCNPDD 
                   
               
               
                   
                 MARDLEQGDVSETIRVFFEQ 
                   
               
               
                   
                 SKSFPPAAKSLLTIQEVDEF 
                   
               
               
                   
                 LLRLSKLTKEDEQQQALQDI 
                   
               
               
                   
                 ASRCTANDLKCIIRLIKHDL 
                   
               
               
                   
                 KMNSGAKHVLDALDPNAYEA 
                   
               
               
                   
                 FKASRNLQDVVERVLHNAQE 
                   
               
               
                   
                 VEKEPGQRRALSVQASLMTP 
                   
               
               
                   
                 VQPMLAEACKSVEYAMKKCP 
                   
               
               
                   
                 NGMFSEIKYDGERVQVHKNG 
                   
               
               
                   
                 DHFSYFSRSLKPVLPHKVAH 
                   
               
               
                   
                 FKDYIPQAFPGGHSMILDSE 
                   
               
               
                   
                 VLLIDNKTGKPLPFGTLGVH 
                   
               
               
                   
                 KKAAFQDANVCLFVFDCIYF 
                   
               
               
                   
                 NDVSLMDRPLCERRKFLHDN 
                   
               
               
                   
                 MVEIPNRIMFSEMKRVTKAL 
                   
               
               
                   
                 DLADMITRVIQEGLEGLVLK 
                   
               
               
                   
                 DVKGTYEPGKRHWLKVKKDY 
                   
               
               
                   
                 LNEGAMADTADLVVLGAFYG 
                   
               
               
                   
                 QGSKGGMMSIFLMGCYDPGS 
                   
               
               
                   
                 QKWCTVTKCAGGHDDATLAR 
                   
               
               
                   
                 LQNELDMVKISKDPSKIPSW 
                   
               
               
                   
                 LKVNKIYYPDFIVPDPKKAA 
                   
               
               
                   
                 VWEITGAEFSKSEAHTADGI 
                   
               
               
                   
                 SIRFPRCTRIRDDKDWKSAT 
                   
               
               
                   
                 NLPQLKELYQLSKEKADFTV 
                   
               
               
                   
                 VAGDEGSSTTGGSSEENKGP 
                   
               
               
                   
                 SGSAVSRKAPSKPSASTKKA 
                   
               
               
                   
                 EGKLSNSNSKDGNMQTAKPS 
                   
               
               
                   
                 AMKVGEKLATKSSPVKVGEK 
                   
               
               
                   
                 RKAADETLCQTKRRPASEQR 
                   
               
               
                   
                 GRTVPAGRR 
                   
               
               
                   
               
               
                 hLIG3 isoform 3 
                 MAEQRFCVDYAKRGTAGCKK 
                 61 
               
               
                   
                 CKEKIVKGVCRIGKVVPNPF 
                   
               
               
                   
                 SESGGDMKEWYHIKCMFEKL 
                   
               
               
                   
                 ERARATTKKIEDLTELEGWE 
                   
               
               
                   
                 ELEDNEKEQITQHIADLSSK 
                   
               
               
                   
                 AAGTPKKKAVVQAKLTTTGQ 
                   
               
               
                   
                 VTSPVKGASFVTSTNPRKFS 
                   
               
               
                   
                 GFSAKPNNSGEAPSSPTPKR 
                   
               
               
                   
                 SLSSSKCDPRHKDCLLREFR 
                   
               
               
                   
                 KLCAMVADNPSYNTKTQIIQ 
                   
               
               
                   
                 DFLRKGSAGDGFHGDVYLTV 
                   
               
               
                   
                 KLLLPGVIKTVYNLNDKQIV 
                   
               
               
                   
                 KLFSRIFNCNPDDMARDLEQ 
                   
               
               
                   
                 GDVSETIRVFFEQSKSFPPA 
                   
               
               
                   
                 AKSLLTIQEVDEFLLRLSKL 
                   
               
               
                   
                 TKEDEQQQALQDIASRCTAN 
                   
               
               
                   
                 DLKCIIRLIKHDLKMNSGAK 
                   
               
               
                   
                 HVLDALDPNAYEAFKASRNL 
                   
               
               
                   
                 QDVVERVLHNAQEVEKEPGQ 
                   
               
               
                   
                 RRALSVQASLMTPVQPMLAE 
                   
               
               
                   
                 ACKSVEYAMKKCPNGMFSEI 
                   
               
               
                   
                 KYDGERVQVHKNGDHFSYFS 
                   
               
               
                   
                 RSLKPVLPHKVAHFKDYIPQ 
                   
               
               
                   
                 AFPGGHSMILDSEVLLIDNK 
                   
               
               
                   
                 TGKPLPFGTLGVHKKAAFQD 
                   
               
               
                   
                 ANVCLFVFDCIYFNDVSLMD 
                   
               
               
                   
                 RPLCERRKFLHDNMVEIPNR 
                   
               
               
                   
                 IMFSEMKRVTKALDLADMIT 
                   
               
               
                   
                 RVIQEGLEGLVLKDVKGTYE 
                   
               
               
                   
                 PGKRHWLKVKKDYLNEGAMA 
                   
               
               
                   
                 DTADLVVLGAFYGQGSKGGM 
                   
               
               
                   
                 MSIFLMGCYDPGSQKWCTVT 
                   
               
               
                   
                 KCAGGHDDATLARLQNELDM 
                   
               
               
                   
                 VKISKDPSKIPSWLKVNKIY 
                   
               
               
                   
                 YPDFIVPDPKKAAVWEITGA 
                   
               
               
                   
                 EFSKSEAHTADGISIRFPRC 
                   
               
               
                   
                 TRIRDDKDWKSATNLPQLKE 
                   
               
               
                   
                 LYQLSKEKADFTVVAGDEGS 
                   
               
               
                   
                 STTGGSSEENKGPSGSAVSR 
                   
               
               
                   
                 KAPSKPSASTKKAEGKLSNS 
                   
               
               
                   
                 NSKDGNMQTAKPSAMKVGEK 
                   
               
               
                   
                 LATKSSPVKVGEKRKAADET 
                   
               
               
                   
                 LCQTKVLLDIFTGVRLYLPP 
                   
               
               
                   
                 STPDFSRLRRYFVAFDGDLV 
                   
               
               
                   
                 QEFDMTSATHVLGSRDKNPA 
                   
               
               
                   
                 AQQVSPEWIWACIRKRRLVA 
                   
               
               
                   
                 PC 
                   
               
               
                   
               
               
                 hLIG3 isoform 4 
                 MAEQRFCVDYAKRGTAGCKK 
                 62 
               
               
                   
                 CKEKIVKGVCRIGKVVPNPF 
                   
               
               
                   
                 SESGGDMKEWYHIKCMFEKL 
                   
               
               
                   
                 ERARATTKKIEDLTELEGWE 
                   
               
               
                   
                 ELEDNEKEQITQHIADLSSK 
                   
               
               
                   
                 AAGTPKKKAVVQAKLTTTGQ 
                   
               
               
                   
                 VTSPVKGASFVTSTNPRKFS 
                   
               
               
                   
                 GFSAKPNNSGEAPSSPTPKR 
                   
               
               
                   
                 SLSSSKCDPRHKDCLLREFR 
                   
               
               
                   
                 KLCAMVADNPSYNTKTQIIQ 
                   
               
               
                   
                 DFLRKGSAGDGFHGDVYLTV 
                   
               
               
                   
                 KLLLPGVIKTVYNLNDKQIV 
                   
               
               
                   
                 KLFSRIFNCNPDDMARDLEQ 
                   
               
               
                   
                 GDVSETIRVFFEQSKSFPPA 
                   
               
               
                   
                 AKSLLTIQEVDEFLLRLSKL 
                   
               
               
                   
                 TKEDEQQQALQDIASRCTAN 
                   
               
               
                   
                 DLKCIIRLIKHDLKMNSGAK 
                   
               
               
                   
                 HVLDALDPNAYEAFKASRNL 
                   
               
               
                   
                 QDVVERVLHNAQEVEKEPGQ 
                   
               
               
                   
                 RRALSVQASLMTPVQPMLAE 
                   
               
               
                   
                 ACKSVEYAMKKCPNGMFSEI 
                   
               
               
                   
                 KYDGERVQVHKNGDHFSYFS 
                   
               
               
                   
                 RSLKPVLPHKVAHFKDYIPQ 
                   
               
               
                   
                 AFPGGHSMILDSEVLLIDNK 
                   
               
               
                   
                 TGKPLPFGTLGVHKKAAFQD 
                   
               
               
                   
                 ANVCLFVFDCIYFNDVSLMD 
                   
               
               
                   
                 RPLCERRKFLHDNMVEIPNR 
                   
               
               
                   
                 IMFSEMKRVTKALDLADMIT 
                   
               
               
                   
                 RVIQEGLEGLVLKDVKGTYE 
                   
               
               
                   
                 PGKRHWLKVKKDYLNEGAMA 
                   
               
               
                   
                 DTADLVVLGAFYGQGSKGGM 
                   
               
               
                   
                 MSIFLMGCYDPGSQKWCTVT 
                   
               
               
                   
                 KCAGGHDDATLARLQNELDM 
                   
               
               
                   
                 VKISKDPSKIPSWLKVNKIY 
                   
               
               
                   
                 YPDFIVPDPKKAAVWEITGA 
                   
               
               
                   
                 EFSKSEAHTADGISIRFPRC 
                   
               
               
                   
                 TRIRDDKDWKSATNLPQLKE 
                   
               
               
                   
                 LYQLSKEKADFTVVAGDEGS 
                   
               
               
                   
                 STTGGSSEENKGPSGSAVSR 
                   
               
               
                   
                 KAPSKPSASTKKAEGKLSNS 
                   
               
               
                   
                 NSKDGNMQTAKPSAMKVGEK 
                   
               
               
                   
                 LATKSSPVKVGEKRKAADET 
                   
               
               
                   
                 LCQTKRRPASEQRGRTVPAG 
                   
               
               
                   
                 RR 
                   
               
               
                   
               
               
                 hLIG4 
                 MAASQTSQTVASHVPFADLC 
                 63 
               
               
                   
                 STLERIQKSKGRAEKIRHFR 
                   
               
               
                   
                 EFLDSWRKFHDALHKNHKDV 
                   
               
               
                   
                 TDSFYPAMRLILPQLERERM 
                   
               
               
                   
                 AYGIKETMLAKLYIELLNLP 
                   
               
               
                   
                 RDGKDALKLLNYRTPTGTHG 
                   
               
               
                   
                 DAGDFAMIAYFVLKPRCLQK 
                   
               
               
                   
                 GSLTIQQVNDLLDSIASNNS 
                   
               
               
                   
                 AKRKDLIKKSLLQLITQSSA 
                   
               
               
                   
                 LEQKWLIRMIIKDLKLGVSQ 
                   
               
               
                   
                 QTIFSVFHNDAAELHNVTTD 
                   
               
               
                   
                 LEKVCRQLHDPSVGLSDISI 
                   
               
               
                   
                 TLFSAFKPMLAAIADIEHIE 
                   
               
               
                   
                 KDMKHQSFYIETKLDGERMQ 
                   
               
               
                   
                 MHKDGDVYKYFSRNGYNYTD 
                   
               
               
                   
                 QFGASPTEGSLTPFIHNAFK 
                   
               
               
                   
                 ADIQICILDGEMMAYNPNTQ 
                   
               
               
                   
                 TFMQKGTKFDIKRMVEDSDL 
                   
               
               
                   
                 QTCYCVFDVLMVNNKKLGHE 
                   
               
               
                   
                 TLRKRYEILSSIFTPIPGRI 
                   
               
               
                   
                 EIVQKTQAHTKNEVIDALNE 
                   
               
               
                   
                 AIDKREEGIMVKQPLSIYKP 
                   
               
               
                   
                 DKRGEGWLKIKPEYVSGLMD 
                   
               
               
                   
                 ELDILIVGGYWGKGSRGGMM 
                   
               
               
                   
                 SHFLCAVAEKPPPGEKPSVF 
                   
               
               
                   
                 HTLSRVGSGCTMKELYDLGL 
                   
               
               
                   
                 KLAKYWKPFHRKAPPSSILC 
                   
               
               
                   
                 GTEKPEVYIEPCNSVIVQIK 
                   
               
               
                   
                 AAEIVPSDMYKTGCTLRFPR 
                   
               
               
                   
                 IEKIRDDKEWHECMTLDDLE 
                   
               
               
                   
                 QLRGKASGKLASKHLYIGGD 
                   
               
               
                   
                 DEPQEKKRKAAPKMKKVIGI 
                   
               
               
                   
                 IEHLKAPNLTNVNKISNIFE 
                   
               
               
                   
                 DVEFCVMSGTDSQPKPDLEN 
                   
               
               
                   
                 RIAEFGGYIVQNPGPDTYCV 
                   
               
               
                   
                 IAGSENIRVKNIILSNKHDV 
                   
               
               
                   
                 VKPAWLLECFKTKSFVPWQP 
                   
               
               
                   
                 RFMIHMCPSTKEHFAREYDC 
                   
               
               
                   
                 YGDSYFIDTDLNQLKEVFSG 
                   
               
               
                   
                 IKNSNEQTPEEMASLIADLE 
                   
               
               
                   
                 YRYSWDCSPLSMFRRHTVYL 
                   
               
               
                   
                 DSYAVINDLSTKNEGTRLAI 
                   
               
               
                   
                 KALELRFHGAKVVSCLAEGV 
                   
               
               
                   
                 SHVIIGEDHSRVADFKAFRR 
                   
               
               
                   
                 TFKRKFKILKESWVTDSIDK 
                   
               
               
                   
                 CELQEENQYLI 
                   
               
               
                   
               
               
                 T4 Ligase 
                 MILKILNEIASIGSTKQKQA 
                 64 
               
               
                   
                 ILEKNKDNELLKRVYRLTYS 
                   
               
               
                   
                 RGLQYYIKKWPKPGIATQSF 
                   
               
               
                   
                 GMLTLTDMLDFIEFTLATRK 
                   
               
               
                   
                 LTGNAAIEELTGYITDGKKD 
                   
               
               
                   
                 DVEVLRRVMMRDLECGASVS 
                   
               
               
                   
                 IANKVWPGLIPEQPQMLASS 
                   
               
               
                   
                 YDEKGINKNIKFPAFAQLKA 
                   
               
               
                   
                 DGARCFAEVRGDELDDVRLL 
                   
               
               
                   
                 SRAGNEYLGLDLLKEELIKM 
                   
               
               
                   
                 TAEARQIHPEGVLIDGELVY 
                   
               
               
                   
                 HEQVKKEPEGLDFLFDAYPE 
                   
               
               
                   
                 NSKAKEFAEVAESRTASNGI 
                   
               
               
                   
                 ANKSLKGTISEKEAQCMKFQ 
                   
               
               
                   
                 VWDYVPLVEIYSLPAFRLKY 
                   
               
               
                   
                 DVRFSKLEQMTSGYDKVILI 
                   
               
               
                   
                 ENQVVNNLDEAKVIYKKYID 
                   
               
               
                   
                 QGLEGIILKNIDGLWENARS 
                   
               
               
                   
                 KNLYKFKEVIDVDLKIVGIY 
                   
               
               
                   
                 PHRKDPTKAGGFILESECGK 
                   
               
               
                   
                 IKVNAGSGLKDKAGVKSHEL 
                   
               
               
                   
                 DRTRIMENQNYYIGKILECE 
                   
               
               
                   
                 CNGWLKSDGRTDYVKLFLPI 
                   
               
               
                   
                 AIRLREDKTKANTFEDVFGD 
                   
               
               
                   
                 FHEVTGL 
                   
               
               
                   
               
               
                 T7 Ligase 
                 MMNIKTNPFKAVSFVESAIK 
                 65 
               
               
                   
                 KALDNAGYLIAEIKYDGVRG 
                   
               
               
                   
                 NICVDNTANSYWLSRVSKTI 
                   
               
               
                   
                 PALEHLNGFDVRWKRLLNDD 
                   
               
               
                   
                 RCFYKDGFMLDGELMVKGVD 
                   
               
               
                   
                 FNTGSGLLRTKWTDTKNQEF 
                   
               
               
                   
                 HEELFVEPIRKKDKVPFKLH 
                   
               
               
                   
                 TGHLHIKLYAILPLHIVESG 
                   
               
               
                   
                 EDCDVMTLLMQEHVKNMLPL 
                   
               
               
                   
                 LQEYFPEIEWQAAESYEVYD 
                   
               
               
                   
                 MVELQQLYEQKRAEGHEGLI 
                   
               
               
                   
                 VKDPMCIYKRGKKSGWWKMK 
                   
               
               
                   
                 PENEADGIIQGLVWGTKGLA 
                   
               
               
                   
                 NEGKVIGFEVLLESGRLVNA 
                   
               
               
                   
                 TNISRALMDEFTETVKEATL 
                   
               
               
                   
                 SQWGFFSPYGIGDNDACTIN 
                   
               
               
                   
                 PYDGWACQISYMEETPDGSL 
                   
               
               
                   
                 RHPSFVMFRGTEDNPQEKM 
                   
               
               
                   
               
               
                 Taq Ligase 
                 MTLEAARRRVNELRDLIRYH 
                 66 
               
               
                   
                 NYLYYVLDAPEISDAEYDRL 
                   
               
               
                   
                 LRELKELEERFPELQSPDSP 
                   
               
               
                   
                 TEQVGARPLESTFRPVRHPT 
                   
               
               
                   
                 RMYSLDNAFSLDEVRAFEER 
                   
               
               
                   
                 IERALGRKGPFLYTVEHKVD 
                   
               
               
                   
                 GLSVNLYYEEGILVFGATRG 
                   
               
               
                   
                 DGETGEEVTQNLLTIRTIPR 
                   
               
               
                   
                 RLTGVPDRLEVRGEVYMPIE 
                   
               
               
                   
                 AFLRLNQELEEAGERIFKNP 
                   
               
               
                   
                 RNAAAGSLRQKDPRVTARRG 
                   
               
               
                   
                 LRATFYALGLGLEETGLKSQ 
                   
               
               
                   
                 HDLLLWLRERGFPVEHGFTR 
                   
               
               
                   
                 ALGAEGVEEVYQAWLKERRK 
                   
               
               
                   
                 LPFEADGVVVKLDDLALWRE 
                   
               
               
                   
                 LGYTARAPRFALAYKFPAEE 
                   
               
               
                   
                 KETRLLSVAFQVGRTGRITP 
                   
               
               
                   
                 VGVLEPVFIEGSEVSRVTLH 
                   
               
               
                   
                 NESFIEELDVRIGDWVLVHK 
                   
               
               
                   
                 AGGVIPEVLRVLKERRTGEE 
                   
               
               
                   
                 KPILWPENCPECGHALIKEG 
                   
               
               
                   
                 KVHRCPNPLCPAKRFEAIRH 
                   
               
               
                   
                 YASRKAMDIQGLGEKLIEKL 
                   
               
               
                   
                 LEKGLVRDVADLYRLKKEDL 
                   
               
               
                   
                 VNLERMGEKSAENLLRQIEE 
                   
               
               
                   
                 SKGRGLERLLYALGLPGVGE 
                   
               
               
                   
                 VLARNLALRFGHMDRLLEAG 
                   
               
               
                   
                 LEDLLEVEGVGELTARAILN 
                   
               
               
                   
                 TLKDPEFRDLVRRLKEAGVE 
                   
               
               
                   
                 MEAKEREGEALKGLTFVITG 
                   
               
               
                   
                 ELSRPREEVKALLRRLGAKV 
                   
               
               
                   
                 TDSVSRKTGFLVVGENPGSK 
                   
               
               
                   
                 LEKARALGVPTLSEEELYRL 
                   
               
               
                   
                 IEERTGKDPRALTA 
                   
               
               
                   
               
               
                 T3 Ligase 
                 MNIFNTNPFKAVSFVESAVK 
                 67 
               
               
                   
                 KALETSGYLIADCKYDGVRG 
                   
               
               
                   
                 NIVVDNVAEAAWLSRVSKFI 
                   
               
               
                   
                 PALEHLNGFDKRWQQLLNDD 
                   
               
               
                   
                 RCIFPDGFMLDGELMVKGVD 
                   
               
               
                   
                 FNTGSGLLRTKWVKRDNMGF 
                   
               
               
                   
                 HLTNVPTKLTPKGREVIDGK 
                   
               
               
                   
                 FEFHLDPKRLSVRLYAVMPI 
                   
               
               
                   
                 HIAESGEDYDVQNLLMPYHV 
                   
               
               
                   
                 EAMRSLLVEYFPEIEWLIAE 
                   
               
               
                   
                 TYEVYDMDSLTELYEEKRAE 
                   
               
               
                   
                 GHEGLIVKDPQGIYKRGKKS 
                   
               
               
                   
                 GWWKLKPECEADGIIQGVNW 
                   
               
               
                   
                 GTEGLANEGKVIGFSVLLET 
                   
               
               
                   
                 GRLVDANNISRALMDEFTSN 
                   
               
               
                   
                 VKAHGEDFYNGWACQVNYME 
                   
               
               
                   
                 ATPDGSLRHPSFEKFRGTED 
                   
               
               
                   
                 NPQEKM 
                   
               
               
                   
               
               
                 NAD-dependent 
                 MESIEQQLTELRTTLRHHEY 
                 68 
               
               
                 
                   E coli 
                 
                 LYHVMDAPEIPDAEYDRLMR 
                   
               
               
                 DNA ligase 
                 ELRELETKHPELITPDSPTQ 
                   
               
               
                 LigA 
                 RVGAAPLAAFSQIRHEVPML 
                   
               
               
                   
                 SLDNVFDEESFLAFNKRVQD 
                   
               
               
                   
                 RLKNNEKVTWCCELKLDGLA 
                   
               
               
                   
                 VSILYENGVLVSAATRGDGT 
                   
               
               
                   
                 TGEDITSNVRTIRAIPLKLH 
                   
               
               
                   
                 GENIPARLEVRGEVFLPQAG 
                   
               
               
                   
                 FEKINEDARRTGGKVFANPR 
                   
               
               
                   
                 NAAAGSLRQLDPRITAKRPL 
                   
               
               
                   
                 TFFCYGVGVLEGGELPDTHL 
                   
               
               
                   
                 GRLLQFKKWGLPVSDRVTLC 
                   
               
               
                   
                 ESAEEVLAFYHKVEEDRPTL 
                   
               
               
                   
                 GFDIDGVVIKVNSLAQQEQL 
                   
               
               
                   
                 GFVARAPRWAVAFKFPAQEQ 
                   
               
               
                   
                 MTFVRDVEFQVGRTGAITPV 
                   
               
               
                   
                 ARLEPVHVAGVLVSNATLHN 
                   
               
               
                   
                 ADEIERLGLRIGDKVVIRRA 
                   
               
               
                   
                 GDVIPQVVNVVLSERPEDTR 
                   
               
               
                   
                 EVVFPTHCPVCGSDVERVEG 
                   
               
               
                   
                 EAVARCTGGLICGAQRKESL 
                   
               
               
                   
                 KHFVSRRAMDVDGMGDKIID 
                   
               
               
                   
                 QLVEKEYVHTPADLFKLTAG 
                   
               
               
                   
                 KLTGLERMGPKSAQNVVNAL 
                   
               
               
                   
                 EKAKETTFARFLYALGIREV 
                   
               
               
                   
                 GEATAAGLAAYFGTLEALEA 
                   
               
               
                   
                 ASIEELQKVPDVGIVVASHV 
                   
               
               
                   
                 HNFFAEESNRNVISELLAEG 
                   
               
               
                   
                 VHWPAPIVINAEEIDSPFAG 
                   
               
               
                   
                 KTVVLTGSLSQMSRDDAKAR 
                   
               
               
                   
                 LVELGAKVAGSVSKKTDLVI 
                   
               
               
                   
                 AGEAAGSKLAKAQELGIEVI 
                   
               
               
                   
                 DEAEMLRLLGS 
                   
               
               
                   
               
               
                 
                   Thermococcus 
                 
                 MRYSELADLYRRLEKTTLKT 
                 69 
               
               
                   kodakarensis  DNA 
                 LKTKFVADFLKKTPDELLEI 
                   
               
               
                 ligase 
                 VPYLILGKVFPDWDERELGV 
                   
               
               
                   
                 GEKLLIKAVSMATGVPEKEI 
                   
               
               
                   
                 EDSVRDTGDLGESVALAIKK 
                   
               
               
                   
                 KKQKSFFSQPLTIKRVYDTF 
                   
               
               
                   
                 VKIAEAQGEGSQDRKMKYLA 
                   
               
               
                   
                 NLFMDAEPEEGKYLARTVLG 
                   
               
               
                   
                 TMRTGVAEGILRDAIAEAFR 
                   
               
               
                   
                 VKPELVERAYMLTSDFGYVA 
                   
               
               
                   
                 KIAKLEGNEGLSKVRIQIGK 
                   
               
               
                   
                 PIRPMLAQNAASVKDALIEM 
                   
               
               
                   
                 GGEAAFEIKYDGARVQVHKD 
                   
               
               
                   
                 GDKVIVYSRRLENVTRSIPE 
                   
               
               
                   
                 VIEAIKAALKPEKAIVEGEL 
                   
               
               
                   
                 VAVGENGRPRPFQYVLRRFR 
                   
               
               
                   
                 RKYNIDEMIEKIPLELNLFD 
                   
               
               
                   
                 VMFVDGESLIETKFIDRRNK 
                   
               
               
                   
                 LEEIVKESEKIKLAEQLITK 
                   
               
               
                   
                 KVEEAEAFYRRALELGHEGL 
                   
               
               
                   
                 MAKRLDSIYEPGNRGKKWLK 
                   
               
               
                   
                 IKPTMENLDLVIIGAEWGEG 
                   
               
               
                   
                 RRAHLLGSFLVAAYDPHSGE 
                   
               
               
                   
                 FLPVGKVGSGFTDEDLVEFT 
                   
               
               
                   
                 KMLKPYIVRQEGKFVEIEPK 
                   
               
               
                   
                 FVIEVTYQEIQKSPKYKSGF 
                   
               
               
                   
                 ALRFPRYVALREDKSPEEAD 
                   
               
               
                   
                 TIERVAELYELQERFKAKK 
                   
               
               
                   
               
               
                 African swine 
                 MLNQFPGQYSNNIFCFPPIE 
                 70 
               
               
                 fever virus 
                 SETKSGKKASWIICVQVVQH 
                   
               
               
                 DNA ligase 
                 NTIIPITDEMFSTDVKDAVA 
                   
               
               
                   
                 EIFTKFFVEEGAVRISKMTR 
                   
               
               
                   
                 VTEGKNLGKKNATTVVHQAF 
                   
               
               
                   
                 KDALSKYNRHARQKRGAHTN 
                   
               
               
                   
                 RGMIPPMLVKYFNIIPKTFF 
                   
               
               
                   
                 EEETDPIVQRKRNGVRAVAC 
                   
               
               
                   
                 QQGDGCILLYSRTEKEFLGL 
                   
               
               
                   
                 DNIKKELKQLYLFIDVRVYL 
                   
               
               
                   
                 DGELYLHRKPLQWIAGQANA 
                   
               
               
                   
                 KTDSSELHFYVFDCFWSDQL 
                   
               
               
                   
                 QMPSNKRQQLLTNIFKQKED 
                   
               
               
                   
                 LTFIHQVENFSVKNVDEALR 
                   
               
               
                   
                 LKAQFIKEGYEGAIVRNANG 
                   
               
               
                   
                 PYEPGYNNYHSAHLAKLKPL 
                   
               
               
                   
                 LDAEFILVDYTQGKKGKDLG 
                   
               
               
                   
                 AILWVCELPNKKRFVVTPKH 
                   
               
               
                   
                 LTYADRYALFQKLTPALFKK 
                   
               
               
                   
                 HLYGKELTVEYAELSPKTGI 
                   
               
               
                   
                 PLQARAVGFREPINVLEII 
                   
               
               
                   
               
               
                 
                   Vaccinia 
                 
                 MTSLREFRKLCCDIYHASGY 
                 71 
               
               
                 virus DNA 
                 KEKSKLIRDFITDRDDKYLI 
                   
               
               
                 ligase 
                 IKLLLPGLDDRIYNMNDKQI 
                   
               
               
                 (strain Western 
                 IKLYSIIFKQSQEDMLQDLG 
                   
               
               
                 Reserve) 
                 YGYIGDTIRTFFKENTEIRP 
                   
               
               
                   
                 RDKSILTLEDVDSFLTTLSS 
                   
               
               
                   
                 VTKESHQIKLLTDIASVCTC 
                   
               
               
                   
                 NDLKCVVMLIDKDLKIKAGP 
                   
               
               
                   
                 RYVLNAISPNAYDVFRKSNN 
                   
               
               
                   
                 LKEIIENASKQNLDSISISV 
                   
               
               
                   
                 MTPINPMLAESCDSVNKAFK 
                   
               
               
                   
                 KFPSGMFAEVKYDGERVQVH 
                   
               
               
                   
                 KNNNEFAFFSRNMKPVLSHK 
                   
               
               
                   
                 VDYLKEYIPKAFKKATSIVL 
                   
               
               
                   
                 DSEIVLVDEHNVPLPFGSLG 
                   
               
               
                   
                 IHKKKEYKNSNMCLFVFDCL 
                   
               
               
                   
                 YFDGFDMTDIPLYERRSFLK 
                   
               
               
                   
                 DVMVEIPNRIVFSELTNISN 
                   
               
               
                   
                 ESQLTDVLDDALTRKLEGLV 
                   
               
               
                   
                 LKDINGVYEPGKRRWLKIKR 
                   
               
               
                   
                 DYLNEGSMADSADLVVLGAY 
                   
               
               
                   
                 YGKGAKGGIMAVFLMGCYDD 
                   
               
               
                   
                 ESGKWKTVTKCSGHDDNTLR 
                   
               
               
                   
                 VLQDQLTMVKINKDPKKIPE 
                   
               
               
                   
                 WLVVNKIYIPDFVVEDPKQS 
                   
               
               
                   
                 QIWEISGAEFTSSKSHTANG 
                   
               
               
                   
                 ISIRFPRFTRIREDKTWKES 
                   
               
               
                   
                 THLNDLVNLTKS 
                   
               
               
                   
               
               
                 
                   Vaccinia 
                 
                 MTSLREFRKLCCDIYHASGY 
                 72 
               
               
                 virus DNA 
                 KEKSKLIRDFITDRDDKYLI 
                   
               
               
                 ligase 
                 IKLLLPGLDDRIYNMNDKQI 
                   
               
               
                 (strain Ankara) 
                 IKLYSIIFKQSQEDMLQDLG 
                   
               
               
                   
                 YGYIGDTIRTFFKENTEIRP 
                   
               
               
                   
                 RDKSILTLEEVDSFLTTLSS 
                   
               
               
                   
                 VTKESHQIKLLTDIASVCTC 
                   
               
               
                   
                 NDLKCVVMLIDKDLKIKAGP 
                   
               
               
                   
                 RYVLNAISPHAYDVFRKSNN 
                   
               
               
                   
                 LKEIIENASKQNLDSISISV 
                   
               
               
                   
                 MTPINPMLAESCDSVNKAFK 
                   
               
               
                   
                 KFPSGMFAEVKYDGERVQVH 
                   
               
               
                   
                 KNNNEFAFFSRNMKPVLSHK 
                   
               
               
                   
                 VDYLKEYIPKAFKKATSIVL 
                   
               
               
                   
                 DSEIVLVDEHNVPLPFGSLG 
                   
               
               
                   
                 IHKKKEYKNSNMCLFVFDCL 
                   
               
               
                   
                 YFDGFDMTDIPLYERRSFLK 
                   
               
               
                   
                 DVMVEIPNRIVFSELTNISN 
                   
               
               
                   
                 ESQLTDVLDDALTRKLEGLV 
                   
               
               
                   
                 LKDINGVYEPGKRRWLKIKR 
                   
               
               
                   
                 DYLNEGSMADSADLVVLGAY 
                   
               
               
                   
                 YGKGAKGGIMAVFLMGCYDD 
                   
               
               
                   
                 ESGKWKTVTKCSGHDDNTLR 
                   
               
               
                   
                 ELQDQLKMIKINKDPKKIPE 
                   
               
               
                   
                 WLVVNKIYIPDFVVEDPKQS 
                   
               
               
                   
                 QIWEISGAEFTSSKSHTANG 
                   
               
               
                   
                 ISIRFPRFTRIREDKTWKES 
                   
               
               
                   
                 THLNDLVNLTKS 
                   
               
               
                   
               
               
                 
                   Burkholderia 
                 
                 MARSPVEPPASQPAKRAAWL 
                 73 
               
               
                   pseudomallei  DNA 
                 RAELERANYAYYVLDQPDLP 
                   
               
               
                 ligase 
                 DAEYDRLFVELQRIEAEHPD 
                   
               
               
                   
                 LVTPDSPTQRVGGEAASGFT 
                   
               
               
                   
                 PVVHDKPMLSLNNGFADEDV 
                   
               
               
                   
                 IAFDKRVADGLDKATDLAGT 
                   
               
               
                   
                 VTEPVEYACELKFDGLAISL 
                   
               
               
                   
                 RYENGRFVQASTRGDGTTGE 
                   
               
               
                   
                 DVTENIRTIRAIPLTLKGKR 
                   
               
               
                   
                 IPRMLDVRGEVLMFKRDFAR 
                   
               
               
                   
                 LNERQRAAGQREFANPRNAA 
                   
               
               
                   
                 AGSLRQLDSKITASRPLSFF 
                   
               
               
                   
                 AYGIGVLDGADMPDTHSGLL 
                   
               
               
                   
                 DWYETLGLPVNRERAVVRGA 
                   
               
               
                   
                 AGLLAFFHSVGERRESLPYD 
                   
               
               
                   
                 IDGVVYKVNRRDEQDRLGFV 
                   
               
               
                   
                 SRAPRFALAHKFPAQEALTK 
                   
               
               
                   
                 LIAIDVQVGRTGAITPVARL 
                   
               
               
                   
                 EPVFVGGATVTNATLHNEDE 
                   
               
               
                   
                 VRRKDIRIGDTVIVRRAGDV 
                   
               
               
                   
                 IPEVVSAVLDRRPADAQEFV 
                   
               
               
                   
                 MPTECPECGSRIERLPDEAI 
                   
               
               
                   
                 ARCTGGLFCPAQRKQALWHF 
                   
               
               
                   
                 AQRRALDIDGLGEKIIDQLV 
                   
               
               
                   
                 EQNLVRTPADLFNLGFSTLV 
                   
               
               
                   
                 GLDRFAEKSARNLIDSLEKA 
                   
               
               
                   
                 KHTTLARFIYALGIRHVGES 
                   
               
               
                   
                 TAKDLAKHFGSLDPIMDAPI 
                   
               
               
                   
                 DALLEVNDVGPIVAESIHQF 
                   
               
               
                   
                 FAEEHNRTVIEQLRARGKVT 
                   
               
               
                   
                 WPEGPPAPRAPQGVLAGKTV 
                   
               
               
                   
                 VLTGTLPTLTREAAKEMLEA 
                   
               
               
                   
                 AGAKVAGSVSKKTDYVVAGA 
                   
               
               
                   
                 DAGSKLAKAEELGIPVLDEA 
                   
               
               
                   
                 GMHTLLEGHAR 
                   
               
               
                   
               
               
                 
                   Alteromonas 
                 
                 MQFFLTVFCLLLITAVTHVN 
                 74 
               
               
                   mediterranea  DNA 
                 AEDKLDIVDGLQLAKQYSHS 
                   
               
               
                 ligase 
                 RQDINIAEYWVSEKLDGIRA 
                   
               
               
                   
                 RWDGTELRTRNNNKIAAPAW 
                   
               
               
                   
                 FTANWPKATIDGELWIARGQ 
                   
               
               
                   
                 FERTASIVLSKLTSVAPHSV 
                   
               
               
                   
                 AGSLPRTESTVGAMTATHSL 
                   
               
               
                   
                 PSKRWAKIRFMAFDMPVAGQ 
                   
               
               
                   
                 SFDSRLNMLNNLKEATPNPT 
                   
               
               
                   
                 FAVVSQFTLSSVNALEEKLE 
                   
               
               
                   
                 QVTLSGGEGLMLHHKKAFYH 
                   
               
               
                   
                 SGRSDKLLKVKQFEDAEAKV 
                   
               
               
                   
                 LAHLPGKGKFKGMMGSLLVE 
                   
               
               
                   
                 TPAGVQFKLGTGFSEKERQA 
                   
               
               
                   
                 PPAIGSWVTFKFYGVTKNGK 
                   
               
               
                   
                 PRFASFLRVRPPSDLPK 
                   
               
               
                   
               
               
                 Yeast DNA 
                 MRRLLTGCLLSSARPLKSRL 
                 75 
               
               
                 ligase 1 
                 PLLMSSSLPSSAGKKPKQAT 
                   
               
               
                 (Cdc9p) 
                 LARFFTSMKNKPTEGTPSPK 
                   
               
               
                   
                 KSSKHMLEDRMDNVSGEEEY 
                   
               
               
                   
                 ATKKLKQTAVTHTVAAPSSM 
                   
               
               
                   
                 GSNFSSIPSSAPSSGVADSP 
                   
               
               
                   
                 QQSQRLVGEVEDALSSNNND 
                   
               
               
                   
                 HYSSNIPYSEVCEVFNKIEA 
                   
               
               
                   
                 ISSRLEIIRICSDFFIKIMK 
                   
               
               
                   
                 QSSKNLIPTTYLFINRLGPD 
                   
               
               
                   
                 YEAGLELGLGENLLMKTISE 
                   
               
               
                   
                 TCGKSMSQIKLKYKDIGDLG 
                   
               
               
                   
                 EIAMGARNVQPTMFKPKPLT 
                   
               
               
                   
                 VGEVFKNLRAIAKTQGKDSQ 
                   
               
               
                   
                 LKKMKLIKRMLTACKGIEAK 
                   
               
               
                   
                 FLIRSLESKLRIGLAEKTVL 
                   
               
               
                   
                 ISLSKALLLHDENREDSPDK 
                   
               
               
                   
                 DVPMDVLESAQQKIRDAFCQ 
                   
               
               
                   
                 VPNYEIVINSCLEHGIMNLD 
                   
               
               
                   
                 KYCTLRPGIPLKPMLAKPTK 
                   
               
               
                   
                 AINEVLDRFQGETFTSEYKY 
                   
               
               
                   
                 DGERAQVHLLNDGTMRIYSR 
                   
               
               
                   
                 NGENMTERYPEINITDFIQD 
                   
               
               
                   
                 LDTTKNLILDCEAVAWDKDQ 
                   
               
               
                   
                 GKILPFQVLSTRKRKDVELN 
                   
               
               
                   
                 DVKVKVCLFAFDILCYNDER 
                   
               
               
                   
                 LINKSLKERREYLTKVTKVV 
                   
               
               
                   
                 PGEFQYATQITTNNLDELQK 
                   
               
               
                   
                 FLDESVNHSCEGLMVKMLEG 
                   
               
               
                   
                 PESHYEPSKRSRNWLKLKKD 
                   
               
               
                   
                 YLEGVGDSLDLCVLGAYYGR 
                   
               
               
                   
                 GKRTGTYGGFLLGCYNQDTG 
                   
               
               
                   
                 EFETCCKIGTGFSDEMLQLL 
                   
               
               
                   
                 HDRLTPTIIDGPKATFVFDS 
                   
               
               
                   
                 SAEPDVWFEPTTLFEVLTAD 
                   
               
               
                   
                 LSLSPIYKAGSATFDKGVSL 
                   
               
               
                   
                 RFPRFLRIREDKGVEDATSS 
                   
               
               
                   
                 DQIVELYENQSHMQN 
                   
               
               
                   
               
               
                 Yeast DNA 
                 MISALDSIPEPQNFAPSPDF 
                 76 
               
               
                 ligase IV 
                 KWLCEELFVKIHEVQINGTA 
                   
               
               
                   
                 GTGKSRSFKYYEIISNFVEM 
                   
               
               
                   
                 WRKTVGNNIYPALVLALPYR 
                   
               
               
                   
                 DRRIYNIKDYVLIRTICSYL 
                   
               
               
                   
                 KLPKNSATEQRLKDWKQRVG 
                   
               
               
                   
                 KGGNLSSLLVEEIAKRRAEP 
                   
               
               
                   
                 SSKAITIDNVNHYLDSLSGD 
                   
               
               
                   
                 RFASGRGFKSLVKSKPFLHC 
                   
               
               
                   
                 VENMSFVELKYFFDIVLKNR 
                   
               
               
                   
                 VIGGQEHKLLNCWHPDAQDY 
                   
               
               
                   
                 LSVISDLKVVTSKLYDPKVR 
                   
               
               
                   
                 LKDDDLSIKVGFAFAPQLAK 
                   
               
               
                   
                 KVNLSYEKICRTLHDDFLVE 
                   
               
               
                   
                 EKMDGERIQVHYMNYGESIK 
                   
               
               
                   
                 FFSRRGIDYTYLYGASLSSG 
                   
               
               
                   
                 TISQHLRFTDSVKECVLDGE 
                   
               
               
                   
                 MVTFDAKRRVILPFGLVKGS 
                   
               
               
                   
                 AKEALSFNSINNVDFHPLYM 
                   
               
               
                   
                 VFDLLYLNGTSLTPLPLHQR 
                   
               
               
                   
                 KQYLNSILSPLKNIVEIVRS 
                   
               
               
                   
                 SRCYGVESIKKSLEVAISLG 
                   
               
               
                   
                 SEGVVLKYYNSSYNVASRNN 
                   
               
               
                   
                 NWIKVKPEYLEEFGENLDLI 
                   
               
               
                   
                 VIGRDSGKKDSFMLGLLVLD 
                   
               
               
                   
                 EEEYKKHQGDSSEIVDHSSQ 
                   
               
               
                   
                 EKHIQNSRRRVKKILSFCSI 
                   
               
               
                   
                 ANGISQEEFKEIDRKTRGHW 
                   
               
               
                   
                 KRTSEVAPPASILEFGSKIP 
                   
               
               
                   
                 AEWIDPSESIVLEIKSRSLD 
                   
               
               
                   
                 NTETNMQKYATNCTLYGGYC 
                   
               
               
                   
                 KRIRYDKEWTDCYTLNDLYE 
                   
               
               
                   
                 SRTVKSNPSYQAERSQLGLI 
                   
               
               
                   
                 RKKRKRVLISDSFHQNRKQL 
                   
               
               
                   
                 PISNIFAGLLFYVLSDYVTE 
                   
               
               
                   
                 DTGIRITRAELEKTIVEHGG 
                   
               
               
                   
                 KLIYNVILKRHSIGDVRLIS 
                   
               
               
                   
                 CKTTTECKALIDRGYDILHP 
                   
               
               
                   
                 NWVLDCIAYKRLILIEPNYC 
                   
               
               
                   
                 FNVSQKMRAVAEKRVDCLGD 
                   
               
               
                   
                 SFENDISETKLSSLYKSQLS 
                   
               
               
                   
                 LPPMGELEIDSEVRRFPLFL 
                   
               
               
                   
                 FSNRIAYVPRRKISTEDDII 
                   
               
               
                   
                 EMKIKLFGGKITDQQSLCNL 
                   
               
               
                   
                 IIIPYTDPILRKDCMNEVHE 
                   
               
               
                   
                 KIKEQIKASDTIPKIARVVA 
                   
               
               
                   
                 PEWVDHSINENCQVPEEDFP 
                   
               
               
                   
                 VVNY 
                   
               
               
                   
               
               
                 T6 ligase 
                 MILKILNEIASIGSTKQKQA 
                 77 
               
               
                   
                 ILEKNKDNELLKRVYRLTYS 
                   
               
               
                   
                 RGLQYYIKKWPKPGIATQSF 
                   
               
               
                   
                 GMLTLTDMLDFIEFTLATRK 
                   
               
               
                   
                 LTGNAAIEELTGYITDGKKD 
                   
               
               
                   
                 DVEVLRRVMMRDLECGASVS 
                   
               
               
                   
                 IANKVWPGLIPEQPQMLASS 
                   
               
               
                   
                 YDEKGINKNIKFPAFAQLKA 
                   
               
               
                   
                 DGARCFAEVRGDELDDVRLL 
                   
               
               
                   
                 SRAGNEYLGLDLLKEELIKM 
                   
               
               
                   
                 TAEARQIHPEGVLIDGELVY 
                   
               
               
                   
                 HEQVKKEPEGLDFLFDAHPE 
                   
               
               
                   
                 NSKVKDFTEVAESRTASNGI 
                   
               
               
                   
                 ANKSLKGTISEKEAQCMKFQ 
                   
               
               
                   
                 VWDYVPLVEVYGLPAFRLKY 
                   
               
               
                   
                 DVRFSKLEQMTSGYDKVILI 
                   
               
               
                   
                 ENQVVNNLDEAKVIYKKYID 
                   
               
               
                   
                 QGLEGIILKNIDGLWENARS 
                   
               
               
                   
                 KNLYKFKEVIDVDLKIVGIY 
                   
               
               
                   
                 PHRKDPTKAGGFILESECGK 
                   
               
               
                   
                 IKVNAGSGLKDKAGVKSHEL 
                   
               
               
                   
                 DRTRIMENQNYYIGKILECE 
                   
               
               
                   
                 CNGWLKSDGRTDYVKLFLPI 
                   
               
               
                   
                 AIRLREDKTKANTFEDVFGD 
                   
               
               
                   
                 FHEVTGL 
                   
               
               
                   
               
               
                 Mouse DNA 
                 MQRSIMSFFQPTKEGKAKKP 
                 78 
               
               
                 ligase 1 
                 EKETPSSIREKEPPPKVALK 
                   
               
               
                   
                 ERNQVVPESDSPVKRTGRKV 
                   
               
               
                   
                 AQVLSCEGEDEDEAPGTPKV 
                   
               
               
                   
                 QKPVSDSEQSSPPSPDTCPE 
                   
               
               
                   
                 NSPVFNCSSPMDISPSGFPK 
                   
               
               
                   
                 RRTARKQLPKRTIQDTLEEQ 
                   
               
               
                   
                 NEDKTKTAKKRKKEEETPKE 
                   
               
               
                   
                 SLAEAEDIKQKEEKEGDQLI 
                   
               
               
                   
                 VPSEPTKSPESVTLTKTENI 
                   
               
               
                   
                 PVCKAGVKLKPQEEEQSKPP 
                   
               
               
                   
                 ARGAKTLSSFFTPRKPAVKT 
                   
               
               
                   
                 EVKQEESGTLRKEETKGTLD 
                   
               
               
                   
                 PANYNPSKNNYHPIEDACWK 
                   
               
               
                   
                 HGQKVPFLAVARTFEKIEEV 
                   
               
               
                   
                 SARLKMVETLSNLLRSVVAL 
                   
               
               
                   
                 SPPDLLPVLYLSLNRLGPPQ 
                   
               
               
                   
                 QGLELGVGDGVLLKAVAQAT 
                   
               
               
                   
                 GRQLESIRAEVAEKGDVGLV 
                   
               
               
                   
                 AENSRSTQRLMLPPPPLTIS 
                   
               
               
                   
                 GVFTKFCDIARLTGSASMAK 
                   
               
               
                   
                 KMDIIKGLFVACRHSEARYI 
                   
               
               
                   
                 ARSLSGRLRLGLAEQSVLAA 
                   
               
               
                   
                 LAQAVSLTPPGQEFPTVVVD 
                   
               
               
                   
                 AGKGKTAEARKMWLEEQGMI 
                   
               
               
                   
                 LKQTFCEVPDLDRIIPVLLE 
                   
               
               
                   
                 HGLERLPEHCKLSPGVPLKP 
                   
               
               
                   
                 MLAHPTRGVSEVLKRFEEVD 
                   
               
               
                   
                 FTCEYKYDGQRAQIHVLEGG 
                   
               
               
                   
                 EVKIFSRNQEDNTGKYPDII 
                   
               
               
                   
                 SRIPKIKHPSVTSFILDTEA 
                   
               
               
                   
                 VAWDREKKQIQPFQVLTTRK 
                   
               
               
                   
                 RKEVDASEIQVQVCLYAFDL 
                   
               
               
                   
                 IYLNGESLVRQPLSRRRQLL 
                   
               
               
                   
                 RENFVETEGEFVFTTSLDTK 
                   
               
               
                   
                 DTEQIAEFLEQSVKDSCEGL 
                   
               
               
                   
                 MVKTLDVDATYEIAKRSHNW 
                   
               
               
                   
                 LKLKKDYLDGVGDTLDLVVI 
                   
               
               
                   
                 GAYLGRGKRAGRYGGFLLAA 
                   
               
               
                   
                 YDEESEELQAICKLGTGFSD 
                   
               
               
                   
                 EELEEHHQSLQALVLPTPRP 
                   
               
               
                   
                 YVRIDGAVAPDHWLDPSIVW 
                   
               
               
                   
                 EVKCADLSLSPIYPAARGLV 
                   
               
               
                   
                 DKEKGISLRFPRFIRVRKDK 
                   
               
               
                   
                 QPEQATTSNQVASLYRKQSQ 
                   
               
               
                   
                 IQNQQSSDLDSDVEDY 
                   
               
               
                   
               
               
                 Mouse DNA 
                 MASSQTSQTVAAHVPFADLC 
                 79 
               
               
                 ligase IV 
                 STLERIQKGKDRAEKIRHFK 
                   
               
               
                   
                 EFLDSWRKFHDALHKNRKDV 
                   
               
               
                   
                 TDSFYPAMRLILPQLERERM 
                   
               
               
                   
                 AYGIKETMLAKLYIELLNLP 
                   
               
               
                   
                 REGKDAQKLLNYRTPSGART 
                   
               
               
                   
                 DAGDFAMIAYFVLKPRCLQK 
                   
               
               
                   
                 GSLTIQQVNELLDLVASNNS 
                   
               
               
                   
                 GKKKDLVKKSLLQLITQSSA 
                   
               
               
                   
                 LEQKWLIRMIIKDLKLGISQ 
                   
               
               
                   
                 QTIFSIFHNDAVELHNVTTD 
                   
               
               
                   
                 LEKVCRQLHDPSVGLSDISI 
                   
               
               
                   
                 TLFSAFKPMLAAVADVERVE 
                   
               
               
                   
                 KDMKQQSFYIETKLDGERMQ 
                   
               
               
                   
                 MHKDGALYRYFSRNGYNYTD 
                   
               
               
                   
                 QFGESPQEGSLTPFIHNAFG 
                   
               
               
                   
                 TDVQACILDGEMMAYNPTTQ 
                   
               
               
                   
                 TFMQKGVKFDIKRMVEDSGL 
                   
               
               
                   
                 QTCYSVFDVLMVNKKKLGRE 
                   
               
               
                   
                 TLRKRYEILSSTFTPIQGRI 
                   
               
               
                   
                 EIVQKTQAHTKKEVVDALND 
                   
               
               
                   
                 AIDKREEGIMVKHPLSIYKP 
                   
               
               
                   
                 DKRGEGWLKIKPEYVSGLMD 
                   
               
               
                   
                 ELDVLIVGGYWGKGSRGGMM 
                   
               
               
                   
                 SHFLCAVAETPPPGDRPSVF 
                   
               
               
                   
                 HTLCRVGSGYTMKELYDLGL 
                   
               
               
                   
                 KLAKYWKPFHKKSPPSSILC 
                   
               
               
                   
                 GTEKPEVYIEPQNSVIVQIK 
                   
               
               
                   
                 AAEIVPSDMYKTGSTLRFPR 
                   
               
               
                   
                 IEKIRDDKEWHECMTLGDLE 
                   
               
               
                   
                 QLRGKASGKLATKHLHVGDD 
                   
               
               
                   
                 DEPREKRRKPISKTKKAIRI 
                   
               
               
                   
                 IEHLKAPNLSNVNKVSNVFE 
                   
               
               
                   
                 DVEFCVMSGLDGYPKADLEN 
                   
               
               
                   
                 RIAEFGGYIVQNPGPDTYCV 
                   
               
               
                   
                 IAGSENVRVKNIISSDKNDV 
                   
               
               
                   
                 VKPEWLLECFKTKTCVPWQP 
                   
               
               
                   
                 RFMIHMCPSTKQHFAREYDC 
                   
               
               
                   
                 YGDSYFVDTDLDQLKEVFLG 
                   
               
               
                   
                 IKPSEQQTPEEMAPVIADLE 
                   
               
               
                   
                 CRYSWDHSPLSMFRHYTIYL 
                   
               
               
                   
                 DLYAVINDLSSRIEATRLGI 
                   
               
               
                   
                 TALELRFHGAKVVSCLSEGV 
                   
               
               
                   
                 SHVIIGEDQRRVTDFKIFRR 
                   
               
               
                   
                 MLKKKFKILQESWVSDSVDK 
                   
               
               
                   
                 GELQEENQYLL 
                   
               
               
                   
               
               
                 
                   Arabidopsis 
                 
                 MLAIRSSNYLRCIPSLCTKT 
                 80 
               
               
                 DNA ligase 
                 QISQFSSVLISFSRQISHLR 
                   
               
               
                 I 
                 LSSCHRAMSSSRPSAFDALM 
                   
               
               
                   
                 SNARAAAKKKTPQTTNLSRS 
                   
               
               
                   
                 PNKRKIGETQDANLGKTIVS 
                   
               
               
                   
                 EGTLPKTEDLLEPVSDSANP 
                   
               
               
                   
                 RSDTSSIAEDSKTGAKKAKT 
                   
               
               
                   
                 LSKTDEMKSKIGLLKKKPND 
                   
               
               
                   
                 FDPEKMSCWEKGERVPFLFV 
                   
               
               
                   
                 ALAFDLISNESGRIVITDIL 
                   
               
               
                   
                 CNMLRTVIATTPEDLVATVY 
                   
               
               
                   
                 LSANEIAPAHEGVELGIGES 
                   
               
               
                   
                 TIIKAISEAFGRTEDHVKKQ 
                   
               
               
                   
                 NTELGDLGLVAKGSRSTQTM 
                   
               
               
                   
                 MFKPEPLTVVKVFDTFRQIA 
                   
               
               
                   
                 KESGKDSNEKKKNRMKALLV 
                   
               
               
                   
                 ATTDCEPLYLTRLLQAKLRL 
                   
               
               
                   
                 GFSGQTVLAALGQAAVYNEE 
                   
               
               
                   
                 HSKPPPNTKSPLEEAAKIVK 
                   
               
               
                   
                 QVFTVLPVYDIIVPALLSGG 
                   
               
               
                   
                 VWNLPKTCNFTLGVPIGPML 
                   
               
               
                   
                 AKPTKGVAEILNKFQDIVFT 
                   
               
               
                   
                 CEYKYDGERAQIHFMEDGTF 
                   
               
               
                   
                 EIYSRNAERNTGKYPDVALA 
                   
               
               
                   
                 LSRLKKPSVKSFILDCEVVA 
                   
               
               
                   
                 FDREKKKILPFQILSTRARK 
                   
               
               
                   
                 NVNVNDIKVGVCIFAFDMLY 
                   
               
               
                   
                 LNGQQLIQENLKIRREKLYE 
                   
               
               
                   
                 SFEEDPGYFQFATAVTSNDI 
                   
               
               
                   
                 DEIQKFLDASVDVGCEGLII 
                   
               
               
                   
                 KTLDSDATYEPAKRSNNWLK 
                   
               
               
                   
                 LKKDYMDSIGDSVDLVPIAA 
                   
               
               
                   
                 FHGRGKRTGVYGAFLLACYD 
                   
               
               
                   
                 VDKEEFQSICKIGTGFSDAM 
                   
               
               
                   
                 LDERSSSLRSQVIATPKQYY 
                   
               
               
                   
                 RVGDSLNPDVWFEPTEVWEV 
                   
               
               
                   
                 KAADLTISPVHRAATGIVDP 
                   
               
               
                   
                 DKGISLRFPRLLRVREDKKP 
                   
               
               
                   
                 EEATSSEQIADLYQAQKHNH 
                   
               
               
                   
                 PSNEVKGDDD 
                   
               
               
                   
               
               
                 
                   Arabidopsis 
                 
                 MTEEIKFSVLVSLFNWIQKS 
                 81 
               
               
                 DNA ligase 
                 KTSSQKRSKFRKFLDTYCKP 
                   
               
               
                 IV 
                 SDYFVAVRLIIPSLDRERGS 
                   
               
               
                   
                 YGLKESVLATCLIDALGISR 
                   
               
               
                   
                 DAPDAVRLLNWRKGGTAKAG 
                   
               
               
                   
                 ANAGNFSLIAAEVLQRRQGM 
                   
               
               
                   
                 ASGGLTIKELNDLLDRLASS 
                   
               
               
                   
                 ENRAEKTLVLSTLIQKTNAQ 
                   
               
               
                   
                 EMKWVIRIILKDLKLGMSEK 
                   
               
               
                   
                 SIFQEFHPDAEDLFNVTCDL 
                   
               
               
                   
                 KLVCEKLRDRHQRHKRQDIE 
                   
               
               
                   
                 VGKAVRPQLAMRIGDVNAAW 
                   
               
               
                   
                 KKLHGKDVVAECKFDGDRIQ 
                   
               
               
                   
                 IHKNGTDIHYFSRNFLDHSE 
                   
               
               
                   
                 YAHAMSDLIVQNILVDKCIL 
                   
               
               
                   
                 DGEMLVWDTSLNRFAEFGSN 
                   
               
               
                   
                 QEIAKAAREGLDSHKQLCYV 
                   
               
               
                   
                 AFDVLYVGDTSVIHQSLKER 
                   
               
               
                   
                 HELLKKVVKPLKGRLEVLVP 
                   
               
               
                   
                 EGGLNVHRPSGEPSWSIVVH 
                   
               
               
                   
                 AAADVERFFKETVENRDEGI 
                   
               
               
                   
                 VLKDLESKWEPGDRSGKWMK 
                   
               
               
                   
                 LKPEYIRAGADLDVLIIGGY 
                   
               
               
                   
                 YGSGRRGGEVAQFLVALADR 
                   
               
               
                   
                 AEANVYPRRFMSFCRVGTGL 
                   
               
               
                   
                 SDDELNTVVSKLKPYFRKNE 
                   
               
               
                   
                 HPKKAPPSFYQVTNHSKERP 
                   
               
               
                   
                 DVWIDSPEKSIILSITSDIR 
                   
               
               
                   
                 TIRSEVFVAPYSLRFPRIDK 
                   
               
               
                   
                 VRYDKPWHECLDVQAFVELV 
                   
               
               
                   
                 NSSNGTTQKQKESESTQDNP 
                   
               
               
                   
                 KVNKSSKRGEKKNVSLVPSQ 
                   
               
               
                   
                 FIQTDVSDIKGKTSIFSNMI 
                   
               
               
                   
                 FYFVNVPRSHSLETFHKMVV 
                   
               
               
                   
                 ENGGKFSMNLNNSVTHCIAA 
                   
               
               
                   
                 ESSGIKYQAAKRQRDVIHFS 
                   
               
               
                   
                 WVLDCCSRNKMLPLLPKYFL 
                   
               
               
                   
                 HLTDASRTKLQDDIDEFSDS 
                   
               
               
                   
                 YYWDLDLEGLKQVLSNAKQS 
                   
               
               
                   
                 EDSKSIDYYKKKLCPEKRWS 
                   
               
               
                   
                 CLLSCCVYFYPYSQTLSTEE 
                   
               
               
                   
                 EALLGIMAKRLMLEVLMAGG 
                   
               
               
                   
                 KVSNNLAHASHLVVLAMAEE 
                   
               
               
                   
                 PLDFTLVSKSFSEMEKRLLL 
                   
               
               
                   
                 KKRLHVVSSHWLEESLQREE 
                   
               
               
                   
                 KLCEDVYTLRPKYMEESDTE 
                   
               
               
                   
                 ESDKSEHDTTEVASQGSAQT 
                   
               
               
                   
                 KEPASSKIAITSSRGRSNTR 
                   
               
               
                   
                 AVKRGRSSTNSLQRVQRRRG 
                   
               
               
                   
                 KQPSKISGDETEESDASEEK 
                   
               
               
                   
                 VSTRLSDIAEETDSFGEAQR 
                   
               
               
                   
                 NSSRGKCAKRGKSRVGQTQR 
                   
               
               
                   
                 VQRSRRGKKAAKIGGDESDE 
                   
               
               
                   
                 NDELDGNNNVSADAEEGNAA 
                   
               
               
                   
                 GRSVENEETREPDIAKYTES 
                   
               
               
                   
                 QQRDNTVAVEEALQDSRNAK 
                   
               
               
                   
                 TEMDMKEKLQIHEDPLQAML 
                   
               
               
                   
                 MKMFPIPSQKTTETSNRTTG 
                   
               
               
                   
                 EYRKANVSGECESSEKRKLD 
                   
               
               
                   
                 AETDNTSVNAGAESDVVPPL 
                   
               
               
                   
                 VKKKKVSYRDVAGELLKDW 
                   
               
               
                   
               
               
                 
                   Arabidopsis 
                 
                 MASDSAGATISGNFSNSDNS 
                 82 
               
               
                 DNA ligase 
                 ETLNLNTTKLYSSAISSISP 
                   
               
               
                 6 
                 QFPSPKPTSSCPSIPNSKRI 
                   
               
               
                   
                 PNTNFIVDLFRLPHQSSSVA 
                   
               
               
                   
                 FFLSHFHSDHYSGLSSSWSK 
                   
               
               
                   
                 GIIYCSHKTARLVAEILQVP 
                   
               
               
                   
                 SQFVFALPMNQMVKIDGSEV 
                   
               
               
                   
                 VLIEANHCPGAVQFLFKVKL 
                   
               
               
                   
                 ESSGFEKYVHTGDFRFCDEM 
                   
               
               
                   
                 RFDPFLNGFVGCDGVFLDTT 
                   
               
               
                   
                 YCNPKFVFPSQEESVGYVVS 
                   
               
               
                   
                 VIDKISEEKVLFLVATYVVG 
                   
               
               
                   
                 KEKILVEIARRCKRKIVVDA 
                   
               
               
                   
                 RKMSMLSVLGCGEEGMFTED 
                   
               
               
                   
                 ENESDVHVVGWNVLGETWPY 
                   
               
               
                   
                 FRPNFVKMNEIMVEKGYDKV 
                   
               
               
                   
                 VGFVPTGWTYEVKRNKFAVR 
                   
               
               
                   
                 FKDSMEIHLVPYSEHSNYDE 
                   
               
               
                   
                 LREFIKFLKPKRVIPTVGVD 
                   
               
               
                   
                 IEKFDCKEVNKMQKHFSGLV 
                   
               
               
                   
                 DEMANKKDFLLGFYRQSYQK 
                   
               
               
                   
                 NEKSDVDVVSHSAEVYEEEE 
                   
               
               
                   
                 KNACEDGGENVPSSRGPILH 
                   
               
               
                   
                 DTTPSSDSRLLIKLRDSLPA 
                   
               
               
                   
                 WVTEEQMLDLIKKHAGNPVD 
                   
               
               
                   
                 IVSNFYEYEAELYKQASLPT 
                   
               
               
                   
                 PSLNNQAVLFDDDVTDLQPN 
                   
               
               
                   
                 PVKGICPDVQAIQKGFDLPR 
                   
               
               
                   
                 KMNLTKGTISPGKRGKSSGS 
                   
               
               
                   
                 KSNKKAKKDPKSKPVGPGQP 
                   
               
               
                   
                 TLFKFFNKVLDGGSNSVSVG 
                   
               
               
                   
                 SETEECNTDKKMVHIDASEA 
                   
               
               
                   
                 YKEVTDQFIDIVNGSESLRD 
                   
               
               
                   
                 YAASIIDEAKGDISRALNIY 
                   
               
               
                   
                 YSKPREIPGDHAGERGLSSK 
                   
               
               
                   
                 TIQYPKCSEACSSQEDKKAS 
                   
               
               
                   
                 ENSGHAVNICVQTSAEESVD 
                   
               
               
                   
                 KNYVSLPPEKYQPKEHACWR 
                   
               
               
                   
                 EGQPAPYIHLVRTFASVESE 
                   
               
               
                   
                 KGKIKAMSMLCNMFRSLFAL 
                   
               
               
                   
                 SPEDVLPAVYLCTNKIAADH 
                   
               
               
                   
                 ENIELNIGGSLISSALEEAC 
                   
               
               
                   
                 GISRSTVRDMYNSLGDLGDV 
                   
               
               
                   
                 AQLCRQTQKLLVPPPPLLVR 
                   
               
               
                   
                 DVFSTLRKISVQTGTGSTRL 
                   
               
               
                   
                 KKNLIVKLMRSCREKEIKFL 
                   
               
               
                   
                 VRTLARNLRIGAMLRTVLPA 
                   
               
               
                   
                 LGRAIVMNSFWNDHNKELSE 
                   
               
               
                   
                 SCFREKLEGVSAAVVEAYNI 
                   
               
               
                   
                 LPSLDVVVPSLMDKDIEFST 
                   
               
               
                   
                 STLSMVPGIPIKPMLAKIAK 
                   
               
               
                   
                 GVQEFFNLSQEKAFTCEYKY 
                   
               
               
                   
                 DGQRAQIHKLLDGTVCIFSR 
                   
               
               
                   
                 NGDETTSRFPDLVDVIKQFS 
                   
               
               
                   
                 CPAAETFMLDAEVVATDRIN 
                   
               
               
                   
                 GNKLMSFQELSTRERGSKDA 
                   
               
               
                   
                 LITTESIKVEVCVFVFDIMF 
                   
               
               
                   
                 VNGEQLLALPLRERRRRLKE 
                   
               
               
                   
                 VFPETRPGYLEYAKEITVGA 
                   
               
               
                   
                 EEASLNNHDTLSRINAFLEE 
                   
               
               
                   
                 AFQSSCEGIMVKSLDVNAGY 
                   
               
               
                   
                 CPTKRSDSWLKVKRDYVDGL 
                   
               
               
                   
                 GDTLDLVPIGAWYGNGRKAG 
                   
               
               
                   
                 WYSPFLMACFNPETEEFQSV 
                   
               
               
                   
                 CRVMSGFSDAFYIEMKEFYS 
                   
               
               
                   
                 EDKILAKKPPYYRTGETPDM 
                   
               
               
                   
                 WFSAEVVWEIRGADFTVSPV 
                   
               
               
                   
                 HSASLGLVHPSRGISVRFPR 
                   
               
               
                   
                 FISKVTDRNPEECSTATDIA 
                   
               
               
                   
                 EMFHAQTRKMNITSQH 
                   
               
               
                   
               
               
                 
                   Bacillus 
                 
                 MDKETAKQRAEELRRTINKY 
                 83 
               
               
                   subtilis  DNA 
                 SYEYYTLDEPSVPDAEYDRL 
                   
               
               
                 ligase 
                 MQELIAIEEEHPDLRTPDSP 
                   
               
               
                   
                 TQRVGGAVLEAFQKVTHGTP 
                   
               
               
                   
                 MLSLGNAFNADDLRDFDRRV 
                   
               
               
                   
                 RQSVGDDVAYNVELKIDGLA 
                   
               
               
                   
                 VSLRYEDGYFVRGATRGDGT 
                   
               
               
                   
                 TGEDITENLKTIRNIPLKMN 
                   
               
               
                   
                 RELSIEVRGEAYMPKRSFEA 
                   
               
               
                   
                 LNEERIKNEEEPFANPRNAA 
                   
               
               
                   
                 AGSLRQLDPKIAAKRNLDIF 
                   
               
               
                   
                 VYSIAELDEMGVETQSQGLD 
                   
               
               
                   
                 FLDELGFKTNQERKKCGSIE 
                   
               
               
                   
                 EVITLIDELQAKRADLPYEI 
                   
               
               
                   
                 DGIVIKVDSLDQQEELGFTA 
                   
               
               
                   
                 KSPRWAIAYKFPAEEVVTKL 
                   
               
               
                   
                 LDIELNVGRTGVITPTAILE 
                   
               
               
                   
                 PVKVAGTTVSRASLHNEDLI 
                   
               
               
                   
                 KEKDIRILDKVVVKKAGDII 
                   
               
               
                   
                 PEVVNVLVDQRTGEEKEFSM 
                   
               
               
                   
                 PTECPECGSELVRIEGEVAL 
                   
               
               
                   
                 RCINPECPAQIREGLIHFVS 
                   
               
               
                   
                 RNAMNIDGLGERVITQLFEE 
                   
               
               
                   
                 NLVRNVADLYKLTKERVIQL 
                   
               
               
                   
                 ERMGEKSTENLISSIQKSKE 
                   
               
               
                   
                 NSLERLLFGLGIRFIGSKAA 
                   
               
               
                   
                 KTLAMHFESLENLKKASKEE 
                   
               
               
                   
                 LLAVDEIGEKMADAVITYFH 
                   
               
               
                   
                 KEEMLELLNELQELGVNTLY 
                   
               
               
                   
                 KGPKKVKAEDSDSYFAGKTI 
                   
               
               
                   
                 VLTGKLEELSRNEAKAQIEA 
                   
               
               
                   
                 LGGKLTGSVSKNTDLVIAGE 
                   
               
               
                   
                 AAGSKLTKAQELNIEVWNEE 
                   
               
               
                   
                 QLMGELKK 
                   
               
               
                   
               
               
                 
                   Bacillus 
                 
                 MDRQQAERRAAELRELLNRY 
                 84 
               
               
                 
                   stearothermophilus 
                 
                 GYEYYVLDRPSVPDAEYDRL 
                   
               
               
                   
                 MQELIAIEEQYPELKTSDSP 
                   
               
               
                   
                 TQRIGGPPLEAFRKVAHRVP 
                   
               
               
                   
                 MMSLANAFGEGDLRDFDRRV 
                   
               
               
                   
                 RQEVGEAAYVCELKIDGLAV 
                   
               
               
                   
                 SVRYEDGYFVQGATRGDGTT 
                   
               
               
                   
                 GEDITENLKTIRSLPLRLKE 
                   
               
               
                   
                 PVSLEARGEAFMPKASFLRL 
                   
               
               
                   
                 NEERKARGEELFANPRNAAA 
                   
               
               
                   
                 GSLRQLDPKVAASRQLDLFV 
                   
               
               
                   
                 YGLADAEALGIASHSEALDY 
                   
               
               
                   
                 LQALGFKVNPERRRCANIDE 
                   
               
               
                   
                 VIAFVSEWHDKRPQLPYEID 
                   
               
               
                   
                 GIVIKVDSFAQQRALGATAK 
                   
               
               
                   
                 SPRWAIAYKFPAEEVVTTLI 
                   
               
               
                   
                 GIEVNVGRTGVVTPTAILEP 
                   
               
               
                   
                 VRVAGTTVQRATLHNEDFIR 
                   
               
               
                   
                 EKDIRIGDAVIIKKAGDIIP 
                   
               
               
                   
                 EVVGVVVDRRDGDETPFAMP 
                   
               
               
                   
                 THCPECESELVRLEGEVALR 
                   
               
               
                   
                 CLNPNCPAQLRERLIHFASR 
                   
               
               
                   
                 AAMNIEGLGEKVVTQLFNAG 
                   
               
               
                   
                 LVRDVADLYCLTKEQLVGLE 
                   
               
               
                   
                 RMGEKSAANLLAAIEASKQN 
                   
               
               
                   
                 SLERLLFGLGIRYVGAKAAQ 
                   
               
               
                   
                 LLAEHFETMERLERATKEEL 
                   
               
               
                   
                 MAVPEIGEKMADAITAFFAQ 
                   
               
               
                   
                 PEATELLQELRAYGVNMAYK 
                   
               
               
                   
                 GPKRSAEAPADSAFAGKTVV 
                   
               
               
                   
                 LTGKLASMSRNEAKEQIERL 
                   
               
               
                   
                 GGRVTGSVSRSTDLVIAGED 
                   
               
               
                   
                 AGSKLEKAQQLGIEIWDESR 
                   
               
               
                   
                 FLQEINRGKR 
                   
               
               
                   
               
               
                 
                   Haemophilus 
                 
                 MKFYRTLLLFFASSFAFANS 
                 85 
               
               
                 
                   influenzae 
                 
                 DLMLLHTYNNQPIEGWVMSE 
                   
               
               
                 Rd 
                 KLDGVRGYWNGKQLLTRQGQ 
                   
               
               
                   
                 RLSPPAYFIKDFPPFAIDGE 
                   
               
               
                   
                 LFSERNHFEEISTITKSFKG 
                   
               
               
                   
                 DGWEKLKLYVFDVPDAEGNL 
                   
               
               
                   
                 FERLAKLKAHLLEHPTTYIE 
                   
               
               
                   
                 IIEQIPVKDKTHLYQFLAQV 
                   
               
               
                   
                 ENLQGEGVVVRNPNAPYERK 
                   
               
               
                   
                 RSSQILKLKTARGEECTVIA 
                   
               
               
                   
                 HHKGKGQFENVMGALTCKNH 
                   
               
               
                   
                 RGEFKIGSGFNLNERENPPP 
                   
               
               
                   
                 IGSVITYKYRGITNSGKPRF 
                   
               
               
                   
                 ATYWREKK 
                   
               
               
                   
               
               
                 
                   Pseudoalteromonas 
                 
                 MSSSISEQVNHLRIILEQHN 
                 86 
               
               
                 
                   haloplanktis 
                 
                 YNYYVLDTPSIPDSEYDRLL 
                   
               
               
                   
                 RELSALETEHPEFLTADSPT 
                   
               
               
                   
                 QKVGGAALSKFEQVAHQVPM 
                   
               
               
                   
                 LSLDNAFSEEEFTAFNRRIK 
                   
               
               
                   
                 ERLMSTDELTFCCEPKLDGL 
                   
               
               
                   
                 AVSIIYRDGVLVQAATRGDG 
                   
               
               
                   
                 FTGENITQNVKTIRNVPLKL 
                   
               
               
                   
                 RGDYPKELEVRGEVFMDSAG 
                   
               
               
                   
                 FDKLNTEAEKRGEKVFVNPR 
                   
               
               
                   
                 NAAAGSLRQLDSKITAKRPL 
                   
               
               
                   
                 MFYAYSTGLVADGNIPEDHY 
                   
               
               
                   
                 QQLEKLTDWGLPLCPETKLV 
                   
               
               
                   
                 EGPKAALAYYRDILTRRSEL 
                   
               
               
                   
                 KYEIDGVVIKINQKTLQERL 
                   
               
               
                   
                 GFVARAPRWAIAYKFPAQEE 
                   
               
               
                   
                 ITQLLDVDFQVGRTGAITPV 
                   
               
               
                   
                 ARLKPVFVGGVTVSNATLHN 
                   
               
               
                   
                 SDEVARLGVKVGDTVIIRRA 
                   
               
               
                   
                 GDVIPQITQVVLERRPDDAR 
                   
               
               
                   
                 DIEFPTTCPICDSHVEKVEG 
                   
               
               
                   
                 EAVARCTGGLVCPAQRKQAI 
                   
               
               
                   
                 KHFASRKALDIDGLGDKIVD 
                   
               
               
                   
                 QLVDRELIKTPADLFILKQG 
                   
               
               
                   
                 HFESLERMGPKSAKNLVTAL 
                   
               
               
                   
                 EEAKGTTLAKFLYSLGIREA 
                   
               
               
                   
                 GEATAQNLANHFLTLENVIN 
                   
               
               
                   
                 ASIDSLTQVSDVGEIVAAHV 
                   
               
               
                   
                 RGFFDEEHNLAVVNALIDQG 
                   
               
               
                   
                 VNWPALSAPSEEEQPLAGLT 
                   
               
               
                   
                 YVLTGTLNTLNRNDAKARLQ 
                   
               
               
                   
                 QLGAKVSGSVSAKTDALVAG 
                   
               
               
                   
                 EKAGSKLTKAQDLGIDILTE 
                   
               
               
                   
                 DELIELLIKHNG 
                   
               
               
                   
               
               
                 
                   Rhodothermus 
                 
                 METHTAPQTAEARLLEATHT 
                 87 
               
               
                 
                   marinus 
                 
                 LLQTVRQRDLEAIDRKEAEA 
                   
               
               
                   
                 LAARLREVLNQHAYRYYVLD 
                   
               
               
                   
                 NPLIPDADYDLLMQALRKLE 
                   
               
               
                   
                 ARFPELVTPDSPTQRVGGPP 
                   
               
               
                   
                 LGRFEKVRHPEPLLSLNNAF 
                   
               
               
                   
                 GEEDVRVWYERCCRMLAERL 
                   
               
               
                   
                 GQPVQPAVTAELKIDGLAMA 
                   
               
               
                   
                 LTYENGVLSVGATRGDGIEG 
                   
               
               
                   
                 ENVTQNVRTIPAIPLRIPVD 
                   
               
               
                   
                 PAVGPPPTRLEVRGEVYMRK 
                   
               
               
                   
                 RDFERLNEQLQARGERPFAN 
                   
               
               
                   
                 PRNAAAGSVRQLNPQVTALR 
                   
               
               
                   
                 PLSFFAYGIGPVEGAEVPDS 
                   
               
               
                   
                 QYEVLQWLGRLGFPVNEHAR 
                   
               
               
                   
                 RFEHLDDVLEYCRYWTEHRD 
                   
               
               
                   
                 ELDYEIDGVVLKIDHRPWQA 
                   
               
               
                   
                 LLGAISNAPRWAVAYKFPAR 
                   
               
               
                   
                 EAITRLLDIMVSVGRTGVVK 
                   
               
               
                   
                 PVAVLEPVEVGGVTVSQATL 
                   
               
               
                   
                 HNEDYVRSRDIRIGDLVVVI 
                   
               
               
                   
                 RAGDVIPQVVRPVVEARTGN 
                   
               
               
                   
                 ERPWRMPERCPSCGSQLVRL 
                   
               
               
                   
                 PGEADYYCVASDCPAQFVRL 
                   
               
               
                   
                 LEHFAGRDAMDIEGMGSQVA 
                   
               
               
                   
                 RQLAESGLVRPLSDLYRLKL 
                   
               
               
                   
                 EDLLKLEGFAETRARNLLRA 
                   
               
               
                   
                 IEASKQRPLSRLLFGLGIRH 
                   
               
               
                   
                 VGKTTAELLVQRFASIDELA 
                   
               
               
                   
                 AATIDELAALEGVGPITAES 
                   
               
               
                   
                 IANWFRVEDNRRLIEELKEL 
                   
               
               
                   
                 GVNTQRLPEEAPAAESPVRG 
                   
               
               
                   
                 KTFVLTGALPHLTRKEAEEL 
                   
               
               
                   
                 IKRAGGRVASSVSRNTDYVV 
                   
               
               
                   
                 VGENPGSKYDRARQLGIPML 
                   
               
               
                   
                 DEDGLLRLLGMK 
                   
               
               
                   
               
               
                 
                   Thermus 
                 
                 MTREEARRRINELRDLIRYH 
                 88 
               
               
                 
                   filiformis 
                 
                 NYRYYVLADPEISDAEYDRL 
                   
               
               
                   
                 LRELKELEERFPEFKSPDSP 
                   
               
               
                   
                 TEQVGARPLEPTFRPVRHPT 
                   
               
               
                   
                 RMYSLDNAFTYEEVLAFEER 
                   
               
               
                   
                 LERALGRKRPFLYTVEHKVD 
                   
               
               
                   
                 GLSVNLYYEEGVLVFGATRG 
                   
               
               
                   
                 DGEVGEEVTQNLLTIPTIPR 
                   
               
               
                   
                 RLKGVPDRLEVRGEVYMPIE 
                   
               
               
                   
                 AFLRLNEELEERGEKVFKNP 
                   
               
               
                   
                 RNAAAGSLRQKDPRVTAKRG 
                   
               
               
                   
                 LRATFYALGLGLEESGLKSQ 
                   
               
               
                   
                 YELLLWLKEKGFPVEHGYEK 
                   
               
               
                   
                 ALGAEGVEEVYRRFLAQRHA 
                   
               
               
                   
                 LPFEADGVVVKLDDLALWRE 
                   
               
               
                   
                 LGYTARAPRFALAYKFPAEE 
                   
               
               
                   
                 KETRLLDVVFQVGRTGRVTP 
                   
               
               
                   
                 VGVLEPVFIEGSEVSRVTLH 
                   
               
               
                   
                 NESYIEELDIRIGDWVLVHK 
                   
               
               
                   
                 AGGVIPEVLRVLKERRTGEE 
                   
               
               
                   
                 RPIRWPETCPECGHRLVKEG 
                   
               
               
                   
                 KVHRCPNPLCPAKRFEAIRH 
                   
               
               
                   
                 YASRKAMDIEGLGEKLIERL 
                   
               
               
                   
                 LEKGLVRDVADLYHLRKEDL 
                   
               
               
                   
                 LGLERMGEKSAQNLLRQIEE 
                   
               
               
                   
                 SKHRGLERLLYALGLPGVGE 
                   
               
               
                   
                 VLARNLARRFGTMDRLLEAS 
                   
               
               
                   
                 LEELLEVEEVGELTARAILE 
                   
               
               
                   
                 TLKDPAFRDLVRRLKEAGVS 
                   
               
               
                   
                 MESKEEVSDLLSGLTFVLTG 
                   
               
               
                   
                 ELSRPREEVKALLQRLGAKV 
                   
               
               
                   
                 TDSVSRKTSYLVVGENPGSK 
                   
               
               
                   
                 LEKARALGVAVLTEEEFWRF 
                   
               
               
                   
                 LKEKGAPVPA 
                   
               
               
                   
               
               
                 
                   Thermus 
                 
                 MTLEEARKRVNELRDLIRYH 
                 89 
               
               
                 
                   scotoductus 
                 
                 NYRYYVLADPEISDAEYDRL 
                   
               
               
                   
                 LRELKELEERFPELKSPDSP 
                   
               
               
                   
                 TEQVGAKPLEATFRPIRHPT 
                   
               
               
                   
                 RMYSLDNAFNFDELKAFEER 
                   
               
               
                   
                 IGRALGREGPFAYTVEHKVD 
                   
               
               
                   
                 GLSVNLYYEDGVLVWGATRG 
                   
               
               
                   
                 DGEVGEEVTQNLLTIPTIPR 
                   
               
               
                   
                 RVKGVPERLEVRGEVYMPIE 
                   
               
               
                   
                 AFLRLNEELEEKGEKIFKNP 
                   
               
               
                   
                 RNAAAGSLRQKDPRITARRG 
                   
               
               
                   
                 LRATFYALGLGLEESGLKTQ 
                   
               
               
                   
                 LDLLHWLREKGFPVEHGFAR 
                   
               
               
                   
                 AEGAEGVERIYQGWLKERRS 
                   
               
               
                   
                 LPFEADGVVVKLDELSLWRE 
                   
               
               
                   
                 LGYTARAPRFAIAYKFPAEE 
                   
               
               
                   
                 KETRLLQVVFQVGRTGRVTP 
                   
               
               
                   
                 VGILEPVFIEGSVVSRVTLH 
                   
               
               
                   
                 NESYIEELDVRIGDWVLVHK 
                   
               
               
                   
                 AGGVIPEVLRVLKEKRTGEE 
                   
               
               
                   
                 RPIRWPETCPECGHRLVKEG 
                   
               
               
                   
                 KVHRCPNPLCPAKRFEAIRH 
                   
               
               
                   
                 YASRKAMDIGGLGEKLIEKL 
                   
               
               
                   
                 LEKGLVKDVADLYRLKKEDL 
                   
               
               
                   
                 LGLERMGEKSAQNLLRQIEE 
                   
               
               
                   
                 SKGRGLERLLYALGLPGVGE 
                   
               
               
                   
                 VLARNLAAHFGTMDRLLEAS 
                   
               
               
                   
                 LEELLQVEEVGELTARGIYE 
                   
               
               
                   
                 TLQDPAFRDLVRRLKEAGVV 
                   
               
               
                   
                 MEAKERGEEALKGLTFVITG 
                   
               
               
                   
                 ELSRPREEVKALLRRLGAKV 
                   
               
               
                   
                 TDSVSRKTSYLVVGENPGSK 
                   
               
               
                   
                 LEKARALGVPTLTEEELYRL 
                   
               
               
                   
                 IEERTGKPVETLAS 
                   
               
               
                   
               
               
                   Thermus  species 
                 MTLEEARRRVNELRDLIRYH 
                 90 
               
               
                 AK16D 
                 NYLYYVLDAPEISDAEYDRL 
                   
               
               
                   
                 LRELKELEERFPELKSPDSP 
                   
               
               
                   
                 TEQVGARPLEATFRPVRHPT 
                   
               
               
                   
                 RMYSLDNAFSLDEVRAFEER 
                   
               
               
                   
                 IERALGRKGPFLYTVERKVD 
                   
               
               
                   
                 GLSVNLYYEEGILVFGATRG 
                   
               
               
                   
                 DGETGEEVTQNLLTIPTIPR 
                   
               
               
                   
                 RLTGVPDRLEVRGEVYMPIE 
                   
               
               
                   
                 AFLRLNQELEEAGERIFKNP 
                   
               
               
                   
                 RNAAAGSLRQKDPRVTARRG 
                   
               
               
                   
                 LRATFYALGLGLEETGLKSQ 
                   
               
               
                   
                 HDLLLWLRERGFPVEHGFTR 
                   
               
               
                   
                 ALGAEGVEEVYQAWLKERRK 
                   
               
               
                   
                 LPFEADGVVVKLDDLALWRE 
                   
               
               
                   
                 LGYTARTPRFALAYKFPAEE 
                   
               
               
                   
                 KETRLLSVAFQVGRTGRITP 
                   
               
               
                   
                 VGVLEPVFIEGSEVSRVTLH 
                   
               
               
                   
                 NESFIEELDVRIGDWVLVHK 
                   
               
               
                   
                 AGGVIPEVLRVLKERRTGEE 
                   
               
               
                   
                 KPIIWPENCPECGHALIKEG 
                   
               
               
                   
                 KVHRCPNPLCPAKRFEAIRH 
                   
               
               
                   
                 YASRKAMDIQGLGEKLIEKL 
                   
               
               
                   
                 LEKGLVRDVADLYRLKKEDL 
                   
               
               
                   
                 VNLERMGEKSAENLLRQIEE 
                   
               
               
                   
                 SKGRGLERLLYALGLPGVGE 
                   
               
               
                   
                 VLARNLALRFGHMDRLLEAG 
                   
               
               
                   
                 LEDLLEVEGVGELTARAILN 
                   
               
               
                   
                 TLKDPEFRDLVRRLKEAGVE 
                   
               
               
                   
                 MEAKEREGEALKGLTFVITG 
                   
               
               
                   
                 ELSRPREEVKALLRRLGAKV 
                   
               
               
                   
                 TDSVSRKTSFLVVGENPGSK 
                   
               
               
                   
                 LEKARALGVPTLSEEELYRL 
                   
               
               
                   
                 IEERTGKDPRALTA 
                   
               
               
                   
               
               
                 
                   Thermus 
                 
                 MTLEEARKRVNELRDLIRYH 
                 91 
               
               
                 
                   thermophilus 
                 
                 NYRYYVLADPEISDAEYDRL 
                   
               
               
                 HB8 
                 LRELKELEERFPELKSPDSP 
                   
               
               
                   
                 TLQVGARPLEATFRPVRHPT 
                   
               
               
                   
                 RMYSLDNAFNLDELKAFEER 
                   
               
               
                   
                 IERALGRKGPFAYTVEHKVD 
                   
               
               
                   
                 GLSVNLYYEEGVLVYGATRG 
                   
               
               
                   
                 DGEVGEEVTQNLLTIPTIPR 
                   
               
               
                   
                 RLKGVPERLEVRGEVYMPIE 
                   
               
               
                   
                 AFLRLNEELEERGERIFKNP 
                   
               
               
                   
                 RNAAAGSLRQKDPRITAKRG 
                   
               
               
                   
                 LRATFYALGLGLEEVEREGV 
                   
               
               
                   
                 ATQFALLHWLKEKGFPVEHG 
                   
               
               
                   
                 YARAVGAEGVEAVYQDWLKK 
                   
               
               
                   
                 RRALPFEADGVVVKLDELAL 
                   
               
               
                   
                 WRELGYTARAPRFAIAYKFP 
                   
               
               
                   
                 AEEKETRLLDVVFQVGRTGR 
                   
               
               
                   
                 VTPVGILEPVFLEGSEVSRV 
                   
               
               
                   
                 TLHNESYIEELDIRIGDWVL 
                   
               
               
                   
                 VHKAGGVIPEVLRVLKERRT 
                   
               
               
                   
                 GEERPIRWPETCPECGHRLL 
                   
               
               
                   
                 KEGKVHRCPNPLCPAKRFEA 
                   
               
               
                   
                 IRHFASRKAMDIQGLGEKLI 
                   
               
               
                   
                 ERLLEKGLVKDVADLYRLRK 
                   
               
               
                   
                 EDLVGLERMGEKSAQNLLRQ 
                   
               
               
                   
                 IEESKKRGLERLLYALGLPG 
                   
               
               
                   
                 VGEVLARNLAARFGNMDRLL 
                   
               
               
                   
                 EASLEELLEVEEVGELTARA 
                   
               
               
                   
                 ILETLKDPAFRDLVRRLKEA 
                   
               
               
                   
                 GVEMEAKEKGGEALKGLTFV 
                   
               
               
                   
                 ITGELSRPREEVKALLRRLG 
                   
               
               
                   
                 AKVTDSVSRKTSYLVVGENP 
                   
               
               
                   
                 GSKLEKARALGVPTLTEEEL 
                   
               
               
                   
                 YRLLEARTGKKAEELV 
                   
               
               
                   
               
               
                 
                   Zymomonas 
                 
                 MNADIDLFSYLNPEKQDLSA 
                 92 
               
               
                 
                   mobilis 
                 
                 LAPKDLSREQAVIELERLAK 
                   
               
               
                   
                 LISHYDHLYHDKDNPAVPDS 
                   
               
               
                   
                 EYDALVLRNRRIEQFFPDLI 
                   
               
               
                   
                 RPDSPSKKVGSRPNSRLPKI 
                   
               
               
                   
                 AHRAAMLSLDNGFLDQDVED 
                   
               
               
                   
                 FLGRVRRFFNLKENQAVICT 
                   
               
               
                   
                 VEPKIDGLSCSLRYEKGILT 
                   
               
               
                   
                 QAVTRGDGVIGEDVTPNVRV 
                   
               
               
                   
                 IDDIPKTLKGDNWPEIIEIR 
                   
               
               
                   
                 GEVYMAKSDFAALNARQTEE 
                   
               
               
                   
                 NKKLFANPRNAAAGSLRQLD 
                   
               
               
                   
                 PNITARRSLRFLAHGWGEAT 
                   
               
               
                   
                 SLPADTQYGMMKVIESYGLS 
                   
               
               
                   
                 VSNLLARADDIGQMLDFYQK 
                   
               
               
                   
                 IEAERADLDFDIDGVVYKLD 
                   
               
               
                   
                 QLDWQQRFGFSARAPRFALA 
                   
               
               
                   
                 HKFPAEKAQTTLLDIEIQVG 
                   
               
               
                   
                 RTGVLTPVAKLEPVTVGGVV 
                   
               
               
                   
                 VSSATLHNSDEIERLGVRPG 
                   
               
               
                   
                 DRVLVQRAGDVIPQIVENLT 
                   
               
               
                   
                 PDVDRPIWRFPHRCPVCDSV 
                   
               
               
                   
                 ARREEGEVAWRCTGGLICPA 
                   
               
               
                   
                 QRVERLCHFVSRTAFEIEGL 
                   
               
               
                   
                 GKSHIESFFADKLIETPADI 
                   
               
               
                   
                 FRLFQKRQLLIEREGWGELS 
                   
               
               
                   
                 VDNLISAIDKRRKVPFDRFL 
                   
               
               
                   
                 FALGIRHVGAVTARDLAKSY 
                   
               
               
                   
                 QTWDNFKAAIDEAAHLRTIL 
                   
               
               
                   
                 QPSSEESEEKYQKRVDKELI 
                   
               
               
                   
                 SFFHIPNMGGKIIRSLLDFF 
                   
               
               
                   
                 AETHNSDVVSDLLQEVQIEP 
                   
               
               
                   
                 LYFELASSPLSGKIIVFTGS 
                   
               
               
                   
                 LQKITRDEAKRQAENLGAKV 
                   
               
               
                   
                 ASSVSKKTNLVVAGEAAGSK 
                   
               
               
                   
                 LSKAKELDISIIDEDRWHRI 
                   
               
               
                   
                 VENDGQDSIKI 
                   
               
               
                   
               
               
                 
                   Campylobacter 
                 
                 MKKEEYLEKVALANLWMRAY 
                 93 
               
               
                 
                   jejuni 
                 
                 YEKDEPLASDEEYDVLIREL 
                   
               
               
                   
                 RVFEEQNKDEISKDSPTQKI 
                   
               
               
                   
                 APTIQSEFKKIAHLKRMWSM 
                   
               
               
                   
                 EDVFDESELRAWAKRAKCEK 
                   
               
               
                   
                 NFFIEPKFDGASLNLLYENG 
                   
               
               
                   
                 KLVSGATRGDGEVGEDITLN 
                   
               
               
                   
                 VFEIENIPKNIAYKERIEIR 
                   
               
               
                   
                 GEVVILKDDFEKINEKRALL 
                   
               
               
                   
                 NQSLFANPRNAASGSLRQLD 
                   
               
               
                   
                 TSITKERNLKFYPWGVGENT 
                   
               
               
                   
                 LNFTKHSEVMQFIRELGFLK 
                   
               
               
                   
                 DDFIKLCANLDEVLKAYDEL 
                   
               
               
                   
                 LALREKKPMMMDGMVVRIDD 
                   
               
               
                   
                 LALCEELGYTVKFPKFMAAF 
                   
               
               
                   
                 KFPALEKTTRLIGVNLQVGR 
                   
               
               
                   
                 SGVITPVAVLEPVNLDGVVV 
                   
               
               
                   
                 KSATLHNFDEIARLDVKIND 
                   
               
               
                   
                 FVSVIRSGDVIPKITKVFKD 
                   
               
               
                   
                 RREGLEMEISRPKLCPTCQS 
                   
               
               
                   
                 ELLDEGTLIKCQNIDCEDRL 
                   
               
               
                   
                 VNSIIHFVSKKCLNIDGLGE 
                   
               
               
                   
                 NIVELLYKHKKITTLESIFH 
                   
               
               
                   
                 LKFSDFEGLEGFKEKKINNL 
                   
               
               
                   
                 LNAIEQARECELFRFITALG 
                   
               
               
                   
                 IEHIGEVAAKKLSLSFGKEW 
                   
               
               
                   
                 HKQSFEAYANLEGFGEQMAL 
                   
               
               
                   
                 SLCEFTRVNHVRIDEFYKLL 
                   
               
               
                   
                 NLKIEKLEIKSDGVIFGKTF 
                   
               
               
                   
                 VITGTLSRPRDEFKALIEKL 
                   
               
               
                   
                 GGKVSSSVSKKTDYVLFGEE 
                   
               
               
                   
                 AGSKLIKAKELEVKCIDESA 
                   
               
               
                   
                 FNELVKE 
                   
               
               
                   
               
               
                 
                   Mycobacterium 
                 
                 MSSPDADQTAPEVLRQWQAL 
                 94 
               
               
                 
                   tuberculosis 
                 
                 AEEVREHQFRYYVRDAPIIS 
                   
               
               
                 ligA 
                 DAEFDELLRRLEALEEQHPE 
                   
               
               
                   
                 LRTPDSPTQLVGGAGFATDF 
                   
               
               
                   
                 EPVDHLERMLSLDNAFTADE 
                   
               
               
                   
                 LAAWAGRIHAEVGDAAHYLC 
                   
               
               
                   
                 ELKIDGVALSLVYREGRLTR 
                   
               
               
                   
                 ASTRGDGRTGEDVTLNARTI 
                   
               
               
                   
                 ADVPERLTPGDDYPVPEVLE 
                   
               
               
                   
                 VRGEVFFRLDDFQALNASLV 
                   
               
               
                   
                 EEGKAPFANPRNSAAGSLRQ 
                   
               
               
                   
                 KDPAVTARRRLRMICHGLGH 
                   
               
               
                   
                 VEGFRPATLHQAYLALRAWG 
                   
               
               
                   
                 LPVSEHTTLATDLAGVRERI 
                   
               
               
                   
                 DYWGEHRHEVDHEIDGVVVK 
                   
               
               
                   
                 VDEVALQRRLGSTSRAPRWA 
                   
               
               
                   
                 IAYKYPPEEAQTKLLDIRVN 
                   
               
               
                   
                 VGRTGRITPFAFMTPVKVAG 
                   
               
               
                   
                 STVGQATLHNASEIKRKGVL 
                   
               
               
                   
                 IGDTVVIRKAGDVIPEVLGP 
                   
               
               
                   
                 VVELRDGSEREFIMPTTCPE 
                   
               
               
                   
                 CGSPLAPEKEGDADIRCPNA 
                   
               
               
                   
                 RGCPGQLRERVFHVASRNGL 
                   
               
               
                   
                 DIEVLGYEAGVALLQAKVIA 
                   
               
               
                   
                 DEGELFALTERDLLRTDLFR 
                   
               
               
                   
                 TKAGELSANGKRLLVNLDKA 
                   
               
               
                   
                 KAAPLWRVLVALSIRHVGPT 
                   
               
               
                   
                 AARALATEFGSLDAIAAAST 
                   
               
               
                   
                 DQLAAVEGVGPTIAAAVTEW 
                   
               
               
                   
                 FAVDWHREIVDKWRAAGVRM 
                   
               
               
                   
                 VDERDESVPRTLAGLTIVVT 
                   
               
               
                   
                 GSLTGFSRDDAKEAIVARGG 
                   
               
               
                   
                 KAAGSVSKKTNYVVAGDSPG 
                   
               
               
                   
                 SKYDKAVELGVPILDEDGFR 
                   
               
               
                   
                 RLLADGPASRT 
                   
               
               
                   
               
               
                 
                   Emiliania 
                 
                 MEAMCTECEDRDARLDVIDI 
                 95 
               
               
                   huxleyi  virus 
                 QLFHALNPKSCNRTTWEQVP 
                   
               
               
                 DNA ligase 
                 KIMGKQGDFVAEGKLDGERD 
                   
               
               
                   
                 ASHLYGESMEDVLCECVRED 
                   
               
               
                   
                 VTSLLLDGEMMVVDLETGRY 
                   
               
               
                   
                 LPFGENRSLKDFGTSMRHCF 
                   
               
               
                   
                 VAFDLLLYNGRSMTGATLAE 
                   
               
               
                   
                 RSELLRKAVRTKQHALTLIE 
                   
               
               
                   
                 RFEVGERGAGATTAVMRQLD 
                   
               
               
                   
                 VMMSRGLEGVVFKSLSSKYD 
                   
               
               
                   
                 PGSRDKSWIKLKPDFVDGMG 
                   
               
               
                   
                 DTLDLLILGGYYGEGRRRSG 
                   
               
               
                   
                 AVSTFLMGVRAPPEAAKRVG 
                   
               
               
                   
                 GAAHPLFYPFCKVGTGYSLP 
                   
               
               
                   
                 QLRELRERLMPASLTRRRGN 
                   
               
               
                   
                 ALGHGASLTAVSCEQVSHEW 
                   
               
               
                   
                 KNSRRPAHLCHWEPSKRDDI 
                   
               
               
                   
                 PDYWFEPEASVVLELTAFEI 
                   
               
               
                   
                 ITPRESFLPANYTLRFPRVK 
                   
               
               
                   
                 RVRYDKGWEGAETFERVVEL 
                   
               
               
                   
                 FKECDGRLSANKRRAEEIAA 
                   
               
               
                   
                 SRASAGPAAKKRAAGVAPTV 
                   
               
               
                   
                 GVPWHLKLSADLANTAVECY 
                   
               
               
                   
                 ALDGVVAVVKGTLSRRPGVE 
                   
               
               
                   
                 TQIKRLGGKVHKNMTSLTTH 
                   
               
               
                   
                 LVDAPGAEVLAEVERARRGG 
                   
               
               
                   
                 GSFEVVTAAWVDECSRVHAR 
                   
               
               
                   
                 VTLEPRYVRHVSEATREQIE 
                   
               
               
                   
                 AIMDEWGDNYTIAADPESLV 
                   
               
               
                   
                 DSMRLVREQRSAGGNCGDSP 
                   
               
               
                   
                 LAREAHVADALRDLDDETAV 
                   
               
               
                   
                 ALRTRYAMLRGVVAYVPRGS 
                   
               
               
                   
                 VALRLRLRLLGAQTVDEPSA 
                   
               
               
                   
                 DSTHAVLSASTSADERQRLR 
                   
               
               
                   
                 DKFTEDRVRDGRPSCGRHIV 
                   
               
               
                   
                 SDRWLAECERRGQREPEAQE 
                   
               
               
                   
                 DAWFGDRVGIRDRAL 
                   
               
               
                   
               
               
                   Lymantria   dispar   
                 MENHDSFYKFCQLCQSLYDA 
                 96 
               
               
                 multicapsid nuclear 
                 DDHQEKRDALERHFADFRGS 
                   
               
               
                 polyhedrosis virus 
                 AFMWRELLAPAESDAAADRE 
                   
               
               
                   
                 LTLIFETILSIERTEQENVT 
                   
               
               
                   
                 RNLKCTIDGAAVPLSRESRI 
                   
               
               
                   
                 TVPQVYEFINDLRGSGSRQE 
                   
               
               
                   
                 RLRLIGQFAAGCTDEDLLTV 
                   
               
               
                   
                 FRVVSDHAHAGLSAEDVMEL 
                   
               
               
                   
                 VEPWERFQKPVPPALAQPCR 
                   
               
               
                   
                 RLASVLVKHPEGALAEVKYD 
                   
               
               
                   
                 GERVQVHKAGSRFKFFSRTL 
                   
               
               
                   
                 KPVPEHKVAGCREHLTRAFP 
                   
               
               
                   
                 RARNFILDAEIVMVDGSGEA 
                   
               
               
                   
                 LPFGTLGRLKQMEHADGHVC 
                   
               
               
                   
                 MYIFDCLRYNGVSYLNATPL 
                   
               
               
                   
                 DFRRRVLQDEIVPIEGRVVL 
                   
               
               
                   
                 SAMERTNTLSELRRFVHRTL 
                   
               
               
                   
                 ATGAEGVVLKGRLSSYAPNK 
                   
               
               
                   
                 RDWFKMKKEHLCDGALVDTL 
                   
               
               
                   
                 DLVVLGAYYGTGRNCRKMSV 
                   
               
               
                   
                 FLMGCLDRESNVWTTVTKVH 
                   
               
               
                   
                 SGLADAALTALSKELRPLMA 
                   
               
               
                   
                 APRDDLPEWFDCNESMVPHL 
                   
               
               
                   
                 LAADPEKMPVWEIACSEMKA 
                   
               
               
                   
                 NIGAHTAGVTMRFPRVKRFR 
                   
               
               
                   
                 PDKDWSTATDLQEAEQLIRN 
                   
               
               
                   
                 SQENTKKTFARLATTYDGPS 
                   
               
               
                   
                 PNKKLKLN 
               
               
                   
               
            
           
         
       
     
     Some aspects include a DNA ligase that ligates DNA strands base paired to a DNA splint. In some embodiments, the DNA ligase ligates DNA strands base paired to an RNA splint. In some embodiments, the DNA ligase comprises an amino acid sequence at least 80% identical to the amino acid sequence of any one of SEQ ID NOS: 55-96, or a functional fragment thereof. 
     In some aspects, the ligases comprises at least one NLS (e.g., any one of the NLS in Table 2). In some aspects, the ligase comprises at least one additional domain. In some aspects, the at least one additional domain is a dimerization domain (e.g., any one of the dimerization domain in Table 3). In some aspects, the ligase comprising a dimerization domain can be dimerized with an endonuclease to form a heterodimer. In some aspects, the at least one additional domain is a functional domain. For example, the functional domain can comprises a chromatin modifying domain (e.g., any one of the chromatin modifying domain in Table 4) or a cell penetrating peptide (e.g., any one of the cell penetrating peptide in Table 5). In some aspects, the ligase comprises a linker, where the linker can covalently connect the ligase with another polypeptide (e.g., the endonuclease). In some aspects, the linker covalently connects the ligase to the at least one additional domain. In some aspects, the ligase comprises a tag (e.g., any one of the tag in Table 6), where the tag can be used for increasing expression, identifying, or purifying the ligase. A linker may separate the ligase from a nuclear localization signal, a chromatin modifying domain, a cell penetrating peptide, or a tag polypeptide. Any linker described herein may be included. 
     The ligase may comprise a binding motif for binding to a nucleic acid motif (e.g., a hairpin motif). In some aspects, the ligase (e.g. DNA ligase) comprises an MS2 coat protein (MCP) peptide. The ligase may include a hairpin binding motif such as an MCP peptide. The MCP peptide may be useful for recruiting the ligase to a guide nucleic acid comprising an MS2 hairpin. A benefit of using a MCP peptide and MS2 hairpin is to separate the ligase and endonuclease such as a Cas nickase (or a portion of them), and allow fitting within separate vectors such as AAV vectors. In some aspects, the ligase comprises a loop region. In some aspects, the loop region is a 2a loop or a 3a loop. The loop region may comprise a 2a loop. The loop region may comprise a 3a loop. 
     Fusion Proteins 
     Disclosed herein are fusion proteins. Some aspects include a nucleic acid (e.g. an expression vector) encoding a fusion protein. The fusion protein may include an endonuclease. The fusion protein may include a ligase. The fusion protein may include a linker. The endonuclease and ligase may be connected through a linker. The fusion protein may be an example of a covalently coupled endonuclease and DNA ligase. The fusion protein may comprise an endonuclease such as an RNA-guided endonuclease fused to a DNA ligase. 
     The fusion protein may be non-naturally occurring. The fusion protein may be engineered. The fusion protein may be synthetic. The fusion protein may be pre-synthetized. The fusion protein may be added to a subject or a cell. The fusion protein may be encoded by a nucleic acid. The encoding nucleic acid may be engineered, synthetic, or added to a subject or a cell. 
     The fusion protein may include one of various orientations. For example, the fusion protein may include an RNA-guided endonuclease upstream (e.g. N-terminal or in the N-direction) or downstream (e.g. C-terminal or in the C-direction) relative to the DNA ligase. The fusion protein may include an RNA-guided endonuclease amino (N)-terminal to the DNA ligase. The fusion protein may include an RNA-guided endonuclease carboxy (C)-terminal to the DNA ligase. The endonuclease may be in the amino direction within the fusion polypeptide relative to the ligase. The endonuclease may be in the carboxy direction within the fusion polypeptide relative to the ligase. The endonuclease may be N-terminal. The endonuclease may be C-terminal. The ligase may be N-terminal. The ligase may be C-terminal. 
     The fusion protein may include a nuclear localization signal, chromatin modifying domain, cell penetrating peptide, tag polypeptide, or exonuclease. The fusion protein may include a nuclear localization signal. The fusion protein may include a chromatin modifying domain. The fusion protein may include a cell penetrating peptide. The fusion protein may include a tag polypeptide. The fusion protein may include an exonuclease. Any of the nuclear localization signal, chromatin modifying domain, cell penetrating peptide, tag polypeptide, or exonuclease, endonuclease, or ligase may be directly connected to another or to the endonuclease or ligase. Any of the nuclear localization signal, chromatin modifying domain, cell penetrating peptide, tag polypeptide, or exonuclease, endonuclease, or ligase may be connected by a linker to another or to the endonuclease or ligase. Multiple linkers may be included in the fusion protein. The fusion protein may exclude a polymerase. 
     A linker may include an amino acid linker. The amino acid linker may include a length of residues. The length may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 residues, or a range of residues defined by any two of the aforementioned integers. The length may include at least 1 residue, at least 2 residues, at least 3 residues, at least 4 residues, at least 5 residues, at least 6 residues, at least 7 residues, at least 8 residues, at least 9 residues, at least 10 residues, at least 15 residues, at least 20 residues, at least 25 residues, at least 30 residues, at least 40 residues, at least 50 residues, at least 60 residues, at least 70 residues, at least 80 residues, at least 90 residues, or at least 100 residues. In some aspects, the length may include less than 2 residues, less than 3 residues, less than 4 residues, less than 5 residues, less than 6 residues, less than 7 residues, less than 8 residues, less than 9 residues, less than 10 residues, less than 15 residues, less than 20 residues, less than 25 residues, less than 30 residues, less than 40 residues, less than 50 residues, less than 60 residues, less than 70 residues, less than 80 residues, less than 90 residues, or less than 100 residues. Examples of residues may include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine, or any combination thereof. The linker may be non-enzymatic, or may lack any enzymatic activity. 
     A connection may be covalent. A covalent connection may include a peptide bond. The peptide bond may include amide bond. A connection may be between an N-terminus and another N-terminus. A connection may be between a C-terminus and another C-terminus. A connection may be between an N-terminus and a C-terminus. A connection may be between a C-terminus and an N-terminus. 
     The fusion protein may include connections in various orientations. The endonuclease may be connected at its C-terminus. The endonuclease may be connected at its N-terminus. The ligase may be connected at its C-terminus. The ligase may be connected at its N-terminus. 
       FIG.  7    illustrates some examples of fusion protein. The figure includes examples of arrangements and orientations of the endonuclease, linker, ligase, or nuclear localization signal. Other aspects may be incorporated into the examples shown. 
     Non-Covalently Coupled Proteins 
     Disclosed herein are non-covalently coupled proteins. Some aspects relate to a nucleic acid (e.g. an expression vector) encoding a protein, or encoding at least part of a protein. The proteins may include an endonuclease such as an RNA-guided endonuclease. A protein of the non-covalently coupled proteins may include a portion of an endonuclease. A protein of the non-covalently coupled proteins may include a portion of a ligase. The proteins may include a ligase such as a DNA ligase. A protein of the non-covalently coupled proteins may include a fusion protein. 
     The non-covalently coupled proteins may be bound together through heterodimerization domains. Examples of heterodimerization domains may include a leucine zipper, PDZ domain, streptavidin, streptavidin binding protein, foldon domain, hydrophobic moiety, or a functional binding fragment thereof. A heterodimerization domain may include a leucine zipper. A heterodimerization domain may include a PDZ domain. A heterodimerization domain may include a streptavidin. A heterodimerization domain may include a streptavidin binding protein. A heterodimerization domain may include a foldon domain. A heterodimerization domain may include a hydrophobic moiety. A heterodimerization domain may include an antibody or antibody fragment. The non-covalently coupled proteins may be bound together through inteins. 
     The endonuclease and ligase may be coupled together by a separate molecule. The separate molecule may comprise a nucleic acid (e.g. a guide nucleic acid). The ligase may include a hairpin binding motif, where the RNA-guided endonuclease and the DNA ligase are coupled with the nucleic acid. The nucleic acid may include a scaffold that binds the RNA-guided endonuclease and a hairpin that binds to the hairpin binding motif. The hairpin binding motif may include an MS2 coat protein (MCP) peptide. The hairpin may include an MS2 hairpin. 
     The endonuclease and ligase may be coupled together by a heterobifunctional molecule. The heterobifunctional molecule may include an endonuclease binding domain and a DNA ligase binding domain. The heterobifunctional molecule may include an endonuclease binding domain. The endonuclease binding domain may include a heterodimerization domain. The endonuclease binding domain may include an antibody or antibody binding fragment. The heterobifunctional molecule may include a ligase binding domain such as a DNA ligase binding domain. The DNA ligase binding domain may include a heterodimerization domain. The DNA ligase binding domain may include an antibody or antibody binding fragment. The heterobifunctional molecule may include a small molecule. The small molecule may comprise a proteolysis targeting chimera (PROTAC), or a related heterobifunctional molecule. 
     Some aspects include a protein complex, comprising: an RNA-guided endonuclease bound to a DNA ligase. The endonuclease and the DNA ligase may be bound together through heterodimerization domains. The protein complex of embodiment 75, wherein the heterodimerization domains may comprise leucine zippers, PDZ domains, streptavidin and streptavidin binding protein, foldon domains, hydrophobic polypeptides, an antibody that binds the Cas nickase, or an antibody that binds the DNA ligase, or one or more binding fragments thereof. The protein complex may be included in a cell. The cell may further include a heterologous RNA-guided endonuclease and a DNA ligase that that was introduced into the cell. The cell may further include a nuclease that is different from the RNA-guided endonuclease. 
     Guide Nucleic Acids 
     Disclosed herein are guide nucleic acids. The guide nucleic acid may be included in a composition, system or method disclosed herein. Some aspects relate to a nucleic acid (e.g. DNA or an expression vector) that encodes a guide nucleic acid such as a guide RNA. Provided herein are guide nucleic acids (e.g., gRNAs) that direct a programmable endonuclease (e.g., a nCas9) to a target nucleic acid (e.g. a genomic locus). The guide nucleic acid may guide an RNA-guided endonuclease to a target nucleic acid locus for nucleic acid replacement or gene editing at the locus. A guide nucleic acid of the present disclosure may facilitate a donor strand to be inserted into a target site of the target nucleic acid. A guide nucleic acid of the present disclosure may facilitate editing of a nucleic acid sequence at a target site of the target nucleic acid. The guide nucleic acid may, in some instances, also act as a splint for a DNA ligase described herein, such as for ligating two nucleic acid strands base paired to a portion of the guide nucleic acid. The guide nucleic acid may be single stranded. The guide nucleic acid may include RNA. The guide nucleic acid may be RNA. The guide nucleic acid may include a guide RNA (gRNA). In some cases, a guide nucleic acid may include DNA. 
     The guide nucleic acid may be non-naturally occurring. The guide nucleic acid may be engineered. The guide nucleic acid may be synthetic. The guide nucleic acid may be pre-synthetized. The guide nucleic acid may be added to a subject or a cell. In some aspects, the guide nucleic acid does not include a template for a polymerase. 
     The guide nucleic acid may include an integrating nucleic acid binding site. The integrating nucleic acid binding site may be referred to as a “donor binding site.” 
     Disclosed herein are guide nucleic acids, comprising: a spacer reverse complementary to a first region of a target nucleic acid; a scaffold configured to bind to an endonuclease; and an integrating nucleic acid binding site and optionally a flap binding site reverse complementary to a nucleic acid flap. 
     In some aspects, the guide nucleic acid comprises a spacer complementary to a genomic locus in a cell; a scaffold for complexing with the at least one endonuclease; a donor binding site that is at least partially complementary to a donor strand; a flap binding site that is at least partially identical or complementary to a genomic flap at or adjacent to the genomic locus; or a combination thereof. In some aspects, the guide nucleic acid can direct the at least one endonuclease to cleave at least one strand of the genomic locus. In some aspects, the guide nucleic acid can be at least partially complementary to the donor strand or at least partially complementary to a genomic flap (e.g., a genomic nucleic acid sequence that is displaced and become single-stranded when the guide nucleic acid recruits the endonuclease to the genomic locus). In some aspects, the guide nucleic acid, being at least partially complementary to the donor strand or at least partially complementary to a genomic flap, brings the donor strand to close proximity of the cleaving of the genomic locus. 
     Disclosed herein, in some embodiments, are guide nucleic acids comprising a scaffold. The scaffold may bind a nuclease. The scaffold may bind a Cas nuclease. The scaffold may bind a nickase. The scaffold may bind a Cas nickase. The scaffold may bind an  S. Pyogenes  Cas9 nuclease. The scaffold may bind an  S. Pyogenes  Cas9 nickase. The scaffold may include a scaffold nucleic acid sequence. A system described herein may include a first guide nucleic acid. The system can include a second guide nucleic acid. The first guide nucleic acid may bind to a first Cas nickase. The second guide nucleic acid may bind to a second Cas nickase. 
     A guide nucleic acid may include any aspect of (i)-(iv): (i) a spacer complementary to a region of a genomic locus of a genomic strand, (ii) a scaffold for complexing with an RNA-guided endonuclease, (iii) a donor binding site that is at least partially complementary to an integrating nucleic acid, or (iv) a flap binding site that is at least partially identical or complementary to a genomic flap at or adjacent to the genomic locus. A guide nucleic acid may include any aspect of (i)-(iii): (i) a spacer complementary to a region of a genomic locus of a genomic strand, (ii) a scaffold for complexing with an RNA-guided endonuclease, or (iii) a donor binding site that is at least partially complementary to a splinting nucleic acid. A component of (i), (ii), or (iii) may be included in a single guide nucleic acid, or may be split between or collectively included among multiple guide nucleic acids. 
     In some aspects, the guide nucleic acid comprises a modified internucleoside linkage. In some aspects, the modified internucleoside linkage comprises a phosphorothioate linkage. In some aspects, the modified internucleoside linkage is between any of the 4 terminal nucleosides at a 5′ end or at a 3′ end of the guide nucleic acid. The guide nucleic acid may include multiple modified internucleoside linkages. For example, the guide nucleic acid may include modified internucleoside linkages at nucleic acids of the 5′ and 3′ ends of the guide nucleic acid, such as between the last 4 nucleic acids at the 5′ end and between the last 4 nucleic acids at the 3′ end. In some aspects, the guide nucleic acid comprises a modified nucleoside. In some aspects, the modified nucleoside comprises a locked nucleic acid (LNA), a 2′ fluoro, a 2′ O-alkyl, or a combination thereof. The modified nucleoside may include an LNA, a 2′fluoro, a 2′ O-alkyl, a methylated cytosine, an inverted thymidine, or a combination thereof. The modified nucleoside may include an LNA. The modified nucleoside may include a 2′fluoro. The modified nucleoside may include a 2′ O-alkyl. The modified nucleoside may include a methylated cytosine. In some aspects, the modified nucleoside is any of the 3 terminal nucleosides at a 5′ end or at a 3′ end of the guide nucleic acid. The guide nucleic acid may include multiple modified nucleosides. For example, the guide nucleic acid may include modified nucleosides at nucleic acids of the 5′ and 3′ ends of the guide nucleic acid, such as the last 3 nucleic acids at the 5′ end and the last 3 nucleic acids at the 3′ end. 
     In some aspects, the guide nucleic acid comprises at least one nucleic acid modification. In some aspect, the at least nucleic acid modification comprises modifying a backbone, a sugar, a base, or a combination thereof of the guide nucleic acid. In some aspects, the at least one nucleic acid modification can increase resistance of the guide nucleic acid to degradation (e.g., against nuclease degradation or hydrolysis). In some aspects, the at least one nucleic acid modification can increase the complexing of the guide nucleic acid to the at least one endonuclease. In some aspects, the at least one nucleic acid modification can increase the complexing of the guide nucleic acid to the donor strand. In some aspects, the at least one nucleic acid modification can increase the complexing of the guide nucleic acid to the genomic locus via by being complementary to the genomic flap. 
     In some aspects, the guide nucleic acid comprises at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more nucleic acid modifications. In some aspects, nucleic acid modification can occur at 3′OH group, 5′OH group, at the backbone, at the sugar component, or at the nucleotide base. Nucleic acid modification can include non-naturally occurring linker molecules of interstrand or intrastrand cross links. In one aspect, the modified nucleic acid comprises modification of one or more of the 3′OH or 5′OH group, the backbone, the sugar component, or the nucleotide base, or addition of non-naturally occurring linker molecules. In some aspects, modified backbone comprises a backbone other than a phosphodiester backbone. In some aspects, a modified sugar comprises a sugar other than deoxyribose (in modified DNA) or other than ribose (modified RNA). In some aspects, a modified base comprises a base other than adenine, guanine, cytosine, thymine or uracil. In some aspects, the guide nucleic acid comprises at least one modified base. In some instances, the guide nucleic acid comprises at least one, two, three, four, five, six, seven, eight, nine, 10, 15, 20, or more modified bases. In some cases, the nucleic acid modifications to the base moiety include natural and synthetic modifications of adenine, guanine, cytosine, thymine, or uracil, and purine or pyrimidine bases. 
     In some aspects, the at least one nucleic acid modification of the guide nucleic acid comprises a modification of any one of or any combination of: 2′ modified nucleotide comprising 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-0-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA); modification of one or both of the non-linking phosphate oxygens in the phosphodiester backbone linkage; modification of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage; modification of a constituent of the ribose sugar; replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring nucleobase; modification of the ribose-phosphate backbone; modification of 5′ end of polynucleotide; modification of 3′ end of polynucleotide; modification of the deoxyribose phosphate backbone; substitution of the phosphate group; modification of the ribophosphate backbone; modifications to the sugar of a nucleotide; modifications to the base of a nucleotide; or stereopure of nucleotide. Non limiting examples of nucleic acid modification to the guide nucleic acid can include: modification of one or both of non-linking or linking phosphate oxygens in the phosphodiester backbone linkage (e.g., sulfur (S), selenium (Se), BR3 (wherein R can be, e.g., hydrogen, alkyl, or aryl), C (e.g., an alkyl group, an aryl group, and the like), H, NR2, wherein R can be, e.g., hydrogen, alkyl, or aryl, or wherein R can be, e.g., alkyl or aryl); replacement of the phosphate moiety with “dephospho” linkers (e.g., replacement with methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, or methyleneoxymethylimino); modification or replacement of a naturally occurring nucleobase with nucleic acid analog; modification of deoxyribose-phosphate or ribose-phosphate backbone (e.g., modifying the ribose-phosphate backbone to incorporate phosphorothioate, phosphonothioacetate, phosphoroselenates, boranophosphates, borano phosphate esters, hydrogen phosphonates, phosphonocarboxylate, phosphoroamidates, alkyl or aryl phosphonates, phosphonoacetate, or phosphotriesters; modification of 5′ end (e.g., 5′ cap or modification of 5′ cap —OH) or 3′ end of the nucleic acid sequence (3′ tail or modification of 3′ end —OH); substitution of the phosphate group with methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, or methyleneoxymethylimino; modification of the ribophosphate backbone to incorporate morpholino (phosphorodiamidate morpholino oligomer PMO), cyclobutyl, pyrrolidine, or peptide nucleic acid (PNA) nucleoside surrogates; modifications to the sugar of a nucleotide to incorporate locked nucleic acid (LNA), unlocked nucleic acid (UNA), ethylene nucleic acid (ENA), constrained ethyl (cEt) sugar, or bridged nucleic acid (BNA); modification of a constituent of the ribose sugar (e.g., 2′-O-methyl, 2′-O-methoxy-ethyl (2′-MOE), 2′-fluoro, 2′-aminoethyl, 2′-deoxy-2′-fuloarabinou-cleic acid, 2′-deoxy, 2′-O-methyl, 3′-phosphorothioate, 3′-phosphonoacetate (PACE), or 3′-phosphonothioacetate (thioPACE)); modification to the base of a nucleotide (of A, T, C, G, or U); and stereopure of nucleotide (e.g., S conformation of phosphorothioate or R conformation of phosphorothioate). 
     In some aspects, the nucleic acid modification comprises at least one substitution of one or both of non-linking phosphate oxygen atoms in a phosphodiester backbone linkage of the guide nucleic acid. In some aspects, the at least one nucleic acid modification of the guide nucleic acid comprises a substitution of one or more of linking phosphate oxygen atoms in a phosphodiester backbone linkage of the guide nucleic acid. A non-limiting example of a nucleic acid modification of a phosphate oxygen atom is a sulfur atom. In some aspects, the nucleic acid modification comprises at least one modification to a sugar. In some aspects, the nucleic acid modification comprises at least one nucleic acid modification to the sugar comprising a modification of a constituent of the sugar, where the sugar is a ribose sugar. In some aspects, the nucleic acid modification of the guide nucleic acid comprises at least one modification to the constituent of the ribose sugar of the nucleotide of the guide nucleic acid comprising a 2′-O-Methyl group. In some aspects, the nucleic acid modification comprises at least one modification comprising replacement of a phosphate moiety of the guide nucleic acid with a dephospho linker. In some aspects, the nucleic acid modification of comprises at least one modification of a phosphate backbone. In some aspects, the modification comprises a phosphorothioate group. In some aspects, the nucleic acid modifications comprises at least one modification comprising a modification to a base of a nucleotide of the guide nucleic acid. In some aspects, the nucleic acid modifications comprises at least one modification comprising an unnatural base of a nucleotide. In some aspects, the nucleic acid modifications comprises at least one modification comprising at least one stereopure nucleic acid. In some aspects, the at least one nucleic acid modification can be positioned proximal to a 5′ end of the guide nucleic acid. In some aspects, the at least one nucleic acid modification can be positioned proximal to a 3′ end of the guide nucleic acid. In some aspects, the at least one nucleic acid modification can be positioned proximal to both 5′ and 3′ ends of the guide nucleic acid. 
     In some aspects, the guide nucleic acid described herein comprises a backbone comprising a plurality of sugar and phosphate moieties covalently linked together. In some cases, a backbone of the guide nucleic acid comprises a phosphodiester bond linkage between a first hydroxyl group in a phosphate group on a 5′ carbon of a deoxyribose in DNA or ribose in RNA and a second hydroxyl group on a 3′ carbon of a deoxyribose in DNA or ribose in RNA. In some aspects, a backbone of the guide nucleic acid can lack a 5′ reducing hydroxyl, a 3′ reducing hydroxyl, or both, capable of being exposed to a solvent. In some aspects, a backbone of the guide nucleic acid can lack a 5′ reducing hydroxyl, a 3′ reducing hydroxyl, or both, capable of being exposed to nucleases. In some aspects, a backbone of the guide nucleic acid can lack a 5′ reducing hydroxyl, a 3′ reducing hydroxyl, or both, capable of being exposed to hydrolytic enzymes. In some instances, a backbone of the guide nucleic acid can be represented as a polynucleotide sequence in a circular 2-dimensional format with one nucleotide after the other. In some instances, a backbone of the guide nucleic acid can be represented as a polynucleotide sequence in a looped 2-dimensional format with one nucleotide after the other. In some cases, a 5′ hydroxyl, a 3′ hydroxyl, or both, are joined through a phosphorus-oxygen bond. In some cases, a 5′ hydroxyl, a 3′ hydroxyl, or both, are modified into a phosphoester with a phosphorus-containing moiety. In some aspects, the guide nucleic acid comprises at least one nucleic acid modification comprising any one of: 5′ adenylate, 5′ guanosine-triphosphate cap, 5′N7-Methylguanosine-triphosphate cap, 5′triphosphate cap, 3′phosphate, 3′thiophosphate, 5′phosphate, 5′thiophosphate, Cis-Syn thymidine dimer, trimers, C12 spacer, C3 spacer, C6 spacer, dSpacer, PC spacer, rSpacer, Spacer 18, Spacer 9,3′-3′ modifications, 5′-5′ modifications, abasic, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-Biotin, dual biotin, PC biotin, psoralen C2, psoralen C6, TINA, 3′DABCYL, black hole quencher 1, black hole quencher 2, DABCYL SE, dT-DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, carboxyl linker, thiol linkers, 2′deoxyribonucleoside analog purine, 2′deoxyribonucleoside analog pyrimidine, ribonucleoside analog, 2′-O-methyl ribonucleoside analog, sugar modified analogs, wobble/universal bases, fluorescent dye label, 2′fluoro RNA, 2′O-methyl RNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA, phosphothioate DNA, phosphorothioate RNA, UNA, LNA, cEt, pseudouridine-5′-triphosphate, 5-methylcytidine-5′-triphosphate, 2-O-methyl-phosphorothioate or any combinations thereof. 
     A nucleic acid modification can also be a phosphorothioate substitute. In some cases, a natural phosphodiester bond can be susceptible to rapid degradation by cellular nucleases and; a modification of internucleotide linkage using phosphorothioate (PS) bond substitutes can be more stable towards hydrolysis by cellular degradation. A modification can increase stability in a polynucleic acid. A modification can also enhance biological activity. In some cases, a phosphorothioate enhanced RNA polynucleic acid can inhibit RNase A, RNase T1, calf serum nucleases, or any combinations thereof. These properties can allow the use of PS-RNA polynucleic acids to be used in applications where exposure to nucleases is of high probability in vivo or in vitro. For example, phosphorothioate (PS) bonds can be introduced between the last 3-5 nucleotides at the 5′- or 3′-end of a polynucleic acid which can inhibit exonuclease degradation. In some cases, phosphorothioate bonds can be added throughout an entire polynucleic acid to reduce attack by endonucleases. In some aspects, the guide nucleic acid comprises at least one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 100, or more internucleotide linkage comprising PS bond. In some aspects, the guide nucleic acid comprises only PS bond as the internucleotide linkage modification. In some aspects, all internucleotide linkages of the guide nucleic acid herein are fully PS-modified or include phosphorothioate internucleotide linkages. 
     The guide nucleic acid may include a hairpin. The hairpin may bind to a hairpin binding motif such as a hairpin binding motif on a DNA ligase. The hairpin may include an MS2 hairpin A hairpin such as an MS2 hairpin may be useful for recruiting a DNA ligase that includes an MCP peptide. 
     The guide nucleic acid may include any aspect included in  FIG.  1 A- 6 C . Table 8 illustrates non-limiting examples of some of the guide nucleic acids described herein. Some of the guide nucleic acids in the table include nucleic acid modifications. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Examples of nucleic acid sequences 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Nucleic acid sequence 
                 SEQ ID 
               
               
                   
                 Name 
                 (5′ to 3′) 
                 NO: 
               
               
                   
                   
               
               
                   
                 BFP 
                 atggtgagcaagggcgagga 
                 97 
               
               
                   
                   
                 gctgttcaccggggtggtgc 
                   
               
               
                   
                   
                 ccatcctggtcgagctggac 
                   
               
               
                   
                   
                 ggcgacgtaaacggccacaa 
                   
               
               
                   
                   
                 gttcagcgtgtccggcgagg 
                   
               
               
                   
                   
                 gcgagggcgatgccacctac 
                   
               
               
                   
                   
                 ggcaagctgaccctgaagtt 
                   
               
               
                   
                   
                 catctgcaccaccggcaagc 
                   
               
               
                   
                   
                 tgccGgtgccctggcccacc 
                   
               
               
                   
                   
                 CTCgtgaccaccctgaccCA 
                   
               
               
                   
                   
                 Tggcgtgcagtgcttcagcc 
                   
               
               
                   
                   
                 gctaccccgaccacatgaag 
                   
               
               
                   
                   
                 cagcacgacttcttcaagtc 
                   
               
               
                   
                   
                 cgccatgcccgaaggctacg 
                   
               
               
                   
                   
                 tccaggagcgcaccatcttc 
                   
               
               
                   
                   
                 ttcaaggacgacggcaacta 
                   
               
               
                   
                   
                 caagacccgcgccgaggtga 
                   
               
               
                   
                   
                 agttcgagggcgacaccctg 
                   
               
               
                   
                   
                 gtgaaccgcatcgagctgaa 
                   
               
               
                   
                   
                 gggcatcgacttcaaggagg 
                   
               
               
                   
                   
                 acggcaacatcctggggcac 
                   
               
               
                   
                   
                 aagctggagtacaactacaa 
                   
               
               
                   
                   
                 cagccacaacgtctatatca 
                   
               
               
                   
                   
                 tggccgacaagcagaagaac 
                   
               
               
                   
                   
                 ggcatcaaggtgaacttcaa 
                   
               
               
                   
                   
                 gatccgccacaacatcgagg 
                   
               
               
                   
                   
                 acggcagcgtgcagctcgcc 
                   
               
               
                   
                   
                 gaccactaccagcagaacac 
                   
               
               
                   
                   
                 ccccatcggcgacggccccg 
                   
               
               
                   
                   
                 tgctgctgcccgacaaccac 
                   
               
               
                   
                   
                 tacctgagcacccagtccaa 
                   
               
               
                   
                   
                 gctgagcaaagaccccaacg 
                   
               
               
                   
                   
                 agaagcgcgatcacatggtc 
                   
               
               
                   
                   
                 ctgctggagttcgtgaccgc 
                   
               
               
                   
                   
                 cgccgggatcactctcggca 
                   
               
               
                   
                   
                 tggacgagctgtacaagTAA 
                   
               
               
                   
                   
               
               
                   
                 Rep1. 
                 mC*mU*mG*AAGUUCAUCUG 
                 98 
               
               
                   
                 BFP. 
                 CACCACGUUUAAGAGCUAUG 
                   
               
               
                   
                 FwdGuide 
                 CUGGAAACAGCAUAGCAAGU 
                   
               
               
                   
                   
                 UUAAAUAAGGCUAGUCCGUU 
                   
               
               
                   
                   
                 AUCAACUUGAAAAAGUGGCA 
                   
               
               
                   
                   
                 CCGAGUCGGUGCGUGGGCCA 
                   
               
               
                   
                   
                 GGGCACCGGCAGCUUGCCGG 
                   
               
               
                   
                   
                 UGGUGCAGAUGmA*mA*mC* 
                   
               
               
                   
                   
                 U 
                   
               
               
                   
                   
               
               
                   
                 Rep1. 
                 mG*mA*mC*GUAGCCUUCGG 
                 99 
               
               
                   
                 BFP. 
                 GCAUGGGUUUAAGAGCUAUG 
                   
               
               
                   
                 RevGuide 
                 CUGGAAACAGCAUAGCAAGU 
                   
               
               
                   
                   
                 UUAAAUAAGGCUAGUCCGUU 
                   
               
               
                   
                   
                 AUCAACUUGAAAAAGUGGCA 
                   
               
               
                   
                   
                 CCGAGUCGGUGCUGAAGCAG 
                   
               
               
                   
                   
                 CACGACUUCUUCAAGUCCGC 
                   
               
               
                   
                   
                 CAUGCCCGAAGmG*mC*mU* 
                   
               
               
                   
                   
                 A 
                   
               
               
                   
                   
               
               
                   
                 Rep1. 
                 mC*mU*mG*AAGUUCAUCUG 
                 100 
               
               
                   
                 BFP. 
                 CACCACGUUUAAGAGCUAUG 
                   
               
               
                   
                 FwdGuide. 
                 CUGGAAACAGCAUAGCAAGU 
                   
               
               
                   
                 SpPAMmut 
                 UUAAAUAAGGCUAGUCCGUU 
                   
               
               
                   
                   
                 AUCAACUUGAAAAAGUGGCA 
                   
               
               
                   
                   
                 CCGAGUCGGUGCGUGGGCCA 
                   
               
               
                   
                   
                 GGGCACCGGCAGCUUGCCGG 
                   
               
               
                   
                   
                 UUGUGCAGAUGmA*mA*mC* 
                   
               
               
                   
                   
                 U 
                   
               
               
                   
                   
               
               
                   
                 Rep1. 
                 mG*mA*mC*GUAGCCUUCGG 
                 101 
               
               
                   
                 BFP. 
                 GCAUGGGUUUAAGAGCUAUG 
                   
               
               
                   
                 RevGuide. 
                 CUGGAAACAGCAUAGCAAGU 
                   
               
               
                   
                 SpPAMmut 
                 UUAAAUAAGGCUAGUCCGUU 
                   
               
               
                   
                   
                 AUCAACUUGAAAAAGUGGCA 
                   
               
               
                   
                   
                 CCGAGUCGGUGCUGAAGCAG 
                   
               
               
                   
                   
                 CACGACUUCUUCAAGUCAGC 
                   
               
               
                   
                   
                 CAUGCCCGAAGmG*mC*mU* 
                   
               
               
                   
                   
                 A 
                   
               
               
                   
                   
               
               
                   
                 Rep1. 
                 /5Phos/caccggcaagctg 
                 102 
               
               
                   
                 BFP2GFP. 
                 ccGgtgccctggcccaccCT 
                   
               
               
                   
                 TopDonor. 
                 CgtgaccaccctgaccTACg 
                   
               
               
                   
                 5P 
                 gcgtgcagtgcttcagccgc 
                   
               
               
                   
                   
                 taccccgaccaca 
                   
               
               
                   
                   
               
               
                   
                 Rep1. 
                 /5Phos/tggcggacttgaa 
                 103 
               
               
                   
                 BFP2GFP. 
                 gaagtcgtgctgcttcatgt 
                   
               
               
                   
                 BotDonor. 
                 ggtcggggtagcggctgaag 
                   
               
               
                   
                 5P 
                 cactgcacgccGTAggtcag 
                   
               
               
                   
                   
                 ggtggtcacGAGg 
                   
               
               
                   
                   
               
               
                   
                 Rep1. 
                 /5Phos/caccggcaagctg 
                 104 
               
               
                   
                 BFP2GFP. 
                 ccGgtgccctggcccacTCT 
                   
               
               
                   
                 TopDonor. 
                 TGTGACCACCTTGACCtACG 
                   
               
               
                   
                 Recoded. 
                 GTGTCCAGTGTTTTAGCAGG 
                   
               
               
                   
                 5P 
                 TATCCGGATCACA 
                   
               
               
                   
                   
               
               
                   
                 Rep1. 
                 /5Phos/tggcggacttgaa 
                 105 
               
               
                   
                 BFP2GFP. 
                 gaagtcgtgctgcttcaTGT 
                   
               
               
                   
                 BotDonor. 
                 GATCCGGATACCTGCTAAAA 
                   
               
               
                   
                 Recoded. 
                 CACTGGACACCGTaGGTCAA 
                   
               
               
                   
                 5P 
                 GGTGGTCACAAGA 
                   
               
               
                   
                   
               
               
                   
                 Rep1. 
                 /5Phos/Aaccggcaagctg 
                 106 
               
               
                   
                 BFP2GFP. 
                 ccGgtgccctggcccacTCT 
                   
               
               
                   
                 TopDonor. 
                 TGTGACCACCTTGACCtACG 
                   
               
               
                   
                 SpP AMmut. 
                 GTGTCCAGTGTTTTAGCAGG 
                   
               
               
                   
                 Recoded. 
                 TATCCGGATCACA 
                   
               
               
                   
                 5P 
                   
                   
               
               
                   
                   
               
               
                   
                 Rep1. 
                 /5Phos/tggcTgacttgaa 
                 107 
               
               
                   
                 BFP2GFP. 
                 gaagtcgtgctgcttcaTGT 
                   
               
               
                   
                 BotDonor. 
                 GATCCGGATACCTGCTAAAA 
                   
               
               
                   
                 SpPAMmut. 
                 CACTGGACACCGTaGGTCAA 
                   
               
               
                   
                 Recoded. 
                 GGTGGTCACAAGA 
                   
               
               
                   
                 5P 
                   
                   
               
               
                   
                   
               
               
                   
                 Rep1. 
                 /5Phos/c*a*c*cggcaag 
                 108 
               
               
                   
                 BFP2GFP. 
                 ctgccGgtgccctggcccac 
                   
               
               
                   
                 TopDonor. 
                 TCTTGTGACCACCTTGACCt 
                   
               
               
                   
                 Recoded. 
                 ACGGTGTCCAGTGTTTTAGC 
                   
               
               
                   
                 5P. 
                 AGGTATCCGGATC*A*C*A 
                   
               
               
                   
                 endPhos 
                   
                   
               
               
                   
                   
               
               
                   
                 Rep1. 
                 /5Phos/t*g*g*cggactt 
                 109 
               
               
                   
                 BFP2GFP. 
                 gaagaagtcgtgctgcttca 
                   
               
               
                   
                 BotDonor. 
                 TGTGATCCGGATACCTGCTA 
                   
               
               
                   
                 Recoded. 
                 AAACACTGGACACCGTaGGT 
                   
               
               
                   
                 5P. 
                 CAAGGTGGTCACA*A*G*A 
                   
               
               
                   
                 endPhos 
                   
                   
               
               
                   
                   
               
               
                   
                 Rep2. 
                 mC*mU*mG*AAGUUCAUCUG 
                 110 
               
               
                   
                 BFP. 
                 CACCACGUUUAAGAGCUAUG 
                   
               
               
                   
                 FwdGuide 
                 CUGGAAACAGCAUAGCAAGU 
                   
               
               
                   
                   
                 UUAAAUAAGGCUAGUCCGUU 
                   
               
               
                   
                   
                 AUCAACUUGAAAAAGUGGCA 
                   
               
               
                   
                   
                 CCGAGUCGGUGCCUACGGCA 
                   
               
               
                   
                   
                 AGCUGACCmC*mU*mG*A 
                   
               
               
                   
                   
               
               
                   
                 Rep2. 
                 mG*mA*mC*GUAGCCUUCGG 
                 ill 
               
               
                   
                 BFP. 
                 GCAUGGGUUUAAGAGCUAUG 
                   
               
               
                   
                 RevGuide 
                 CUGGAAACAGCAUAGCAAGU 
                   
               
               
                   
                   
                 UUAAAUAAGGCUAGUCCGUU 
                   
               
               
                   
                   
                 AUCAACUUGAAAAAGUGGCA 
                   
               
               
                   
                   
                 CCGAGUCGGUGCAGAUGGUG 
                   
               
               
                   
                   
                 CGCUCCUGmG*mA*mC*G 
                   
               
               
                   
                   
               
               
                   
                 Rep2. 
                 /5Phos/Aaccggcaagctg 
                 112 
               
               
                   
                 BFP2GFP. 
                 ccGgtgccctggcccaccCT 
                   
               
               
                   
                 TopDonor. 
                 CgtgaccaccctgaccTACg 
                   
               
               
                   
                 SpP AMmut. 
                 gcgtgcagtgcttcagccgc 
                   
               
               
                   
                 5P 
                 taccccgaccacatgaagca 
                   
               
               
                   
                   
                 gcacgacttcttcaagtcAg 
                   
               
               
                   
                   
                 ccatgcccgaaggctacgtc 
                   
               
               
                   
                   
                 caggagcgcaccatct 
                   
               
               
                   
                   
               
               
                   
                 Rep2. 
                 /5Phos/tggcTgacttgaa 
                 113 
               
               
                   
                 BFP2GFP. 
                 gaagtcgtgctgcttcatgt 
                   
               
               
                   
                 BotDonor. 
                 ggtcggggtagcggctgaag 
                   
               
               
                   
                 SpPAMmut. 
                 cactgcacgccGTAggtcag 
                   
               
               
                   
                 5P 
                 ggtggtcacGAGggtgggcc 
                   
               
               
                   
                   
                 agggcacCggcagcttgccg 
                   
               
               
                   
                   
                 gtTgtgcagatgaacttcag 
                   
               
               
                   
                   
                 ggtcagcttgccgtag 
                   
               
               
                   
                   
               
               
                   
                 Rep2. 
                 /5Phos/AACCGGTAAGTTG 
                 114 
               
               
                   
                 BFP2GFP. 
                 CCAGTCCCGTGGCCTACTCT 
                   
               
               
                   
                 TopDonor. 
                 TGTGACCACCTTGACCtACG 
                   
               
               
                   
                 SpP AMmut. 
                 GTGTCCAGTGTTTTAGCAGG 
                   
               
               
                   
                 Recoded. 
                 TATCCGGATCACATGAAACA 
                   
               
               
                   
                 5P 
                 GCATGACTTCTTTAAATCAG 
                   
               
               
                   
                   
                 CTATGcccgaaggctacgtc 
                   
               
               
                   
                   
                 caggagcgcaccatct 
                   
               
               
                   
                   
               
               
                   
                 Rep2. 
                 /5Phos/TAGCTGATTTAAA 
                 115 
               
               
                   
                 BFP2GFP. 
                 GAAGTCATGCTGTTTCATGT 
                   
               
               
                   
                 BotDonor. 
                 GATCCGGATACCTGCTAAAA 
                   
               
               
                   
                 SpPAMmut. 
                 CACTGGACACCGTaGGTCAA 
                   
               
               
                   
                 Recoded. 
                 GGTGGTCACAAGAGTAGGCC 
                   
               
               
                   
                 5P 
                 ACGGGACTGGCAACTTACCG 
                   
               
               
                   
                   
                 GTTgtgcagatgaacttcag 
                   
               
               
                   
                   
                 ggtcagcttgccgtag 
                   
               
               
                   
                   
               
               
                   
                 Rep2. 
                 /5Phos/caccggcaagctg 
                 116 
               
               
                   
                 BFP2GFP. 
                 ccGgtgccctggcccaccCT 
                   
               
               
                   
                 TopDonor. 
                 CgtgaccaccctgaccTACg 
                   
               
               
                   
                 5P 
                 gcgtgcagtgcttcagccgc 
                   
               
               
                   
                   
                 taccccgaccacatgaagca 
                   
               
               
                   
                   
                 gcacgacttcttcaagtccg 
                   
               
               
                   
                   
                 ccatgcccgaaggctacgtc 
                   
               
               
                   
                   
                 caggagcgcaccatct 
                   
               
               
                   
                   
               
               
                   
                 Rep2. 
                 /5Phos/tggcggacttgaa 
                 117 
               
               
                   
                 BFP2GFP. 
                 gaagtcgtgctgcttcatgt 
                   
               
               
                   
                 BotDonor. 
                 ggtcggggtagcggctgaag 
                   
               
               
                   
                 5P 
                 cactgcacgccGTAggtcag 
                   
               
               
                   
                   
                 ggtggtcacGAGggtgggcc 
                   
               
               
                   
                   
                 agggcacCggcagcttgccg 
                   
               
               
                   
                   
                 gtggtgcagatgaacttcag 
                   
               
               
                   
                   
                 ggtcagcttgccgtag 
                   
               
               
                   
                   
               
               
                   
                 Rep2. 
                 /5Phos/A*A*C*CGGTAAG 
                 118 
               
               
                   
                 BFP2GFP. 
                 TTGCCAGTCCCGTGGCCTAC 
                   
               
               
                   
                 TopDonor. 
                 TCTTGTGACCACCTTGACCt 
                   
               
               
                   
                 SpP AMmut. 
                 ACGGTGTCCAGTGTTTTAGC 
                   
               
               
                   
                 Recoded. 
                 AGGTATCCGGATCACATGAA 
                   
               
               
                   
                 5P. 
                 ACAGCATGACTTCTTTAAAT 
                   
               
               
                   
                 endPhos 
                 CAGCTATGcccgaaggctac 
                   
               
               
                   
                   
                 gtccaggagcgcacca*t*c 
                   
               
               
                   
                   
                 *t 
                   
               
               
                   
                   
               
               
                   
                 Rep2. 
                 /5Phos/T*A*G*CTGATTT 
                 119 
               
               
                   
                 BFP2GFP. 
                 AAAGAAGTCATGCTGTTTCA 
                   
               
               
                   
                 B 
                 TGTGATCCGGATACCTGCTA 
                   
               
               
                   
                   
                 AAACACTGGACACCGTaGGT 
                   
               
               
                   
                   
                 CAAGGTGGTCACAAGAGTAG 
                   
               
               
                   
                   
                 GCCACGGGACTGGCAACTTA 
                   
               
               
                   
                 otDonor. 
                 CCGGTTgtgcagatgaactt 
                   
               
               
                   
                 SpPAMmut. 
                 cagggtcagcttgccg*t*a 
                   
               
               
                   
                 Recoded. 
                 *g 
                   
               
               
                   
                 5P. 
                   
                   
               
               
                   
                 endPhos 
                   
                   
               
               
                   
                   
               
               
                   
                 Rep2. 
                 mG*mA*mA*AGCUGGCGGGC 
                 120 
               
               
                   
                 CBXl. 
                 ACUAUGGUUUAAGAGCUAUG 
                   
               
               
                   
                 FwdGuide 
                 CUGGAAACAGCAUAGCAAGU 
                   
               
               
                   
                   
                 UUAAAUAAGGCUAGUCCGUU 
                   
               
               
                   
                   
                 AUCAACUUGAAAAAGUGGCA 
                   
               
               
                   
                   
                 CCGAGUCGGUGCGUCACCCU 
                   
               
               
                   
                   
                 UUACACCAmG*mA*mA*A 
                   
               
               
                   
                   
               
               
                   
                 Rep2. 
                 mC*mU*mU*UGCCCUUUACC 
                 121 
               
               
                   
                 CBXl. 
                 ACUCGAGUUUAAGAGCUAUG 
                   
               
               
                   
                 RevGuide 
                 CUGGAAACAGCAUAGCAAGU 
                   
               
               
                   
                   
                 UUAAAUAAGGCUAGUCCGUU 
                   
               
               
                   
                   
                 AUCAACUUGAAAAAGUGGCA 
                   
               
               
                   
                   
                 CCGAGUCGGUGCUUUAGGAG 
                   
               
               
                   
                   
                 GUACUCCAmC*mU*mU*U 
                   
               
               
                   
                   
               
               
                   
                 Rep2. 
                 ATGgtgagcaagggcgagga 
                 122 
               
               
                   
                 mGL-CBX1. 
                 gctgttcaccggggtggtgc 
                   
               
               
                   
                 TopDonor. 
                 ccatcctggtcgagctggac 
                   
               
               
                   
                 SpPAMmut. 
                 ggcgacgtaaacggccacaa 
                   
               
               
                   
                 5P 
                 gttcagcgtccgcggcgagg 
                   
               
               
                   
                   
                 gcgagggcgatgccaccaac 
                   
               
               
                   
                   
                 ggcaagctgaccctgaagtt 
                   
               
               
                   
                   
                 catctgcaccaccggcaagc 
                   
               
               
                   
                   
                 tgcccgtgccctggcccacc 
                   
               
               
                   
                   
                 ctcgtgaccaccttaggcta 
                   
               
               
                   
                   
                 cggcgtggcctgcttcgccc 
                   
               
               
                   
                   
                 gctaccccgaccacatgaag 
                   
               
               
                   
                   
                 cagcacgacttcttcaagtc 
                   
               
               
                   
                   
                 cgccatgcccgaaggctacg 
                   
               
               
                   
                   
                 tccaggagcgcaccatctct 
                   
               
               
                   
                   
                 ttcaaggacgacggtaccta 
                   
               
               
                   
                   
                 caagacccgcgccgaggtga 
                   
               
               
                   
                   
                 agttcgagggcgacaccctg 
                   
               
               
                   
                   
                 gtgaaccgcatcgtgctgaa 
                   
               
               
                   
                   
                 gggcatcgacttcaaggagg 
                   
               
               
                   
                   
                 acggcaacatcctggggcac 
                   
               
               
                   
                   
                 aagctggagtacaacttcaa 
                   
               
               
                   
                   
                 cagccacaaggtctatatca 
                   
               
               
                   
                   
                 cggccgacaagcagaagaac 
                   
               
               
                   
                   
                 ggcatcaaggctaacttcaa 
                   
               
               
                   
                   
                 gacccgccacaacgttgagg 
                   
               
               
                   
                   
                 acggcggcgtgcagctcgcc 
                   
               
               
                   
                   
                 gaccactaccagcagaacac 
                   
               
               
                   
                   
                 ccccatcggcgacggccccg 
                   
               
               
                   
                   
                 tgctgctgcccgacaaccac 
                   
               
               
                   
                   
                 tacctgagccatcagtccaa 
                   
               
               
                   
                   
                 actgagcaaagaccccaacg 
                   
               
               
                   
                   
                 agaagcgcgatcacatggtc 
                   
               
               
                   
                   
                 ctgaaggagagggtgaccgc 
                   
               
               
                   
                   
                 cgccgggattacacatgaca 
                   
               
               
                   
                   
                 tggacgagctgtacaagtct 
                   
               
               
                   
                   
                 ggaggatctagcggaggatc 
                   
               
               
                   
                   
                 cGGGAAGAAACAAAACAAGA 
                   
               
               
                   
                   
                 AGAAAGTGGAGGAGGTGCTA 
                   
               
               
                   
                   
                 GAAGAGGAGGAAGAGGAATA 
                   
               
               
                   
                   
                 TGTGGTGGAAAAAGTTCTCG 
                   
               
               
                   
                   
                 AtCGTCGAGTGGTAAAGGGC 
                   
               
               
                   
                   
                 AAAGTGGAGTACCTCCTAAA 
                   
               
               
                   
                   
               
               
                   
                 Rep2. 
                 CGACGaTCGAGAACTTTTTC 
                 123 
               
               
                   
                 mGL-CBX1. 
                 CACCACATATTCCTCTTCCT 
                   
               
               
                   
                 BotDonor. 
                 CCTCTTCTAGCACCTCCTCC 
                   
               
               
                   
                 SpPAMmut. 
                 ACTTTCTTCTTGTTTTGTTT 
                   
               
               
                   
                 5P 
                 CTTCCCggatcctccgctag 
                   
               
               
                   
                   
                 atcctccagacttgtacagc 
                   
               
               
                   
                   
                 tcgtccatgtcatgtgtaat 
                   
               
               
                   
                   
                 cccggcggcggtcaccctct 
                   
               
               
                   
                   
                 ccttcaggaccatgtgatcg 
                   
               
               
                   
                   
                 cgcttctcgttggggtcttt 
                   
               
               
                   
                   
                 gctcagtttggactgatggc 
                   
               
               
                   
                   
                 tcaggtagtggttgtcgggc 
                   
               
               
                   
                   
                 agcagcacggggccgtcgcc 
                   
               
               
                   
                   
                 gatgggggtgttctgctggt 
                   
               
               
                   
                   
                 agtggtcggcgagctgcacg 
                   
               
               
                   
                   
                 ccgccgtcctcaacgttgtg 
                   
               
               
                   
                   
                 gcgggtcttgaagttagcct 
                   
               
               
                   
                   
                 tgatgccgttcttctgcttg 
                   
               
               
                   
                   
                 tcggccgtgatatagacctt 
                   
               
               
                   
                   
                 gtggctgttgaagttgtact 
                   
               
               
                   
                   
                 ccagcttgtgccccaggatg 
                   
               
               
                   
                   
                 ttgccgtcctccttgaagtc 
                   
               
               
                   
                   
                 gatgcccttcagcacgatgc 
                   
               
               
                   
                   
                 ggttcaccagggtgtcgccc 
                   
               
               
                   
                   
                 tcgaacttcacctcggcgcg 
                   
               
               
                   
                   
                 ggtcttgtaggtaccgtcgt 
                   
               
               
                   
                   
                 ccttgaaagagatggtgcgc 
                   
               
               
                   
                   
                 tcctggacgtagccttcggg 
                   
               
               
                   
                   
                 catggcggacttgaagaagt 
                   
               
               
                   
                   
                 cgtgctgcttcatgtggtcg 
                   
               
               
                   
                   
                 gggtagcgggcgaagcaggc 
                   
               
               
                   
                   
                 cacgccgtagcctaaggtgg 
                   
               
               
                   
                   
                 tcacgagggtgggccagggc 
                   
               
               
                   
                   
                 acgggcagcttgccggtggt 
                   
               
               
                   
                   
                 gcagatgaacttcagggtca 
                   
               
               
                   
                   
                 gcttgccgttggtggcatcg 
                   
               
               
                   
                   
                 ccctcgccctcgccgcggac 
                   
               
               
                   
                   
                 gctgaacttgtggccgttta 
                   
               
               
                   
                   
                 cgtcgccgtccagctcgacc 
                   
               
               
                   
                   
                 aggatgggcaccaccccggt 
                   
               
               
                   
                   
                 gaacagctcctcgcccttgc 
                   
               
               
                   
                   
                 tcacCATAGTGCCCGCCAGC 
                   
               
               
                   
                   
                 TTTCTGGTGTAAAGGGTGAC 
                   
               
               
                   
                   
               
               
                   
                 CBX1-001 
                 CAGCGTCACCCTTTACACCA 
                 124 
               
               
                   
                 Exon 2 
                 GAAAGCTGGCGGGCACTATG 
                   
               
               
                   
                 (includes 
                 GGGAAAAAACAAAACAAGAA 
                   
               
               
                   
                 beginning 
                 GAAAGTGGAGGAGGTGCTAG 
                   
               
               
                   
                 of 
                 AAGAGGAGGAAGAGGAATAT 
                   
               
               
                   
                 ORF) 
                 GTGGTGGAAAAAGTTCTCGA 
                   
               
               
                   
                   
                 CCGTCGAGTGGTAAAGGGCA 
                   
               
               
                   
                   
                 AAGTGGAGTACCTCCTAAAG 
                   
               
               
                   
                   
                 TGGAAGGGATTCTCAGA 
               
               
                   
                   
               
            
           
         
       
     
     The guide nucleic acid may include a sequence of linking nucleic acids (e.g. linking RNA or DNA nucleotides) between components of the guide nucleic acid. For example, the guide nucleic acid may include a sequence of linking nucleic acids between any of the following components: a spacer, a scaffold, a donor binding site, or a flap binding site. The guide nucleic acid may include a sequence of linking nucleic acids between a spacer, a scaffold, or a donor binding site. The guide nucleic acid include a sequence of linking nucleic acids between the scaffold and the donor binding site The guide nucleic acid may include a sequence of linking nucleic acids between a spacer and a scaffold. The guide nucleic acid may include multiple sequences of linking nucleic acids between components. 
     The sequence of linking nucleic acids may include any base, such as A, U, T, G, or C, or a combination thereof. The sequence of linking nucleic acids may include A, T, G, or C, or a combination thereof. The sequence of linking nucleic acids may include A, U, G, or C, or a combination thereof. The sequence of linking nucleic acids may include a series of As. The sequence of linking nucleic acids may include a series of Ts. The sequence of linking nucleic acids may include a series of Us. The sequence of linking nucleic acids may include a series of Cs. The sequence of linking nucleic acids may include a series of Gs. 
     The sequence of linking nucleic acids may include a length, such as a number of nucleotides. The length may include 1, 2, 3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides, or a range defined by any two of the aforementioned numbers of nucleotides. The length may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 nucleotides. In some aspects, the length may be less than 2, less than 3, less than 4, less than 5, less than 6, less than 7, less than 8, less than 9 10, less than 11, less than 12, less than 13, less than 14, less than 15, less than 16, less than 17, less than 18, less than 19, less than 20, less than 21, less than 22, less than 23, less than 24, less than 25, less than 30, less than 35, less than 40, less than 45, less than 50, less than 55, less than 60, less than 65, less than 70, less than 75, less than 80, less than 85, less than 90, less than 95, or less than 100 nucleotides. 
     Some aspects relate to a guide nucleic acid comprising: a spacer that is at least partially complementary to a genomic locus in a cell; a scaffold for complexing with a RNA-guided endonuclease; and a donor binding site that is at least partially complementary to an integrating nucleic acid. The guide nucleic acid may further comprise a flap binding site that is at least partially complementary to a genomic sequence of the genomic locus. The guide nucleic acid may further comprise at least one nucleic acid modification. The at least one nucleic acid modification may comprise a modification to a backbone, a sugar, a base, or a combination thereof. The guide nucleic acid may comprise RNA. 
     Some aspects include a guide nucleic acid, comprising: a spacer at least partially reverse complementary to a first region of a target nucleic acid; a scaffold configured to bind to an endonuclease; and a flap binding site at least partially reverse complementary to a nucleic acid flap, and an integrating nucleic acid binding site. 
     Integrating Nucleic Acids 
     Disclosed herein are integrating nucleic acids. The integrating nucleic acid may be included in a composition, system, or method disclosed herein. Some aspects relate to a nucleic acid that encodes an integrating nucleic acid. Provided herein are integrating nucleic acids that are inserted into a target nucleic acid such as a host genome at a genetic locus. For example, the integrating nucleic acid may replace a nucleic acid in the target nucleic acid. The integrating nucleic acid may be referred to as a “donor nucleic acid,” “donor” or “donor strand.” Where a genomic locus is described, a genetic locus may be included, or vice versa. For example, the locus may be part of a host genome or may be a part of a non-genome nucleic acid. The donor may include DNA. Likewise, the target nucleic acid may include DNA. In some cases, the donor may include RNA, for example when a target nucleic acid includes RNA. The integrating nucleic acid may include any insert, such as a gene or a regulatory element, to be inserted at a genomic locus of a target nucleic acid. The donor strand may include a sequence that is at least partially homologous to the genomic locus. The integrating nucleic acid may, in some instances, also act as a splint for a DNA ligase described herein, such as for ligating two nucleic acid strands base paired to a portion of the splinting integrating nucleic acid. In some cases, the splint includes one strand of the integrating nucleic acid, and the portion being ligated may be another strand of the integrating nucleic acid. In some cases, the splint includes a strand of the integrating nucleic acid, and the portion being ligated may be an upstream or downstream portion of the same strand of the integrating nucleic acid. The integrating nucleic acid may be single stranded. The integrating nucleic acid may be double stranded. The integrating nucleic acid may be delivered as two strands. The integrating nucleic acid may be delivered as multiple strands, e.g. 2 strands. 
     The integrating nucleic acid may be non-naturally occurring. The integrating nucleic acid may be engineered. The integrating nucleic acid may be synthetic. The integrating nucleic acid may be pre-synthetized. The integrating nucleic acid may be added to a subject or a cell. In some aspects, the integrating nucleic acid does not include a template for a polymerase. 
     Disclosed herein are integrating nucleic acids, comprising: a double-stranded DNA region to be inserted into a target nucleic acid, wherein the double-stranded DNA region is flanked by at least one overhang comprising a flap binding site and/or guide binding site. 
     The integrating nucleic acid may be ligated into a target nucleic acid such as a genomic strand. The integrating nucleic acid may include a 5′ end that may be ligated to a 3′ terminus of a genomic strand generated by an RNA-guided endonuclease. 
     The donor may include any aspect included in  FIG.  1 A- 6 C . For example, the donor may include an aspect such as a guide binding site, a flap binding site, or an overhang. The donor may include a guide binding site. The donor may include 2 guide binding sites. The donor may include a flap binding site. The donor may include 2 flap binding sites. The donor may include an overhang. The donor may include 2 overhangs. The aspects may be included at a 5′ end or a 3′ end of the donor, or at both ends. A guide binding site or a flap binding site may be in an internal region of the donor. 
     Some aspects include an integrating nucleic acid, comprising: a double-stranded DNA region to be inserted into a target nucleic acid, wherein the double-stranded DNA region is flanked by at least one overhang comprising a flap binding site or guide binding site. 
     In some aspects, the integrating nucleic acid comprises a modified internucleoside linkage. In some aspects, the modified internucleoside linkage comprises a phosphorothioate linkage. In some aspects, the modified internucleoside linkage is between any of the 4 terminal nucleosides at a 5′ end or at a 3′ end of the integrating nucleic acid. The integrating nucleic acid may include multiple modified internucleoside linkages. For example, the integrating nucleic acid may include modified internucleoside linkages at nucleic acids of the 5′ and 3′ ends of the integrating nucleic acid, such as between the last 4 nucleic acids at the 5′ end and between the last 4 nucleic acids at the 3′ end. In some aspects, the integrating nucleic acid comprises a modified nucleoside. In some aspects, the modified nucleoside comprises a locked nucleic acid (LNA), a 2′ fluoro, a 2′ O-alkyl, a 5′ O-methyl, a 2′-O-methyl, or a combination thereof. The modified nucleoside may include an LNA, a 2′fluoro, a 2′ O-alkyl, a methylated cytosine, an inverted thymidine, or a combination thereof. The modified nucleoside may include an LNA. The modified nucleoside may include a 2′fluoro. The modified nucleoside may include a 2′ O-alkyl. The modified nucleoside may include a methylated cytosine. In some aspects, the modified nucleoside is any of the 3 terminal nucleosides at a 5′ end or at a 3′ end of the integrating nucleic acid. The integrating nucleic acid may include multiple modified nucleosides. For example, the integrating nucleic acid may include modified nucleosides at nucleic acids of the 5′ and 3′ ends of the integrating nucleic acid, such as the last 3 nucleic acids at the 5′ end and the last 3 nucleic acids at the 3′ end. The integrating nucleic acid may include any modification such as a modified nucleoside or modified internucleoside linkage described in relation to guide nucleic acids, insofar as it does not interfere with the function of the integrating nucleic acid after it is ligated into a target nucleic acid such as a host genome. The integrating nucleic acid may include any number or combination of modifications such as a number or combination described in relation to guide nucleic acids, insofar as it does not interfere with a function of the integrating nucleic acid. Table 8 includes some examples of integrating nucleic acid sequences. 
     The integrating nucleic acid may include a methylated nucleotide. The integrating nucleic acid may include an unmethylated nucleotide. An example of a methylated nucleotide may include a nucleotide including methylated cytosine. The cytosine may be methylated at a C-5 position of the cytosine ring. An example of an unmethylated nucleotide may include an unmethylated cytosine. The unmethylated nucleotide may include a cytosine that is not methylated at a C-5 position of the cytosine ring. 
     Target Nucleic Acids 
     Disclosed herein are target nucleic acids. The target nucleic acid may include DNA. The target nucleic acid may be DNA. The target nucleic acid may include RNA. The target nucleic acid may be in a cell. The target nucleic acid may be methylated. The target nucleic acid may be unmethylated. The target nucleic acid may comprise a genome. The target nucleic acid may comprise genomic DNA. The target nucleic acid may comprise a chromosome. The target nucleic acid may comprise a gene. 
     The target nucleic acid may be in a subject. The target nucleic acid may be in a cell. The target nucleic acid may be in a test tube. 
     The target nucleic acid may be edited. The target nucleic acid may be edited in vitro. The target nucleic acid may be edited in vivo. 
     Systems 
     Described herein are systems for nucleic acid editing (also known as gene editing). The editing system may include an endonuclease such as an RNA-guided endonuclease, a guide nucleic acid, and an integrating nucleic acid. Where gene editing is described, it is contemplated that the editing may be of a gene, regulatory element, or any sequence of a nucleic acid. Also, where genome editing is described, such as genome editing at a genetic locus, it is contemplated that nucleic acid editing not comprising a genome may also be performed. For example, genome editing may refer to editing of a genome of an organism, or may include editing of a nucleic acid that is not part of a genome. The systems described herein may be used in gene editing methods. 
     Described herein, in some aspects, is a system comprising at least one endonuclease; at least one guide nucleic acid; at least one ligase; at least one donor strand; or a combination thereof. In some aspects, the guide nucleic acid directs the endonuclease to the genomic locus for cleaving at least one strand of the genomic locus, where, after cleavage, the donor strand is ligated and thus incorporated into the genomic locus by the ligase. In some aspects, the system comprises: a first endonuclease to be complexed with a first guide nucleic acid, where the first endonuclease can be operatively coupled to a first ligase; and a second endonuclease to be complexed with a second guide nucleic acid, where the second endonuclease can be operatively coupled to a second ligase. In such system each of the first endonuclease and the second endonuclease can each cleave at least one strand of the genomic locus for incorporation of the donor strand. 
     In some aspects, the system comprises one, two, three, or more endonucleases. In some aspects, the system comprises one endonucleases. In some aspects, the two endonucleases can each be complexed with a different guide nucleic acid. In some aspects, the two endonucleases can each be operatively coupled to a ligase. In some aspects, the endonuclease is a programmable endonuclease. In some aspects, the endonuclease comprises a RNA-guided endonuclease, where the guide nucleic acid comprises a guide RNA. In some aspects, the endonuclease comprises a nickase, where the endonuclease only cleaves one strand (as opposed to making a double-stranded break). In some aspects, the endonuclease comprises a localization signal sequence to increase the accumulation of the endonuclease in the proximity of the genomic locus (e.g., in the nucleus). In some aspects, the endonuclease comprises at least one additional domain. In some aspects, the at least one additional domain is a dimerization domain. In some aspects, the endonuclease comprising a dimerization domain can be dimerized with a ligase to form a heterodimer. In some aspects, the at least one additional domain is a functional domain. For example, the functional domain can comprises a chromatin modifying domain or a cell penetrating peptide. In some aspects, the endonuclease comprises a linker, where the linker can covalently connect the endonuclease with another polypeptide (e.g., the ligase). In some aspects, the linker covalently connects the endonuclease to the at least one additional domain. In some aspects, the endonuclease comprises a tag, where the tag can be used for increasing expression, identifying, or purifying the endonuclease. 
     In some aspects, the system comprises one, two, three, or more guide nucleic acids. In some aspects, the system comprises one guide nucleic acid, where the one guide nucleic acid can be complexed with at least one endonuclease. In some aspects, the system comprises two guide nucleic acids, where the two guide nucleic acids can each be complexed with the at least one endonuclease. In some aspects, the guide nucleic acid comprises a spacer complementary to a genomic locus in a cell; a scaffold for complexing with the at least one endonuclease; a donor binding site that is at least partially complementary to a donor strand; a flap binding site that is at least partially identical or complementary to a genomic flap at or adjacent to the genomic locus; or a combination thereof. In some aspects, the guide nucleic acid can direct the at least one endonuclease to cleave at least one strand of the genomic locus. In some aspects, the guide nucleic acid can be at least partially complementary to the donor strand or at least partially complementary to a genomic flap (e.g., a genomic nucleic acid sequence that is displaced and become single-stranded when the guide nucleic acid recruits the endonuclease to the genomic locus). In some aspects, the guide nucleic acid, being at least partially complementary to the donor strand or at least partially complementary to a genomic flap, brings the donor strand to close proximity of the cleaving of the genomic locus. In some aspects, the guide nucleic acid comprises at least one nucleic acid modification. In some aspect, the at least nucleic acid modification comprises modifying a backbone, a sugar, a base, or a combination thereof of the guide nucleic acid. In some aspects, the at least one nucleic acid modification can increase resistance of the guide nucleic acid to degradation (e.g., against nuclease degradation or hydrolysis). In some aspects, the at least one nucleic acid modification can increase the complexing of the guide nucleic acid to the at least one endonuclease. In some aspects, the at least one nucleic acid modification can increase the complexing of the guide nucleic acid to the donor strand. In some aspects, the at least one nucleic acid modification can increase the complexing of the guide nucleic acid to the genomic locus via by being complementary to the genomic flap. 
     In some aspects, the system comprises one, two, three, or more ligase. In some aspects, the system comprises one ligase. In some aspects, the one ligase is operatively coupled with at least one endonuclease, where the ligase can ligate at least one end of the donor strand to the cleaved genomic locus, thus incorporating the donor strand into the genomic locus. In some aspects, the system comprises two ligases. In some aspects, the two ligases can each be operatively coupled to a different endonuclease, where the genomic locus is cleaved at two or more locations. In such scenario, the two ligases can each ligate one end of the donor strand to the cleaved genomic locus, thus incorporating the donor strand into the genomic locus. In some aspects, the ligase comprises a ligase that can ligate a substrate comprising DNA. In some aspects, the ligase comprises a ligase that can ligate a substrate comprising a DNA splint. In some aspects, the ligase comprises a ligase that can ligate a substrate comprising a DNA/RNA. In some aspects, the ligase comprises a ligase that can ligate a substrate comprising a RNA splint. In some aspects, the ligase comprises at least one additional domain. In some aspects, the at least one additional domain is a dimerization domain. In some aspects, the ligase comprising a dimerization domain can be dimerized with a endonuclease to form a heterodimer. In some aspects, the at least one additional domain is a functional domain. For example, the functional domain can comprises a chromatin modifying domain or a cell penetrating peptide. In some aspects, the ligase comprises a linker, where the linker can covalently connect the ligase with another polypeptide (e.g., the endonuclease). In some aspects, the linker covalently connects the ligase to the at least one additional domain. In some aspects, the ligase comprises a tag, where the tag can be used for increasing expression, identifying, or purifying the ligase. 
     Disclosed herein are fusion proteins comprising: an RNA-guided endonuclease fused to a ligase. Table 9 illustrates non-limiting examples of polypeptide and nucleic acid sequences encoding a fusion polypeptide comprising components (e.g., a endonuclease fused to a ligase) of a system described herein. SEQ ID NO: 125 illustrates a nucleic acid sequence encoding the polypeptide sequence of SEQ ID NO: 126, where SEQ ID NO: 126 illustrates a fusion protein (NLS-nCas9-linker-hLIG1(119-919)-bpNLS) comprising a N-terminus NLS followed by a endonuclease (nCas9) covalently connected to a ligase (hLIG1, 119-919 fragment) via a linker followed by a C-terminus NLS. SEQ ID NO: 127 illustrates a nucleic acid sequence encoding the polypeptide sequence of SEQ ID NO: 128, where SEQ ID NO: 128 illustrates a fusion protein (NLS-nCas9-linker-hLIG1(233-919)-bpNLS) comprising a N-terminus NLS followed by a endonuclease (nCas9) covalently connected to a ligase (hLIG1, 233-919 fragment) via a linker followed by a C-terminus NLS. SEQ ID NO: 129 illustrates a nucleic acid sequence encoding the polypeptide sequence of SEQ ID NO: 130, where SEQ ID NO: 130 illustrates a fusion protein (NLS-nCas9-linker-SplintR-bpNLS) comprising a N-terminus NLS followed by a endonuclease (nCas9) covalently connected to a ligase (SplintR) via a linker followed by a C-terminus NLS. SEQ ID NO: 13 illustrates a nucleic acid sequence encoding the polypeptide sequence of SEQ ID NO: 132, where SEQ ID NO: 132 illustrates a fusion protein (NLS-nCas9-linker-T4LIG-bpNLS) comprising a N-terminus NLS followed by a endonuclease (nCas9) covalently connected to a ligase (T4LIG) via a linker followed by a C-terminus NLS. SEQ ID NO: 133 illustrates a nucleic acid sequence encoding a endonuclease (nCas9) comprising a N-terminus NLS and a leucine zipper (LZ) dimerization domain. SEQ ID NO: 134 illustrates a fusion protein (NLS1-hFEN1-linker1-nCas9-linker2-T4LIG-NLS2) comprising first NLS (NLS1) at N-terminus followed by a exonuclease (hFEN1) covalently connected to a endonuclease (nCas9) via linker1 and further covalently connected to a ligase (T4LIG) via linker 2 followed by a second NLS (NLS2) at C-terminus. SEQ ID NO: 135 illustrates a fusion protein (NLS1-hFEN1-linker1-T4LIG-linker2-nCas9-NLS2) comprising a N-terminus NLS1 followed by a exonuclease (hFEN1) covalently connected to a ligase (T4LIG) via linker 1 and further covalently connected to a endonuclease (nCas9) via linker 2 followed by a C-terminus NLS2. SEQ ID NO: 136 illustrates a fusion protein (NLS1-nCas9-linker1-hFEN1-linker2-T4LIG-NLS2) comprising a N-terminus NLS1 followed by a endonuclease (nCas9) covalently connected to a exonuclease (hFEN1) via linker 1 and further covalently connected to a ligase (T4LIG) via linker 2 followed by a C-terminus NLS2. SEQ ID NO: 137 illustrates a fusion protein (NLS1-T4LIG-linker1-nCas9-linker2-hFEN1-NLS2) comprising a N-terminus NLS1 followed by a ligase (T4LIG) covalently connected to a endonuclease (nCas9) via linker 1 and further covalently connected to a exonuclease (hFEN1) via linker 2 followed by a C-terminus NLS2. SEQ ID NO: 138 illustrates a fusion protein (NLS1-nCas9-linker1-T4LIG-linker2-hFEN1-NLS2) comprising a N-terminus NLS1 followed by a endonuclease (nCas9) covalently connected to a ligase (T4LIG) via linker 1 and further covalently connected to a exonuclease (hFEN1) via linker 2 followed by a C-terminus NLS2. SEQ ID NO: 139 illustrates a fusion protein (NLS1-T4LIG-linker1-hFEN1-linker2-nCas9-NLS2) comprising a N-terminus NLS1 followed by a ligase (T4LIG) covalently connected to a exonuclease (hFEN1) via linker 1 and further covalently connected to a endonuclease (nCas9) via linker 2 followed by a C-terminus NLS2. SEQ ID NO: 140 illustrates a fusion protein (NLS1-T5 EXO-linker1-nCas9-linker2-T4LIG-NLS2) comprising a N-terminus NLS1 followed by a exonuclease (EXO) covalently connected to a endonuclease (nCas9) via linker 1 and further covalently connected to a ligase (T4LIG) via linker 2 followed by a C-terminus NLS2. SEQ ID NO: 141 illustrates a nucleic acid sequence encoding a fusion protein (LZ-SplintR-bpNLS) comprising a ligase (SplintR) fused to a dimerization domain (LZ) and a NLS. SEQ ID NO: 142 illustrates a nucleic acid sequence encoding a fusion protein (LZ-T4LIG-bpNLS) comprising a ligase (T4LIG) fused to a dimerization domain (LZ) and a NLS. SEQ ID NO: 143 illustrates a nucleic acid sequence encoding a fusion protein (LZ-hLIG 233-919 polypeptide fragment-bpNLS) comprising a ligase (hLIG) fused to a dimerization domain (LZ) and a NLS. SEQ ID NO: 144 illustrates a nucleic acid sequence encoding a fusion protein (LZ-hLIG1 119-919 polypeptide fragment-bpNLS) comprising a ligase (hLIG) fused to a dimerization domain (LZ) and a NLS. SEQ ID NO: 145 illustrates a nucleic acid sequence encoding a fusion protein (T4-LZ) comprising a ligase (T4) fused to a dimerization domain (LZ) and a NLS. SEQ ID NO: 146 illustrates a nucleic acid sequence encoding a fusion protein (LZ-hLIG4(1-620)) comprising a ligase polypeptide fragment (hLIG4(1-620)) fused to a dimerization domain (LZ) and a NLS. SEQ ID NO: 147 illustrates a nucleic acid sequence encoding a fusion protein (LZ-nCas9) comprising an endonuclease (nCas9) fused to a dimerization domain (LZ) and a NLS. SEQ ID NO: 148 illustrates a nucleic acid sequence encoding a fusion protein (SplintR-LZ) comprising a ligase (SplintR) fused to a dimerization domain (LZ) and a NLS. SEQ ID NO: 149 illustrates a nucleic acid sequence encoding a fusion protein (hLIG4(1-620)-LZ) comprising a ligase polypeptide fragment (hLIG4(1-620)) fused to a dimerization domain (LZ) and a NLS. SEQ ID NO: 150 illustrates a nucleic acid sequence encoding a fusion protein (nCas9-hLIG4(1-620)) comprising a ligase polypeptide fragment (hLIG4(1-620)) fused to an endonuclease (nCas9) and a NLS. SEQ ID NO: 151 illustrates a nucleic acid sequence encoding a fusion protein (T4-nCas9) comprising a ligase (T4) fused to an endonuclease (nCas9) and a NLS. SEQ ID NO: 152 illustrates a nucleic acid sequence encoding a fusion protein (SplintR-nCas9) comprising a ligase (SplintR) fused to an endonuclease (nCas9) and a NLS. SEQ ID NO: 153 illustrates a nucleic acid sequence encoding a fusion protein (hLIG4(1-620)-nCas9) comprising a ligase polypeptide fragment (hLIG4(1-620)) fused to an endonuclease (nCas9) and a NLS. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Non-limiting examples of fusion protein polypeptide sequence or 
               
               
                 nucleic acid sequence encoding the fusion protein 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ 
               
               
                 Name 
                 Fusion protein polypeptide sequence or nucleic acid sequence 
                 ID NO: 
               
               
                   
               
               
                 NLS- 
                 atgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcg 
                 125 
               
               
                 nCas9- 
                 gaaagtcgacaagaagtacagcatcggcctggacatcggcaccaactctg 
                   
               
               
                 linker- 
                 tgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattc 
                   
               
               
                 hLIG1(119 
                 aaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcgg 
                   
               
               
                 -919)- 
                 agccctgctgttcgacagcggcgaaacagccgaggccacccggctgaaga 
                   
               
               
                 bpNLS 
                 gaaccgccagaagaagatacaccagacggaagaaccggatctgctatctg 
                   
               
               
                   
                 caagagatcttcagcaacgagatggccaaggtggacgacagcttcttcca 
                   
               
               
                   
                 cagactggaagagtccttcctggtggaagaggataagaagcacgagcggc 
                   
               
               
                   
                 accccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtac 
                   
               
               
                   
                 cccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggc 
                   
               
               
                   
                 cgacctgcggctgatctatctggccctggcccacatgatcaagttccggg 
                   
               
               
                   
                 gccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggac 
                   
               
               
                   
                 aagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaa 
                   
               
               
                   
                 ccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagac 
                   
               
               
                   
                 tgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgag 
                   
               
               
                   
                 aagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgac 
                   
               
               
                   
                 ccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagc 
                   
               
               
                   
                 tgagcaaggacacctacgacgacgacctggacaacctgctggcccagatc 
                   
               
               
                   
                 ggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgc 
                   
               
               
                   
                 catcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggccc 
                   
               
               
                   
                 ccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctg 
                   
               
               
                   
                 accctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaaga 
                   
               
               
                   
                 gattttcttcgaccagagcaagaacggctacgccggctacattgacggcg 
                   
               
               
                   
                 gagccagccaggaagagttctacaagttcatcaagcccatcctggaaaag 
                   
               
               
                   
                 atggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgct 
                   
               
               
                   
                 gcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacc 
                   
               
               
                   
                 tgggagagctgcacgccattctgcggcggcaggaagatttttacccattc 
                   
               
               
                   
                 ctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatccc 
                   
               
               
                   
                 ctactacgtgggccctctggccaggggaaacagcagattcgcctggatga 
                   
               
               
                   
                 ccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtg 
                   
               
               
                   
                 gacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcga 
                   
               
               
                   
                 taagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacg 
                   
               
               
                   
                 agtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgag 
                   
               
               
                   
                 ggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgt 
                   
               
               
                   
                 ggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaag 
                   
               
               
                   
                 aggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggc 
                   
               
               
                   
                 gtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaa 
                   
               
               
                   
                 aattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattc 
                   
               
               
                   
                 tggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatc 
                   
               
               
                   
                 gaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaa 
                   
               
               
                   
                 gcagctgaagcggcggagatacaccggctggggcaggctgagccggaagc 
                   
               
               
                   
                 tgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttc 
                   
               
               
                   
                 ctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacga 
                   
               
               
                   
                 cgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggcc 
                   
               
               
                   
                 agggcgatagcctgcacgagcacattgccaatctggccggcagccccgcc 
                   
               
               
                   
                 attaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaa 
                   
               
               
                   
                 agtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagag 
                   
               
               
                   
                 agaaccagaccacccagaagggacagaagaacagccgcgagagaatgaag 
                   
               
               
                   
                 cggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaaca 
                   
               
               
                   
                 ccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacc 
                   
               
               
                   
                 tgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccgg 
                   
               
               
                   
                 ctgtccgactacgatgtggacgctatcgtgcctcagagctttctgaagga 
                   
               
               
                   
                 cgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggca 
                   
               
               
                   
                 agagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactac 
                   
               
               
                   
                 tggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaa 
                   
               
               
                   
                 tctgaccaaggccgagagaggcggcctgagcgaactggataaggccggct 
                   
               
               
                   
                 tcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggca 
                   
               
               
                   
                 cagatcctggactcccggatgaacactaagtacgacgagaatgacaagct 
                   
               
               
                   
                 gatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatt 
                   
               
               
                   
                 tccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccac 
                   
               
               
                   
                 cacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaa 
                   
               
               
                   
                 aaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgt 
                   
               
               
                   
                 acgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggct 
                   
               
               
                   
                 accgccaagtacttcttctacagcaacatcatgaactttttcaagaccga 
                   
               
               
                   
                 gattaccctggccaacggcgagatccggaagcggcctctgatcgagacaa 
                   
               
               
                   
                 acggcgaaaccggggagatcgtgtgggataagggccgggattttgccacc 
                   
               
               
                   
                 gtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccga 
                   
               
               
                   
                 ggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaaca 
                   
               
               
                   
                 gcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggc 
                   
               
               
                   
                 ggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagt 
                   
               
               
                   
                 ggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctgggga 
                   
               
               
                   
                 tcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctg 
                   
               
               
                   
                 gaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcc 
                   
               
               
                   
                 taagtactccctgttcgagctggaaaacggccggaagagaatgctggcct 
                   
               
               
                   
                 ctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatat 
                   
               
               
                   
                 gtgaacttcctgtacctggccagccactatgagaagctgaagggctcccc 
                   
               
               
                   
                 cgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacc 
                   
               
               
                   
                 tggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctg 
                   
               
               
                   
                 gccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccggga 
                   
               
               
                   
                 taagcccatcagagagcaggccgagaatatcatccacctgtttaccctga 
                   
               
               
                   
                 ccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgac 
                   
               
               
                   
                 cggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatcca 
                   
               
               
                   
                 ccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgg 
                   
               
               
                   
                 gaggtgacTCCGGCGGAAGCTCTGGTGGCAGCAAGCGGACCGCCGACGGC 
                   
               
               
                   
                 TCTGAATTCGAGAGCCCTAAGAAGAAAAGAAAGGTGAGCGGAGGCTCTAG 
                   
               
               
                   
                 CGGCGGAAGCCCGAAGCGCCGGACTGCACGAAAGCAACTGCCAAAACGGA 
                   
               
               
                   
                 CTATACAAGAAGTCCTGGAAGAACAAAGCGAAGATGAGGATCGCGAAGCC 
                   
               
               
                   
                 AAGCGCAAGAAAGAGGAAGAGGAAGAAGAGACTCCAAAGGAGTCCTTGAC 
                   
               
               
                   
                 CGAAGCAGAAGTCGCAACGGAGAAGGAAGGTGAGGATGGGGATCAGCCAA 
                   
               
               
                   
                 CAACCCCGCCTAAACCTCTGAAAACCTCTAAGGCGGAGACACCAACTGAG 
                   
               
               
                   
                 AGTGTCAGCGAACCGGAGGTAGCCACGAAACAAGAGCTTCAGGAGGAAGA 
                   
               
               
                   
                 AGAACAGACAAAGCCACCTCGGCGGGCTCCCAAAACCCTTAGCTCCTTCT 
                   
               
               
                   
                 TCACGCCTCGAAAGCCAGCAGTGAAGAAAGAAGTGAAGGAGGAGGAACCT 
                   
               
               
                   
                 GGCGCCCCTGGAAAGGAGGGCGCAGCCGAGGGCCCGCTGGACCCTTCAGG 
                   
               
               
                   
                 GTATAACCCGGCAAAAAATAATTACCACCCGGTCGAGGACGCTTGTTGGA 
                   
               
               
                   
                 AACCAGGCCAAAAGGTACCTTACCTCGCCGTCGCTAGGACCTTTGAGAAG 
                   
               
               
                   
                 ATAGAGGAAGTTAGTGCTAGGTTGAGAATGGTCGAAACCCTTAGTAACCT 
                   
               
               
                   
                 TCTCAGGTCCGTAGTCGCCCTTAGTCCCCCAGACCTGCTTCCGGTGCTGT 
                   
               
               
                   
                 ACCTGTCCCTGAACCATCTCGGTCCCCCCCAACAGGGACTGGAGTTGGGC 
                   
               
               
                   
                 GTCGGTGACGGCGTTCTCCTGAAAGCGGTTGCACAAGCTACAGGAAGGCA 
                   
               
               
                   
                 ACTGGAATCTGTCCGGGCTGAGGCTGCAGAGAAAGGTGACGTGGGGCTTG 
                   
               
               
                   
                 TGGCAGAGAATAGTCGGTCAACACAGCGGCTGATGCTGCCACCGCCCCCG 
                   
               
               
                   
                 CTTACGGCTAGTGGGGTATTCTCCAAATTTAGAGATATAGCACGGCTGAC 
                   
               
               
                   
                 GGGATCAGCTTCCACTGCGAAGAAGATCGATATCATTAAGGGTTTGTTCG 
                   
               
               
                   
                 TGGCTTGCAGGCATTCCGAAGCACGCTTCATTGCACGCTCCCTTTCAGGG 
                   
               
               
                   
                 AGACTCAGACTTGGGCTGGCCGAGCAATCTGTACTGGCGGCCCTGTCTCA 
                   
               
               
                   
                 GGCGGTGAGCCTTACGCCGCCCGGGCAAGAGTTCCCTCCTGCGATGGTCG 
                   
               
               
                   
                 ATGCTGGGAAGGGAAAAACCGCCGAAGCTCGAAAAACATGGCTGGAGGAG 
                   
               
               
                   
                 CAAGGAATGATTTTGAAGCAGACGTTCTGTGAAGTACCGGACTTGGATCG 
                   
               
               
                   
                 CATCATACCTGTGCTTCTCGAACATGGTTTGGAGCGGCTCCCCGAGCATT 
                   
               
               
                   
                 GCAAACTCTCTCCGGGCATCCCCCTCAAGCCAATGCTCGCCCACCCCACG 
                   
               
               
                   
                 CGCGGAATCAGTGAGGTACTGAAACGCTTTGAAGAGGCAGCGTTTACTTG 
                   
               
               
                   
                 TGAATACAAGTACGATGGCCAAAGGGCACAAATTCATGCACTTGAAGGCG 
                   
               
               
                   
                 GGGAAGTTAAGATATTCAGCAGGAATCAGGAGGACAACACGGGAAAATAT 
                   
               
               
                   
                 CCTGACATAATATCTCGAATCCCTAAAATTAAGTTGCCTAGCGTAACCAG 
                   
               
               
                   
                 CTTCATCCTGGATACCGAAGCCGTGGCGTGGGATAGGGAGAAAAAGCAAA 
                   
               
               
                   
                 TACAGCCATTTCAGGTGCTTACAACTAGAAAACGAAAAGAGGTGGACGCT 
                   
               
               
                   
                 AGTGAAATCCAAGTCCAGGTATGTCTTTATGCCTTCGATTTGATATACCT 
                   
               
               
                   
                 TAATGGTGAGTCCCTTGTACGGGAACCGCTTAGTAGGAGGCGGCAGTTGC 
                   
               
               
                   
                 TGAGGGAAAATTTTGTCGAAACTGAGGGAGAGTTTGTATTTGCAACGTCA 
                   
               
               
                   
                 TTGGATACAAAGGACATAGAACAAATAGCAGAATTTCTGGAGCAGTCAGT 
                   
               
               
                   
                 AAAAGACTCCTGCGAGGGCCTGATGGTGAAAACTCTTGATGTGGACGCCA 
                   
               
               
                   
                 CTTATGAAATCGCAAAAAGGTCACACAATTGGCTGAAACTTAAAAAGGAT 
                   
               
               
                   
                 TACTTGGACGGGGTCGGGGATACCCTCGATCTCGTCGTAATCGGAGCTTA 
                   
               
               
                   
                 TCTCGGTAGGGGGAAGCGAGCCGGGCGATACGGAGGCTTTCTCTTGGCTA 
                   
               
               
                   
                 GTTATGACGAAGATTCCGAAGAGCTGCAGGCCATATGCAAGCTTGGAACG 
                   
               
               
                   
                 GGTTTCAGCGATGAGGAATTGGAGGAGCATCATCAGAGCTTGAAGGCACT 
                   
               
               
                   
                 GGTGCTCCCCTCTCCTAGGCCGTACGTTAGAATAGACGGAGCAGTGATAC 
                   
               
               
                   
                 CCGATCATTGGCTCGATCCGTCAGCTGTTTGGGAGGTGAAGTGTGCAGAC 
                   
               
               
                   
                 CTGTCCCTCTCTCCTATTTACCCTGCAGCACGCGGTCTGGTTGACTCTGA 
                   
               
               
                   
                 CAAAGGGATTAGCTTGAGGTTCCCTAGATTTATTCGGGTGCGCGAAGATA 
                   
               
               
                   
                 AACAGCCTGAACAGGCGACAACGTCCGCGCAGGTCGCATGCCTTTATCGA 
                   
               
               
                   
                 AAACAGAGTCAGATCCAGAATCAACAAGGAGAAGATTCAGGGAGTGACCC 
                   
               
               
                   
                 GGAGGACACTTATAGTGGCGGCTCAAAACGAACCGCCGATAGTCAGCATT 
                   
               
               
                   
                 CAACACCTCCAAAAACTAAAAGGAAAGTCGAGTTTGAGCCAAAGAAGAAG 
                   
               
               
                   
                 CGCAAAGTCTAA 
                   
               
               
                   
               
               
                 NLS- 
                 MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKF 
                 126 
               
               
                 nCas9- 
                 KVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL 
                   
               
               
                 linker- 
                 QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY 
                   
               
               
                 hLIG1(119 
                 PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVD 
                   
               
               
                 -919)- 
                 KLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGE 
                   
               
               
                 bpNLS 
                 KKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI 
                   
               
               
                   
                 GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDL 
                   
               
               
                   
                 TLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEK 
                   
               
               
                   
                 MDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPF 
                   
               
               
                   
                 LKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV 
                   
               
               
                   
                 DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE 
                   
               
               
                   
                 GMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG 
                   
               
               
                   
                 VEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI 
                   
               
               
                   
                 EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF 
                   
               
               
                   
                 LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA 
                   
               
               
                   
                 IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMK 
                   
               
               
                   
                 RIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR 
                   
               
               
                   
                 LSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY 
                   
               
               
                   
                 WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVA 
                   
               
               
                   
                 QILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYH 
                   
               
               
                   
                 HAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKA 
                   
               
               
                   
                 TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
                   
               
               
                   
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYG 
                   
               
               
                   
                 GFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFL 
                   
               
               
                   
                 EAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKY 
                   
               
               
                   
                 VNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL 
                   
               
               
                   
                 ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTID 
                   
               
               
                   
                 RKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSKRTADG 
                   
               
               
                   
                 SEFESPKKKRKVSGGSSGGSPKRRTARKQLPKRTIQEVLEEQSEDEDREA 
                   
               
               
                   
                 KRKKEEEEEETPKESLTEAEVATEKEGEDGDQPTTPPKPLKTSKAETPTE 
                   
               
               
                   
                 SVSEPEVATKQELQEEEEQTKPPRRAPKTLSSFFTPRKPAVKKEVKEEEP 
                   
               
               
                   
                 GAPGKEGAAEGPLDPSGYNPAKNNYHPVEDACWKPGQKVPYLAVARTFEK 
                   
               
               
                   
                 IEEVSARLRMVETLSNLLRSVVALSPPDLLPVLYLSLNHLGPPQQGLELG 
                   
               
               
                   
                 VGDGVLLKAVAQATGRQLESVRAEAAEKGDVGLVAENSRSTQRLMLPPPP 
                   
               
               
                   
                 LTASGVFSKFRDIARLTGSASTAKKIDIIKGLFVACRHSEARFIARSLSG 
                   
               
               
                   
                 RLRLGLAEQSVLAALSQAVSLTPPGQEFPPAMVDAGKGKTAEARKTWLEE 
                   
               
               
                   
                 QGMILKQTFCEVPDLDRIIPVLLEHGLERLPEHCKLSPGIPLKPMLAHPT 
                   
               
               
                   
                 RGISEVLKRFEEAAFTCEYKYDGQRAQIHALEGGEVKIFSRNQEDNTGKY 
                   
               
               
                   
                 PDIISRIPKIKLPSVTSFILDTEAVAWDREKKQIQPFQVLTTRKRKEVDA 
                   
               
               
                   
                 SEIQVQVCLYAFDLIYLNGESLVREPLSRRRQLLRENFVETEGEFVFATS 
                   
               
               
                   
                 LDTKDIEQIAEFLEQSVKDSCEGLMVKTLDVDATYEIAKRSHNWLKLKKD 
                   
               
               
                   
                 YLDGVGDTLDLVVIGAYLGRGKRAGRYGGFLLASYDEDSEELQAICKLGT 
                   
               
               
                   
                 GFSDEELEEHHQSLKALVLPSPRPYVRIDGAVIPDHWLDPSAVWEVKCAD 
                   
               
               
                   
                 LSLSPIYPAARGLVDSDKGISLRFPRFIRVREDKQPEQATTSAQVACLYR 
                   
               
               
                   
                 KQSQIQNQQGEDSGSDPEDTYSGGSKRTADSQHSTPPKTKRKVEFEPKKK 
                   
               
               
                   
                 RKV* 
                   
               
               
                   
               
               
                 NLS- 
                 atgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcg 
                 127 
               
               
                 nCas9- 
                 gaaagtcgacaagaagtacagcatcggcctggacatcggcaccaactctg 
                   
               
               
                 linker- 
                 tgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattc 
                   
               
               
                 hLIG1(233 
                 aaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcgg 
                   
               
               
                 -919)- 
                 agccctgctgttcgacagcggcgaaacagccgaggccacccggctgaaga 
                   
               
               
                 bpNLS 
                 gaaccgccagaagaagatacaccagacggaagaaccggatctgctatctg 
                   
               
               
                   
                 caagagatcttcagcaacgagatggccaaggtggacgacagcttcttcca 
                   
               
               
                   
                 cagactggaagagtccttcctggtggaagaggataagaagcacgagcggc 
                   
               
               
                   
                 accccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtac 
                   
               
               
                   
                 cccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggc 
                   
               
               
                   
                 cgacctgcggctgatctatctggccctggcccacatgatcaagttccggg 
                   
               
               
                   
                 gccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggac 
                   
               
               
                   
                 aagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaa 
                   
               
               
                   
                 ccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagac 
                   
               
               
                   
                 tgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgag 
                   
               
               
                   
                 aagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgac 
                   
               
               
                   
                 ccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagc 
                   
               
               
                   
                 tgagcaaggacacctacgacgacgacctggacaacctgctggcccagatc 
                   
               
               
                   
                 ggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgc 
                   
               
               
                   
                 catcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggccc 
                   
               
               
                   
                 ccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctg 
                   
               
               
                   
                 accctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaaga 
                   
               
               
                   
                 gattttcttcgaccagagcaagaacggctacgccggctacattgacggcg 
                   
               
               
                   
                 gagccagccaggaagagttctacaagttcatcaagcccatcctggaaaag 
                   
               
               
                   
                 atggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgct 
                   
               
               
                   
                 gcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacc 
                   
               
               
                   
                 tgggagagctgcacgccattctgcggcggcaggaagatttttacccattc 
                   
               
               
                   
                 ctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatccc 
                   
               
               
                   
                 ctactacgtgggccctctggccaggggaaacagcagattcgcctggatga 
                   
               
               
                   
                 ccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtg 
                   
               
               
                   
                 gacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcga 
                   
               
               
                   
                 taagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacg 
                   
               
               
                   
                 agtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgag 
                   
               
               
                   
                 ggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgt 
                   
               
               
                   
                 ggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaag 
                   
               
               
                   
                 aggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggc 
                   
               
               
                   
                 gtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaa 
                   
               
               
                   
                 aattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattc 
                   
               
               
                   
                 tggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatc 
                   
               
               
                   
                 gaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaa 
                   
               
               
                   
                 gcagctgaagcggcggagatacaccggctggggcaggctgagccggaagc 
                   
               
               
                   
                 tgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttc 
                   
               
               
                   
                 ctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacga 
                   
               
               
                   
                 cgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggcc 
                   
               
               
                   
                 agggcgatagcctgcacgagcacattgccaatctggccggcagccccgcc 
                   
               
               
                   
                 attaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaa 
                   
               
               
                   
                 agtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagag 
                   
               
               
                   
                 agaaccagaccacccagaagggacagaagaacagccgcgagagaatgaag 
                   
               
               
                   
                 cggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaaca 
                   
               
               
                   
                 ccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacc 
                   
               
               
                   
                 tgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccgg 
                   
               
               
                   
                 ctgtccgactacgatgtggacgctatcgtgcctcagagctttctgaagga 
                   
               
               
                   
                 cgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggca 
                   
               
               
                   
                 agagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactac 
                   
               
               
                   
                 tggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaa 
                   
               
               
                   
                 tctgaccaaggccgagagaggcggcctgagcgaactggataaggccggct 
                   
               
               
                   
                 tcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggca 
                   
               
               
                   
                 cagatcctggactcccggatgaacactaagtacgacgagaatgacaagct 
                   
               
               
                   
                 gatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatt 
                   
               
               
                   
                 tccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccac 
                   
               
               
                   
                 cacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaa 
                   
               
               
                   
                 aaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgt 
                   
               
               
                   
                 acgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggct 
                   
               
               
                   
                 accgccaagtacttcttctacagcaacatcatgaactttttcaagaccga 
                   
               
               
                   
                 gattaccctggccaacggcgagatccggaagcggcctctgatcgagacaa 
                   
               
               
                   
                 acggcgaaaccggggagatcgtgtgggataagggccgggattttgccacc 
                   
               
               
                   
                 gtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccga 
                   
               
               
                   
                 ggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaaca 
                   
               
               
                   
                 gcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggc 
                   
               
               
                   
                 ggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagt 
                   
               
               
                   
                 ggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctgggga 
                   
               
               
                   
                 tcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctg 
                   
               
               
                   
                 gaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcc 
                   
               
               
                   
                 taagtactccctgttcgagctggaaaacggccggaagagaatgctggcct 
                   
               
               
                   
                 ctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatat 
                   
               
               
                   
                 gtgaacttcctgtacctggccagccactatgagaagctgaagggctcccc 
                   
               
               
                   
                 cgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacc 
                   
               
               
                   
                 tggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctg 
                   
               
               
                   
                 gccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccggga 
                   
               
               
                   
                 taagcccatcagagagcaggccgagaatatcatccacctgtttaccctga 
                   
               
               
                   
                 ccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgac 
                   
               
               
                   
                 cggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatcca 
                   
               
               
                   
                 ccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgg 
                   
               
               
                   
                 gaggtgacTCCGGCGGAAGCTCTGGTGGCAGCAAGCGGACCGCCGACGGC 
                   
               
               
                   
                 TCTGAATTCGAGAGCCCTAAGAAGAAAAGAAAGGTGAGCGGAGGCTCTAG 
                   
               
               
                   
                 CGGCGGAAGCACACCCAGGAAACCAGCCGTGAAAAAAGAGGTTAAAGAAG 
                   
               
               
                   
                 AGGAACCTGGGGCTCCGGGAAAGGAGGGAGCAGCGGAAGGTCCGCTCGAC 
                   
               
               
                   
                 CCTTCAGGATACAACCCAGCCAAAAACAACTACCACCCCGTAGAGGATGC 
                   
               
               
                   
                 TTGCTGGAAGCCAGGCCAAAAGGTGCCCTATTTGGCCGTTGCTAGGACTT 
                   
               
               
                   
                 TCGAAAAAATTGAGGAGGTGAGCGCGCGACTCAGAATGGTAGAGACTCTG 
                   
               
               
                   
                 TCTAACCTCCTTCGCTCCGTAGTGGCTCTTTCACCTCCAGATCTTCTTCC 
                   
               
               
                   
                 AGTGCTGTACCTGAGCCTGAACCACTTGGGCCCTCCCCAGCAGGGACTGG 
                   
               
               
                   
                 AACTGGGCGTAGGGGACGGAGTATTGCTGAAGGCTGTTGCTCAGGCAACC 
                   
               
               
                   
                 GGACGACAGCTCGAGTCTGTGCGAGCAGAAGCTGCAGAAAAGGGGGACGT 
                   
               
               
                   
                 CGGGTTGGTTGCCGAAAATTCAAGATCTACCCAACGATTGATGTTGCCAC 
                   
               
               
                   
                 CGCCGCCTCTGACTGCGTCAGGTGTATTCTCCAAGTTCCGGGATATTGCC 
                   
               
               
                   
                 AGGCTTACGGGTAGCGCTTCCACTGCTAAAAAGATCGACATAATAAAAGG 
                   
               
               
                   
                 TCTGTTCGTCGCTTGTCGCCATTCAGAGGCGAGGTTTATAGCCAGATCCC 
                   
               
               
                   
                 TTTCCGGACGACTTCGACTCGGCTTGGCTGAGCAGTCAGTACTGGCAGCT 
                   
               
               
                   
                 TTGTCTCAAGCTGTATCACTCACGCCCCCCGGACAAGAATTTCCACCCGC 
                   
               
               
                   
                 CATGGTTGACGCAGGCAAGGGTAAGACTGCTGAGGCAAGAAAGACGTGGC 
                   
               
               
                   
                 TGGAGGAACAAGGTATGATACTTAAACAAACGTTTTGCGAAGTTCCGGAC 
                   
               
               
                   
                 TTGGACCGGATCATACCTGTGTTGCTGGAGCACGGCCTCGAGCGCTTGCC 
                   
               
               
                   
                 CGAACACTGTAAACTGTCTCCAGGAATACCTCTCAAACCCATGTTGGCTC 
                   
               
               
                   
                 ATCCTACGAGGGGAATCTCAGAGGTACTTAAACGGTTTGAAGAAGCCGCT 
                   
               
               
                   
                 TTCACGTGCGAATACAAGTATGATGGTCAGAGAGCGCAAATCCACGCATT 
                   
               
               
                   
                 GGAAGGGGGTGAGGTAAAGATTTTTTCAAGGAATCAGGAGGACAATACAG 
                   
               
               
                   
                 GGAAGTACCCCGATATCATCAGTCGGATTCCTAAAATTAAGCTTCCATCA 
                   
               
               
                   
                 GTCACGTCCTTCATACTGGACACTGAGGCAGTGGCTTGGGACCGAGAGAA 
                   
               
               
                   
                 GAAGCAGATACAACCCTTTCAGGTACTTACAACCAGAAAGCGCAAGGAAG 
                   
               
               
                   
                 TCGACGCTTCTGAGATTCAAGTACAAGTCTGCCTTTATGCGTTTGACCTG 
                   
               
               
                   
                 ATCTATCTTAATGGAGAGAGTTTGGTGAGAGAACCCTTGAGCAGACGACG 
                   
               
               
                   
                 GCAGCTCTTGAGAGAAAATTTCGTAGAAACTGAGGGGGAGTTCGTCTTTG 
                   
               
               
                   
                 CGACTAGTCTCGACACCAAAGACATTGAGCAAATCGCGGAATTCCTCGAA 
                   
               
               
                   
                 CAGTCAGTTAAAGACTCCTGCGAAGGTCTGATGGTTAAGACTCTTGACGT 
                   
               
               
                   
                 GGATGCTACCTACGAGATAGCTAAGCGGTCACACAATTGGCTGAAACTGA 
                   
               
               
                   
                 AAAAGGACTATCTGGATGGAGTTGGGGACACGCTGGATTTGGTCGTTATC 
                   
               
               
                   
                 GGGGCCTATCTGGGACGCGGTAAGCGGGCAGGGAGATATGGTGGATTCCT 
                   
               
               
                   
                 CCTCGCTTCATACGATGAGGACTCTGAAGAGCTGCAGGCTATATGCAAAC 
                   
               
               
                   
                 TTGGGACGGGTTTTTCCGATGAAGAATTGGAGGAACATCATCAGTCACTG 
                   
               
               
                   
                 AAGGCCCTTGTATTGCCAAGTCCACGCCCATACGTACGAATCGATGGAGC 
                   
               
               
                   
                 AGTAATCCCTGACCACTGGCTTGACCCGTCCGCCGTCTGGGAAGTAAAGT 
                   
               
               
                   
                 GCGCGGATCTCTCTCTCAGTCCGATCTACCCAGCCGCACGGGGGCTGGTT 
                   
               
               
                   
                 GACAGTGACAAGGGTATCAGCCTGCGATTTCCTCGATTCATACGCGTCCG 
                   
               
               
                   
                 GGAAGACAAGCAACCGGAACAGGCTACGACCTCTGCACAGGTCGCATGTT 
                   
               
               
                   
                 TGTATAGAAAACAGAGCCAAATTCAGAATCAACAAGGCGAAGACAGTGGG 
                   
               
               
                   
                 TCCGATCCTGAAGATACCTACTCAGGCGGCAGTAAACGGACAGCTGATAG 
                   
               
               
                   
                 CCAACACTCAACTCCTCCGAAGACTAAAAGGAAGGTAGAGTTCGAACCAA 
                   
               
               
                   
                 AAAAGAAAAGGAAAGTGTAA 
                   
               
               
                   
               
               
                 NLS- 
                 MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKF 
                 128 
               
               
                 nCas9- 
                 KVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL 
                   
               
               
                 linker- 
                 QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY 
                   
               
               
                 hLIG1(233 
                 PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVD 
                   
               
               
                 -919)- 
                 KLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGE 
                   
               
               
                 bpNLS 
                 KKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI 
                   
               
               
                   
                 GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDL 
                   
               
               
                   
                 TLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEK 
                   
               
               
                   
                 MDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPF 
                   
               
               
                   
                 LKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV 
                   
               
               
                   
                 DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE 
                   
               
               
                   
                 GMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG 
                   
               
               
                   
                 VEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI 
                   
               
               
                   
                 EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF 
                   
               
               
                   
                 LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA 
                   
               
               
                   
                 IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMK 
                   
               
               
                   
                 RIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR 
                   
               
               
                   
                 LSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY 
                   
               
               
                   
                 WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVA 
                   
               
               
                   
                 QILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYH 
                   
               
               
                   
                 HAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKA 
                   
               
               
                   
                 TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
                   
               
               
                   
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYG 
                   
               
               
                   
                 GFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFL 
                   
               
               
                   
                 EAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKY 
                   
               
               
                   
                 VNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL 
                   
               
               
                   
                 ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTID 
                   
               
               
                   
                 RKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSKRTADG 
                   
               
               
                   
                 SEFESPKKKRKVSGGSSGGSTPRKPAVKKEVKEEEPGAPGKEGAAEGPLD 
                   
               
               
                   
                 PSGYNPAKNNYHPVEDACWKPGQKVPYLAVARTFEKIEEVSARLRMVETL 
                   
               
               
                   
                 SNLLRSVVALSPPDLLPVLYLSLNHLGPPQQGLELGVGDGVLLKAVAQAT 
                   
               
               
                   
                 GRQLESVRAEAAEKGDVGLVAENSRSTQRLMLPPPPLTASGVFSKFRDIA 
                   
               
               
                   
                 RLTGSASTAKKIDIIKGLFVACRHSEARFIARSLSGRLRLGLAEQSVLAA 
                   
               
               
                   
                 LSQAVSLTPPGQEFPPAMVDAGKGKTAEARKTWLEEQGMILKQTFCEVPD 
                   
               
               
                   
                 LDRIIPVLLEHGLERLPEHCKLSPGIPLKPMLAHPTRGISEVLKRFEEAA 
                   
               
               
                   
                 FTCEYKYDGQRAQIHALEGGEVKIFSRNQEDNTGKYPDIISRIPKIKLPS 
                   
               
               
                   
                 VTSFILDTEAVAWDREKKQIQPFQVLTTRKRKEVDASEIQVQVCLYAFDL 
                   
               
               
                   
                 IYLNGESLVREPLSRRRQLLRENFVETEGEFVFATSLDTKDIEQIAEFLE 
                   
               
               
                   
                 QSVKDSCEGLMVKTLDVDATYEIAKRSHNWLKLKKDYLDGVGDTLDLVVI 
                   
               
               
                   
                 GAYLGRGKRAGRYGGFLLASYDEDSEELQAICKLGTGFSDEELEEHHQSL 
                   
               
               
                   
                 KALVLPSPRPYVRIDGAVIPDHWLDPSAVWEVKCADLSLSPIYPAARGLV 
                   
               
               
                   
                 DSDKGISLRFPRFIRVREDKQPEQATTSAQVACLYRKQSQIQNQQGEDSG 
                   
               
               
                   
                 SDPEDTYSGGSKRTADSQHSTPPKTKRKVEFEPKKKRKV* 
                   
               
               
                   
               
               
                 NLS- 
                 atgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcg 
                 129 
               
               
                 nCas9- 
                 gaaagtcgacaagaagtacagcatcggcctggacatcggcaccaactctg 
                   
               
               
                 linker- 
                 tgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattc 
                   
               
               
                 SplintR- 
                 aaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcgg 
                   
               
               
                 bpNLS 
                 agccctgctgttcgacagcggcgaaacagccgaggccacccggctgaaga 
                   
               
               
                   
                 gaaccgccagaagaagatacaccagacggaagaaccggatctgctatctg 
                   
               
               
                   
                 caagagatcttcagcaacgagatggccaaggtggacgacagcttcttcca 
                   
               
               
                   
                 cagactggaagagtccttcctggtggaagaggataagaagcacgagcggc 
                   
               
               
                   
                 accccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtac 
                   
               
               
                   
                 cccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggc 
                   
               
               
                   
                 cgacctgcggctgatctatctggccctggcccacatgatcaagttccggg 
                   
               
               
                   
                 gccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggac 
                   
               
               
                   
                 aagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaa 
                   
               
               
                   
                 ccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagac 
                   
               
               
                   
                 tgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgag 
                   
               
               
                   
                 aagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgac 
                   
               
               
                   
                 ccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagc 
                   
               
               
                   
                 tgagcaaggacacctacgacgacgacctggacaacctgctggcccagatc 
                   
               
               
                   
                 ggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgc 
                   
               
               
                   
                 catcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggccc 
                   
               
               
                   
                 ccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctg 
                   
               
               
                   
                 accctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaaga 
                   
               
               
                   
                 gattttcttcgaccagagcaagaacggctacgccggctacattgacggcg 
                   
               
               
                   
                 gagccagccaggaagagttctacaagttcatcaagcccatcctggaaaag 
                   
               
               
                   
                 atggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgct 
                   
               
               
                   
                 gcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacc 
                   
               
               
                   
                 tgggagagctgcacgccattctgcggcggcaggaagatttttacccattc 
                   
               
               
                   
                 ctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatccc 
                   
               
               
                   
                 ctactacgtgggccctctggccaggggaaacagcagattcgcctggatga 
                   
               
               
                   
                 ccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtg 
                   
               
               
                   
                 gacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcga 
                   
               
               
                   
                 taagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacg 
                   
               
               
                   
                 agtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgag 
                   
               
               
                   
                 ggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgt 
                   
               
               
                   
                 ggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaag 
                   
               
               
                   
                 aggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggc 
                   
               
               
                   
                 gtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaa 
                   
               
               
                   
                 aattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattc 
                   
               
               
                   
                 tggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatc 
                   
               
               
                   
                 gaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaa 
                   
               
               
                   
                 gcagctgaagcggcggagatacaccggctggggcaggctgagccggaagc 
                   
               
               
                   
                 tgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttc 
                   
               
               
                   
                 ctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacga 
                   
               
               
                   
                 cgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggcc 
                   
               
               
                   
                 agggcgatagcctgcacgagcacattgccaatctggccggcagccccgcc 
                   
               
               
                   
                 attaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaa 
                   
               
               
                   
                 agtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagag 
                   
               
               
                   
                 agaaccagaccacccagaagggacagaagaacagccgcgagagaatgaag 
                   
               
               
                   
                 cggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaaca 
                   
               
               
                   
                 ccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacc 
                   
               
               
                   
                 tgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccgg 
                   
               
               
                   
                 ctgtccgactacgatgtggacgctatcgtgcctcagagctttctgaagga 
                   
               
               
                   
                 cgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggca 
                   
               
               
                   
                 agagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactac 
                   
               
               
                   
                 tggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaa 
                   
               
               
                   
                 tctgaccaaggccgagagaggcggcctgagcgaactggataaggccggct 
                   
               
               
                   
                 tcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggca 
                   
               
               
                   
                 cagatcctggactcccggatgaacactaagtacgacgagaatgacaagct 
                   
               
               
                   
                 gatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatt 
                   
               
               
                   
                 tccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccac 
                   
               
               
                   
                 cacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaa 
                   
               
               
                   
                 aaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgt 
                   
               
               
                   
                 acgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggct 
                   
               
               
                   
                 accgccaagtacttcttctacagcaacatcatgaactttttcaagaccga 
                   
               
               
                   
                 gattaccctggccaacggcgagatccggaagcggcctctgatcgagacaa 
                   
               
               
                   
                 acggcgaaaccggggagatcgtgtgggataagggccgggattttgccacc 
                   
               
               
                   
                 gtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccga 
                   
               
               
                   
                 ggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaaca 
                   
               
               
                   
                 gcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggc 
                   
               
               
                   
                 ggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagt 
                   
               
               
                   
                 ggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctgggga 
                   
               
               
                   
                 tcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctg 
                   
               
               
                   
                 gaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcc 
                   
               
               
                   
                 taagtactccctgttcgagctggaaaacggccggaagagaatgctggcct 
                   
               
               
                   
                 ctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatat 
                   
               
               
                   
                 gtgaacttcctgtacctggccagccactatgagaagctgaagggctcccc 
                   
               
               
                   
                 cgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacc 
                   
               
               
                   
                 tggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctg 
                   
               
               
                   
                 gccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccggga 
                   
               
               
                   
                 taagcccatcagagagcaggccgagaatatcatccacctgtttaccctga 
                   
               
               
                   
                 ccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgac 
                   
               
               
                   
                 cggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatcca 
                   
               
               
                   
                 ccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgg 
                   
               
               
                   
                 gaggtgacTCCGGCGGAAGCTCTGGTGGCAGCAAGCGGACCGCCGACGGC 
                   
               
               
                   
                 TCTGAATTCGAGAGCCCTAAGAAGAAAAGAAAGGTGAGCGGAGGCTCTAG 
                   
               
               
                   
                 CGGCGGAAGCATGGCAATCACTAAGCCCCTCTTGGCGGCGACTTTGGAAA 
                   
               
               
                   
                 ACATCGAGGATGTGCAATTCCCGTGCCTTGCCACACCAAAGATAGACGGG 
                   
               
               
                   
                 ATCCGATCAGTGAAGCAAACGCAGATGCTCTCTAGAACGTTCAAGCCTAT 
                   
               
               
                   
                 TAGAAACTCAGTGATGAATCGGCTCTTGACTGAGCTGTTGCCGGAAGGCA 
                   
               
               
                   
                 GCGATGGGGAAATATCTATCGAGGGAGCCACATTTCAAGACACTACGAGC 
                   
               
               
                   
                 GCCGTAATGACTGGACATAAGATGTATAATGCTAAATTCTCCTACTATTG 
                   
               
               
                   
                 GTTTGACTATGTCACTGATGACCCTCTTAAGAAATATATAGACCGAGTGG 
                   
               
               
                   
                 AGGATATGAAAAATTATATTACTGTACACCCGCATATTCTGGAACATGCC 
                   
               
               
                   
                 CAAGTTAAGATTATTCCTCTCATTCCCGTCGAGATTAATAATATCACAGA 
                   
               
               
                   
                 ACTGCTTCAGTATGAGCGCGACGTATTGTCCAAAGGCTTTGAAGGGGTTA 
                   
               
               
                   
                 TGATACGCAAACCGGACGGCAAGTACAAGTTCGGAAGAAGCACATTGAAA 
                   
               
               
                   
                 GAGGGTATATTGCTGAAGATGAAGCAGTTTAAGGATGCTGAGGCAACAAT 
                   
               
               
                   
                 AATCAGCATGACAGCACTTTTTAAAAATACCAACACGAAAACTAAGGACA 
                   
               
               
                   
                 ATTTTGGTTATAGTAAGCGGTCAACGCACAAAAGTGGGAAGGTAGAAGAA 
                   
               
               
                   
                 GACGTAATGGGTAGCATTGAGGTGGATTATGACGGGGTGGTTTTCAGCAT 
                   
               
               
                   
                 AGGGACTGGGTTTGATGCAGATCAACGGAGGGACTTTTGGCAGAACAAAG 
                   
               
               
                   
                 AATCATATATAGGCAAAATGGTAAAGTTCAAATACTTCGAAATGGGAAGT 
                   
               
               
                   
                 AAAGACTGCCCCAGATTCCCTGTATTCATTGGCATCAGGCACGAGGAGGA 
                   
               
               
                   
                 CAGGAGTGGGGGATCAAAGCGGACTGCTGATAGTCAGCATAGTACTCCAC 
                   
               
               
                   
                 CCAAGACCAAGCGGAAAGTTGAGTTTGAGCCGAAGAAAAAGCGAAAAGTG 
                   
               
               
                   
                 TAA 
                   
               
               
                   
               
               
                 NLS- 
                 MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKF 
                 130 
               
               
                 nCas9- 
                 KVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL 
                   
               
               
                 linker- 
                 QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY 
                   
               
               
                 SplintR- 
                 PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVD 
                   
               
               
                 bpNLS 
                 KLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGE 
                   
               
               
                   
                 KKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI 
                   
               
               
                   
                 GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDL 
                   
               
               
                   
                 TLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEK 
                   
               
               
                   
                 MDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPF 
                   
               
               
                   
                 LKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV 
                   
               
               
                   
                 DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE 
                   
               
               
                   
                 GMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG 
                   
               
               
                   
                 VEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI 
                   
               
               
                   
                 EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF 
                   
               
               
                   
                 LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA 
                   
               
               
                   
                 IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMK 
                   
               
               
                   
                 RIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR 
                   
               
               
                   
                 LSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY 
                   
               
               
                   
                 WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVA 
                   
               
               
                   
                 QILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYH 
                   
               
               
                   
                 HAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKA 
                   
               
               
                   
                 TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
                   
               
               
                   
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYG 
                   
               
               
                   
                 GFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFL 
                   
               
               
                   
                 EAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKY 
                   
               
               
                   
                 VNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL 
                   
               
               
                   
                 ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTID 
                   
               
               
                   
                 RKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSKRTADG 
                   
               
               
                   
                 SEFESPKKKRKVSGGSSGGSMAITKPLLAATLENIEDVQFPCLATPKIDG 
                   
               
               
                   
                 IRSVKQTQMLSRTFKPIRNSVMNRLLTELLPEGSDGEISIEGATFQDTTS 
                   
               
               
                   
                 AVMTGHKMYNAKFSYYWFDYVTDDPLKKYIDRVEDMKNYITVHPHILEHA 
                   
               
               
                   
                 QVKIIPLIPVEINNITELLQYERDVLSKGFEGVMIRKPDGKYKFGRSTLK 
                   
               
               
                   
                 EGILLKMKQFKDAEATIISMTALFKNTNTKTKDNFGYSKRSTHKSGKVEE 
                   
               
               
                   
                 DVMGSIEVDYDGVVFSIGTGFDADQRRDFWQNKESYIGKMVKFKYFEMGS 
                   
               
               
                   
                 KDCPRFPVFIGIRHEEDRSGGSKRTADSQHSTPPKTKRKVEFEPKKKRKV 
                   
               
               
                   
                 * 
                   
               
               
                   
               
               
                 NLS- 
                 atgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcg 
                 131 
               
               
                 nCas9- 
                 gaaagtcgacaagaagtacagcatcggcctggacatcggcaccaactctg 
                   
               
               
                 linker- 
                 tgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattc 
                   
               
               
                 T4LIG- 
                 aaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcgg 
                   
               
               
                 bpNLS 
                 agccctgctgttcgacagcggcgaaacagccgaggccacccggctgaaga 
                   
               
               
                   
                 gaaccgccagaagaagatacaccagacggaagaaccggatctgctatctg 
                   
               
               
                   
                 caagagatcttcagcaacgagatggccaaggtggacgacagcttcttcca 
                   
               
               
                   
                 cagactggaagagtccttcctggtggaagaggataagaagcacgagcggc 
                   
               
               
                   
                 accccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtac 
                   
               
               
                   
                 cccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggc 
                   
               
               
                   
                 cgacctgcggctgatctatctggccctggcccacatgatcaagttccggg 
                   
               
               
                   
                 gccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggac 
                   
               
               
                   
                 aagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaa 
                   
               
               
                   
                 ccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagac 
                   
               
               
                   
                 tgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgag 
                   
               
               
                   
                 aagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgac 
                   
               
               
                   
                 ccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagc 
                   
               
               
                   
                 tgagcaaggacacctacgacgacgacctggacaacctgctggcccagatc 
                   
               
               
                   
                 ggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgc 
                   
               
               
                   
                 catcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggccc 
                   
               
               
                   
                 ccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctg 
                   
               
               
                   
                 accctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaaga 
                   
               
               
                   
                 gattttcttcgaccagagcaagaacggctacgccggctacattgacggcg 
                   
               
               
                   
                 gagccagccaggaagagttctacaagttcatcaagcccatcctggaaaag 
                   
               
               
                   
                 atggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgct 
                   
               
               
                   
                 gcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacc 
                   
               
               
                   
                 tgggagagctgcacgccattctgcggcggcaggaagatttttacccattc 
                   
               
               
                   
                 ctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatccc 
                   
               
               
                   
                 ctactacgtgggccctctggccaggggaaacagcagattcgcctggatga 
                   
               
               
                   
                 ccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtg 
                   
               
               
                   
                 gacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcga 
                   
               
               
                   
                 taagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacg 
                   
               
               
                   
                 agtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgag 
                   
               
               
                   
                 ggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgt 
                   
               
               
                   
                 ggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaag 
                   
               
               
                   
                 aggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggc 
                   
               
               
                   
                 gtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaa 
                   
               
               
                   
                 aattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattc 
                   
               
               
                   
                 tggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatc 
                   
               
               
                   
                 gaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaa 
                   
               
               
                   
                 gcagctgaagcggcggagatacaccggctggggcaggctgagccggaagc 
                   
               
               
                   
                 tgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttc 
                   
               
               
                   
                 ctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacga 
                   
               
               
                   
                 cgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggcc 
                   
               
               
                   
                 agggcgatagcctgcacgagcacattgccaatctggccggcagccccgcc 
                   
               
               
                   
                 attaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaa 
                   
               
               
                   
                 agtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagag 
                   
               
               
                   
                 agaaccagaccacccagaagggacagaagaacagccgcgagagaatgaag 
                   
               
               
                   
                 cggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaaca 
                   
               
               
                   
                 ccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacc 
                   
               
               
                   
                 tgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccgg 
                   
               
               
                   
                 ctgtccgactacgatgtggacgctatcgtgcctcagagctttctgaagga 
                   
               
               
                   
                 cgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggca 
                   
               
               
                   
                 agagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactac 
                   
               
               
                   
                 tggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaa 
                   
               
               
                   
                 tctgaccaaggccgagagaggcggcctgagcgaactggataaggccggct 
                   
               
               
                   
                 tcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggca 
                   
               
               
                   
                 cagatcctggactcccggatgaacactaagtacgacgagaatgacaagct 
                   
               
               
                   
                 gatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatt 
                   
               
               
                   
                 tccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccac 
                   
               
               
                   
                 cacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaa 
                   
               
               
                   
                 aaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgt 
                   
               
               
                   
                 acgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggct 
                   
               
               
                   
                 accgccaagtacttcttctacagcaacatcatgaactttttcaagaccga 
                   
               
               
                   
                 gattaccctggccaacggcgagatccggaagcggcctctgatcgagacaa 
                   
               
               
                   
                 acggcgaaaccggggagatcgtgtgggataagggccgggattttgccacc 
                   
               
               
                   
                 gtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccga 
                   
               
               
                   
                 ggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaaca 
                   
               
               
                   
                 gcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggc 
                   
               
               
                   
                 ggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagt 
                   
               
               
                   
                 ggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctgggga 
                   
               
               
                   
                 tcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctg 
                   
               
               
                   
                 gaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcc 
                   
               
               
                   
                 taagtactccctgttcgagctggaaaacggccggaagagaatgctggcct 
                   
               
               
                   
                 ctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatat 
                   
               
               
                   
                 gtgaacttcctgtacctggccagccactatgagaagctgaagggctcccc 
                   
               
               
                   
                 cgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacc 
                   
               
               
                   
                 tggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctg 
                   
               
               
                   
                 gccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccggga 
                   
               
               
                   
                 taagcccatcagagagcaggccgagaatatcatccacctgtttaccctga 
                   
               
               
                   
                 ccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgac 
                   
               
               
                   
                 cggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatcca 
                   
               
               
                   
                 ccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgg 
                   
               
               
                   
                 gaggtgacTCCGGCGGAAGCTCTGGTGGCAGCAAGCGGACCGCCGACGGC 
                   
               
               
                   
                 TCTGAATTCGAGAGCCCTAAGAAGAAAAGAAAGGTGAGCGGAGGCTCTAG 
                   
               
               
                   
                 CGGCGGAAGCATGATTCTTAAAATTCTTAACGAGATTGCGAGTATTGGCA 
                   
               
               
                   
                 GCACGAAACAAAAGCAGGCCATATTGGAAAAGAATAAGGACAATGAGTTG 
                   
               
               
                   
                 CTTAAACGCGTGTATAGGCTCACTTACTCTCGCGGACTGCAATACTATAT 
                   
               
               
                   
                 TAAAAAATGGCCTAAGCCCGGCATCGCTACTCAAAGCTTCGGAATGCTTA 
                   
               
               
                   
                 CGCTGACAGATATGCTCGACTTCATCGAGTTTACTCTCGCAACAAGGAAG 
                   
               
               
                   
                 TTGACTGGCAACGCCGCGATTGAAGAATTGACGGGTTATATCACGGACGG 
                   
               
               
                   
                 GAAGAAGGATGATGTTGAGGTGCTGAGGCGCGTTATGATGCGCGACCTCG 
                   
               
               
                   
                 AATGTGGTGCCTCAGTTTCCATAGCCAATAAAGTTTGGCCAGGCTTGATC 
                   
               
               
                   
                 CCGGAGCAGCCACAGATGCTGGCCAGTAGCTACGACGAGAAGGGTATTAA 
                   
               
               
                   
                 CAAAAATATCAAGTTTCCAGCGTTTGCACAACTTAAAGCGGATGGGGCGC 
                   
               
               
                   
                 GGTGTTTCGCCGAAGTCCGGGGTGACGAATTGGACGATGTGCGCCTTCTG 
                   
               
               
                   
                 AGTCGCGCAGGAAATGAATATCTGGGGCTTGACCTCTTGAAGGAGGAGCT 
                   
               
               
                   
                 GATTAAGATGACAGCAGAAGCCAGGCAGATCCATCCAGAGGGGGTACTTA 
                   
               
               
                   
                 TTGATGGTGAACTCGTATACCATGAGCAGGTTAAGAAGGAGCCAGAGGGT 
                   
               
               
                   
                 TTGGATTTCCTCTTTGACGCCTATCCCGAGAATTCAAAGGCAAAGGAGTT 
                   
               
               
                   
                 CGCCGAGGTTGCAGAATCAAGAACGGCTTCCAACGGCATAGCGAATAAAT 
                   
               
               
                   
                 CACTCAAAGGAACTATATCTGAAAAGGAGGCACAGTGTATGAAATTCCAA 
                   
               
               
                   
                 GTGTGGGACTATGTGCCGCTTGTCGAGATTTACAGCTTGCCTGCTTTCCG 
                   
               
               
                   
                 ATTGAAGTACGATGTACGGTTTAGTAAGCTCGAGCAAATGACTTCAGGTT 
                   
               
               
                   
                 ACGATAAAGTCATCTTGATTGAGAACCAGGTCGTTAATAATCTTGACGAG 
                   
               
               
                   
                 GCGAAGGTCATATATAAGAAATATATAGATCAAGGGCTCGAGGGTATCAT 
                   
               
               
                   
                 TCTGAAGAATATAGATGGCTTGTGGGAAAACGCCAGGTCCAAAAACCTGT 
                   
               
               
                   
                 ATAAGTTTAAGGAAGTAATAGATGTAGATTTGAAAATAGTTGGAATTTAC 
                   
               
               
                   
                 CCCCATCGGAAGGACCCCACGAAAGCGGGTGGGTTTATCCTCGAGAGCGA 
                   
               
               
                   
                 GTGTGGGAAGATAAAAGTGAATGCCGGCTCCGGATTGAAGGACAAGGCAG 
                   
               
               
                   
                 GTGTGAAAAGTCATGAGCTCGATCGGACGAGAATAATGGAAAACCAGAAT 
                   
               
               
                   
                 TACTACATTGGAAAGATTTTGGAATGCGAGTGTAACGGCTGGTTGAAGAG 
                   
               
               
                   
                 CGACGGACGCACCGATTACGTGAAACTCTTTCTGCCAATTGCAATCAGGT 
                   
               
               
                   
                 TGAGAGAGGATAAGACTAAGGCCAATACTTTCGAGGACGTCTTCGGAGAC 
                   
               
               
                   
                 TTTCACGAAGTCACTGGGCTTTCTGGGGGTAGTAAGAGAACTGCAGATAG 
                   
               
               
                   
                 CCAGCATTCAACGCCGCCAAAAACAAAGCGAAAGGTAGAATTCGAACCAA 
                   
               
               
                   
                 AGAAAAAGCGGAAAGTATAA 
                   
               
               
                   
               
               
                 NLS- 
                 MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKF 
                 132 
               
               
                 nCas9- 
                 KVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL 
                   
               
               
                 linker- 
                 QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY 
                   
               
               
                 T4LIG- 
                 PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVD 
                   
               
               
                 bpNLS 
                 KLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGE 
                   
               
               
                   
                 KKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI 
                   
               
               
                   
                 GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDL 
                   
               
               
                   
                 TLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEK 
                   
               
               
                   
                 MDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPF 
                   
               
               
                   
                 LKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV 
                   
               
               
                   
                 DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE 
                   
               
               
                   
                 GMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG 
                   
               
               
                   
                 VEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI 
                   
               
               
                   
                 EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF 
                   
               
               
                   
                 LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA 
                   
               
               
                   
                 IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMK 
                   
               
               
                   
                 RIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR 
                   
               
               
                   
                 LSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY 
                   
               
               
                   
                 WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVA 
                   
               
               
                   
                 QILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYH 
                   
               
               
                   
                 HAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKA 
                   
               
               
                   
                 TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
                   
               
               
                   
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYG 
                   
               
               
                   
                 GFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFL 
                   
               
               
                   
                 EAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKY 
                   
               
               
                   
                 VNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL 
                   
               
               
                   
                 ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTID 
                   
               
               
                   
                 RKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSKRTADG 
                   
               
               
                   
                 SEFESPKKKRKVSGGSSGGSMILKILNEIASIGSTKQKQAILEKNKDNEL 
                   
               
               
                   
                 LKRVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLTDMLDFIEFTLATRK 
                   
               
               
                   
                 LTGNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGASVSIANKVWPGLI 
                   
               
               
                   
                 PEQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLL 
                   
               
               
                   
                 SRAGNEYLGLDLLKEELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEG 
                   
               
               
                   
                 LDFLFDAYPENSKAKEFAEVAESRTASNGIANKSLKGTISEKEAQCMKFQ 
                   
               
               
                   
                 VWDYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKVILIENQVVNNLDE 
                   
               
               
                   
                 AKVIYKKYIDQGLEGIILKNIDGLWENARSKNLYKFKEVIDVDLKIVGIY 
                   
               
               
                   
                 PHRKDPTKAGGFILESECGKIKVNAGSGLKDKAGVKSHELDRTRIMENQN 
                   
               
               
                   
                 YYIGKILECECNGWLKSDGRTDYVKLFLPIAIRLREDKTKANTFEDVFGD 
                   
               
               
                   
                 FHEVTGLSGGSKRTADSQHSTPPKTKRKVEFEPKKKRKV* 
                   
               
               
                   
               
               
                 NLS- 
                 atgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcg 
                 133 
               
               
                 nCas9-LZ 
                 gaaagtcgacaagaagtacagcatcggcctggacatcggcaccaactctg 
                   
               
               
                   
                 tgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattc 
                   
               
               
                   
                 aaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcgg 
                   
               
               
                   
                 agccctgctgttcgacagcggcgaaacagccgaggccacccggctgaaga 
                   
               
               
                   
                 gaaccgccagaagaagatacaccagacggaagaaccggatctgctatctg 
                   
               
               
                   
                 caagagatcttcagcaacgagatggccaaggtggacgacagcttcttcca 
                   
               
               
                   
                 cagactggaagagtccttcctggtggaagaggataagaagcacgagcggc 
                   
               
               
                   
                 accccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtac 
                   
               
               
                   
                 cccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggc 
                   
               
               
                   
                 cgacctgcggctgatctatctggccctggcccacatgatcaagttccggg 
                   
               
               
                   
                 gccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggac 
                   
               
               
                   
                 aagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaa 
                   
               
               
                   
                 ccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagac 
                   
               
               
                   
                 tgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgag 
                   
               
               
                   
                 aagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgac 
                   
               
               
                   
                 ccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagc 
                   
               
               
                   
                 tgagcaaggacacctacgacgacgacctggacaacctgctggcccagatc 
                   
               
               
                   
                 ggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgc 
                   
               
               
                   
                 catcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggccc 
                   
               
               
                   
                 ccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctg 
                   
               
               
                   
                 accctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaaga 
                   
               
               
                   
                 gattttcttcgaccagagcaagaacggctacgccggctacattgacggcg 
                   
               
               
                   
                 gagccagccaggaagagttctacaagttcatcaagcccatcctggaaaag 
                   
               
               
                   
                 atggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgct 
                   
               
               
                   
                 gcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacc 
                   
               
               
                   
                 tgggagagctgcacgccattctgcggcggcaggaagatttttacccattc 
                   
               
               
                   
                 ctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatccc 
                   
               
               
                   
                 ctactacgtgggccctctggccaggggaaacagcagattcgcctggatga 
                   
               
               
                   
                 ccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtg 
                   
               
               
                   
                 gacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcga 
                   
               
               
                   
                 taagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacg 
                   
               
               
                   
                 agtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgag 
                   
               
               
                   
                 ggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgt 
                   
               
               
                   
                 ggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaag 
                   
               
               
                   
                 aggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggc 
                   
               
               
                   
                 gtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaa 
                   
               
               
                   
                 aattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattc 
                   
               
               
                   
                 tggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatc 
                   
               
               
                   
                 gaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaa 
                   
               
               
                   
                 gcagctgaagcggcggagatacaccggctggggcaggctgagccggaagc 
                   
               
               
                   
                 tgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttc 
                   
               
               
                   
                 ctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacga 
                   
               
               
                   
                 cgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggcc 
                   
               
               
                   
                 agggcgatagcctgcacgagcacattgccaatctggccggcagccccgcc 
                   
               
               
                   
                 attaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaa 
                   
               
               
                   
                 agtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagag 
                   
               
               
                   
                 agaaccagaccacccagaagggacagaagaacagccgcgagagaatgaag 
                   
               
               
                   
                 cggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaaca 
                   
               
               
                   
                 ccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacc 
                   
               
               
                   
                 tgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccgg 
                   
               
               
                   
                 ctgtccgactacgatgtggacgctatcgtgcctcagagctttctgaagga 
                   
               
               
                   
                 cgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggca 
                   
               
               
                   
                 agagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactac 
                   
               
               
                   
                 tggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaa 
                   
               
               
                   
                 tctgaccaaggccgagagaggcggcctgagcgaactggataaggccggct 
                   
               
               
                   
                 tcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggca 
                   
               
               
                   
                 cagatcctggactcccggatgaacactaagtacgacgagaatgacaagct 
                   
               
               
                   
                 gatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatt 
                   
               
               
                   
                 tccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccac 
                   
               
               
                   
                 cacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaa 
                   
               
               
                   
                 aaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgt 
                   
               
               
                   
                 acgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggct 
                   
               
               
                   
                 accgccaagtacttcttctacagcaacatcatgaactttttcaagaccga 
                   
               
               
                   
                 gattaccctggccaacggcgagatccggaagcggcctctgatcgagacaa 
                   
               
               
                   
                 acggcgaaaccggggagatcgtgtgggataagggccgggattttgccacc 
                   
               
               
                   
                 gtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccga 
                   
               
               
                   
                 ggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaaca 
                   
               
               
                   
                 gcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggc 
                   
               
               
                   
                 ggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagt 
                   
               
               
                   
                 ggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctgggga 
                   
               
               
                   
                 tcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctg 
                   
               
               
                   
                 gaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcc 
                   
               
               
                   
                 taagtactccctgttcgagctggaaaacggccggaagagaatgctggcct 
                   
               
               
                   
                 ctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatat 
                   
               
               
                   
                 gtgaacttcctgtacctggccagccactatgagaagctgaagggctcccc 
                   
               
               
                   
                 cgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacc 
                   
               
               
                   
                 tggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctg 
                   
               
               
                   
                 gccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccggga 
                   
               
               
                   
                 taagcccatcagagagcaggccgagaatatcatccacctgtttaccctga 
                   
               
               
                   
                 ccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgac 
                   
               
               
                   
                 cggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatcca 
                   
               
               
                   
                 ccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgg 
                   
               
               
                   
                 gaggtgacggctcaaaaagaaccgccgacggcagcgaattcgagcccaag 
                   
               
               
                   
                 aagaagaggaaagtcGGAGGAGGAGGCAGTGGTGGGCGACTTGAAATTAG 
                   
               
               
                   
                 AGCCGCGTTCCTGCGCCAGAGGAATACGGCTCTCCGCACGGAGGTAGCCG 
                   
               
               
                   
                 AACTTGAGCAAGAAGTACAGAGATTGGAGAACGAGGTTTCACAGTATGAG 
                   
               
               
                   
                 ACACGATATGGCCCCCTTGGCGGCGGAAAGtaa 
                   
               
               
                   
               
               
                 NLS1- 
                 KRTADGSEFESPKKKRKVMGIQGLAKLIADVAPSAIRENDIKSYFGRKVA 
                 134 
               
               
                 hFEN1- 
                 IDASMSIYQFLIAVRQGGDVLQNEEGETTSHLMGMFYRTIRMMENGIKPV 
                   
               
               
                 linker1- 
                 YVFDGKPPQLKSGELAKRSERRAEAEKQLQQAQAAGAEQEVEKFTKRLVK 
                   
               
               
                 nCas9- 
                 VTKQHNDECKHLLSLMGIPYLDAPSEAEASCAALVKAGKVYAAATEDMDC 
                   
               
               
                 linker2- 
                 LTFGSPVLMRHLTASEAKKLPIQEFHLSRILQELGLNQEQFVDLCILLGS 
                   
               
               
                 T4LIG- 
                 DYCESIRGIGPKRAVDLIQKHKSIEEIVRRLDPNKYPVPENWLHKEAHQL 
                   
               
               
                 NLS2 
                 FLEPEVLDPESVELKWSEPNEEELIKFMCGEKQFSEERIRSGVKRLSKSR 
                   
               
               
                   
                 QGSTQGRLDDFFKVTGSLSSAKRKEPEPKGSTKKKAKTGAAGKFKRGKSG 
                   
               
               
                   
                 GSSGGSSGSETPGTSESATPESSGGSSGGSSMDKKYSIGLDIGTNSVGWA 
                   
               
               
                   
                 VITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR 
                   
               
               
                   
                 RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF 
                   
               
               
                   
                 GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFL 
                   
               
               
                   
                 IEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKS 
                   
               
               
                   
                 RRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKD 
                   
               
               
                   
                 TYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSA 
                   
               
               
                   
                 SMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQ 
                   
               
               
                   
                 EEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGEL 
                   
               
               
                   
                 HAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKS 
                   
               
               
                   
                 EETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFT 
                   
               
               
                   
                 VYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYF 
                   
               
               
                   
                 KKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDI 
                   
               
               
                   
                 VLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLING 
                   
               
               
                   
                 IRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDS 
                   
               
               
                   
                 LHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQT 
                   
               
               
                   
                 TQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNG 
                   
               
               
                   
                 RDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDN 
                   
               
               
                   
                 VPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKR 
                   
               
               
                   
                 QLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKD 
                   
               
               
                   
                 FQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVR 
                   
               
               
                   
                 KMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGET 
                   
               
               
                   
                 GEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKL 
                   
               
               
                   
                 IARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIM 
                   
               
               
                   
                 ERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGE 
                   
               
               
                   
                 LQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI 
                   
               
               
                   
                 IEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLG 
                   
               
               
                   
                 APAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDS 
                   
               
               
                   
                 GGSSGGSKRTADGSEFESPKKKRKVSGGSSGGSMILKILNEIASIGSTKQ 
                   
               
               
                   
                 KQAILEKNKDNELLKRVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLTD 
                   
               
               
                   
                 MLDFIEFTLATRKLTGNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGA 
                   
               
               
                   
                 SVSIANKVWPGLIPEQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFA 
                   
               
               
                   
                 EVRGDELDDVRLLSRAGNEYLGLDLLKEELIKMTAEARQIHPEGVLIDGE 
                   
               
               
                   
                 LVYHEQVKKEPEGLDFLFDAYPENSKAKEFAEVAESRTASNGIANKSLKG 
                   
               
               
                   
                 TISEKEAQCMKFQVWDYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKV 
                   
               
               
                   
                 ILIENQVVNNLDEAKVIYKKYIDQGLEGIILKNIDGLWENARSKNLYKFK 
                   
               
               
                   
                 EVIDVDLKIVGIYPHRKDPTKAGGFILESECGKIKVNAGSGLKDKAGVKS 
                   
               
               
                   
                 HELDRTRIMENQNYYIGKILECECNGWLKSDGRTDYVKLFLPIAIRLRED 
                   
               
               
                   
                 KTKANTFEDVFGDFHEVTGLSGGSKRTADSQHSTPPKTKRKVEFEPKKKR 
                   
               
               
                   
                 KV 
                   
               
               
                   
               
               
                 NLS1- 
                 KRTADGSEFESPKKKRKVMILKILNEIASIGSTKQKQAILEKNKDNELLK 
                 135 
               
               
                 hFEN1- 
                 RVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLTDMLDFIEFTLATRKLT 
                   
               
               
                 linker1- 
                 GNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGASVSIANKVWPGLIPE 
                   
               
               
                 T4LIG- 
                 QPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLLSR 
                   
               
               
                 linker2- 
                 AGNEYLGLDLLKEELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEGLD 
                   
               
               
                 nCas9- 
                 FLFDAYPENSKAKEFAEVAESRTASNGIANKSLKGTISEKEAQCMKFQVW 
                   
               
               
                 NLS2 
                 DYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKVILIENQVVNNLDEAK 
                   
               
               
                   
                 VIYKKYIDQGLEGIILKNIDGLWENARSKNLYKFKEVIDVDLKIVGIYPH 
                   
               
               
                   
                 RKDPTKAGGFILESECGKIKVNAGSGLKDKAGVKSHELDRTRIMENQNYY 
                   
               
               
                   
                 IGKILECECNGWLKSDGRTDYVKLFLPIAIRLREDKTKANTFEDVFGDFH 
                   
               
               
                   
                 EVTGLSGGSSGGSSGSETPGTSESATPESSGGSSGGSSMGIQGLAKLIAD 
                   
               
               
                   
                 VAPSAIRENDIKSYFGRKVAIDASMSIYQFLIAVRQGGDVLQNEEGETTS 
                   
               
               
                   
                 HLMGMFYRTIRMMENGIKPVYVFDGKPPQLKSGELAKRSERRAEAEKQLQ 
                   
               
               
                   
                 QAQAAGAEQEVEKFTKRLVKVTKQHNDECKHLLSLMGIPYLDAPSEAEAS 
                   
               
               
                   
                 CAALVKAGKVYAAATEDMDCLTFGSPVLMRHLTASEAKKLPIQEFHLSRI 
                   
               
               
                   
                 LQELGLNQEQFVDLCILLGSDYCESIRGIGPKRAVDLIQKHKSIEEIVRR 
                   
               
               
                   
                 LDPNKYPVPENWLHKEAHQLFLEPEVLDPESVELKWSEPNEEELIKFMCG 
                   
               
               
                   
                 EKQFSEERIRSGVKRLSKSRQGSTQGRLDDFFKVTGSLSSAKRKEPEPKG 
                   
               
               
                   
                 STKKKAKTGAAGKFKRGKSGGSSGGSKRTADGSEFESPKKKRKVSGGSSG 
                   
               
               
                   
                 GSMDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI 
                   
               
               
                   
                 GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFF 
                   
               
               
                   
                 HRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDK 
                   
               
               
                   
                 ADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE 
                   
               
               
                   
                 NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGL 
                   
               
               
                   
                 TPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSD 
                   
               
               
                   
                 AILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYK 
                   
               
               
                   
                 EIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDL 
                   
               
               
                   
                 LRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI 
                   
               
               
                   
                 PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNF 
                   
               
               
                   
                 DKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAI 
                   
               
               
                   
                 VDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLL 
                   
               
               
                   
                 KIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVM 
                   
               
               
                   
                 KQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
                   
               
               
                   
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELV 
                   
               
               
                   
                 KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKE 
                   
               
               
                   
                 HPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLK 
                   
               
               
                   
                 DDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFD 
                   
               
               
                   
                 NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDK 
                   
               
               
                   
                 LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALI 
                   
               
               
                   
                 KKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT 
                   
               
               
                   
                 EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKT 
                   
               
               
                   
                 EVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAK 
                   
               
               
                   
                 VEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKL 
                   
               
               
                   
                 PKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGS 
                   
               
               
                   
                 PEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR 
                   
               
               
                   
                 DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI 
                   
               
               
                   
                 HQSITGLYETRIDLSQLGGDSGGSKRTADSQHSTPPKTKRKVEFEPKKKR 
                   
               
               
                   
                 KV 
                   
               
               
                   
               
               
                 NLS1- 
                 KRTADGSEFESPKKKRKVMDKKYSIGLDIGTNSVGWAVITDEYKVPSKKF 
                 136 
               
               
                 nCas9- 
                 KVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL 
                   
               
               
                 linker1- 
                 QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY 
                   
               
               
                 hFENI- 
                 PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVD 
                   
               
               
                 linker2- 
                 KLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGE 
                   
               
               
                 T4LIG- 
                 KKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI 
                   
               
               
                 NLS2 
                 GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDL 
                   
               
               
                   
                 TLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEK 
                   
               
               
                   
                 MDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPF 
                   
               
               
                   
                 LKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV 
                   
               
               
                   
                 DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE 
                   
               
               
                   
                 GMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG 
                   
               
               
                   
                 VEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI 
                   
               
               
                   
                 EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF 
                   
               
               
                   
                 LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA 
                   
               
               
                   
                 IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMK 
                   
               
               
                   
                 RIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR 
                   
               
               
                   
                 LSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY 
                   
               
               
                   
                 WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVA 
                   
               
               
                   
                 QILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYH 
                   
               
               
                   
                 HAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKA 
                   
               
               
                   
                 TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
                   
               
               
                   
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYG 
                   
               
               
                   
                 GFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFL 
                   
               
               
                   
                 EAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKY 
                   
               
               
                   
                 VNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL 
                   
               
               
                   
                 ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTID 
                   
               
               
                   
                 RKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSSGSETP 
                   
               
               
                   
                 GTSESATPESSGGSSGGSSMGIQGLAKLIADVAPSAIRENDIKSYFGRKV 
                   
               
               
                   
                 AIDASMSIYQFLIAVRQGGDVLQNEEGETTSHLMGMFYRTIRMMENGIKP 
                   
               
               
                   
                 VYVFDGKPPQLKSGELAKRSERRAEAEKQLQQAQAAGAEQEVEKFTKRLV 
                   
               
               
                   
                 KVTKQHNDECKHLLSLMGIPYLDAPSEAEASCAALVKAGKVYAAATEDMD 
                   
               
               
                   
                 CLTFGSPVLMRHLTASEAKKLPIQEFHLSRILQELGLNQEQFVDLCILLG 
                   
               
               
                   
                 SDYCESIRGIGPKRAVDLIQKHKSIEEIVRRLDPNKYPVPENWLHKEAHQ 
                   
               
               
                   
                 LFLEPEVLDPESVELKWSEPNEEELIKFMCGEKQFSEERIRSGVKRLSKS 
                   
               
               
                   
                 RQGSTQGRLDDFFKVTGSLSSAKRKEPEPKGSTKKKAKTGAAGKFKRGKS 
                   
               
               
                   
                 GGSSGGSKRTADGSEFESPKKKRKVSGGSSGGSMILKILNEIASIGSTKQ 
                   
               
               
                   
                 KQAILEKNKDNELLKRVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLTD 
                   
               
               
                   
                 MLDFIEFTLATRKLTGNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGA 
                   
               
               
                   
                 SVSIANKVWPGLIPEQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFA 
                   
               
               
                   
                 EVRGDELDDVRLLSRAGNEYLGLDLLKEELIKMTAEARQIHPEGVLIDGE 
                   
               
               
                   
                 LVYHEQVKKEPEGLDFLFDAYPENSKAKEFAEVAESRTASNGIANKSLKG 
                   
               
               
                   
                 TISEKEAQCMKFQVWDYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKV 
                   
               
               
                   
                 ILIENQVVNNLDEAKVIYKKYIDQGLEGIILKNIDGLWENARSKNLYKFK 
                   
               
               
                   
                 EVIDVDLKIVGIYPHRKDPTKAGGFILESECGKIKVNAGSGLKDKAGVKS 
                   
               
               
                   
                 HELDRTRIMENQNYYIGKILECECNGWLKSDGRTDYVKLFLPIAIRLRED 
                   
               
               
                   
                 KTKANTFEDVFGDFHEVTGLSGGSKRTADSQHSTPPKTKRKVEFEPKKKR 
                   
               
               
                   
                 KV 
                   
               
               
                   
               
               
                 NLS1- 
                 KRTADGSEFESPKKKRKVMILKILNEIASIGSTKQKQAILEKNKDNELLK 
                 137 
               
               
                 T4LIG- 
                 RVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLTDMLDFIEFTLATRKLT 
                   
               
               
                 linker1- 
                 GNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGASVSIANKVWPGLIPE 
                   
               
               
                 nCas9- 
                 QPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLLSR 
                   
               
               
                 linker2- 
                 AGNEYLGLDLLKEELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEGLD 
                   
               
               
                 hFEN1- 
                 FLFDAYPENSKAKEFAEVAESRTASNGIANKSLKGTISEKEAQCMKFQVW 
                   
               
               
                 NLS2 
                 DYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKVILIENQVVNNLDEAK 
                   
               
               
                   
                 VIYKKYIDQGLEGIILKNIDGLWENARSKNLYKFKEVIDVDLKIVGIYPH 
                   
               
               
                   
                 RKDPTKAGGFILESECGKIKVNAGSGLKDKAGVKSHELDRTRIMENQNYY 
                   
               
               
                   
                 IGKILECECNGWLKSDGRTDYVKLFLPIAIRLREDKTKANTFEDVFGDFH 
                   
               
               
                   
                 EVTGLSGGSSGGSSGSETPGTSESATPESSGGSSGGSSMDKKYSIGLDIG 
                   
               
               
                   
                 TNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEAT 
                   
               
               
                   
                 RLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK 
                   
               
               
                   
                 HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI 
                   
               
               
                   
                 KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAIL 
                   
               
               
                   
                 SARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDA 
                   
               
               
                   
                 KLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEI 
                   
               
               
                   
                 TKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGY 
                   
               
               
                   
                 IDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPH 
                   
               
               
                   
                 QIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRF 
                   
               
               
                   
                 AWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS 
                   
               
               
                   
                 LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK 
                   
               
               
                   
                 QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN 
                   
               
               
                   
                 EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRL 
                   
               
               
                   
                 SRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQ 
                   
               
               
                   
                 VSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
                   
               
               
                   
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY 
                   
               
               
                   
                 LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDK 
                   
               
               
                   
                 NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELD 
                   
               
               
                   
                 KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL 
                   
               
               
                   
                 VSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGD 
                   
               
               
                   
                 YKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPL 
                   
               
               
                   
                 IETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILP 
                   
               
               
                   
                 KRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKE 
                   
               
               
                   
                 LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKR 
                   
               
               
                   
                 MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQH 
                   
               
               
                   
                 KHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHL 
                   
               
               
                   
                 FTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDL 
                   
               
               
                   
                 SQLGGDSGGSSGGSKRTADGSEFESPKKKRKVSGGSSGGSMGIQGLAKLI 
                   
               
               
                   
                 ADVAPSAIRENDIKSYFGRKVAIDASMSIYQFLIAVRQGGDVLQNEEGET 
                   
               
               
                   
                 TSHLMGMFYRTIRMMENGIKPVYVFDGKPPQLKSGELAKRSERRAEAEKQ 
                   
               
               
                   
                 LQQAQAAGAEQEVEKFTKRLVKVTKQHNDECKHLLSLMGIPYLDAPSEAE 
                   
               
               
                   
                 ASCAALVKAGKVYAAATEDMDCLTFGSPVLMRHLTASEAKKLPIQEFHLS 
                   
               
               
                   
                 RILQELGLNQEQFVDLCILLGSDYCESIRGIGPKRAVDLIQKHKSIEEIV 
                   
               
               
                   
                 RRLDPNKYPVPENWLHKEAHQLFLEPEVLDPESVELKWSEPNEEELIKFM 
                   
               
               
                   
                 CGEKQFSEERIRSGVKRLSKSRQGSTQGRLDDFFKVTGSLSSAKRKEPEP 
                   
               
               
                   
                 KGSTKKKAKTGAAGKFKRGKSGGSKRTADSQHSTPPKTKRKVEFEPKKKR 
                   
               
               
                   
                 KV 
                   
               
               
                   
               
               
                 NLS1- 
                 KRTADGSEFESPKKKRKVMDKKYSIGLDIGTNSVGWAVITDEYKVPSKKF 
                 138 
               
               
                 nCas9- 
                 KVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL 
                   
               
               
                 linker1- 
                 QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY 
                   
               
               
                 T4LIG- 
                 PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVD 
                   
               
               
                 linker2- 
                 KLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGE 
                   
               
               
                 hFEN1- 
                 KKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI 
                   
               
               
                 NLS2 
                 GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDL 
                   
               
               
                   
                 TLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEK 
                   
               
               
                   
                 MDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPF 
                   
               
               
                   
                 LKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV 
                   
               
               
                   
                 DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE 
                   
               
               
                   
                 GMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG 
                   
               
               
                   
                 VEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI 
                   
               
               
                   
                 EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF 
                   
               
               
                   
                 LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA 
                   
               
               
                   
                 IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMK 
                   
               
               
                   
                 RIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR 
                   
               
               
                   
                 LSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY 
                   
               
               
                   
                 WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVA 
                   
               
               
                   
                 QILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYH 
                   
               
               
                   
                 HAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKA 
                   
               
               
                   
                 TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
                   
               
               
                   
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYG 
                   
               
               
                   
                 GFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFL 
                   
               
               
                   
                 EAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKY 
                   
               
               
                   
                 VNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL 
                   
               
               
                   
                 ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTID 
                   
               
               
                   
                 RKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSSGSETP 
                   
               
               
                   
                 GTSESATPESSGGSSGGSSMILKILNEIASIGSTKQKQAILEKNKDNELL 
                   
               
               
                   
                 KRVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLTDMLDFIEFTLATRKL 
                   
               
               
                   
                 TGNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGASVSIANKVWPGLIP 
                   
               
               
                   
                 EQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLLS 
                   
               
               
                   
                 RAGNEYLGLDLLKEELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEGL 
                   
               
               
                   
                 DFLFDAYPENSKAKEFAEVAESRTASNGIANKSLKGTISEKEAQCMKFQV 
                   
               
               
                   
                 WDYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKVILIENQVVNNLDEA 
                   
               
               
                   
                 KVIYKKYIDQGLEGIILKNIDGLWENARSKNLYKFKEVIDVDLKIVGIYP 
                   
               
               
                   
                 HRKDPTKAGGFILESECGKIKVNAGSGLKDKAGVKSHELDRTRIMENQNY 
                   
               
               
                   
                 YIGKILECECNGWLKSDGRTDYVKLFLPIAIRLREDKTKANTFEDVFGDF 
                   
               
               
                   
                 HEVTGLSGGSSGGSKRTADGSEFESPKKKRKVSGGSSGGSMGIQGLAKLI 
                   
               
               
                   
                 ADVAPSAIRENDIKSYFGRKVAIDASMSIYQFLIAVRQGGDVLQNEEGET 
                   
               
               
                   
                 TSHLMGMFYRTIRMMENGIKPVYVFDGKPPQLKSGELAKRSERRAEAEKQ 
                   
               
               
                   
                 LQQAQAAGAEQEVEKFTKRLVKVTKQHNDECKHLLSLMGIPYLDAPSEAE 
                   
               
               
                   
                 ASCAALVKAGKVYAAATEDMDCLTFGSPVLMRHLTASEAKKLPIQEFHLS 
                   
               
               
                   
                 RILQELGLNQEQFVDLCILLGSDYCESIRGIGPKRAVDLIQKHKSIEEIV 
                   
               
               
                   
                 RRLDPNKYPVPENWLHKEAHQLFLEPEVLDPESVELKWSEPNEEELIKFM 
                   
               
               
                   
                 CGEKQFSEERIRSGVKRLSKSRQGSTQGRLDDFFKVTGSLSSAKRKEPEP 
                   
               
               
                   
                 KGSTKKKAKTGAAGKFKRGKSGGSKRTADSQHSTPPKTKRKVEFEPKKKR 
                   
               
               
                   
                 KV 
                   
               
               
                   
               
               
                 NLS1- 
                 KRTADGSEFESPKKKRKVMILKILNEIASIGSTKQKQAILEKNKDNELLK 
                 139 
               
               
                 T4LIG- 
                 RVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLTDMLDFIEFTLATRKLT 
                   
               
               
                 linker1- 
                 GNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGASVSIANKVWPGLIPE 
                   
               
               
                 hFENl- 
                 QPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLLSR 
                   
               
               
                 linker2- 
                 AGNEYLGLDLLKEELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEGLD 
                   
               
               
                 nCas9- 
                 FLFDAYPENSKAKEFAEVAESRTASNGIANKSLKGTISEKEAQCMKFQVW 
                   
               
               
                 NLS2 
                 DYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKVILIENQVVNNLDEAK 
                   
               
               
                   
                 VIYKKYIDQGLEGIILKNIDGLWENARSKNLYKFKEVIDVDLKIVGIYPH 
                   
               
               
                   
                 RKDPTKAGGFILESECGKIKVNAGSGLKDKAGVKSHELDRTRIMENQNYY 
                   
               
               
                   
                 IGKILECECNGWLKSDGRTDYVKLFLPIAIRLREDKTKANTFEDVFGDFH 
                   
               
               
                   
                 EVTGLSGGSSGGSSGSETPGTSESATPESSGGSSGGSSMGIQGLAKLIAD 
                   
               
               
                   
                 VAPSAIRENDIKSYFGRKVAIDASMSIYQFLIAVRQGGDVLQNEEGETTS 
                   
               
               
                   
                 HLMGMFYRTIRMMENGIKPVYVFDGKPPQLKSGELAKRSERRAEAEKQLQ 
                   
               
               
                   
                 QAQAAGAEQEVEKFTKRLVKVTKQHNDECKHLLSLMGIPYLDAPSEAEAS 
                   
               
               
                   
                 CAALVKAGKVYAAATEDMDCLTFGSPVLMRHLTASEAKKLPIQEFHLSRI 
                   
               
               
                   
                 LQELGLNQEQFVDLCILLGSDYCESIRGIGPKRAVDLIQKHKSIEEIVRR 
                   
               
               
                   
                 LDPNKYPVPENWLHKEAHQLFLEPEVLDPESVELKWSEPNEEELIKFMCG 
                   
               
               
                   
                 EKQFSEERIRSGVKRLSKSRQGSTQGRLDDFFKVTGSLSSAKRKEPEPKG 
                   
               
               
                   
                 STKKKAKTGAAGKFKRGKSGGSSGGSKRTADGSEFESPKKKRKVSGGSSG 
                   
               
               
                   
                 GSMDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI 
                   
               
               
                   
                 GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFF 
                   
               
               
                   
                 HRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDK 
                   
               
               
                   
                 ADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE 
                   
               
               
                   
                 NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGL 
                   
               
               
                   
                 TPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSD 
                   
               
               
                   
                 AILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYK 
                   
               
               
                   
                 EIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDL 
                   
               
               
                   
                 LRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI 
                   
               
               
                   
                 PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNF 
                   
               
               
                   
                 DKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAI 
                   
               
               
                   
                 VDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLL 
                   
               
               
                   
                 KIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVM 
                   
               
               
                   
                 KQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
                   
               
               
                   
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELV 
                   
               
               
                   
                 KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKE 
                   
               
               
                   
                 HPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLK 
                   
               
               
                   
                 DDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFD 
                   
               
               
                   
                 NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDK 
                   
               
               
                   
                 LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALI 
                   
               
               
                   
                 KKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT 
                   
               
               
                   
                 EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKT 
                   
               
               
                   
                 EVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAK 
                   
               
               
                   
                 VEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKL 
                   
               
               
                   
                 PKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGS 
                   
               
               
                   
                 PEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR 
                   
               
               
                   
                 DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI 
                   
               
               
                   
                 HQSITGLYETRIDLSQLGGDSGGSKRTADSQHSTPPKTKRKVEFEPKKKR 
                   
               
               
                   
                 KV 
                   
               
               
                   
               
               
                 NLS1-T5 
                 KRTADGSEFESPKKKRKVMSKSWGKFIEEEEAEMASRRNLMIVDGTNLGF 
                 140 
               
               
                 EXO- 
                 RFKHNNSKKPFASSYVSTIQSLAKSYSARTTIVLGDKGKSVFRLEHLPEY 
                   
               
               
                 linker1- 
                 KGNRDEKYAQRTEEEKALDEQFFEYLKDAFELCKTTFPTFTIRGVEADDM 
                   
               
               
                 nCas9- 
                 AAYIVKLIGHLYDHVWLISTDGDWDTLLTDKVSRFSFTTRREYHLRDMYE 
                   
               
               
                 linker2- 
                 HHNVDDVEQFISLKAIMGDLGDNIRGVEGIGAKRGYNIIREFGNVLDIID 
                   
               
               
                 T4LIG- 
                 QLPLPGKQKYIQNLNASEELLFRNLILVDLPTYCVDAIAAVGQDVLDKFT 
                   
               
               
                 NLS2 
                 KDILEIAEQSGGSSGGSSGSETPGTSESATPESSGGSSGGSSMDKKYSIG 
                   
               
               
                   
                 LDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
                   
               
               
                   
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVE 
                   
               
               
                   
                 EDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL 
                   
               
               
                   
                 AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDA 
                   
               
               
                   
                 KAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL 
                   
               
               
                   
                 AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRV 
                   
               
               
                   
                 NTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNG 
                   
               
               
                   
                 YAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG 
                   
               
               
                   
                 SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG 
                   
               
               
                   
                 NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVL 
                   
               
               
                   
                 PKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRK 
                   
               
               
                   
                 VTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLD 
                   
               
               
                   
                 NEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTG 
                   
               
               
                   
                 WGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDI 
                   
               
               
                   
                 QKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPEN 
                   
               
               
                   
                 IVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQN 
                   
               
               
                   
                 EKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLT 
                   
               
               
                   
                 RSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGL 
                   
               
               
                   
                 SELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITL 
                   
               
               
                   
                 KSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEF 
                   
               
               
                   
                 VYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIR 
                   
               
               
                   
                 KRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE 
                   
               
               
                   
                 SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLK 
                   
               
               
                   
                 SVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELEN 
                   
               
               
                   
                 GRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLF 
                   
               
               
                   
                 VEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAEN 
                   
               
               
                   
                 IIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYET 
                   
               
               
                   
                 RIDLSQLGGDSGGSSGGSKRTADGSEFESPKKKRKVSGGSSGGSMILKIL 
                   
               
               
                   
                 NEIASIGSTKQKQAILEKNKDNELLKRVYRLTYSRGLQYYIKKWPKPGIA 
                   
               
               
                   
                 TQSFGMLTLTDMLDFIEFTLATRKLTGNAAIEELTGYITDGKKDDVEVLR 
                   
               
               
                   
                 RVMMRDLECGASVSIANKVWPGLIPEQPQMLASSYDEKGINKNIKFPAFA 
                   
               
               
                   
                 QLKADGARCFAEVRGDELDDVRLLSRAGNEYLGLDLLKEELIKMTAEARQ 
                   
               
               
                   
                 IHPEGVLIDGELVYHEQVKKEPEGLDFLFDAYPENSKAKEFAEVAESRTA 
                   
               
               
                   
                 SNGIANKSLKGTISEKEAQCMKFQVWDYVPLVEIYSLPAFRLKYDVRFSK 
                   
               
               
                   
                 LEQMTSGYDKVILIENQVVNNLDEAKVIYKKYIDQGLEGIILKNIDGLWE 
                   
               
               
                   
                 NARSKNLYKFKEVIDVDLKIVGIYPHRKDPTKAGGFILESECGKIKVNAG 
                   
               
               
                   
                 SGLKDKAGVKSHELDRTRIMENQNYYIGKILECECNGWLKSDGRTDYVKL 
                   
               
               
                   
                 FLPIAIRLREDKTKANTFEDVFGDFHEVTGLSGGSKRTADSQHSTPPKTK 
                   
               
               
                   
                 RKVEFEPKKKRKV 
                   
               
               
                   
               
               
                 LZ- 
                 ATGTTGGAGATAGAGGCTGCTTTCCTTGAACGGGAGAATACGGCCCTCGA 
                 141 
               
               
                 SplintR- 
                 GACTAGGGTTGCTGAGCTTAGACAGCGAGTCCAAAGACTGCGAAACCGGG 
                   
               
               
                 bpNLS 
                 TGTCCCAATATAGGACCAGATACGGACCTCTGGGTGGAGGGAAATCCGGT 
                   
               
               
                   
                 GGGAGTAGCGGCGGGTCTAGTGGCTCAGAGACACCTGGCACGAGCGAGAG 
                   
               
               
                   
                 TGCGACTCCTGAAAGCTCCGGCGGCAGCAGTGGGGGAAGTTCCATGGCAA 
                   
               
               
                   
                 TCACTAAGCCCCTCTTGGCGGCGACTTTGGAAAACATCGAGGATGTGCAA 
                   
               
               
                   
                 TTCCCGTGCCTTGCCACACCAAAGATAGACGGGATCCGATCAGTGAAGCA 
                   
               
               
                   
                 AACGCAGATGCTCTCTAGAACGTTCAAGCCTATTAGAAACTCAGTGATGA 
                   
               
               
                   
                 ATCGGCTCTTGACTGAGCTGTTGCCGGAAGGCAGCGATGGGGAAATATCT 
                   
               
               
                   
                 ATCGAGGGAGCCACATTTCAAGACACTACGAGCGCCGTAATGACTGGACA 
                   
               
               
                   
                 TAAGATGTATAATGCTAAATTCTCCTACTATTGGTTTGACTATGTCACTG 
                   
               
               
                   
                 ATGACCCTCTTAAGAAATATATAGACCGAGTGGAGGATATGAAAAATTAT 
                   
               
               
                   
                 ATTACTGTACACCCGCATATTCTGGAACATGCCCAAGTTAAGATTATTCC 
                   
               
               
                   
                 TCTCATTCCCGTCGAGATTAATAATATCACAGAACTGCTTCAGTATGAGC 
                   
               
               
                   
                 GCGACGTATTGTCCAAAGGCTTTGAAGGGGTTATGATACGCAAACCGGAC 
                   
               
               
                   
                 GGCAAGTACAAGTTCGGAAGAAGCACATTGAAAGAGGGTATATTGCTGAA 
                   
               
               
                   
                 GATGAAGCAGTTTAAGGATGCTGAGGCAACAATAATCAGCATGACAGCAC 
                   
               
               
                   
                 TTTTTAAAAATACCAACACGAAAACTAAGGACAATTTTGGTTATAGTAAG 
                   
               
               
                   
                 CGGTCAACGCACAAAAGTGGGAAGGTAGAAGAAGACGTAATGGGTAGCAT 
                   
               
               
                   
                 TGAGGTGGATTATGACGGGGTGGTTTTCAGCATAGGGACTGGGTTTGATG 
                   
               
               
                   
                 CAGATCAACGGAGGGACTTTTGGCAGAACAAAGAATCATATATAGGCAAA 
                   
               
               
                   
                 ATGGTAAAGTTCAAATACTTCGAAATGGGAAGTAAAGACTGCCCCAGATT 
                   
               
               
                   
                 CCCTGTATTCATTGGCATCAGGCACGAGGAGGACAGGAGTGGGGGATCAA 
                   
               
               
                   
                 AGCGGACTGCTGATAGTCAGCATAGTACTCCACCCAAGACCAAGCGGAAA 
                   
               
               
                   
                 GTTGAGTTTGAGCCGAAGAAAAAGCGAAAAGTGTAA 
                   
               
               
                   
               
               
                 LZ-T4LIG- 
                 ATGCTTGAGATCGAGGCGGCGTTCCTCGAAAGAGAGAACACCGCACTTGA 
                 142 
               
               
                 bpNLS 
                 AACTCGCGTGGCAGAATTGAGGCAGCGGGTGCAAAGACTTAGAAATAGAG 
                   
               
               
                   
                 TCTCTCAGTATCGGACCCGATATGGTCCTCTGGGAGGCGGGAAGTCTGGA 
                   
               
               
                   
                 GGTTCAAGCGGAGGCAGTTCCGGGAGTGAGACACCGGGAACTTCTGAGAG 
                   
               
               
                   
                 TGCAACTCCTGAGAGCTCTGGTGGATCATCCGGAGGCTCCAGTATGATTC 
                   
               
               
                   
                 TTAAAATTCTTAACGAGATTGCGAGTATTGGCAGCACGAAACAAAAGCAG 
                   
               
               
                   
                 GCCATATTGGAAAAGAATAAGGACAATGAGTTGCTTAAACGCGTGTATAG 
                   
               
               
                   
                 GCTCACTTACTCTCGCGGACTGCAATACTATATTAAAAAATGGCCTAAGC 
                   
               
               
                   
                 CCGGCATCGCTACTCAAAGCTTCGGAATGCTTACGCTGACAGATATGCTC 
                   
               
               
                   
                 GACTTCATCGAGTTTACTCTCGCAACAAGGAAGTTGACTGGCAACGCCGC 
                   
               
               
                   
                 GATTGAAGAATTGACGGGTTATATCACGGACGGGAAGAAGGATGATGTTG 
                   
               
               
                   
                 AGGTGCTGAGGCGCGTTATGATGCGCGACCTCGAATGTGGTGCCTCAGTT 
                   
               
               
                   
                 TCCATAGCCAATAAAGTTTGGCCAGGCTTGATCCCGGAGCAGCCACAGAT 
                   
               
               
                   
                 GCTGGCCAGTAGCTACGACGAGAAGGGTATTAACAAAAATATCAAGTTTC 
                   
               
               
                   
                 CAGCGTTTGCACAACTTAAAGCGGATGGGGCGCGGTGTTTCGCCGAAGTC 
                   
               
               
                   
                 CGGGGTGACGAATTGGACGATGTGCGCCTTCTGAGTCGCGCAGGAAATGA 
                   
               
               
                   
                 ATATCTGGGGCTTGACCTCTTGAAGGAGGAGCTGATTAAGATGACAGCAG 
                   
               
               
                   
                 AAGCCAGGCAGATCCATCCAGAGGGGGTACTTATTGATGGTGAACTCGTA 
                   
               
               
                   
                 TACCATGAGCAGGTTAAGAAGGAGCCAGAGGGTTTGGATTTCCTCTTTGA 
                   
               
               
                   
                 CGCCTATCCCGAGAATTCAAAGGCAAAGGAGTTCGCCGAGGTTGCAGAAT 
                   
               
               
                   
                 CAAGAACGGCTTCCAACGGCATAGCGAATAAATCACTCAAAGGAACTATA 
                   
               
               
                   
                 TCTGAAAAGGAGGCACAGTGTATGAAATTCCAAGTGTGGGACTATGTGCC 
                   
               
               
                   
                 GCTTGTCGAGATTTACAGCTTGCCTGCTTTCCGATTGAAGTACGATGTAC 
                   
               
               
                   
                 GGTTTAGTAAGCTCGAGCAAATGACTTCAGGTTACGATAAAGTCATCTTG 
                   
               
               
                   
                 ATTGAGAACCAGGTCGTTAATAATCTTGACGAGGCGAAGGTCATATATAA 
                   
               
               
                   
                 GAAATATATAGATCAAGGGCTCGAGGGTATCATTCTGAAGAATATAGATG 
                   
               
               
                   
                 GCTTGTGGGAAAACGCCAGGTCCAAAAACCTGTATAAGTTTAAGGAAGTA 
                   
               
               
                   
                 ATAGATGTAGATTTGAAAATAGTTGGAATTTACCCCCATCGGAAGGACCC 
                   
               
               
                   
                 CACGAAAGCGGGTGGGTTTATCCTCGAGAGCGAGTGTGGGAAGATAAAAG 
                   
               
               
                   
                 TGAATGCCGGCTCCGGATTGAAGGACAAGGCAGGTGTGAAAAGTCATGAG 
                   
               
               
                   
                 CTCGATCGGACGAGAATAATGGAAAACCAGAATTACTACATTGGAAAGAT 
                   
               
               
                   
                 TTTGGAATGCGAGTGTAACGGCTGGTTGAAGAGCGACGGACGCACCGATT 
                   
               
               
                   
                 ACGTGAAACTCTTTCTGCCAATTGCAATCAGGTTGAGAGAGGATAAGACT 
                   
               
               
                   
                 AAGGCCAATACTTTCGAGGACGTCTTCGGAGACTTTCACGAAGTCACTGG 
                   
               
               
                   
                 GCTTTCTGGGGGTAGTAAGAGAACTGCAGATAGCCAGCATTCAACGCCGC 
                   
               
               
                   
                 CAAAAACAAAGCGAAAGGTAGAATTCGAACCAAAGAAAAAGCGGAAAGTA 
                   
               
               
                   
                 TAA 
                   
               
               
                   
               
               
                 LZ- 
                 ATGCTCGAGATCGAAGCTGCATTTCTGGAGAGGGAGAATACCGCCCTCGA 
                 143 
               
               
                 hLIG1(233 
                 AACCCGGGTGGCTGAATTGCGACAGAGAGTGCAACGGCTCCGGAATAGAG 
                   
               
               
                 -919)- 
                 TATCTCAATATCGAACCCGCTATGGGCCTCTCGGAGGGGGTAAATCTGGC 
                   
               
               
                 bpNLS 
                 GGAAGTTCTGGCGGTAGTTCAGGAAGTGAGACACCGGGAACTAGTGAATC 
                   
               
               
                   
                 CGCGACTCCCGAATCAAGTGGGGGATCATCTGGAGGGTCAAGCACACCCA 
                   
               
               
                   
                 GGAAACCAGCCGTGAAAAAAGAGGTTAAAGAAGAGGAACCTGGGGCTCCG 
                   
               
               
                   
                 GGAAAGGAGGGAGCAGCGGAAGGTCCGCTCGACCCTTCAGGATACAACCC 
                   
               
               
                   
                 AGCCAAAAACAACTACCACCCCGTAGAGGATGCTTGCTGGAAGCCAGGCC 
                   
               
               
                   
                 AAAAGGTGCCCTATTTGGCCGTTGCTAGGACTTTCGAAAAAATTGAGGAG 
                   
               
               
                   
                 GTGAGCGCGCGACTCAGAATGGTAGAGACTCTGTCTAACCTCCTTCGCTC 
                   
               
               
                   
                 CGTAGTGGCTCTTTCACCTCCAGATCTTCTTCCAGTGCTGTACCTGAGCC 
                   
               
               
                   
                 TGAACCACTTGGGCCCTCCCCAGCAGGGACTGGAACTGGGCGTAGGGGAC 
                   
               
               
                   
                 GGAGTATTGCTGAAGGCTGTTGCTCAGGCAACCGGACGACAGCTCGAGTC 
                   
               
               
                   
                 TGTGCGAGCAGAAGCTGCAGAAAAGGGGGACGTCGGGTTGGTTGCCGAAA 
                   
               
               
                   
                 ATTCAAGATCTACCCAACGATTGATGTTGCCACCGCCGCCTCTGACTGCG 
                   
               
               
                   
                 TCAGGTGTATTCTCCAAGTTCCGGGATATTGCCAGGCTTACGGGTAGCGC 
                   
               
               
                   
                 TTCCACTGCTAAAAAGATCGACATAATAAAAGGTCTGTTCGTCGCTTGTC 
                   
               
               
                   
                 GCCATTCAGAGGCGAGGTTTATAGCCAGATCCCTTTCCGGACGACTTCGA 
                   
               
               
                   
                 CTCGGCTTGGCTGAGCAGTCAGTACTGGCAGCTTTGTCTCAAGCTGTATC 
                   
               
               
                   
                 ACTCACGCCCCCCGGACAAGAATTTCCACCCGCCATGGTTGACGCAGGCA 
                   
               
               
                   
                 AGGGTAAGACTGCTGAGGCAAGAAAGACGTGGCTGGAGGAACAAGGTATG 
                   
               
               
                   
                 ATACTTAAACAAACGTTTTGCGAAGTTCCGGACTTGGACCGGATCATACC 
                   
               
               
                   
                 TGTGTTGCTGGAGCACGGCCTCGAGCGCTTGCCCGAACACTGTAAACTGT 
                   
               
               
                   
                 CTCCAGGAATACCTCTCAAACCCATGTTGGCTCATCCTACGAGGGGAATC 
                   
               
               
                   
                 TCAGAGGTACTTAAACGGTTTGAAGAAGCCGCTTTCACGTGCGAATACAA 
                   
               
               
                   
                 GTATGATGGTCAGAGAGCGCAAATCCACGCATTGGAAGGGGGTGAGGTAA 
                   
               
               
                   
                 AGATTTTTTCAAGGAATCAGGAGGACAATACAGGGAAGTACCCCGATATC 
                   
               
               
                   
                 ATCAGTCGGATTCCTAAAATTAAGCTTCCATCAGTCACGTCCTTCATACT 
                   
               
               
                   
                 GGACACTGAGGCAGTGGCTTGGGACCGAGAGAAGAAGCAGATACAACCCT 
                   
               
               
                   
                 TTCAGGTACTTACAACCAGAAAGCGCAAGGAAGTCGACGCTTCTGAGATT 
                   
               
               
                   
                 CAAGTACAAGTCTGCCTTTATGCGTTTGACCTGATCTATCTTAATGGAGA 
                   
               
               
                   
                 GAGTTTGGTGAGAGAACCCTTGAGCAGACGACGGCAGCTCTTGAGAGAAA 
                   
               
               
                   
                 ATTTCGTAGAAACTGAGGGGGAGTTCGTCTTTGCGACTAGTCTCGACACC 
                   
               
               
                   
                 AAAGACATTGAGCAAATCGCGGAATTCCTCGAACAGTCAGTTAAAGACTC 
                   
               
               
                   
                 CTGCGAAGGTCTGATGGTTAAGACTCTTGACGTGGATGCTACCTACGAGA 
                   
               
               
                   
                 TAGCTAAGCGGTCACACAATTGGCTGAAACTGAAAAAGGACTATCTGGAT 
                   
               
               
                   
                 GGAGTTGGGGACACGCTGGATTTGGTCGTTATCGGGGCCTATCTGGGACG 
                   
               
               
                   
                 CGGTAAGCGGGCAGGGAGATATGGTGGATTCCTCCTCGCTTCATACGATG 
                   
               
               
                   
                 AGGACTCTGAAGAGCTGCAGGCTATATGCAAACTTGGGACGGGTTTTTCC 
                   
               
               
                   
                 GATGAAGAATTGGAGGAACATCATCAGTCACTGAAGGCCCTTGTATTGCC 
                   
               
               
                   
                 AAGTCCACGCCCATACGTACGAATCGATGGAGCAGTAATCCCTGACCACT 
                   
               
               
                   
                 GGCTTGACCCGTCCGCCGTCTGGGAAGTAAAGTGCGCGGATCTCTCTCTC 
                   
               
               
                   
                 AGTCCGATCTACCCAGCCGCACGGGGGCTGGTTGACAGTGACAAGGGTAT 
                   
               
               
                   
                 CAGCCTGCGATTTCCTCGATTCATACGCGTCCGGGAAGACAAGCAACCGG 
                   
               
               
                   
                 AACAGGCTACGACCTCTGCACAGGTCGCATGTTTGTATAGAAAACAGAGC 
                   
               
               
                   
                 CAAATTCAGAATCAACAAGGCGAAGACAGTGGGTCCGATCCTGAAGATAC 
                   
               
               
                   
                 CTACTCAGGCGGCAGTAAACGGACAGCTGATAGCCAACACTCAACTCCTC 
                   
               
               
                   
                 CGAAGACTAAAAGGAAGGTAGAGTTCGAACCAAAAAAGAAAAGGAAAGTG 
                   
               
               
                   
                 TAA 
                   
               
               
                   
               
               
                 LZ- 
                 ATGCTCGAGATCGAGGCGGCGTTCCTTGAACGCGAGAACACTGCGCTGGA 
                 144 
               
               
                 hLIG1(119 
                 AACGAGGGTCGCGGAACTCCGCCAGAGGGTTCAACGGTTGAGGAATCGAG 
                   
               
               
                 -919)- 
                 TGAGTCAGTACCGAACCCGATATGGACCACTGGGTGGCGGGAAATCAGGG 
                   
               
               
                 bpNLS 
                 GGCTCATCCGGCGGCTCCAGCGGGAGCGAAACCCCGGGTACCTCAGAATC 
                   
               
               
                   
                 TGCGACGCCAGAAAGCTCAGGCGGATCTAGCGGCGGTAGTTCACCGAAGC 
                   
               
               
                   
                 GCCGGACTGCACGAAAGCAACTGCCAAAACGGACTATACAAGAAGTCCTG 
                   
               
               
                   
                 GAAGAACAAAGCGAAGATGAGGATCGCGAAGCCAAGCGCAAGAAAGAGGA 
                   
               
               
                   
                 AGAGGAAGAAGAGACTCCAAAGGAGTCCTTGACCGAAGCAGAAGTCGCAA 
                   
               
               
                   
                 CGGAGAAGGAAGGTGAGGATGGGGATCAGCCAACAACCCCGCCTAAACCT 
                   
               
               
                   
                 CTGAAAACCTCTAAGGCGGAGACACCAACTGAGAGTGTCAGCGAACCGGA 
                   
               
               
                   
                 GGTAGCCACGAAACAAGAGCTTCAGGAGGAAGAAGAACAGACAAAGCCAC 
                   
               
               
                   
                 CTCGGCGGGCTCCCAAAACCCTTAGCTCCTTCTTCACGCCTCGAAAGCCA 
                   
               
               
                   
                 GCAGTGAAGAAAGAAGTGAAGGAGGAGGAACCTGGCGCCCCTGGAAAGGA 
                   
               
               
                   
                 GGGCGCAGCCGAGGGCCCGCTGGACCCTTCAGGGTATAACCCGGCAAAAA 
                   
               
               
                   
                 ATAATTACCACCCGGTCGAGGACGCTTGTTGGAAACCAGGCCAAAAGGTA 
                   
               
               
                   
                 CCTTACCTCGCCGTCGCTAGGACCTTTGAGAAGATAGAGGAAGTTAGTGC 
                   
               
               
                   
                 TAGGTTGAGAATGGTCGAAACCCTTAGTAACCTTCTCAGGTCCGTAGTCG 
                   
               
               
                   
                 CCCTTAGTCCCCCAGACCTGCTTCCGGTGCTGTACCTGTCCCTGAACCAT 
                   
               
               
                   
                 CTCGGTCCCCCCCAACAGGGACTGGAGTTGGGCGTCGGTGACGGCGTTCT 
                   
               
               
                   
                 CCTGAAAGCGGTTGCACAAGCTACAGGAAGGCAACTGGAATCTGTCCGGG 
                   
               
               
                   
                 CTGAGGCTGCAGAGAAAGGTGACGTGGGGCTTGTGGCAGAGAATAGTCGG 
                   
               
               
                   
                 TCAACACAGCGGCTGATGCTGCCACCGCCCCCGCTTACGGCTAGTGGGGT 
                   
               
               
                   
                 ATTCTCCAAATTTAGAGATATAGCACGGCTGACGGGATCAGCTTCCACTG 
                   
               
               
                   
                 CGAAGAAGATCGATATCATTAAGGGTTTGTTCGTGGCTTGCAGGCATTCC 
                   
               
               
                   
                 GAAGCACGCTTCATTGCACGCTCCCTTTCAGGGAGACTCAGACTTGGGCT 
                   
               
               
                   
                 GGCCGAGCAATCTGTACTGGCGGCCCTGTCTCAGGCGGTGAGCCTTACGC 
                   
               
               
                   
                 CGCCCGGGCAAGAGTTCCCTCCTGCGATGGTCGATGCTGGGAAGGGAAAA 
                   
               
               
                   
                 ACCGCCGAAGCTCGAAAAACATGGCTGGAGGAGCAAGGAATGATTTTGAA 
                   
               
               
                   
                 GCAGACGTTCTGTGAAGTACCGGACTTGGATCGCATCATACCTGTGCTTC 
                   
               
               
                   
                 TCGAACATGGTTTGGAGCGGCTCCCCGAGCATTGCAAACTCTCTCCGGGC 
                   
               
               
                   
                 ATCCCCCTCAAGCCAATGCTCGCCCACCCCACGCGCGGAATCAGTGAGGT 
                   
               
               
                   
                 ACTGAAACGCTTTGAAGAGGCAGCGTTTACTTGTGAATACAAGTACGATG 
                   
               
               
                   
                 GCCAAAGGGCACAAATTCATGCACTTGAAGGCGGGGAAGTTAAGATATTC 
                   
               
               
                   
                 AGCAGGAATCAGGAGGACAACACGGGAAAATATCCTGACATAATATCTCG 
                   
               
               
                   
                 AATCCCTAAAATTAAGTTGCCTAGCGTAACCAGCTTCATCCTGGATACCG 
                   
               
               
                   
                 AAGCCGTGGCGTGGGATAGGGAGAAAAAGCAAATACAGCCATTTCAGGTG 
                   
               
               
                   
                 CTTACAACTAGAAAACGAAAAGAGGTGGACGCTAGTGAAATCCAAGTCCA 
                   
               
               
                   
                 GGTATGTCTTTATGCCTTCGATTTGATATACCTTAATGGTGAGTCCCTTG 
                   
               
               
                   
                 TACGGGAACCGCTTAGTAGGAGGCGGCAGTTGCTGAGGGAAAATTTTGTC 
                   
               
               
                   
                 GAAACTGAGGGAGAGTTTGTATTTGCAACGTCATTGGATACAAAGGACAT 
                   
               
               
                   
                 AGAACAAATAGCAGAATTTCTGGAGCAGTCAGTAAAAGACTCCTGCGAGG 
                   
               
               
                   
                 GCCTGATGGTGAAAACTCTTGATGTGGACGCCACTTATGAAATCGCAAAA 
                   
               
               
                   
                 AGGTCACACAATTGGCTGAAACTTAAAAAGGATTACTTGGACGGGGTCGG 
                   
               
               
                   
                 GGATACCCTCGATCTCGTCGTAATCGGAGCTTATCTCGGTAGGGGGAAGC 
                   
               
               
                   
                 GAGCCGGGCGATACGGAGGCTTTCTCTTGGCTAGTTATGACGAAGATTCC 
                   
               
               
                   
                 GAAGAGCTGCAGGCCATATGCAAGCTTGGAACGGGTTTCAGCGATGAGGA 
                   
               
               
                   
                 ATTGGAGGAGCATCATCAGAGCTTGAAGGCACTGGTGCTCCCCTCTCCTA 
                   
               
               
                   
                 GGCCGTACGTTAGAATAGACGGAGCAGTGATACCCGATCATTGGCTCGAT 
                   
               
               
                   
                 CCGTCAGCTGTTTGGGAGGTGAAGTGTGCAGACCTGTCCCTCTCTCCTAT 
                   
               
               
                   
                 TTACCCTGCAGCACGCGGTCTGGTTGACTCTGACAAAGGGATTAGCTTGA 
                   
               
               
                   
                 GGTTCCCTAGATTTATTCGGGTGCGCGAAGACAAACAGCCTGAACAGGCG 
                   
               
               
                   
                 ACAACGTCCGCGCAGGTCGCATGCCTTTATCGAAAACAGAGTCAGATCCA 
                   
               
               
                   
                 GAATCAACAAGGAGAAGATTCAGGGAGTGACCCGGAGGACACTTATAGTG 
                   
               
               
                   
                 GCGGCTCAAAACGAACCGCCGATAGTCAGCATTCAACACCTCCAAAAACT 
                   
               
               
                   
                 AAAAGGAAAGTCGAGTTTGAGCCAAAGAAGAAGCGCAAAGTCTAA 
                   
               
               
                   
               
               
                 T4-LZ 
                 ATGATCCTTAAGATTCTCAACGAAATCGCTAGTATAGGGTCCACTAAGCA 
                 145 
               
               
                   
                 GAAGCAGGCCATATTGGAAAAAAATAAGGACAATGAACTTTTGAAGAGAG 
                   
               
               
                   
                 TCTATAGACTGACGTACTCTAGGGGGCTCCAGTACTACATCAAGAAATGG 
                   
               
               
                   
                 CCTAAACCTGGCATTGCGACGCAGTCATTCGGTATGCTGACATTGACGGA 
                   
               
               
                   
                 TATGTTGGATTTCATTGAGTTTACGCTGGCCACCAGAAAACTTACGGGTA 
                   
               
               
                   
                 ATGCTGCGATAGAAGAACTTACAGGGTACATAACAGACGGGAAGAAAGAT 
                   
               
               
                   
                 GACGTGGAAGTGCTCAGACGAGTTATGATGCGCGATCTCGAGTGCGGCGC 
                   
               
               
                   
                 TAGCGTGTCAATCGCGAACAAAGTCTGGCCCGGCCTCATACCAGAGCAGC 
                   
               
               
                   
                 CACAGATGCTGGCATCTTCCTATGACGAAAAAGGCATAAACAAGAATATT 
                   
               
               
                   
                 AAATTCCCGGCCTTCGCTCAACTCAAAGCAGATGGTGCCAGGTGTTTTGC 
                   
               
               
                   
                 CGAAGTTCGGGGTGATGAACTTGATGACGTGCGGCTCTTGTCTAGGGCAG 
                   
               
               
                   
                 GTAACGAGTACCTCGGCCTGGACTTGCTTAAAGAGGAACTGATTAAAATG 
                   
               
               
                   
                 ACAGCTGAGGCGCGGCAGATACACCCCGAGGGCGTCCTTATCGACGGGGA 
                   
               
               
                   
                 GCTGGTGTATCACGAACAAGTTAAAAAGGAACCGGAGGGTCTTGATTTTC 
                   
               
               
                   
                 TTTTCGACGCGTATCCTGAGAACAGCAAGGCGAAAGAATTTGCAGAAGTT 
                   
               
               
                   
                 GCAGAAAGCAGGACCGCAAGTAATGGAATCGCTAATAAAAGCCTCAAGGG 
                   
               
               
                   
                 TACCATCAGCGAAAAAGAAGCCCAGTGCATGAAATTTCAAGTTTGGGACT 
                   
               
               
                   
                 ATGTCCCCTTGGTCGAAATTTACTCCCTGCCCGCATTCCGGCTGAAGTAT 
                   
               
               
                   
                 GATGTTCGCTTCAGTAAACTGGAGCAAATGACGAGCGGTTATGATAAGGT 
                   
               
               
                   
                 TATACTTATTGAGAATCAGGTCGTAAATAATTTGGACGAGGCGAAAGTTA 
                   
               
               
                   
                 TATACAAAAAGTATATAGACCAAGGGTTGGAGGGGATCATTTTGAAGAAC 
                   
               
               
                   
                 ATAGACGGACTTTGGGAGAACGCCCGGTCCAAGAATTTGTATAAATTCAA 
                   
               
               
                   
                 AGAAGTCATAGATGTTGACCTCAAGATAGTAGGTATATATCCCCACAGAA 
                   
               
               
                   
                 AGGACCCAACCAAAGCAGGCGGATTCATTTTGGAGTCCGAGTGTGGGAAG 
                   
               
               
                   
                 ATAAAGGTCAATGCTGGATCTGGACTCAAGGACAAAGCTGGTGTGAAGTC 
                   
               
               
                   
                 ACATGAACTGGACCGAACCAGGATTATGGAGAATCAGAACTATTACATCG 
                   
               
               
                   
                 GGAAGATATTGGAGTGTGAATGCAACGGCTGGCTTAAATCAGATGGAAGA 
                   
               
               
                   
                 ACTGATTACGTTAAATTGTTCCTGCCCATAGCCATACGACTCCGCGAGGA 
                   
               
               
                   
                 CAAAACGAAGGCTAACACGTTTGAAGACGTATTCGGAGATTTCCATGAGG 
                   
               
               
                   
                 TGACTGGCCTTAGTGgaggctccaaacggacagcagactcccaacattca 
                   
               
               
                   
                 acacccccaaaaacaaagcggaaggtagagtttgagccaaaaaagaaaag 
                   
               
               
                   
                 aaaggtcGGAGGAGGAGGCAGTGGTGGGCGACTTGAAATTAGAGCCGCGT 
                   
               
               
                   
                 TCCTGCGCCAGAGGAATACGGCTCTCCGCACGGAGGTAGCCGAACTTGAG 
                   
               
               
                   
                 CAAGAAGTACAGAGATTGGAGAACGAGGTTTCACAGTATGAGACACGATA 
                   
               
               
                   
                 TGGCCCCCTTGGCGGCGGAAAGTAA 
                   
               
               
                   
               
               
                 LZ- 
                 ATGCTTGAGATCGAGGCGGCGTTCCTCGAAAGAGAGAACACCGCACTTGA 
                 146 
               
               
                 hLIG4(1- 
                 AACTCGCGTGGCAGAATTGAGGCAGCGGGTGCAAAGACTTAGAAATAGAG 
                   
               
               
                 620) 
                 TCTCTCAGTATCGGACCCGATATGGTCCTCTGGGAGGCGGGAAGTCTGGA 
                   
               
               
                   
                 GGTTCAAGCGGAGGCAGTTCCGGGAGTGAGACACCGGGAACTTCTGAGAG 
                   
               
               
                   
                 TGCAACTCCTGAGAGCTCTGGTGGATCATCCGGAGGCTCCAGTGCAGCTT 
                   
               
               
                   
                 CTCAGACCTCTCAAACAGTAGCCTCTCATGTACCGTTCGCTGACTTGTGT 
                   
               
               
                   
                 TCTACGCTCGAACGCATCCAGAAATCAAAGGGGCGCGCCGAGAAAATCCG 
                   
               
               
                   
                 GCACTTCAGAGAATTCTTGGATTCCTGGAGGAAGTTTCATGATGCTCTCC 
                   
               
               
                   
                 ACAAAAATCACAAAGATGTAACGGATAGTTTCTACCCTGCTATGAGACTT 
                   
               
               
                   
                 ATACTGCCGCAGCTTGAGAGGGAACGCATGGCGTATGGTATAAAGGAGAC 
                   
               
               
                   
                 AATGTTGGCGAAATTGTATATTGAGCTGCTGAACTTGCCAAGAGATGGAA 
                   
               
               
                   
                 AGGACGCGCTCAAACTGCTGAACTATAGAACACCCACGGGTACCCATGGT 
                   
               
               
                   
                 GACGCCGGTGACTTTGCCATGATCGCCTATTTCGTACTGAAACCTCGATG 
                   
               
               
                   
                 TCTTCAAAAAGGTTCTCTTACAATTCAGCAAGTCAACGACCTGCTGGATT 
                   
               
               
                   
                 CAATTGCGAGTAACAACAGCGCTAAGCGAAAGGATCTCATTAAGAAAAGC 
                   
               
               
                   
                 CTCCTGCAGCTGATAACTCAGTCCTCTGCACTCGAACAAAAATGGCTGAT 
                   
               
               
                   
                 TCGGATGATTATCAAGGATTTGAAGTTGGGGGTATCTCAGCAAACTATTT 
                   
               
               
                   
                 TCAGCGTGTTTCACAATGATGCAGCAGAATTGCATAATGTCACAACAGAT 
                   
               
               
                   
                 CTTGAGAAAGTCTGCCGACAGTTGCACGACCCCTCTGTAGGCTTGAGTGA 
                   
               
               
                   
                 CATATCTATAACACTTTTTTCTGCGTTCAAACCCATGTTGGCTGCTATTG 
                   
               
               
                   
                 CGGACATAGAACACATCGAGAAAGACATGAAACATCAGTCATTCTATATA 
                   
               
               
                   
                 GAGACTAAATTGGACGGCGAGAGGATGCAAATGCACAAAGATGGTGATGT 
                   
               
               
                   
                 GTATAAATATTTTTCCCGCAACGGCTACAACTACACTGATCAATTCGGAG 
                   
               
               
                   
                 CGTCCCCAACTGAAGGGTCCCTCACTCCTTTCATACACAATGCGTTTAAG 
                   
               
               
                   
                 GCCGATATTCAGATATGTATCCTCGACGGCGAAATGATGGCGTACAATCC 
                   
               
               
                   
                 CAATACCCAGACCTTCATGCAAAAGGGAACGAAGTTCGATATTAAACGGA 
                   
               
               
                   
                 TGGTTGAAGATTCCGACCTCCAAACATGTTACTGTGTGTTTGATGTCCTG 
                   
               
               
                   
                 ATGGTGAATAACAAAAAACTCGGCCATGAAACCCTTCGAAAGCGATACGA 
                   
               
               
                   
                 AATACTCAGCAGTATATTTACTCCAATACCAGGCCGAATCGAGATCGTAC 
                   
               
               
                   
                 AGAAAACACAAGCCCATACTAAGAATGAAGTTATTGATGCACTGAACGAA 
                   
               
               
                   
                 GCCATAGACAAGAGGGAAGAAGGCATAATGGTCAAGCAGCCTCTGAGTAT 
                   
               
               
                   
                 ATATAAACCTGACAAAAGGGGGGAAGGATGGCTGAAGATAAAGCCAGAAT 
                   
               
               
                   
                 ACGTGTCTGGTCTTATGGACGAATTGGACATTCTCATCGTCGGAGGATAT 
                   
               
               
                   
                 TGGGGTAAGGGTTCCAGGGGGGGGATGATGTCCCACTTTCTGTGTGCGGT 
                   
               
               
                   
                 TGCCGAGAAACCGCCCCCAGGGGAAAAACCATCAGTGTTCCATACGTTGT 
                   
               
               
                   
                 CACGCGTCGGCTCAGGTTGTACGATGAAGGAACTTTACGATCTGGGGTTG 
                   
               
               
                   
                 AAACTCGCCAAATATTGGAAGCCATTCCATCGGAAAGCACCGCCCTCTAG 
                   
               
               
                   
                 TATCTTGTGTGGGACGGAGAAGCCAGAAGTTTATATAGAGCCATGTAACT 
                   
               
               
                   
                 CAGTAATTGTTCAAATCAAAGCCGCAGAGATCGTCCCGTCAGACATGTAC 
                   
               
               
                   
                 AAGACTGGATGCACCCTTAGATTTCCTCGCATCGAAAAAATAAGAGATGA 
                   
               
               
                   
                 TAAAGAGTGGCATGAGTGCATGACTCTTGACGACCTTGAACAGCTCCGCG 
                   
               
               
                   
                 GGAAGGCCAGCGGTAAACTGGCTAGTAAGCACCTCTACATCGGGGGTGAC 
                   
               
               
                   
                 AGTGgaggctccaaacggacagcagactcccaacattcaacacccccaaa 
                   
               
               
                   
                 aacaaagcggaaggtagagtttgagccaaaaaagaaaagaaaggtctaa 
                   
               
               
                   
               
               
                 LZ-nCas9 
                 ATGCTTGAGATCGAGGCGGCGTTCCTCGAAAGAGAGAACACCGCACTTGA 
                 147 
               
               
                   
                 AACTCGCGTGGCAGAATTGAGGCAGCGGGTGCAAAGACTTAGAAATAGAG 
                   
               
               
                   
                 TCTCTCAGTATCGGACCCGATATGGTCCTCTGGGAGGCGGGAAGTCTGGA 
                   
               
               
                   
                 GGTTCAAGCGGAGGCAGTTCCGGGAGTGAGACACCGGGAACTTCTGAGAG 
                   
               
               
                   
                 TGCAACTCCTGAGAGCTCTGGTGGATCATCCGGAGGCTCCAGTaaacgga 
                   
               
               
                   
                 cagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtcgac 
                   
               
               
                   
                 aagaagtacagcatcggcctggacatcggcaccaactctgtgggctgggc 
                   
               
               
                   
                 cgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgg 
                   
               
               
                   
                 gcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctg 
                   
               
               
                   
                 ttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccag 
                   
               
               
                   
                 aagaagatacaccagacggaagaaccggatctgctatctgcaagagatct 
                   
               
               
                   
                 tcagcaacgagatggccaaggtggacgacagcttcttccacagactggaa 
                   
               
               
                   
                 gagtccttcctggtggaagaggataagaagcacgagcggcaccccatctt 
                   
               
               
                   
                 cggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatct 
                   
               
               
                   
                 accacctgagaaagaaactggtggacagcaccgacaaggccgacctgcgg 
                   
               
               
                   
                 ctgatctatctggccctggcccacatgatcaagttccggggccacttcct 
                   
               
               
                   
                 gatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttca 
                   
               
               
                   
                 tccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaac 
                   
               
               
                   
                 gccagcggcgtggacgccaaggccatcctgtctgccagactgagcaagag 
                   
               
               
                   
                 cagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatg 
                   
               
               
                   
                 gcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttc 
                   
               
               
                   
                 aagagcaacttcgacctggccgaggatgccaaactgcagctgagcaagga 
                   
               
               
                   
                 cacctacgacgacgacctggacaacctgctggcccagatcggcgaccagt 
                   
               
               
                   
                 acgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctg 
                   
               
               
                   
                 agcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgc 
                   
               
               
                   
                 ctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctga 
                   
               
               
                   
                 aagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttc 
                   
               
               
                   
                 gaccagagcaagaacggctacgccggctacattgacggcggagccagcca 
                   
               
               
                   
                 ggaagagttctacaagttcatcaagcccatcctggaaaagatggacggca 
                   
               
               
                   
                 ccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcag 
                   
               
               
                   
                 cggaccttcgacaacggcagcatcccccaccagatccacctgggagagct 
                   
               
               
                   
                 gcacgccattctgcggcggcaggaagatttttacccattcctgaaggaca 
                   
               
               
                   
                 accgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtg 
                   
               
               
                   
                 ggccctctggccaggggaaacagcagattcgcctggatgaccagaaagag 
                   
               
               
                   
                 cgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcg 
                   
               
               
                   
                 cttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctg 
                   
               
               
                   
                 cccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcac 
                   
               
               
                   
                 cgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaa 
                   
               
               
                   
                 agcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctg 
                   
               
               
                   
                 ttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactactt 
                   
               
               
                   
                 caagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatc 
                   
               
               
                   
                 ggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaag 
                   
               
               
                   
                 gacaaggacttcctggacaatgaggaaaacgaggacattctggaagatat 
                   
               
               
                   
                 cgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggc 
                   
               
               
                   
                 tgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaag 
                   
               
               
                   
                 cggcggagatacaccggctggggcaggctgagccggaagctgatcaacgg 
                   
               
               
                   
                 catccgggacaagcagtccggcaagacaatcctggatttcctgaagtccg 
                   
               
               
                   
                 acggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctg 
                   
               
               
                   
                 acctttaaagaggacatccagaaagcccaggtgtccggccagggcgatag 
                   
               
               
                   
                 cctgcacgagcacattgccaatctggccggcagccccgccattaagaagg 
                   
               
               
                   
                 gcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggc 
                   
               
               
                   
                 cggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccagac 
                   
               
               
                   
                 cacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaag 
                   
               
               
                   
                 agggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaa 
                   
               
               
                   
                 aacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgg 
                   
               
               
                   
                 gcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgact 
                   
               
               
                   
                 acgatgtggacgctatcgtgcctcagagctttctgaaggacgactccatc 
                   
               
               
                   
                 gacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaa 
                   
               
               
                   
                 cgtgccctccgaagaggtcgtgaagaagatgaagaactactggcggcagc 
                   
               
               
                   
                 tgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaag 
                   
               
               
                   
                 gccgagagaggcggcctgagcgaactggataaggccggcttcatcaagag 
                   
               
               
                   
                 acagctggtggaaacccggcagatcacaaagcacgtggcacagatcctgg 
                   
               
               
                   
                 actcccggatgaacactaagtacgacgagaatgacaagctgatccgggaa 
                   
               
               
                   
                 gtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaagga 
                   
               
               
                   
                 tttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacg 
                   
               
               
                   
                 acgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccct 
                   
               
               
                   
                 aagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcg 
                   
               
               
                   
                 gaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgccaagt 
                   
               
               
                   
                 acttcttctacagcaacatcatgaactttttcaagaccgagattaccctg 
                   
               
               
                   
                 gccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaac 
                   
               
               
                   
                 cggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaag 
                   
               
               
                   
                 tgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagaca 
                   
               
               
                   
                 ggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagct 
                   
               
               
                   
                 gatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgaca 
                   
               
               
                   
                 gccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggc 
                   
               
               
                   
                 aagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcat 
                   
               
               
                   
                 ggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaagg 
                   
               
               
                   
                 gctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactcc 
                   
               
               
                   
                 ctgttcgagctggaaaacggccggaagagaatgctggcctctgccggcga 
                   
               
               
                   
                 actgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcc 
                   
               
               
                   
                 tgtacctggccagccactatgagaagctgaagggctcccccgaggataat 
                   
               
               
                   
                 gagcagaaacagctgtttgtggaacagcacaagcactacctggacgagat 
                   
               
               
                   
                 catcgagcagatcagcgagttctccaagagagtgatcctggccgacgcta 
                   
               
               
                   
                 atctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatc 
                   
               
               
                   
                 agagagcaggccgagaatatcatccacctgtttaccctgaccaatctggg 
                   
               
               
                   
                 agcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggt 
                   
               
               
                   
                 acaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatc 
                   
               
               
                   
                 accggcctgtacgagacacggatcgacctgtctcagctgggaggtgacgg 
                   
               
               
                   
                 ctcaaaaagaaccgccgacggcagcgaattcgagtcacccaagaagaaga 
                   
               
               
                   
                 ggaaagtctaa 
                   
               
               
                   
               
               
                 SplintR-LZ 
                 atggcgataacgaagcccttgttggcagctacgttggaaaatattgagga 
                 148 
               
               
                   
                 cgtacagttcccatgccttgccactccgaagatcgatggaatccgatccg 
                   
               
               
                   
                 tgaaacagacacaaatgcttagcagaacattcaaacccatcaggaacagc 
                   
               
               
                   
                 gtaatgaatagattgcttacggaactcttgcccgaagggtctgacggcga 
                   
               
               
                   
                 gattagcatcgaaggagcgactttccaagatactacctcagcagttatga 
                   
               
               
                   
                 cgggacacaagatgtataatgctaaattctcatattactggtttgactat 
                   
               
               
                   
                 gttactgacgatcctttgaagaaatacatagacagggttgaagatatgaa 
                   
               
               
                   
                 aaattacataactgtccaccctcatatcctggagcatgcacaggtaaaga 
                   
               
               
                   
                 ttatcccgctcataccagtagaaattaacaatataaccgaattgttgcag 
                   
               
               
                   
                 tatgaacgcgatgtgctctctaaaggcttcgagggcgtgatgataaggaa 
                   
               
               
                   
                 gcctgatggcaaatataagttcggtaggtccacattgaaagagggaattc 
                   
               
               
                   
                 tcttgaagatgaaacagtttaaggatgcggaagctactatcattagtatg 
                   
               
               
                   
                 acggcactgtttaaaaacactaacactaaaaccaaggacaactttggcta 
                   
               
               
                   
                 tagtaaaaggagcacacacaaatcaggaaaagttgaggaggacgtaatgg 
                   
               
               
                   
                 gcagtatagaggtagattacgatggtgtggtgtttagcattggaacgggc 
                   
               
               
                   
                 ttcgacgctgaccagcggagggacttttggcagaataaggaaagttacat 
                   
               
               
                   
                 tggcaagatggttaaattcaaatacttcgagatgggctcaaaagactgtc 
                   
               
               
                   
                 cgagatttcctgtgtttattggaatcagacacgaagaggataggAGTGga 
                   
               
               
                   
                 ggctccaaacggacagcagactcccaacattcaacacccccaaaaacaaa 
                   
               
               
                   
                 gcggaaggtagagtttgagccaaaaaagaaaagaaaggtcGGAGGAGGAG 
                   
               
               
                   
                 GCAGTGGTGGGCGACTTGAAATTAGAGCCGCGTTCCTGCGCCAGAGGAAT 
                   
               
               
                   
                 ACGGCTCTCCGCACGGAGGTAGCCGAACTTGAGCAAGAAGTACAGAGATT 
                   
               
               
                   
                 GGAGAACGAGGTTTCACAGTATGAGACACGATATGGCCCCCTTGGCGGCG 
                   
               
               
                   
                 GAAAGtaa 
                   
               
               
                   
               
               
                 hLIG4(1- 
                 ATGGCAGCTTCTCAGACCTCTCAAACAGTAGCCTCTCATGTACCGTTCGC 
                 149 
               
               
                 620)-LZ 
                 TGACTTGTGTTCTACGCTCGAACGCATCCAGAAATCAAAGGGGCGCGCCG 
                   
               
               
                   
                 AGAAAATCCGGCACTTCAGAGAATTCTTGGATTCCTGGAGGAAGTTTCAT 
                   
               
               
                   
                 GATGCTCTCCACAAAAATCACAAAGATGTAACGGATAGTTTCTACCCTGC 
                   
               
               
                   
                 TATGAGACTTATACTGCCGCAGCTTGAGAGGGAACGCATGGCGTATGGTA 
                   
               
               
                   
                 TAAAGGAGACAATGTTGGCGAAATTGTATATTGAGCTGCTGAACTTGCCA 
                   
               
               
                   
                 AGAGATGGAAAGGACGCGCTCAAACTGCTGAACTATAGAACACCCACGGG 
                   
               
               
                   
                 TACCCATGGTGACGCCGGTGACTTTGCCATGATCGCCTATTTCGTACTGA 
                   
               
               
                   
                 AACCTCGATGTCTTCAAAAAGGTTCTCTTACAATTCAGCAAGTCAACGAC 
                   
               
               
                   
                 CTGCTGGATTCAATTGCGAGTAACAACAGCGCTAAGCGAAAGGATCTCAT 
                   
               
               
                   
                 TAAGAAAAGCCTCCTGCAGCTGATAACTCAGTCCTCTGCACTCGAACAAA 
                   
               
               
                   
                 AATGGCTGATTCGGATGATTATCAAGGATTTGAAGTTGGGGGTATCTCAG 
                   
               
               
                   
                 CAAACTATTTTCAGCGTGTTTCACAATGATGCAGCAGAATTGCATAATGT 
                   
               
               
                   
                 CACAACAGATCTTGAGAAAGTCTGCCGACAGTTGCACGACCCCTCTGTAG 
                   
               
               
                   
                 GCTTGAGTGACATATCTATAACACTTTTTTCTGCGTTCAAACCCATGTTG 
                   
               
               
                   
                 GCTGCTATTGCGGACATAGAACACATCGAGAAAGACATGAAACATCAGTC 
                   
               
               
                   
                 ATTCTATATAGAGACTAAATTGGACGGCGAGAGGATGCAAATGCACAAAG 
                   
               
               
                   
                 ATGGTGATGTGTATAAATATTTTTCCCGCAACGGCTACAACTACACTGAT 
                   
               
               
                   
                 CAATTCGGAGCGTCCCCAACTGAAGGGTCCCTCACTCCTTTCATACACAA 
                   
               
               
                   
                 TGCGTTTAAGGCCGATATTCAGATATGTATCCTCGACGGCGAAATGATGG 
                   
               
               
                   
                 CGTACAATCCCAATACCCAGACCTTCATGCAAAAGGGAACGAAGTTCGAT 
                   
               
               
                   
                 ATTAAACGGATGGTTGAAGATTCCGACCTCCAAACATGTTACTGTGTGTT 
                   
               
               
                   
                 TGATGTCCTGATGGTGAATAACAAAAAACTCGGCCATGAAACCCTTCGAA 
                   
               
               
                   
                 AGCGATACGAAATACTCAGCAGTATATTTACTCCAATACCAGGCCGAATC 
                   
               
               
                   
                 GAGATCGTACAGAAAACACAAGCCCATACTAAGAATGAAGTTATTGATGC 
                   
               
               
                   
                 ACTGAACGAAGCCATAGACAAGAGGGAAGAAGGCATAATGGTCAAGCAGC 
                   
               
               
                   
                 CTCTGAGTATATATAAACCTGACAAAAGGGGGGAAGGATGGCTGAAGATA 
                   
               
               
                   
                 AAGCCAGAATACGTGTCTGGTCTTATGGACGAATTGGACATTCTCATCGT 
                   
               
               
                   
                 CGGAGGATATTGGGGTAAGGGTTCCAGGGGGGGGATGATGTCCCACTTTC 
                   
               
               
                   
                 TGTGTGCGGTTGCCGAGAAACCGCCCCCAGGGGAAAAACCATCAGTGTTC 
                   
               
               
                   
                 CATACGTTGTCACGCGTCGGCTCAGGTTGTACGATGAAGGAACTTTACGA 
                   
               
               
                   
                 TCTGGGGTTGAAACTCGCCAAATATTGGAAGCCATTCCATCGGAAAGCAC 
                   
               
               
                   
                 CGCCCTCTAGTATCTTGTGTGGGACGGAGAAGCCAGAAGTTTATATAGAG 
                   
               
               
                   
                 CCATGTAACTCAGTAATTGTTCAAATCAAAGCCGCAGAGATCGTCCCGTC 
                   
               
               
                   
                 AGACATGTACAAGACTGGATGCACCCTTAGATTTCCTCGCATCGAAAAAA 
                   
               
               
                   
                 TAAGAGATGATAAAGAGTGGCATGAGTGCATGACTCTTGACGACCTTGAA 
                   
               
               
                   
                 CAGCTCCGCGGGAAGGCCAGCGGTAAACTGGCTAGTAAGCACCTCTACAT 
                   
               
               
                   
                 CGGGGGTGACAGTGgaggctccaaacggacagcagactcccaacattcaa 
                   
               
               
                   
                 cacccccaaaaacaaagcggaaggtagagtttgagccaaaaaagaaaaga 
                   
               
               
                   
                 aaggtcGGAGGAGGAGGCAGTGGTGGGCGACTTGAAATTAGAGCCGCGTT 
                   
               
               
                   
                 CCTGCGCCAGAGGAATACGGCTCTCCGCACGGAGGTAGCCGAACTTGAGC 
                   
               
               
                   
                 AAGAAGTACAGAGATTGGAGAACGAGGTTTCACAGTATGAGACACGATAT 
                   
               
               
                   
                 GGCCCCCTTGGCGGCGGAAAGtaa 
                   
               
               
                   
               
               
                 nCas9- 
                 atgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcg 
                 150 
               
               
                 hLIG4(1- 
                 gaaagtcgacaagaagtacagcatcggcctggacatcggcaccaactctg 
                   
               
               
                 620) 
                 tgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattc 
                   
               
               
                   
                 aaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcgg 
                   
               
               
                   
                 agccctgctgttcgacagcggcgaaacagccgaggccacccggctgaaga 
                   
               
               
                   
                 gaaccgccagaagaagatacaccagacggaagaaccggatctgctatctg 
                   
               
               
                   
                 caagagatcttcagcaacgagatggccaaggtggacgacagcttcttcca 
                   
               
               
                   
                 cagactggaagagtccttcctggtggaagaggataagaagcacgagcggc 
                   
               
               
                   
                 accccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtac 
                   
               
               
                   
                 cccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggc 
                   
               
               
                   
                 cgacctgcggctgatctatctggccctggcccacatgatcaagttccggg 
                   
               
               
                   
                 gccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggac 
                   
               
               
                   
                 aagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaa 
                   
               
               
                   
                 ccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagac 
                   
               
               
                   
                 tgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgag 
                   
               
               
                   
                 aagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgac 
                   
               
               
                   
                 ccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagc 
                   
               
               
                   
                 tgagcaaggacacctacgacgacgacctggacaacctgctggcccagatc 
                   
               
               
                   
                 ggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgc 
                   
               
               
                   
                 catcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggccc 
                   
               
               
                   
                 ccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctg 
                   
               
               
                   
                 accctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaaga 
                   
               
               
                   
                 gattttcttcgaccagagcaagaacggctacgccggctacattgacggcg 
                   
               
               
                   
                 gagccagccaggaagagttctacaagttcatcaagcccatcctggaaaag 
                   
               
               
                   
                 atggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgct 
                   
               
               
                   
                 gcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacc 
                   
               
               
                   
                 tgggagagctgcacgccattctgcggcggcaggaagatttttacccattc 
                   
               
               
                   
                 ctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatccc 
                   
               
               
                   
                 ctactacgtgggccctctggccaggggaaacagcagattcgcctggatga 
                   
               
               
                   
                 ccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtg 
                   
               
               
                   
                 gacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcga 
                   
               
               
                   
                 taagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacg 
                   
               
               
                   
                 agtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgag 
                   
               
               
                   
                 ggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgt 
                   
               
               
                   
                 ggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaag 
                   
               
               
                   
                 aggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggc 
                   
               
               
                   
                 gtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaa 
                   
               
               
                   
                 aattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattc 
                   
               
               
                   
                 tggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatc 
                   
               
               
                   
                 gaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaa 
                   
               
               
                   
                 gcagctgaagcggcggagatacaccggctggggcaggctgagccggaagc 
                   
               
               
                   
                 tgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttc 
                   
               
               
                   
                 ctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacga 
                   
               
               
                   
                 cgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggcc 
                   
               
               
                   
                 agggcgatagcctgcacgagcacattgccaatctggccggcagccccgcc 
                   
               
               
                   
                 attaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaa 
                   
               
               
                   
                 agtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagag 
                   
               
               
                   
                 agaaccagaccacccagaagggacagaagaacagccgcgagagaatgaag 
                   
               
               
                   
                 cggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaaca 
                   
               
               
                   
                 ccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacc 
                   
               
               
                   
                 tgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccgg 
                   
               
               
                   
                 ctgtccgactacgatgtggacgctatcgtgcctcagagctttctgaagga 
                   
               
               
                   
                 cgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggca 
                   
               
               
                   
                 agagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactac 
                   
               
               
                   
                 tggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaa 
                   
               
               
                   
                 tctgaccaaggccgagagaggcggcctgagcgaactggataaggccggct 
                   
               
               
                   
                 tcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggca 
                   
               
               
                   
                 cagatcctggactcccggatgaacactaagtacgacgagaatgacaagct 
                   
               
               
                   
                 gatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatt 
                   
               
               
                   
                 tccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccac 
                   
               
               
                   
                 cacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaa 
                   
               
               
                   
                 aaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgt 
                   
               
               
                   
                 acgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggct 
                   
               
               
                   
                 accgccaagtacttcttctacagcaacatcatgaactttttcaagaccga 
                   
               
               
                   
                 gattaccctggccaacggcgagatccggaagcggcctctgatcgagacaa 
                   
               
               
                   
                 acggcgaaaccggggagatcgtgtgggataagggccgggattttgccacc 
                   
               
               
                   
                 gtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccga 
                   
               
               
                   
                 ggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaaca 
                   
               
               
                   
                 gcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggc 
                   
               
               
                   
                 ggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagt 
                   
               
               
                   
                 ggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctgggga 
                   
               
               
                   
                 tcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctg 
                   
               
               
                   
                 gaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcc 
                   
               
               
                   
                 taagtactccctgttcgagctggaaaacggccggaagagaatgctggcct 
                   
               
               
                   
                 ctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatat 
                   
               
               
                   
                 gtgaacttcctgtacctggccagccactatgagaagctgaagggctcccc 
                   
               
               
                   
                 cgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacc 
                   
               
               
                   
                 tggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctg 
                   
               
               
                   
                 gccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccggga 
                   
               
               
                   
                 taagcccatcagagagcaggccgagaatatcatccacctgtttaccctga 
                   
               
               
                   
                 ccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgac 
                   
               
               
                   
                 cggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatcca 
                   
               
               
                   
                 ccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgg 
                   
               
               
                   
                 gaggtgacTCCGGTGGCTCCTCAGGGGGATCTAAACGCACGGCCGATGGG 
                   
               
               
                   
                 TCCGAGTTTGAGTCTCCCAAGAAGAAAAGGAAAGTGAGTGGTGGAAGTAG 
                   
               
               
                   
                 CGGCGGTAGCGCAGCTTCTCAGACCTCTCAAACAGTAGCCTCTCATGTAC 
                   
               
               
                   
                 CGTTCGCTGACTTGTGTTCTACGCTCGAACGCATCCAGAAATCAAAGGGG 
                   
               
               
                   
                 CGCGCCGAGAAAATCCGGCACTTCAGAGAATTCTTGGATTCCTGGAGGAA 
                   
               
               
                   
                 GTTTCATGATGCTCTCCACAAAAATCACAAAGATGTAACGGATAGTTTCT 
                   
               
               
                   
                 ACCCTGCTATGAGACTTATACTGCCGCAGCTTGAGAGGGAACGCATGGCG 
                   
               
               
                   
                 TATGGTATAAAGGAGACAATGTTGGCGAAATTGTATATTGAGCTGCTGAA 
                   
               
               
                   
                 CTTGCCAAGAGATGGAAAGGACGCGCTCAAACTGCTGAACTATAGAACAC 
                   
               
               
                   
                 CCACGGGTACCCATGGTGACGCCGGTGACTTTGCCATGATCGCCTATTTC 
                   
               
               
                   
                 GTACTGAAACCTCGATGTCTTCAAAAAGGTTCTCTTACAATTCAGCAAGT 
                   
               
               
                   
                 CAACGACCTGCTGGATTCAATTGCGAGTAACAACAGCGCTAAGCGAAAGG 
                   
               
               
                   
                 ATCTCATTAAGAAAAGCCTCCTGCAGCTGATAACTCAGTCCTCTGCACTC 
                   
               
               
                   
                 GAACAAAAATGGCTGATTCGGATGATTATCAAGGATTTGAAGTTGGGGGT 
                   
               
               
                   
                 ATCTCAGCAAACTATTTTCAGCGTGTTTCACAATGATGCAGCAGAATTGC 
                   
               
               
                   
                 ATAATGTCACAACAGATCTTGAGAAAGTCTGCCGACAGTTGCACGACCCC 
                   
               
               
                   
                 TCTGTAGGCTTGAGTGACATATCTATAACACTTTTTTCTGCGTTCAAACC 
                   
               
               
                   
                 CATGTTGGCTGCTATTGCGGACATAGAACACATCGAGAAAGACATGAAAC 
                   
               
               
                   
                 ATCAGTCATTCTATATAGAGACTAAATTGGACGGCGAGAGGATGCAAATG 
                   
               
               
                   
                 CACAAAGATGGTGATGTGTATAAATATTTTTCCCGCAACGGCTACAACTA 
                   
               
               
                   
                 CACTGATCAATTCGGAGCGTCCCCAACTGAAGGGTCCCTCACTCCTTTCA 
                   
               
               
                   
                 TACACAATGCGTTTAAGGCCGATATTCAGATATGTATCCTCGACGGCGAA 
                   
               
               
                   
                 ATGATGGCGTACAATCCCAATACCCAGACCTTCATGCAAAAGGGAACGAA 
                   
               
               
                   
                 GTTCGATATTAAACGGATGGTTGAAGATTCCGACCTCCAAACATGTTACT 
                   
               
               
                   
                 GTGTGTTTGATGTCCTGATGGTGAATAACAAAAAACTCGGCCATGAAACC 
                   
               
               
                   
                 CTTCGAAAGCGATACGAAATACTCAGCAGTATATTTACTCCAATACCAGG 
                   
               
               
                   
                 CCGAATCGAGATCGTACAGAAAACACAAGCCCATACTAAGAATGAAGTTA 
                   
               
               
                   
                 TTGATGCACTGAACGAAGCCATAGACAAGAGGGAAGAAGGCATAATGGTC 
                   
               
               
                   
                 AAGCAGCCTCTGAGTATATATAAACCTGACAAAAGGGGGGAAGGATGGCT 
                   
               
               
                   
                 GAAGATAAAGCCAGAATACGTGTCTGGTCTTATGGACGAATTGGACATTC 
                   
               
               
                   
                 TCATCGTCGGAGGATATTGGGGTAAGGGTTCCAGGGGGGGGATGATGTCC 
                   
               
               
                   
                 CACTTTCTGTGTGCGGTTGCCGAGAAACCGCCCCCAGGGGAAAAACCATC 
                   
               
               
                   
                 AGTGTTCCATACGTTGTCACGCGTCGGCTCAGGTTGTACGATGAAGGAAC 
                   
               
               
                   
                 TTTACGATCTGGGGTTGAAACTCGCCAAATATTGGAAGCCATTCCATCGG 
                   
               
               
                   
                 AAAGCACCGCCCTCTAGTATCTTGTGTGGGACGGAGAAGCCAGAAGTTTA 
                   
               
               
                   
                 TATAGAGCCATGTAACTCAGTAATTGTTCAAATCAAAGCCGCAGAGATCG 
                   
               
               
                   
                 TCCCGTCAGACATGTACAAGACTGGATGCACCCTTAGATTTCCTCGCATC 
                   
               
               
                   
                 GAAAAAATAAGAGATGATAAAGAGTGGCATGAGTGCATGACTCTTGACGA 
                   
               
               
                   
                 CCTTGAACAGCTCCGCGGGAAGGCCAGCGGTAAACTGGCTAGTAAGCACC 
                   
               
               
                   
                 TCTACATCGGGGGTGACtaa 
                   
               
               
                   
               
               
                 T4-nCas9 
                 atgatgatccttaagattctcaacgaaatcgctagtatagggtccactaa 
                 151 
               
               
                   
                 gcagaagcaggccatattggaaaaaaataaggacaatgaacttttgaaga 
                   
               
               
                   
                 gagtctatagactgacgtactctagggggctccagtactacatcaagaaa 
                   
               
               
                   
                 tggcctaaacctggcattgcgacgcagtcattcggtatgctgacattgac 
                   
               
               
                   
                 ggatatgttggatttcattgagtttacgctggccaccagaaaacttacgg 
                   
               
               
                   
                 gtaatgctgcgatagaagaacttacagggtacataacagacgggaagaaa 
                   
               
               
                   
                 gatgacgtggaagtgctcagacgagttatgatgcgcgatctcgagtgcgg 
                   
               
               
                   
                 cgctagcgtgtcaatcgcgaacaaagtctggcccggcctcataccagagc 
                   
               
               
                   
                 agccacagatgctggcatcttcctatgacgaaaaaggcataaacaagaat 
                   
               
               
                   
                 attaaattcccggccttcgctcaactcaaagcagatggtgccaggtgttt 
                   
               
               
                   
                 tgccgaagttcggggtgatgaacttgatgacgtgcggctcttgtctaggg 
                   
               
               
                   
                 caggtaacgagtacctcggcctggacttgcttaaagaggaactgattaaa 
                   
               
               
                   
                 atgacagctgaggcgcggcagatacaccccgagggcgtccttatcgacgg 
                   
               
               
                   
                 ggagctggtgtatcacgaacaagttaaaaaggaaccggagggtcttgatt 
                   
               
               
                   
                 ttcttttcgacgcgtatcctgagaacagcaaggcgaaagaatttgcagaa 
                   
               
               
                   
                 gttgcagaaagcaggaccgcaagtaatggaatcgctaataaaagcctcaa 
                   
               
               
                   
                 gggtaccatcagcgaaaaagaagcccagtgcatgaaatttcaagtttggg 
                   
               
               
                   
                 actatgtccccttggtcgaaatttactccctgcccgcattccggctgaag 
                   
               
               
                   
                 tatgatgttcgcttcagtaaactggagcaaatgacgagcggttatgataa 
                   
               
               
                   
                 ggttatacttattgagaatcaggtcgtaaataatttggacgaggcgaaag 
                   
               
               
                   
                 ttatatacaaaaagtatatagaccaagggttggaggggatcattttgaag 
                   
               
               
                   
                 aacatagacggactttgggagaacgcccggtccaagaatttgtataaatt 
                   
               
               
                   
                 caaagaagtcatagatgttgacctcaagatagtaggtatatatccccaca 
                   
               
               
                   
                 gaaaggacccaaccaaagcaggcggattcattttggagtccgagtgtggg 
                   
               
               
                   
                 aagataaaggtcaatgctggatctggactcaaggacaaagctggtgtgaa 
                   
               
               
                   
                 gtcacatgaactggaccgaaccaggattatggagaatcagaactattaca 
                   
               
               
                   
                 tcgggaagatattggagtgtgaatgcaacggctggcttaaatcagatgga 
                   
               
               
                   
                 agaactgattacgttaaattgttcctgcccatagccatacgactccgcga 
                   
               
               
                   
                 ggacaaaacgaaggctaacacgtttgaagacgtattcggagatttccatg 
                   
               
               
                   
                 aggtgactggcctttccggtggctcctcagggggatctaaacgcacggcc 
                   
               
               
                   
                 gatgggtccgagtttgagtctcccaagaagaaaaggaaagtgagtggtgg 
                   
               
               
                   
                 aagtagcggcggtagcgacaagaagtacagcatcggcctggacatcggca 
                   
               
               
                   
                 ccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagc 
                   
               
               
                   
                 aagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaa 
                   
               
               
                   
                 cctgatcggagccctgctgttcgacagcggcgaaacagccgaggccaccc 
                   
               
               
                   
                 ggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatc 
                   
               
               
                   
                 tgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacag 
                   
               
               
                   
                 cttcttccacagactggaagagtccttcctggtggaagaggataagaagc 
                   
               
               
                   
                 acgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccac 
                   
               
               
                   
                 gagaagtaccccaccatctaccacctgagaaagaaactggtggacagcac 
                   
               
               
                   
                 cgacaaggccgacctgcggctgatctatctggccctggcccacatgatca 
                   
               
               
                   
                 agttccggggccacttcctgatcgagggcgacctgaaccccgacaacagc 
                   
               
               
                   
                 gacgtggacaagctgttcatccagctggtgcagacctacaaccagctgtt 
                   
               
               
                   
                 cgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgt 
                   
               
               
                   
                 ctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctg 
                   
               
               
                   
                 cccggcgagaagaagaatggcctgttcggaaacctgattgccctgagcct 
                   
               
               
                   
                 gggcctgacccccaacttcaagagcaacttcgacctggccgaggatgcca 
                   
               
               
                   
                 aactgcagctgagcaaggacacctacgacgacgacctggacaacctgctg 
                   
               
               
                   
                 gcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacct 
                   
               
               
                   
                 gtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatca 
                   
               
               
                   
                 ccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccac 
                   
               
               
                   
                 caggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaa 
                   
               
               
                   
                 gtacaaagagattttcttcgaccagagcaagaacggctacgccggctaca 
                   
               
               
                   
                 ttgacggcggagccagccaggaagagttctacaagttcatcaagcccatc 
                   
               
               
                   
                 ctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagaga 
                   
               
               
                   
                 ggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccacc 
                   
               
               
                   
                 agatccacctgggagagctgcacgccattctgcggcggcaggaagatttt 
                   
               
               
                   
                 tacccattcctgaaggacaaccgggaaaagatcgagaagatcctgacctt 
                   
               
               
                   
                 ccgcatcccctactacgtgggccctctggccaggggaaacagcagattcg 
                   
               
               
                   
                 cctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgag 
                   
               
               
                   
                 gaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgac 
                   
               
               
                   
                 caacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcc 
                   
               
               
                   
                 tgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatac 
                   
               
               
                   
                 gtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaa 
                   
               
               
                   
                 ggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagc 
                   
               
               
                   
                 agctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaa 
                   
               
               
                   
                 atctccggcgtggaagatcggttcaacgcctccctgggcacataccacga 
                   
               
               
                   
                 tctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacg 
                   
               
               
                   
                 aggacattctggaagatatcgtgctgaccctgacactgtttgaggacaga 
                   
               
               
                   
                 gagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaa 
                   
               
               
                   
                 agtgatgaagcagctgaagcggcggagatacaccggctggggcaggctga 
                   
               
               
                   
                 gccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatc 
                   
               
               
                   
                 ctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagct 
                   
               
               
                   
                 gatccacgacgacagcctgacctttaaagaggacatccagaaagcccagg 
                   
               
               
                   
                 tgtccggccagggcgatagcctgcacgagcacattgccaatctggccggc 
                   
               
               
                   
                 agccccgccattaagaagggcatcctgcagacagtgaaggtggtggacga 
                   
               
               
                   
                 gctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaa 
                   
               
               
                   
                 tggccagagagaaccagaccacccagaagggacagaagaacagccgcgag 
                   
               
               
                   
                 agaatgaagcggatcgaagagggcatcaaagagctgggcagccagatcct 
                   
               
               
                   
                 gaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacc 
                   
               
               
                   
                 tgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggac 
                   
               
               
                   
                 atcaaccggctgtccgactacgatgtggacgctatcgtgcctcagagctt 
                   
               
               
                   
                 tctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaaga 
                   
               
               
                   
                 accggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatg 
                   
               
               
                   
                 aagaactactggcggcagctgctgaacgccaagctgattacccagagaaa 
                   
               
               
                   
                 gttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggata 
                   
               
               
                   
                 aggccggcttcatcaagagacagctggtggaaacccggcagatcacaaag 
                   
               
               
                   
                 cacgtggcacagatcctggactcccggatgaacactaagtacgacgagaa 
                   
               
               
                   
                 tgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctgg 
                   
               
               
                   
                 tgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaac 
                   
               
               
                   
                 aactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgc 
                   
               
               
                   
                 cctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgact 
                   
               
               
                   
                 acaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatc 
                   
               
               
                   
                 ggcaaggctaccgccaagtacttcttctacagcaacatcatgaacttttt 
                   
               
               
                   
                 caagaccgagattaccctggccaacggcgagatccggaagcggcctctga 
                   
               
               
                   
                 tcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggat 
                   
               
               
                   
                 tttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtgaa 
                   
               
               
                   
                 aaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgccca 
                   
               
               
                   
                 agaggaacagcgataagctgatcgccagaaagaaggactgggaccctaag 
                   
               
               
                   
                 aagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggt 
                   
               
               
                   
                 ggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagc 
                   
               
               
                   
                 tgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatc 
                   
               
               
                   
                 gactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcat 
                   
               
               
                   
                 caagctgcctaagtactccctgttcgagctggaaaacggccggaagagaa 
                   
               
               
                   
                 tgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccc 
                   
               
               
                   
                 tccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaa 
                   
               
               
                   
                 gggctcccccgaggataatgagcagaaacagctgtttgtggaacagcaca 
                   
               
               
                   
                 agcactacctggacgagatcatcgagcagatcagcgagttctccaagaga 
                   
               
               
                   
                 gtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaa 
                   
               
               
                   
                 gcaccgggataagcccatcagagagcaggccgagaatatcatccacctgt 
                   
               
               
                   
                 ttaccctgaccaatctgggagcccctgccgccttcaagtactttgacacc 
                   
               
               
                   
                 accatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccac 
                   
               
               
                   
                 cctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgt 
                   
               
               
                   
                 ctcagctgggaggtgacaaacggacagccgacggaagcgagttcgagtca 
                   
               
               
                   
                 ccaaagaagaagcggaaagtctaa 
                   
               
               
                   
               
               
                 SplintR- 
                 atgatggcgataacgaagcccttgttggcagctacgttggaaaatattga 
                 152 
               
               
                 nCas9 
                 ggacgtacagttcccatgccttgccactccgaagatcgatggaatccgat 
                   
               
               
                   
                 ccgtgaaacagacacaaatgcttagcagaacattcaaacccatcaggaac 
                   
               
               
                   
                 agcgtaatgaatagattgcttacggaactcttgcccgaagggtctgacgg 
                   
               
               
                   
                 cgagattagcatcgaaggagcgactttccaagatactacctcagcagtta 
                   
               
               
                   
                 tgacgggacacaagatgtataatgctaaattctcatattactggtttgac 
                   
               
               
                   
                 tatgttactgacgatcctttgaagaaatacatagacagggttgaagatat 
                   
               
               
                   
                 gaaaaattacataactgtccaccctcatatcctggagcatgcacaggtaa 
                   
               
               
                   
                 agattatcccgctcataccagtagaaattaacaatataaccgaattgttg 
                   
               
               
                   
                 cagtatgaacgcgatgtgctctctaaaggcttcgagggcgtgatgataag 
                   
               
               
                   
                 gaagcctgatggcaaatataagttcggtaggtccacattgaaagagggaa 
                   
               
               
                   
                 ttctcttgaagatgaaacagtttaaggatgcggaagctactatcattagt 
                   
               
               
                   
                 atgacggcactgtttaaaaacactaacactaaaaccaaggacaactttgg 
                   
               
               
                   
                 ctatagtaaaaggagcacacacaaatcaggaaaagttgaggaggacgtaa 
                   
               
               
                   
                 tgggcagtatagaggtagattacgatggtgtggtgtttagcattggaacg 
                   
               
               
                   
                 ggcttcgacgctgaccagcggagggacttttggcagaataaggaaagtta 
                   
               
               
                   
                 cattggcaagatggttaaattcaaatacttcgagatgggctcaaaagact 
                   
               
               
                   
                 gtccgagatttcctgtgtttattggaatcagacacgaagaggataggtCC 
                   
               
               
                   
                 GGTGGCTCCTCAgggggatctaaacgcacggccgatgggtccgagtttga 
                   
               
               
                   
                 gtctcccaagaagaaaaggaaagtgagtggtggaagtagcggcggtagcg 
                   
               
               
                   
                 acaagaagtacagcatcggcctggacatcggcaccaactctgtgggctgg 
                   
               
               
                   
                 gccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgct 
                   
               
               
                   
                 gggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgc 
                   
               
               
                   
                 tgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgcc 
                   
               
               
                   
                 agaagaagatacaccagacggaagaaccggatctgctatctgcaagagat 
                   
               
               
                   
                 cttcagcaacgagatggccaaggtggacgacagcttcttccacagactgg 
                   
               
               
                   
                 aagagtccttcctggtggaagaggataagaagcacgagcggcaccccatc 
                   
               
               
                   
                 ttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccat 
                   
               
               
                   
                 ctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgc 
                   
               
               
                   
                 ggctgatctatctggccctggcccacatgatcaagttccggggccacttc 
                   
               
               
                   
                 ctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgtt 
                   
               
               
                   
                 catccagctggtgcagacctacaaccagctgttcgaggaaaaccccatca 
                   
               
               
                   
                 acgccagcggcgtggacgccaaggccatcctgtctgccagactgagcaag 
                   
               
               
                   
                 agcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaa 
                   
               
               
                   
                 tggcctgttcggaaacctgattgccctgagcctgggcctgacccccaact 
                   
               
               
                   
                 tcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaag 
                   
               
               
                   
                 gacacctacgacgacgacctggacaacctgctggcccagatcggcgacca 
                   
               
               
                   
                 gtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgc 
                   
               
               
                   
                 tgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagc 
                   
               
               
                   
                 gcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgct 
                   
               
               
                   
                 gaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttct 
                   
               
               
                   
                 tcgaccagagcaagaacggctacgccggctacattgacggcggagccagc 
                   
               
               
                   
                 caggaagagttctacaagttcatcaagcccatcctggaaaagatggacgg 
                   
               
               
                   
                 caccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagc 
                   
               
               
                   
                 agcggaccttcgacaacggcagcatcccccaccagatccacctgggagag 
                   
               
               
                   
                 ctgcacgccattctgcggcggcaggaagatttttacccattcctgaagga 
                   
               
               
                   
                 caaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacg 
                   
               
               
                   
                 tgggccctctggccaggggaaacagcagattcgcctggatgaccagaaag 
                   
               
               
                   
                 agcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaaggg 
                   
               
               
                   
                 cgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacc 
                   
               
               
                   
                 tgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttc 
                   
               
               
                   
                 accgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgag 
                   
               
               
                   
                 aaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgc 
                   
               
               
                   
                 tgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactac 
                   
               
               
                   
                 ttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaaga 
                   
               
               
                   
                 tcggttcaacgcctccctgggcacataccacgatctgctgaaaattatca 
                   
               
               
                   
                 aggacaaggacttcctggacaatgaggaaaacgaggacattctggaagat 
                   
               
               
                   
                 atcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacg 
                   
               
               
                   
                 gctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctga 
                   
               
               
                   
                 agcggcggagatacaccggctggggcaggctgagccggaagctgatcaac 
                   
               
               
                   
                 ggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtc 
                   
               
               
                   
                 cgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcc 
                   
               
               
                   
                 tgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgat 
                   
               
               
                   
                 agcctgcacgagcacattgccaatctggccggcagccccgccattaagaa 
                   
               
               
                   
                 gggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgg 
                   
               
               
                   
                 gccggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccag 
                   
               
               
                   
                 accacccagaagggacagaagaacagccgcgagagaatgaagcggatcga 
                   
               
               
                   
                 agagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtgg 
                   
               
               
                   
                 aaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaat 
                   
               
               
                   
                 gggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccga 
                   
               
               
                   
                 ctacgatgtggacgctatcgtgcctcagagctttctgaaggacgactcca 
                   
               
               
                   
                 tcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgac 
                   
               
               
                   
                 aacgtgccctccgaagaggtcgtgaagaagatgaagaactactggcggca 
                   
               
               
                   
                 gctgctgaacgccaagctgattacccagagaaagttcgacaatctgacca 
                   
               
               
                   
                 aggccgagagaggcggcctgagcgaactggataaggccggcttcatcaag 
                   
               
               
                   
                 agacagctggtggaaacccggcagatcacaaagcacgtggcacagatcct 
                   
               
               
                   
                 ggactcccggatgaacactaagtacgacgagaatgacaagctgatccggg 
                   
               
               
                   
                 aagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaag 
                   
               
               
                   
                 gatttccagttttacaaagtgcgcgagatcaacaactaccaccacgccca 
                   
               
               
                   
                 cgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtacc 
                   
               
               
                   
                 ctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtg 
                   
               
               
                   
                 cggaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgccaa 
                   
               
               
                   
                 gtacttcttctacagcaacatcatgaactttttcaagaccgagattaccc 
                   
               
               
                   
                 tggccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaa 
                   
               
               
                   
                 accggggagatcgtgtgggataagggccgggattttgccaccgtgcggaa 
                   
               
               
                   
                 agtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcaga 
                   
               
               
                   
                 caggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataag 
                   
               
               
                   
                 ctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcga 
                   
               
               
                   
                 cagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagg 
                   
               
               
                   
                 gcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatc 
                   
               
               
                   
                 atggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaa 
                   
               
               
                   
                 gggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtact 
                   
               
               
                   
                 ccctgttcgagctggaaaacggccggaagagaatgctggcctctgccggc 
                   
               
               
                   
                 gaactgcagaagggaaacgaactggccctgccctccaaatatgtgaactt 
                   
               
               
                   
                 cctgtacctggccagccactatgagaagctgaagggctcccccgaggata 
                   
               
               
                   
                 atgagcagaaacagctgtttgtggaacagcacaagcactacctggacgag 
                   
               
               
                   
                 atcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgc 
                   
               
               
                   
                 taatctggacaaagtgctgtccgcctacaacaagcaccgggataagccca 
                   
               
               
                   
                 tcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctg 
                   
               
               
                   
                 ggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagag 
                   
               
               
                   
                 gtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagca 
                   
               
               
                   
                 tcaccggcctgtacgagacacggatcgacctgtctcagctgggaggtgac 
                   
               
               
                   
                 aaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaa 
                   
               
               
                   
                 agtctaa 
                   
               
               
                   
               
               
                 hLIG4(1- 
                 ATGGCAGCTTCTCAGACCTCTCAAACAGTAGCCTCTCATGTACCGTTCGC 
                 153 
               
               
                 620)-nCas9 
                 TGACTTGTGTTCTACGCTCGAACGCATCCAGAAATCAAAGGGGCGCGCCG 
                   
               
               
                   
                 AGAAAATCCGGCACTTCAGAGAATTCTTGGATTCCTGGAGGAAGTTTCAT 
                   
               
               
                   
                 GATGCTCTCCACAAAAATCACAAAGATGTAACGGATAGTTTCTACCCTGC 
                   
               
               
                   
                 TATGAGACTTATACTGCCGCAGCTTGAGAGGGAACGCATGGCGTATGGTA 
                   
               
               
                   
                 TAAAGGAGACAATGTTGGCGAAATTGTATATTGAGCTGCTGAACTTGCCA 
                   
               
               
                   
                 AGAGATGGAAAGGACGCGCTCAAACTGCTGAACTATAGAACACCCACGGG 
                   
               
               
                   
                 TACCCATGGTGACGCCGGTGACTTTGCCATGATCGCCTATTTCGTACTGA 
                   
               
               
                   
                 AACCTCGATGTCTTCAAAAAGGTTCTCTTACAATTCAGCAAGTCAACGAC 
                   
               
               
                   
                 CTGCTGGATTCAATTGCGAGTAACAACAGCGCTAAGCGAAAGGATCTCAT 
                   
               
               
                   
                 TAAGAAAAGCCTCCTGCAGCTGATAACTCAGTCCTCTGCACTCGAACAAA 
                   
               
               
                   
                 AATGGCTGATTCGGATGATTATCAAGGATTTGAAGTTGGGGGTATCTCAG 
                   
               
               
                   
                 CAAACTATTTTCAGCGTGTTTCACAATGATGCAGCAGAATTGCATAATGT 
                   
               
               
                   
                 CACAACAGATCTTGAGAAAGTCTGCCGACAGTTGCACGACCCCTCTGTAG 
                   
               
               
                   
                 GCTTGAGTGACATATCTATAACACTTTTTTCTGCGTTCAAACCCATGTTG 
                   
               
               
                   
                 GCTGCTATTGCGGACATAGAACACATCGAGAAAGACATGAAACATCAGTC 
                   
               
               
                   
                 ATTCTATATAGAGACTAAATTGGACGGCGAGAGGATGCAAATGCACAAAG 
                   
               
               
                   
                 ATGGTGATGTGTATAAATATTTTTCCCGCAACGGCTACAACTACACTGAT 
                   
               
               
                   
                 CAATTCGGAGCGTCCCCAACTGAAGGGTCCCTCACTCCTTTCATACACAA 
                   
               
               
                   
                 TGCGTTTAAGGCCGATATTCAGATATGTATCCTCGACGGCGAAATGATGG 
                   
               
               
                   
                 CGTACAATCCCAATACCCAGACCTTCATGCAAAAGGGAACGAAGTTCGAT 
                   
               
               
                   
                 ATTAAACGGATGGTTGAAGATTCCGACCTCCAAACATGTTACTGTGTGTT 
                   
               
               
                   
                 TGATGTCCTGATGGTGAATAACAAAAAACTCGGCCATGAAACCCTTCGAA 
                   
               
               
                   
                 AGCGATACGAAATACTCAGCAGTATATTTACTCCAATACCAGGCCGAATC 
                   
               
               
                   
                 GAGATCGTACAGAAAACACAAGCCCATACTAAGAATGAAGTTATTGATGC 
                   
               
               
                   
                 ACTGAACGAAGCCATAGACAAGAGGGAAGAAGGCATAATGGTCAAGCAGC 
                   
               
               
                   
                 CTCTGAGTATATATAAACCTGACAAAAGGGGGGAAGGATGGCTGAAGATA 
                   
               
               
                   
                 AAGCCAGAATACGTGTCTGGTCTTATGGACGAATTGGACATTCTCATCGT 
                   
               
               
                   
                 CGGAGGATATTGGGGTAAGGGTTCCAGGGGGGGGATGATGTCCCACTTTC 
                   
               
               
                   
                 TGTGTGCGGTTGCCGAGAAACCGCCCCCAGGGGAAAAACCATCAGTGTTC 
                   
               
               
                   
                 CATACGTTGTCACGCGTCGGCTCAGGTTGTACGATGAAGGAACTTTACGA 
                   
               
               
                   
                 TCTGGGGTTGAAACTCGCCAAATATTGGAAGCCATTCCATCGGAAAGCAC 
                   
               
               
                   
                 CGCCCTCTAGTATCTTGTGTGGGACGGAGAAGCCAGAAGTTTATATAGAG 
                   
               
               
                   
                 CCATGTAACTCAGTAATTGTTCAAATCAAAGCCGCAGAGATCGTCCCGTC 
                   
               
               
                   
                 AGACATGTACAAGACTGGATGCACCCTTAGATTTCCTCGCATCGAAAAAA 
                   
               
               
                   
                 TAAGAGATGATAAAGAGTGGCATGAGTGCATGACTCTTGACGACCTTGAA 
                   
               
               
                   
                 CAGCTCCGCGGGAAGGCCAGCGGTAAACTGGCTAGTAAGCACCTCTACAT 
                   
               
               
                   
                 CGGGGGTGACTCCGGTGGCTCCTCAGGGGGATCTAAACGCACGGCCGATG 
                   
               
               
                   
                 GGTCCGAGTTTGAGTCTCCCAAGAAGAAAAGGAAAGTGAGTGGTGGAAGT 
                   
               
               
                   
                 AGCGGCGGTAGCgacaagaagtacagcatcggcctggacatcggcaccaa 
                   
               
               
                   
                 ctctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaaga 
                   
               
               
                   
                 aattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctg 
                   
               
               
                   
                 atcggagccctgctgttcgacagcggcgaaacagccgaggccacccggct 
                   
               
               
                   
                 gaagagaaccgccagaagaagatacaccagacggaagaaccggatctgct 
                   
               
               
                   
                 atctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttc 
                   
               
               
                   
                 ttccacagactggaagagtccttcctggtggaagaggataagaagcacga 
                   
               
               
                   
                 gcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgaga 
                   
               
               
                   
                 agtaccccaccatctaccacctgagaaagaaactggtggacagcaccgac 
                   
               
               
                   
                 aaggccgacctgcggctgatctatctggccctggcccacatgatcaagtt 
                   
               
               
                   
                 ccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacg 
                   
               
               
                   
                 tggacaagctgttcatccagctggtgcagacctacaaccagctgttcgag 
                   
               
               
                   
                 gaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgc 
                   
               
               
                   
                 cagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccg 
                   
               
               
                   
                 gcgagaagaagaatggcctgttcggaaacctgattgccctgagcctgggc 
                   
               
               
                   
                 ctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaact 
                   
               
               
                   
                 gcagctgagcaaggacacctacgacgacgacctggacaacctgctggccc 
                   
               
               
                   
                 agatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtcc 
                   
               
               
                   
                 gacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaa 
                   
               
               
                   
                 ggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccagg 
                   
               
               
                   
                 acctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtac 
                   
               
               
                   
                 aaagagattttcttcgaccagagcaagaacggctacgccggctacattga 
                   
               
               
                   
                 cggcggagccagccaggaagagttctacaagttcatcaagcccatcctgg 
                   
               
               
                   
                 aaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggac 
                   
               
               
                   
                 ctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagat 
                   
               
               
                   
                 ccacctgggagagctgcacgccattctgcggcggcaggaagatttttacc 
                   
               
               
                   
                 cattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgc 
                   
               
               
                   
                 atcccctactacgtgggccctctggccaggggaaacagcagattcgcctg 
                   
               
               
                   
                 gatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaag 
                   
               
               
                   
                 tggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaac 
                   
               
               
                   
                 ttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgct 
                   
               
               
                   
                 gtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtga 
                   
               
               
                   
                 ccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggcc 
                   
               
               
                   
                 atcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagct 
                   
               
               
                   
                 gaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatct 
                   
               
               
                   
                 ccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctg 
                   
               
               
                   
                 ctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgagga 
                   
               
               
                   
                 cattctggaagatatcgtgctgaccctgacactgtttgaggacagagaga 
                   
               
               
                   
                 tgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaaagtg 
                   
               
               
                   
                 atgaagcagctgaagcggcggagatacaccggctggggcaggctgagccg 
                   
               
               
                   
                 gaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctgg 
                   
               
               
                   
                 atttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgatc 
                   
               
               
                   
                 cacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtc 
                   
               
               
                   
                 cggccagggcgatagcctgcacgagcacattgccaatctggccggcagcc 
                   
               
               
                   
                 ccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctc 
                   
               
               
                   
                 gtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggc 
                   
               
               
                   
                 cagagagaaccagaccacccagaagggacagaagaacagccgcgagagaa 
                   
               
               
                   
                 tgaagcggatcgaagagggcatcaaagagctgggcagccagatcctgaaa 
                   
               
               
                   
                 gaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgta 
                   
               
               
                   
                 ctacctgcagaatgggcgggatatgtacgtggaccaggaactggacatca 
                   
               
               
                   
                 accggctgtccgactacgatgtggacgctatcgtgcctcagagctttctg 
                   
               
               
                   
                 aaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccg 
                   
               
               
                   
                 gggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaaga 
                   
               
               
                   
                 actactggcggcagctgctgaacgccaagctgattacccagagaaagttc 
                   
               
               
                   
                 gacaatctgaccaaggccgagagaggcggcctgagcgaactggataaggc 
                   
               
               
                   
                 cggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacg 
                   
               
               
                   
                 tggcacagatcctggactcccggatgaacactaagtacgacgagaatgac 
                   
               
               
                   
                 aagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtc 
                   
               
               
                   
                 cgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaact 
                   
               
               
                   
                 accaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctg 
                   
               
               
                   
                 atcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaa 
                   
               
               
                   
                 ggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggca 
                   
               
               
                   
                 aggctaccgccaagtacttcttctacagcaacatcatgaactttttcaag 
                   
               
               
                   
                 accgagattaccctggccaacggcgagatccggaagcggcctctgatcga 
                   
               
               
                   
                 gacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttg 
                   
               
               
                   
                 ccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaag 
                   
               
               
                   
                 accgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagag 
                   
               
               
                   
                 gaacagcgataagctgatcgccagaaagaaggactgggaccctaagaagt 
                   
               
               
                   
                 acggcggcttcgacagccccaccgtggcctattctgtgctggtggtggcc 
                   
               
               
                   
                 aaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgct 
                   
               
               
                   
                 ggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgact 
                   
               
               
                   
                 ttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaag 
                   
               
               
                   
                 ctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgct 
                   
               
               
                   
                 ggcctctgccggcgaactgcagaagggaaacgaactggccctgccctcca 
                   
               
               
                   
                 aatatgtgaacttcctgtacctggccagccactatgagaagctgaagggc 
                   
               
               
                   
                 tcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagca 
                   
               
               
                   
                 ctacctggacgagatcatcgagcagatcagcgagttctccaagagagtga 
                   
               
               
                   
                 tcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcac 
                   
               
               
                   
                 cgggataagcccatcagagagcaggccgagaatatcatccacctgtttac 
                   
               
               
                   
                 cctgaccaatctgggagcccctgccgccttcaagtactttgacaccacca 
                   
               
               
                   
                 tcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctg 
                   
               
               
                   
                 atccaccagagcatcaccggcctgtacgagacacggatcgacctgtctca 
                   
               
               
                   
                 gctgggaggtgacaaacggacagccgacggaagcgagttcgagtcaccaa 
                   
               
               
                   
                 agaagaagcggaaagtctaa 
               
               
                   
               
            
           
         
       
     
     Disclosed herein are protein complexes comprising: an RNA-guided endonuclease bound to a ligase. The endonuclease and the ligase may be bound together through heterodimerization domains. The heterodimerization domains may include one or more of leucine zippers, PDZ domains, streptavidin and streptavidin binding protein, foldon domains, hydrophobic polypeptides, an antibody that binds the Cas nickase, or an antibody that binds the ligase, or one or more binding fragments thereof. 
     In some aspects, the system comprises at least one donor strand. In some aspects, the donor strand comprises a nucleic acid sequence that is at least partially homologous to the genomic locus targeted by the at least one guide nucleic acid. In some aspects, the donor strand comprises a nucleic acid sequence that is not homologous to the genomic locus targeted by the at least one guide nucleic acid. In some aspects, the donor strand is a single-stranded or a double-stranded nucleic acid. In some aspects, the donor strand comprising double-stranded nucleic acid comprises at least one overhang. In some aspects, the overhang comprises a guide binding site that is at least partially complementary to a guide nucleic acid. In some aspects, the overhang comprises a genomic flap binding site that is at least partially identical or complementary to a genomic flap at or adjacent to the genomic locus. In some aspects, the donor strand comprises two overhangs, where the first overhang: comprises a first guide binding site that is at least partially complementary to a first guide nucleic acid; or a first genomic flap binding site that is at least partially identical or complementary to a first genomic flap at or adjacent to the genomic locus; and the second overhang: comprises a second guide binding site that is at least partially complementary to a second guide nucleic acid; or a second genomic flap binding site that is at least partially identical or complementary to a second genomic flap at or adjacent to the genomic locus. In some aspects, the donor strand corrects at least one genetic mutation in the at least one genomic locus. In some aspects, the donor strand comprises a coding sequence. In some aspects, the coding sequence encodes a full length protein or a fragment thereof. In some aspects, the donor strand comprises a non-coding sequence. In some aspects, the non-coding sequence knocks out an endogenous gene. In some aspects, the non-coding sequence comprises a regulatory element. 
     In some aspects, the system comprises a nuclease. The nuclease may be heterologous. In some aspects, the nuclease comprises an exonuclease for digesting the genomic flap. In some aspects, the exonuclease is a 5′ exonuclease. Non-limiting example of the exonuclease can include a human flap endonuclease 1 (hFEN1), a human exonuclease 5 (hEXO5), a T5 exonuclease, a T7 exonuclease, an exonuclease VIII, a flap endonuclease domain of  E. coli  PolI, a RecJF, a Lambda exonuclease, a Xni (ExoIXI), a SaFEN ( Staphylococcus aureus  FEN), a nuclease BAL-31, or a fragment thereof. In some aspects, the exonuclease comprises an exonuclease in Table 10. In some aspects, the exonuclease comprises a polypeptide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more identical to the polypeptide sequence of any one of the exonuclease in Table 10. 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Non-limiting examples of exonuclease polypeptide sequence 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ   
               
               
                   
                   
                 ID 
               
               
                 Name 
                 Exonuclease polypeptide sequence 
                 NO: 
               
               
                   
               
               
                 hFEN1 
                 MGIQGLAKLIADVAPSAIRENDIKSYFGRKVAIDASMSIYQFLIAVRQGG 
                 212 
               
               
                   
                 DVLQNEEGETTSHLMGMFYRTIRMMENGIKPVYVFDGKPPQLKSGELA 
                   
               
               
                   
                 KRSERRAEAEKQLQQAQAAGAEQEVEKFTKRLVKVTKQHNDECKHLLS 
                   
               
               
                   
                 LMGIPYLDAPSEAEASCAALVKAGKVYAAATEDMDCLTFGSPVLMRHL 
                   
               
               
                   
                 TASEAKKLPIQEFHLSRILQELGLNQEQFVDLCILLGSDYCESIRGIGPKRA 
                   
               
               
                   
                 VDLIQKHKSIEEIVRRLDPNKYPVPENWLHKEAHQLFLEPEVLDPESVEL 
                   
               
               
                   
                 KWSEPNEEELIKFMCGEKQFSEERIRSGVKRLSKSRQGSTQGRLDDFFKV 
                   
               
               
                   
                 TGSLSSAKRKEPEPKGSTKKKAKTGAAGKFKRGK 
                   
               
               
                   
               
               
                 hFen1  
                 MGIQGLAKLIADVAPSAIRENDIKSYFGRKVAIDASMSIYQFLIAVRQGG 
                 213 
               
               
                 (1-333) 
                 DVLQNEEGETTSHLMGMFYRTIRMMENGIKPVYVFDGKPPQLKSGELA 
                   
               
               
                   
                 KRSERRAEAEKQLQQAQAAGAEQEVEKFTKRLVKVTKQHNDECKHLLS 
                   
               
               
                   
                 LMGIPYLDAPSEAEASCAALVKAGKVYAAATEDMDCLTFGSPVLMRHL 
                   
               
               
                   
                 TASEAKKLPIQEFHLSRILQELGLNQEQFVDLCILLGSDYCESIRGIGPKRA 
                   
               
               
                   
                 VDLIQKHKSIEEIVRRLDPNKYPVPENWLHKEAHQLFLEPEVLDPESVEL 
                   
               
               
                   
                 KWSEPNEEELIKFMCGEKQFSEERIRSGVKRLSKSRQ 
                   
               
               
                   
               
               
                 hEXO5 
                 MAETREEETVSAEASGFSDLSDSEFLEFLDLEDAQESKALVNMPGPSSES 
                 214 
               
               
                   
                 LGKDDKPISLQNWKRGLDILSPMERFHLKYLYVTDLATQNWCELQTAY 
                   
               
               
                   
                 GKELPGFLAPEKAAVLDTGASIHLARELELHDLVTVPVTTKEDAWAIKF 
                   
               
               
                   
                 LNILLLIPTLQSEGHIREFPVFGEGEGVLLVGVIDELHYTAKGELELAELK 
                   
               
               
                   
                 TRRRPMLPLEAQKKKDCFQVSLYKYIFDAMVQGKVTPASLIHHTKLCLE 
                   
               
               
                   
                 KPLGPSVLRHAQQGGFSVKSLGDLMELVFLSLTLSDLPVIDILKIEYIHQE 
                   
               
               
                   
                 TATVLGTEIVAFKEKEVRAKVQHYMAYWMGHREPQGVDVEEAWKCR 
                   
               
               
                   
                 TCTYADICEWRKGSGVLSSTLAPQVKKAK 
                   
               
               
                   
               
               
                 T5 EXO 
                 MSKSWGKFIEEEEAEMASRRNLMIVDGTNLGFRFKHNNSKKPFASSYVS 
                 215 
               
               
                   
                 TIQSLAKSYSARTTIVLGDKGKSVFRLEHLPEYKGNRDEKYAQRTEEEK 
                   
               
               
                   
                 ALDEQFFEYLKDAFELCKTTFPTFTIRGVEADDMAAYIVKLIGHLYDHV 
                   
               
               
                   
                 WLISTDGDWDTLLTDKVSRFSFTTRREYHLRDMYEHHNVDDVEQFISLK 
                   
               
               
                   
                 AIMGDLGDNIRGVEGIGAKRGYNIIREFGNVLDIIDQLPLPGKQKYIQNLN 
                   
               
               
                   
                 ASEELLFRNLILVDLPTYCVDAIAAVGQDVLDKFTKDILEIAEQ 
                   
               
               
                   
               
               
                 T7 EXO 
                 MALLDLKQFYELREGCDDKGILVMDGDWLVFQAMSAAEFDASWEEEI 
                 216 
               
               
                   
                 WHRCCDHAKARQILEDSIKSYETRKKAWAGAPIVLAFTDSVNWRKELV 
                   
               
               
                   
                 DPNYKANRKAVKKPVGYFEFLDALFEREEFYCIREPMLEGDDVMGVIAS 
                   
               
               
                   
                 NPSAFGARKAVIISCDKDFKTIPNCDFLWCTTGNILTQTEESADWWHLFQ 
                   
               
               
                   
                 TIKGDITDGYSGIAGWGDTAEDFLNNPFITEPKTSVLKSGKNKGQEVTK 
                   
               
               
                   
                 WVKRDPEPHETLWDCIKSIGAKAGMTEEDIIKQGQMARILRFNEYNFIDK 
                   
               
               
                   
                 EIYLWRP 
                   
               
               
                   
               
               
                 EXO VIII 
                 MSTKPLFLLRKAKKSSGEPDVVLWASNDFESTCATLDYLIVKSGKKLSS 
                 217 
               
               
                 (RecE) 
                 YFKAVATNFPVVNDLPAEGEIDFTWSERYQLSKDSMTWELKPGAAPDN 
                   
               
               
                   
                 AHYQGNTNVNGEDMTEIEENMLLPISGQELPIRWLAQHGSEKPVTHVSR 
                   
               
               
                   
                 DGLQALHIARAEELPAVTALAVSHKTSLLDPLEIRELHKLVRDTDKVFPN 
                   
               
               
                   
                 PGNSNLGLITAFFEAYLNADYTDRGLLTKEWMKGNRVSHITRTASGANA 
                   
               
               
                   
                 GGGNLTDRGEGFVHDLTSLARDVATGVLARSMDLDIYNLHPAHAKRIE 
                   
               
               
                   
                 EIIAENKPPFSVFRDKFITMPGGLDYSRAIVVASVKEAPIGIEVIPAHVTEY 
                   
               
               
                   
                 LNKVLTETDHANPDPEIVDIACGRSSAPMPQRVTEEGKQDDEEKPQPSGT 
                   
               
               
                   
                 TAVEQGEAETMEPDATEHHQDTQPLDAQSQVNSVDAKYQELRAELHEA 
                   
               
               
                   
                 RKNIPSKNPVDDDKLLAASRGEFVDGISDPNDPKWVKGIQTRDCVYQNQ 
                   
               
               
                   
                 PETEKTSPDMNQPEPVVQQEPEIACNACGQTGGDNCPDCGAVMGDATY 
                   
               
               
                   
                 QETFDEESQVEAKENDPEEMEGAEHPHNENAGSDPHRDCSDETGEVAD 
                   
               
               
                   
                 PVIVEDIEPGIYYGISNENYHAGPGISKSQLDDIADTPALYLWRKNAPVDT 
                   
               
               
                   
                 TKTKTLDLGTAFHCRVLEPEEFSNRFIVAPEFNRRTNAGKEEEKAFLMEC 
                   
               
               
                   
                 ASTGKTVITAEEGRKIELMYQSVMALPLGQWLVESAGHAESSIYWEDPE 
                   
               
               
                   
                 TGILCRCRPDKIIPEFHWIMDVKTTADIQRFKTAYYDYRYHVQDAFYSD 
                   
               
               
                   
                 GYEAQFGVQPTFVFLVASTTIECGRYPVEIFMMGEEAKLAGQQEYHRNL 
                   
               
               
                   
                 RTLSDCLNTDEWPAIKTLSLPRWAKEYAND 
                   
               
               
                   
               
               
                 EXO VIII, 
                 EHPHNENAGSDPHRDCSDETGEVADPVIVEDIEPGIYYGISNENYHAGPG 
                 218 
               
               
                 truncated 
                 ISKSQLDDIADTPALYLWRKNAPVDTTKTKTLDLGTAFHCRVLEPEEFSN 
                   
               
               
                   
                 RFIVAPEFNRRTNAGKEEEKAFLMECASTGKTVITAEEGRKIELMYQSV 
                   
               
               
                   
                 MALPLGQWLVESAGHAESSIYWEDPETGILCRCRPDKIIPEFHWIMDVKT 
                   
               
               
                   
                 TADIQRFKTAYYDYRYHVQDAFYSDGYEAQFGVQPTFVFLVASTTIECG 
                   
               
               
                   
                 RYPVEIFMMGEEAKLAGQQEYHRNLRTLSDCLNTDEWPAIKTLSLPRW 
                   
               
               
                   
                 AKEYAND 
                   
               
               
                   
               
               
                 Flap endo 
                 VQIPQNPLILVDGSSYLYRAYHAFPPLTNSAGEPTGAMYGVLNMLRSLI 
                 219 
               
               
                 domain of  
                 MQYKPTHAAVVFDAKGKTFRDELFEHYKSHRPPMPDDLRAQIEPLHAM 
                   
               
               
                 
                   E coli 
                 
                 VKAMGLPLLAVSGVEADDVIGTLAREAEKAGRPVLISTGDKDMAQLVT 
                   
               
               
                 PolI 
                 PNITLINTMTNTILGPEEVVNKYGVPPELIIDFLALMGDSSDNIPGVPGVG 
                   
               
               
                   
                 EKTAQALLQGLGGLDTLYAEPEKIAGLSFRGAKTMAAKLEQNKEVAYL 
                   
               
               
                   
                 SYQLATIKTDVELELTCEQLEVQQPAAEELLGLFKKYEFKRWTADVEAG 
                   
               
               
                   
                 KWLQAKGAKPAAKPQETSVADEAPEVTATVI 
                   
               
               
                   
               
               
                 RecJ 
                 MKQQIQLRRREVDETADLPAELPPLLRRLYASRGVRSAQELERSVKGML 
                 220 
               
               
                   
                 PWQQLSGVEKAVEILYNAFREGTRIIVVGDFDADGATSTALSVLAMRSL 
                   
               
               
                   
                 GCSNIDYLVPNRFEDGYGLSPEVVDQAHARGAQLIVTVDNGISSHAGVE 
                   
               
               
                   
                 HARSLGIPVIVTDHHLPGDTLPAAEAIINPNLRDCNFPSKSLAGVGVAFYL 
                   
               
               
                   
                 MLALRTFLRDQGWFDERNIAIPNLAELLDLVALGTVADVVPLDANNRIL 
                   
               
               
                   
                 TWQGMSRIRAGKCRPGIKALLEVANRDAQKLAASDLGFALGPRLNAAG 
                   
               
               
                   
                 RLDDMSVGVALLLCDNIGEARVLANELDALNQTRKEIEQGMQIEALTLC 
                   
               
               
                   
                 EKLERSRDTLPGGLAMYHPEWHQGVVGILASRIKERFHRPVIAFAPAGD 
                   
               
               
                   
                 GTLKGSGRSIQGLHMRDALERLDTLYPGMMLKFGGHAMAAGLSLEEDK 
                   
               
               
                   
                 FKLFQQRFGELVTEWLDPSLLQGEVVSDGPLSPAEMTMEVAQLLRDAGP 
                   
               
               
                   
                 WGQMFPEPLFDGHFRLLQQRLVGERHLKVMVEPVGGGPLLDGIAFNVD 
                   
               
               
                   
                 TALWPDNGVREVQLAYKLDINEFRGNRSLQIIIDNIWPI 
                   
               
               
                   
               
               
                 Lambda exo 
                 MTPDIILQRTGIDVRAVEQGDDAWHKLRLGVITASEVHNVIAKPRSGKK 
                 154 
               
               
                   
                 WPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTS 
                   
               
               
                   
                 GVNVTESPIIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGG 
                   
               
               
                   
                 FEAIKSAYMAQVQYSMWVTRKNAWYFANYDPRMKREGLHYVVIERDE 
                   
               
               
                   
                 KYMASFDEIVPEFIEKMDEALAEIGFVFGEQWR 
                   
               
               
                   
               
               
                 Xni  
                 MAVHLLIVDALNLIRRIHAVQGSPCVETCQHALDQLIMHSQPTHAVAVF 
                 155 
               
               
                 (ExoIXI) 
                 DDENRSSGWRHQRLPDYKAGRPPMPEELHDEMPALRAAFEQRGVPCWS 
                   
               
               
                 from 
                 TSGNEADDLAATLAVKVTQAGHQATIVSTDKGYCQLLSPTLRIRDYFQK 
                   
               
               
                 
                   E coli 
                 
                 RWLDAPFIDKEFGVQPQQLPDYWGLAGISSSKVPGVAGIGPKSATQLLV 
                   
               
               
                   
                 EFQSLEGIYENLDAVAEKWRKKLETHKEMAFLCRDIARLQTDLHIDGNL 
                   
               
               
                   
                 QQLRLVR 
                   
               
               
                   
               
               
                 SaFEN  
                 MPNKILLVDGMALLFRHFYATSLHKQFMYNSQGVPTNGIQGFVRHIFSAI 
                 156 
               
               
                 ( Staphaureus ) 
                 HEIRPTHVAVCWDMGQSTFRNDMFDGYKQNRSAPPEELIPQFDYVKEIS 
                   
               
               
                   
                 EQFGFVNIGVKNYEADDVIGTLAQQYSTDNDVYIITGDKDLLQCINDNV 
                   
               
               
                   
                 EVWLIKKGFNIYNRYTLHRFNEEYALEPQQLIDIKAFMGDTADGYAGVK 
                   
               
               
                   
                 GIGEKTAIKLIQQYQSVENVVENIDALSAGQRNKINDNLDELYLSKRLAE 
                   
               
               
                   
                 IHTQVPIDSEALFEKMSFATTLNHILSICNEHELHVSGKYISSHF 
               
               
                   
               
            
           
         
       
     
     In some aspects, the system comprises at least one additional endonuclease that is different from the at least one programmable endonuclease described herein. In some aspects, the at least one additional endonuclease can digest the genomic flap. 
     In some aspects, the system comprises a dominant negative MMR peptide to improve genomic editing capability, particularly in cells which overexpress the MMR pathway. In some aspects, the dominant negative MMR peptide can be delivered as a fusion (e.g., fused with any component of the system described herein), recruited, or as separate peptide. Table 11 lists non-limiting examples of the MMR peptide sequences. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Non-limiting examples of MMR polypeptide sequence 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ 
               
               
                   
                   
                 ID 
               
               
                 Name 
                 MMR peptide sequence 
                 NO: 
               
               
                   
               
               
                 MLH1 
                 MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIVKEGGLKLIQ 
                 157 
               
               
                   
                 IQDNGTGIRKEDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTAD 
                   
               
               
                   
                 GKCAYRASYSDGKLKAPPKPCAGNQGTQITVEDLFYNIATRRKALKNPSEEYGKILEV 
                   
               
               
                   
                 VGRYSVHNAGISFSVKKQGETVADVRTLPNASTVDNIRSIFGNAVSRELIEIGCEDKTLA 
                   
               
               
                   
                 FKMNGYISNANYSVKKCIFLLFINHRLVESTSLRKAIETVYAAYLPKNTHPFLYLSLEISP 
                   
               
               
                   
                 QNVDVNVHPTKHEVHFLHEESILERVQQHIESKLLGSNSSRMYFTQTLLPGLAGPSGEM 
                   
               
               
                   
                 VKSTTSLTSSSTSGSSDKVYAHQMVRTDSREQKLDAFLQPLSKPLSSQPQAIVTEDKTDI 
                   
               
               
                   
                 SSGRARQQDEEMLELPAPAEVAAKNQSLEGDTTKGTSEMSEKRGPTSSNPRKRHREDS 
                   
               
               
                   
                 DVEMVEDDSRKEMTAACTPRRRIINLTSVLSLQEEINEQGHEVLREMLHNHSFVGCVN 
                   
               
               
                   
                 PQWALAQHQTKLYLLNTTKLSEELFYQILIYDFANFGVLRLSEPAPLFDLAMLALDSPE 
                   
               
               
                   
                 SGWTEEDGPKEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLP 
                   
               
               
                   
                 IFILRLATEVNWDEEKECFESLSKECAMFYSIRKQYISEESTLSGQQSEVPGSIPNSWKW 
                   
               
               
                   
                 TVEHIVYKALRSHILPPKHFTEDGNILQLANLPDLYKVFERC 
                   
               
               
                   
               
               
                 MLH1 
                 MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKAMIENCLDAKSTSIQVIVKEGGLKLIQ 
                 158 
               
               
                 E34A 
                 IQDNGTGIRKEDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTAD 
                   
               
               
                   
                 GKCAYRASYSDGKLKAPPKPCAGNQGTQITVEDLFYNIATRRKALKNPSEEYGKILEV 
                   
               
               
                   
                 VGRYSVHNAGISFSVKKQGETVADVRTLPNASTVDNIRSIFGNAVSRELIEIGCEDKTLA 
                   
               
               
                   
                 FKMNGYISNANYSVKKCIFLLFINHRLVESTSLRKAIETVYAAYLPKNTHPFLYLSLEISP 
                   
               
               
                   
                 QNVDVNVHPTKHEVHFLHEESILERVQQHIESKLLGSNSSRMYFTQTLLPGLAGPSGEM 
                   
               
               
                   
                 VKSTTSLTSSSTSGSSDKVYAHQMVRTDSREQKLDAFLQPLSKPLSSQPQAIVTEDKTDI 
                   
               
               
                   
                 SSGRARQQDEEMLELPAPAEVAAKNQSLEGDTTKGTSEMSEKRGPTSSNPRKRHREDS 
                   
               
               
                   
                 DVEMVEDDSRKEMTAACTPRRRIINLTSVLSLQEEINEQGHEVLREMLHNHSFVGCVN 
                   
               
               
                   
                 PQWALAQHQTKLYLLNTTKLSEELFYQILIYDFANFGVLRLSEPAPLFDLAMLALDSPE 
                   
               
               
                   
                 SGWTEEDGPKEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLP 
                   
               
               
                   
                 IFILRLATEVNWDEEKECFESLSKECAMFYSIRKQYISEESTLSGQQSEVPGSIPNSWKW 
                   
               
               
                   
                 TVEHIVYKALRSHILPPKHFTEDGNILQLANLPDLYKVFERC 
                   
               
               
                   
               
               
                 MLH1 
                 MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIVKEGGLKLIQ 
                 159 
               
               
                 del 
                 IQDNGTGIRKEDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTAD 
                   
               
               
                 754- 
                 GKCAYRASYSDGKLKAPPKPCAGNQGTQITVEDLFYNIATRRKALKNPSEEYGKILEV 
                   
               
               
                 756 
                 VGRYSVHNAGISFSVKKQGETVADVRTLPNASTVDNIRSIFGNAVSRELIEIGCEDKTLA 
                   
               
               
                   
                 FKMNGYISNANYSVKKCIFLLFINHRLVESTSLRKAIETVYAAYLPKNTHPFLYLSLEISP 
                   
               
               
                   
                 QNVDVNVHPTKHEVHFLHEESILERVQQHIESKLLGSNSSRMYFTQTLLPGLAGPSGEM 
                   
               
               
                   
                 VKSTTSLTSSSTSGSSDKVYAHQMVRTDSREQKLDAFLQPLSKPLSSQPQAIVTEDKTDI 
                   
               
               
                   
                 SSGRARQQDEEMLELPAPAEVAAKNQSLEGDTTKGTSEMSEKRGPTSSNPRKRHREDS 
                   
               
               
                   
                 DVEMVEDDSRKEMTAACTPRRRIINLTSVLSLQEEINEQGHEVLREMLHNHSFVGCVN 
                   
               
               
                   
                 PQWALAQHQTKLYLLNTTKLSEELFYQILIYDFANFGVLRLSEPAPLFDLAMLALDSPE 
                   
               
               
                   
                 SGWTEEDGPKEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLP 
                   
               
               
                   
                 IFILRLATEVNWDEEKECFESLSKECAMFYSIRKQYISEESTLSGQQSEVPGSIPNSWKW 
                   
               
               
                   
                 TVEHIVYKALRSHILPPKHFTEDGNILQLANLPDLYKVF 
                   
               
               
                   
               
               
                 MLH1 
                 MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKAMIENCLDAKSTSIQVIVKEGGLKLIQ 
                 160 
               
               
                 E34A 
                 IQDNGTGIRKEDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTAD 
                   
               
               
                 del 
                 GKCAYRASYSDGKLKAPPKPCAGNQGTQITVEDLFYNIATRRKALKNPSEEYGKILEV 
                   
               
               
                 754- 
                 VGRYSVHNAGISFSVKKQGETVADVRTLPNASTVDNIRSIFGNAVSRELIEIGCEDKTLA 
                   
               
               
                 756 
                 FKMNGYISNANYSVKKCIFLLFINHRLVESTSLRKAIETVYAAYLPKNTHPFLYLSLEISP 
                   
               
               
                   
                 QNVDVNVHPTKHEVHFLHEESILERVQQHIESKLLGSNSSRMYFTQTLLPGLAGPSGEM 
                   
               
               
                   
                 VKSTTSLTSSSTSGSSDKVYAHQMVRTDSREQKLDAFLQPLSKPLSSQPQAIVTEDKTDI 
                   
               
               
                   
                 SSGRARQQDEEMLELPAPAEVAAKNQSLEGDTTKGTSEMSEKRGPTSSNPRKRHREDS 
                   
               
               
                   
                 DVEMVEDDSRKEMTAACTPRRRIINLTSVLSLQEEINEQGHEVLREMLHNHSFVGCVN 
                   
               
               
                   
                 PQWALAQHQTKLYLLNTTKLSEELFYQILIYDFANFGVLRLSEPAPLFDLAMLALDSPE 
                   
               
               
                   
                 SGWTEEDGPKEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLP 
                   
               
               
                   
                 IFILRLATEVNWDEEKECFESLSKECAMFYSIRKQYISEESTLSGQQSEVPGSIPNSWKW 
                   
               
               
                   
                 TVEHIVYKALRSHILPPKHFTEDGNILQLANLPDLYKVF 
                   
               
               
                   
               
               
                 MLH1 
                 MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIVKEGGLKLIQ 
                 161 
               
               
                 1-335 
                 IQDNGTGIRKEDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTAD 
                   
               
               
                   
                 GKCAYRASYSDGKLKAPPKPCAGNQGTQITVEDLFYNIATRRKALKNPSEEYGKILEV 
                   
               
               
                   
                 VGRYSVHNAGISFSVKKQGETVADVRTLPNASTVDNIRSIFGNAVSRELIEIGCEDKTLA 
                   
               
               
                   
                 FKMNGYISNANYSVKKCIFLLFINHRLVESTSLRKAIETVYAAYLPKNTHPFLYLSLEISP 
                   
               
               
                   
                 QNVDVNVHPTKHEVHFLHEESILERVQQHIESKLL 
                   
               
               
                   
               
               
                 MLH1 
                 MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKAMIENCLDAKSTSIQVIVKEGGLKLIQ 
                 162 
               
               
                 1-335 
                 IQDNGTGIRKEDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTAD 
                   
               
               
                 E34A 
                 GKCAYRASYSDGKLKAPPKPCAGNQGTQITVEDLFYNIATRRKALKNPSEEYGKILEV 
                   
               
               
                   
                 VGRYSVHNAGISFSVKKQGETVADVRTLPNASTVDNIRSIFGNAVSRELIEIGCEDKTLA 
                   
               
               
                   
                 FKMNGYISNANYSVKKCIFLLFINHRLVESTSLRKAIETVYAAYLPKNTHPFLYLSLEISP 
                   
               
               
                   
                 QNVDVNVHPTKHEVHFLHEESILERVQQHIESKLL 
                   
               
               
                   
               
               
                 MSH2 
                 MAVQPKETLQLESAAEVGFVRFFQGMPEKPTTTVRLFDRGDFYTAHGEDALLAAREV 
                 163 
               
               
                   
                 FKTQGVIKYMGPAGAKNLQSVVLSKMNFESFVKDLLLVRQYRVEVYKNRAGNKASK 
                   
               
               
                   
                 ENDWYLAYKASPGNLSQFEDILFGNNDMSASIGVVGVKMSAVDGQRQVGVGYVDSIQ 
                   
               
               
                   
                 RKLGLCEFPDNDQFSNLEALLIQIGPKECVLPGGETAGDMGKLRQIIQRGGILITERKKA 
                   
               
               
                   
                 DFSTKDIYQDLNRLLKGKKGEQMNSAVLPEMENQVAVSSLSAVIKFLELLSDDSNFGQ 
                   
               
               
                   
                 FELTTFDFSQYMKLDIAAVRALNLFQGSVEDTTGSQSLAALLNKCKTPQGQRLVNQWI 
                   
               
               
                   
                 KQPLMDKNRIEERLNLVEAFVEDAELRQTLQEDLLRRFPDLNRLAKKFQRQAANLQD 
                   
               
               
                   
                 CYRLYQGINQLPNVIQALEKHEGKHQKLLLAVFVTPLTDLRSDFSKFQEMIETTLDMD 
                   
               
               
                   
                 QVENHEFLVKPSFDPNLSELREIMNDLEKKMQSTLISAARDLGLDPGKQIKLDSSAQFG 
                   
               
               
                   
                 YYFRVTCKEEKVLRNNKNFSTVDIQKNGVKFTNSKLTSLNEEYTKNKTEYEEAQDAIV 
                   
               
               
                   
                 KEIVNISSGYVEPMQTLNDVLAQLDAVVSFAHVSNGAPVPYVRPAILEKGQGRIILKAS 
                   
               
               
                   
                 RHACVEVQDEIAFIPNDVYFEKDKQMFHIITGPNMGGKSTYIRQTGVIVLMAQIGCFVP 
                   
               
               
                   
                 CESAEVSIVDCILARVGAGDSQLKGVSTFMAEMLETASILRSATKDSLIIIDELGRGTST 
                   
               
               
                   
                 YDGFGLAWAISEYIATKIGAFCMFATHFHELTALANQIPTVNNLHVTALTTEETLTMLY 
                   
               
               
                   
                 QVKKGVCDQSFGIHVAELANFPKHVIECAKQKALELEEFQYIGESQGYDIMEPAAKKC 
                   
               
               
                   
                 YLEREQGEKIIQEFLSKVKQMPFTEMSEENITIKLKQLKAEVIAKNNSFVNEIISRIKVTT 
                   
               
               
                   
               
               
                 MSH2 
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                 164 
               
               
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                 ENDWYLAYKASPGNLSQFEDILFGNNDMSASIGVVGVKMSAVDGQRQVGVGYVDSIQ 
                   
               
               
                   
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                 KQPLMDKNRIEERLNLVEAFVEDAELRQTLQEDLLRRFPDLNRLAKKFQRQAANLQD 
                   
               
               
                   
                 CYRLYQGINQLPNVIQALEKHEGKHQKLLLAVFVTPLTDLRSDFSKFQEMIETTLDMD 
                   
               
               
                   
                 QVENHEFLVKPSFDPNLSELREIMNDLEKKMQSTLISAARDLGLDPGKQIKLDSSAQFG 
                   
               
               
                   
                 YYFRVTCKEEKVLRNNKNFSTVDIQKNGVKFTNSKLTSLNEEYTKNKTEYEEAQDAIV 
                   
               
               
                   
                 KEIVNISSGYVEPMQTLNDVLAQLDAVVSFAHVSNGAPVPYVRPAILEKGQGRIILKAS 
                   
               
               
                   
                 RHACVEVQDEIAFIPNDVYFEKDKQMFHIITGPNMGAKSTYIRQTGVIVLMAQIGCFVP 
                   
               
               
                   
                 CESAEVSIVDCILARVGAGDSQLKGVSTFMAEMLETASILRSATKDSLIIIDELGRGTST 
                   
               
               
                   
                 YDGFGLAWAISEYIATKIGAFCMFATHFHELTALANQIPTVNNLHVTALTTEETLTMLY 
                   
               
               
                   
                 QVKKGVCDQSFGIHVAELANFPKHVIECAKQKALELEEFQYIGESQGYDIMEPAAKKC 
                   
               
               
                   
                 YLEREQGEKIIQEFLSKVKQMPFTEMSEENITIKLKQLKAEVIAKNNSFVNEIISRIKVTT 
                   
               
               
                   
               
               
                 MSH2 
                 MAVQPKETLQLESAAEVGFVRFFQGMPEKPTTTVRLFDRGDFYTAHGEDALLAAREV 
                 165 
               
               
                 N671I 
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                 ENDWYLAYKASPGNLSQFEDILFGNNDMSASIGVVGVKMSAVDGQRQVGVGYVDSIQ 
                   
               
               
                   
                 RKLGLCEFPDNDQFSNLEALLIQIGPKECVLPGGETAGDMGKLRQIIQRGGILITERKKA 
                   
               
               
                   
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                 KQPLMDKNRIEERLNLVEAFVEDAELRQTLQEDLLRRFPDLNRLAKKFQRQAANLQD 
                   
               
               
                   
                 CYRLYQGINQLPNVIQALEKHEGKHQKLLLAVFVTPLTDLRSDFSKFQEMIETTLDMD 
                   
               
               
                   
                 QVENHEFLVKPSFDPNLSELREIMNDLEKKMQSTLISAARDLGLDPGKQIKLDSSAQFG 
                   
               
               
                   
                 YYFRVTCKEEKVLRNNKNFSTVDIQKNGVKFTNSKLTSLNEEYTKNKTEYEEAQDAIV 
                   
               
               
                   
                 KEIVNISSGYVEPMQTLNDVLAQLDAVVSFAHVSNGAPVPYVRPAILEKGQGRIILKAS 
                   
               
               
                   
                 RHACVEVQDEIAFIPNDVYFEKDKQMFHIITGPIMGGKSTYIRQTGVIVLMAQIGCFVPC 
                   
               
               
                   
                 ESAEVSIVDCILARVGAGDSQLKGVSTFMAEMLETASILRSATKDSLIIIDELGRGTSTY 
                   
               
               
                   
                 DGFGLAWAISEYIATKIGAFCMFATHFHELTALANQIPTVNNLHVTALTTEETLTMLYQ 
                   
               
               
                   
                 VKKGVCDQSFGIHVAELANFPKHVIECAKQKALELEEFQYIGESQGYDIMEPAAKKCY 
                   
               
               
                   
                 LEREQGEKIIQEFLSKVKQMPFTEMSEENITIKLKQLKAEVIAKNNSFVNEIISRIKVTT 
               
               
                   
               
            
           
         
       
     
     The system may relate to a 1-sided Replacer 1. Some aspects include a system comprising: (a) at least one RNA-guided endonuclease; (b) at least one guide nucleic acid comprising: (i) a spacer complementary to a genomic locus in a cell, (ii) a scaffold for complexing with the at least one RNA-guided endonuclease, (iii) an optional donor binding site that is at least partially complementary to an integrating nucleic acid, and (iv) a flap binding site that is at least partially identical or complementary to a genomic flap at or adjacent to the genomic locus; and (c) at least one DNA ligase; and (d) the integrating nucleic acid, optionally comprising a guide binding site that is at least partially complementary to the at least one guide nucleic acid, wherein the at least one RNA-guided endonuclease cleaves at least one strand of the genomic locus, and wherein the at least one DNA ligase ligates an end of the integrating nucleic acid to the genomic flap site, thereby replacing a region of the genomic locus with the integrating nucleic acid in the cell. The integrating nucleic acid may comprise a single-stranded DNA. 
     The system may relate to a 2-sided Replacer 1. Some aspects include a system comprising: (a) at least one RNA-guided endonuclease comprising a first RNA-guided endonuclease and an optional second RNA-guided endonuclease; (b) at least one guide nucleic acid comprising a first guide nucleic acid and a second guide nucleic acid, the first guide nucleic acid comprising: (i) a first spacer complementary to a first region of a genomic locus in a cell, (ii) a first scaffold for complexing with the first RNA-guided endonuclease, and (iii) an optional first donor binding site that at least partially complementary to an integrating nucleic acid, and (iv) a first flap binding site that is at least partially identical or complementary to a first genomic flap at or adjacent to the genomic locus; and the second guide nucleic acid comprising: (i) a second spacer complementary to a second region of the genomic locus in the cell, (ii) a second scaffold for complexing with the first or second RNA-guided endonuclease, (iii) an optional second donor binding site that at least partially complementary to the integrating nucleic acid, and (iv) a second flap binding site that is at least partially identical or complementary to a second genomic flap at or adjacent to the genomic locus; (c) at least one DNA ligase comprising a first DNA ligase and an optional second DNA ligase; and (d) at least one integrating nucleic acid comprising a first strand and a second strand: (i) wherein the first strand comprises an optional first guide binding site that is at least partially complementary to the first guide nucleic acid, and (ii) wherein the second strand comprises an optional second guide binding site that is at least partially complementary to the second guide nucleic acid, wherein the first RNA-guided endonuclease and/or the second RNA-guided endonuclease each cleaves at least one strand of the genomic locus in the cell; and wherein the first DNA ligase ligates an end of the first strand of the integrating nucleic acid to the first genomic flap; and the first or second DNA ligase ligates an end of the second strand of the integrating nucleic acid to the second genomic flap, thereby replacing a region of the genomic locus with the integrating nucleic acid in the cell. The integrating nucleic acid may comprise a double-stranded DNA duplex region. The integrating nucleic acid may comprise a 5′ overhang optionally comprising the first guide binding site. The integrating nucleic acid may comprise a 5′ overhang optionally comprising the second guide binding site. 
     The system may relate to 1-sided Replacer 2. Some aspects include a system comprising: (a) at least one RNA-guided endonuclease; (b) at least one guide nucleic acid comprising: (i) a spacer complementary to a genomic locus in a cell, (ii) a scaffold for complexing with the at least one RNA-guided endonuclease, and (iii) an optional donor binding site that is at least partially complementary to an integrating nucleic acid; (c) at least one DNA ligase; and (d) the integrating nucleic acid that: (i) comprises an optional guide binding site that is at least partially complementary to the at least one guide nucleic acid, and (ii) comprises a flap binding site that is at least partially identical or complementary to a genomic flap at or adjacent to the genomic locus, wherein the at least one RNA-guided endonuclease cleaves at least one strand of the genomic locus; and wherein the at least one DNA ligase ligates an end of the integrating nucleic acid to the genomic flap, thereby replacing a region of the genomic locus with the integrating nucleic acid in the cell. The integrating nucleic acid may comprise a DNA comprising a 3′ overhang. The 3′ overhang may comprise the guide binding site. The 3′ overhang may comprise the flap binding site. The at least one DNA ligase may ligates a strand of the integrating nucleic acid to the genomic nucleic acid sequence. 
     The system may relate to 2-sided Replacer 2. Some aspects include a system comprising: (a) at least one RNA-guided endonuclease comprising a first RNA-guided endonuclease and an optional second RNA-guided endonuclease; (b) at least one guide nucleic acid comprising a first guide nucleic acid and a second guide nucleic acid, the first guide nucleic acid comprising: (i) a first spacer complementary to a first region of a genomic locus in a cell, (ii) a first scaffold for complexing with the first RNA-guided endonuclease, and (iii) an optional first donor binding site that at least partially complementary to an integrating nucleic acid; and the second guide nucleic acid comprising: (i) a second spacer complementary to a second region of the genomic locus in the cell, (ii) a second scaffold for complexing with the first or second RNA-guided endonuclease, and (iii) an optional second donor binding site that at least partially complementary to the integrating nucleic acid; and at least one DNA ligase comprising a first DNA ligase and an optional second DNA ligase; and the integrating nucleic acid comprising a first strand and a second strand: wherein the first strand comprises an optional first guide binding site that is at least partially complementary to the first guide nucleic acid; wherein the second strand comprises an optional second binding site that is at least partially complementary to the second guide nucleic acid; wherein the first strand comprises a first flap binding site that is at least partially identical or complementary to a first genomic flap at or adjacent to the genomic locus; and wherein the second strand comprises a second flap binding site that is at least partially identical or complementary to a second genomic flap at or adjacent to the genomic locus; wherein the first RNA-guided endonuclease and/or the second RNA-guided endonuclease each cleaves at least one strand of the genomic locus in the cell; and wherein the first DNA ligase ligates an end of the first strand of the integrating nucleic acid to the first genomic flap; and the first or second DNA ligase ligates an end of the second strand of the integrating nucleic acid to the second genomic flap, thereby replacing a region of the genomic locus with the integrating nucleic acid in the cell. The integrating nucleic acid may comprise a double-stranded DNA duplex region. The double-stranded DNA may comprise a 3′ overhang optionally comprising the first guide binding site, and comprising the first flap binding site. The double stranded DNA may comprise a 3′ overhang optionally comprising the second guide binding site, and comprising the second flap binding site. 
     In the system, the at least one RNA-guided endonuclease may comprise a Cas protein or a functional fragment thereof. The Cas protein or the functional fragment thereof may comprise nickase activity The at least one RNA-guided endonuclease may comprise a Cas9 nickase or a functional fragment thereof. The at least one DNA ligase may ligates nucleic acids bound to DNA. The at least one DNA ligase may ligates nucleic acids bound to RNA. The at least one DNA ligase may comprise a PBCV-1 DNA ligase. The at least one DNA ligase may be operatively coupled to the at least one RNA-guided endonuclease. The at least one DNA ligase may be fused to the at least one RNA-guided endonuclease as a fusion polypeptide. The at least one RNA-guided endonuclease and the at least one DNA ligase may comprise a heterodimer domain. The at least one RNA-guided endonuclease and the at least one DNA ligase may form a heterodimer via the heterodimer domain. The at least one RNA-guided endonuclease may comprise a linker. The linker may connect the Cas protein or a functional fragment thereof to the heterodimer domain. The at least one RNA-guided endonuclease may comprise a localization signal sequence. The at least one DNA ligase may comprise a localization signal sequence. The localization signal sequence may comprise a nuclear localization sequence (NLS). The a least one RNA-guided endonuclease or the at least one DNA ligase may be directed to nucleus of the cell by the NLS. The at least one integrating nucleic acid may correct at least one genetic mutation in the at least one genomic locus. The at least one integrating nucleic acid may insert a coding sequence. The coding sequence may encode a full length protein. The at least one integrating nucleic acid may insert a non-coding sequence. The non-coding sequence may knock out an endogenous gene. The non-coding sequence may comprise a regulatory element. The system may further include a nuclease. The nuclease may comprise an exonuclease for digesting the genomic flap. The nuclease may comprise a human flap endonuclease 1 (hFEN1), a human exonuclease 5 (hEXO5), a T5 exonuclease, a T7 exonuclease, an exonuclease VIII, a flap endonuclease domain of  E. coli  PolI, a RecJF, a Lambda exonuclease, a Xni (ExoIXI), a SaFEN ( Staphylococcus aureus  FEN), a nuclease BAL-31, or a fragment thereof. The heterologous nuclease may comprise an endonuclease for digesting the genomic flap, and the endonuclease may be different from the at least one RNA-guided endonuclease. The at least one RNA-guided endonuclease may comprise at least one additional functional domain. The at least one additional functional domain may comprise a chromatin modifying domain. The at least one additional functional domain may comprise a cell penetrating peptide. The at least one guide nucleic acid may comprise at least one nucleic acid modification. The at least one nucleic acid modification may comprise a modification to a backbone, a sugar, a base, or a combination thereof. The at least one RNA-guided endonuclease may be complexed with the at least one guide nucleic acid. The at least one guide nucleic acid may be complexed with the integrating nucleic acid. The at least one RNA-guided endonuclease, the at least one guide nucleic acid, the at least one at least one DNA ligase, the integrating nucleic acid, or a combination thereof may be encoded by a polynucleotide. The polynucleotide may comprise mRNA. The polynucleotide may comprise a vector. The vector may comprise a viral vector. The at least one RNA-guided endonuclease, the at least one guide nucleic acid, the at least one at least one DNA ligase, the integrating nucleic acid, or a combination thereof may be encapsulated by at least one lipid nanoparticle. The cell may comprise a bacterial cell or a prokaryotic cell. The cell may include a prokaryotic cell. The prokaryotic cell may include a bacterial cell. The editing may be performed in a cytoplasm of the bacterial cell. The cell may include a eukaryotic cell. The eukaryotic cell may include an animal cell or a plant cell. The eukaryotic cell may include a plant cell. The eukaryotic cell may include an animal cell. The eukaryotic cell may comprise a mammalian cell. The editing may be performed in a cytoplasm of the eukaryotic cell. The editing may be performed in a nucleus of the eukaryotic cell. The system, or any aspect of the system, may be included in a composition, or in a cell such as a cell line. 
     Some aspects relate to a system that includes nucleic acids. The system may include guide nucleic acids, integrating nucleic acids, or a combination thereof. Some aspects relate to a system of nucleic acids. The system may include a system of guide nucleic acids. The system may include a system of integrating nucleic acids. The system of nucleic acids may further include other aspects such as additional nucleic acids or non-nucleic acid components. 
     The system of nucleic acids may include a guide nucleic acid. The guide nucleic acid may include a spacer. The spacer may be complementary to a region of a locus (e.g. genomic locus) of a target nucleic acid such as a genomic strand. The target nucleic acid may be in a cell. The genomic strand may be in a cell. The target nucleic acid may be in vitro. The guide nucleic acid may include a scaffold. The scaffold may complex with an endonuclease such as an RNA-guided endonuclease. The guide nucleic acid may include a flap binding site. The flap binding site may be complementary or at least partially complementary to a flap such as a genomic flap. The flap binding site may be identical or at least partially identical to a flap such as a genomic flap. The flap may be at the locus. The flap may be adjacent to the locus. The guide nucleic acid may include a donor binding site. The donor binding site may be complementary to an integrating nucleic acid. The donor binding site may be partially complementary to an integrating nucleic acid. The donor binding site may be complementary to a splinting nucleic acid. The donor binding site may be partially complementary to a splinting nucleic acid. Components of the guide nucleic acid may be included in 1 guide nucleic acid. More than one guide nucleic acid may be used. Components of the guide nucleic acid may collectively be included among multiple guide nucleic acids. Components of the guide nucleic acid may split between multiple guide nucleic acids. 
     The system of nucleic acids may include an integrating nucleic acid. The integrating nucleic acid may include a 5′ end to be ligated. The 5′ end may be ligated. The 5′ end may be ligated to a 3′ terminus. The 3′ terminus may be of a target nucleic acid strand (e.g. a genomic strand). The 3′ terminus may be generated by an endonuclease such as an RNA-guided endonuclease. The integrating nucleic acid may include a 5′ end to be ligated to a 3′ terminus of a genomic strand generated by an RNA-guided endonuclease. Components of the integrating nucleic acid may be included in 1 or 2 complementary strands. Components of the integrating nucleic acid may be included in 1 integrating nucleic acid. More than one integrating nucleic acid may be used. Components of the integrating nucleic acid may collectively be included among multiple integrating nucleic acids. Components of the integrating nucleic acid may split between multiple integrating nucleic acids. 
     The system of nucleic acids may include a splinting nucleic acid (also referred to as a “splinting strand”). The splinting strand may hybridize to two nucleic acids comprising ends to be ligated. The splinting nucleic acid may include a flap binding site. The flap binding site may be complementary to a flap. The flap binding site may be partially complementary to a flap. The flap binding site may be identical to a flap. The flap binding site may be partially identical to a flap. The flap may be at a locus of a target nucleic acid. The flap may be adjacent to a locus of a target nucleic acid. The flap may be a genomic flap. The locus may be a genomic locus. The flap binding site may be at least partially identical or complementary to a genomic flap at or adjacent to a genomic locus. The splinting nucleic acid may include a guide binding site. The guide binding site may be complementary to a guide nucleic acid. The guide binding site may be partially complementary to a guide nucleic acid. Components of the splinting nucleic acid may be included in 1 splinting nucleic acid. More than one splinting nucleic acid may be used. The splinting nucleic acid may include a donor binding site. The donor binding site may be complementary to an integrating nucleic acid. The donor binding site may be partially complementary to an integrating nucleic acid. 
     The splinting strand may be or include DNA. The splinting strand may be or include RNA. The splinting nucleic acid may be included as part of an integrating nucleic acid. The splinting nucleic acid may be included as a strand of a double stranded integrating nucleic acid. The splinting nucleic acid may be included as part of a guide nucleic acid. 
     The system of nucleic acids may include: (a) a guide nucleic acid comprising: (i) a spacer complementary to a region of a genomic locus of a genomic strand, (ii) a scaffold for complexing with RNA-guided endonuclease, (iii) an optional donor binding site that is at least partially complementary to an integrating nucleic acid, and (iv) a flap binding site that is at least partially identical or complementary to a genomic flap at or adjacent to the genomic locus; and (b) an integrating nucleic acid comprising a 5′ end to be ligated to a 3′ terminus of the genomic strand generated by an RNA-guided endonuclease. A component of (i), (ii), (iii), or (iv) may be included in a single guide nucleic acid, or may be split between or collectively included among multiple guide nucleic acids. 
     The system of nucleic acids may include: (a) a guide nucleic acid comprising (i) a spacer complementary to a region of a genomic locus of a genomic strand, (ii) a scaffold for complexing with an RNA-guided endonuclease, and (iii) an optional donor binding site that is at least partially complementary to a splinting nucleic acid; (b) an integrating nucleic acid comprising a 5′ end to be ligated to a 3′ terminus of the genomic strand generated by an RNA-guided endonuclease; and (c) a splinting nucleic acid comprising a flap binding site that is at least partially identical or complementary to a genomic flap at or adjacent to the genomic locus, and comprising an optional guide binding site that is at least partially complementary to a guide nucleic acid. A component of (i), (ii), or (iii) may be included in a single guide nucleic acid, or may be split between or collectively included among multiple guide nucleic acids. 
     In some aspects, the system described herein can be delivered into a cell, where one or more of the components of the system can be delivered into the cell together. In some aspects, each component of the system can be delivered into the cell separately. In some aspects, the system can be encoded by a polynucleotide such as a heterologous polynucleotide, where the polynucleotide is delivered into a cell and where the polynucleotide is expressed by the cell to generate the components of the cell. In some aspects, the system can be encoded and delivered into the cell via a polynucleotide comprising mRNA. In some aspects, the system can be encoded and delivered into the cell via a polynucleotide comprising a vector. In some aspects, the vector comprises a viral vector. The system can be encapsulated in a lipid or nanoparticle, or multiple lipids or nanoparticles. In some aspects, the system can be encapsulated in at least one lipid nanoparticle. In some aspects, the system comprises a ribonucleoprotein (RNP). For example, at least one RNA-guided endonuclease described herein (e.g., a Cas9) can be complexed with at least one guide nucleic acid described herein (e.g., forming a CRISPR ribonucleoprotein) for delivery. In some aspects, the system comprises at least one RNP comprising a RNA-guided endonuclease complexed with at least one first guide nucleic acid or with at least one second guide nucleic acid. In some aspects, the system comprises at least one RNP and at least one integrating nucleic acid (e.g., a single-stranded or a double-stranded integrating nucleic acid described herein). In some aspects, the system comprises at least one RNP and at least one integrating nucleic acid. In some aspects, the system comprises at least one RNP and at least one first integrating nucleic acid or at least one second integrating nucleic acid. 
     In some aspects, the system described herein can modify a genomic locus or gene in a cell. In some aspects, the cell comprises a bacterial cell, an eukaryotic cell, or a plant cell. In some aspects, the system described herein can be formulated into a composition, a pharmaceutical composition, a kit, or a combination thereof. In some aspects, the system described herein can be delivered and propagated in a cell line. 
     Some aspects include an editing system, comprising an RNA-guided endonuclease, a guide nucleic acid, and an integrating nucleic acid. Some aspects include an editing method, comprising: contacting a target nucleic acid with the editing system and a DNA ligase. 
     Pharmaceutical Compositions 
     Described herein, in some aspects, is a pharmaceutical composition comprising the system or the composition described herein. The pharmaceutical composition may include a pharmaceutically acceptable excipient, carrier, or diluent. The pharmaceutical composition may include a carrier. The pharmaceutical composition may include an excipient. The pharmaceutical composition may be delivered to a subject. The pharmaceutical composition may be delivered to a cell. The pharmaceutical composition may be used in a method disclosed herein. 
     The pharmaceutical compositions described herein comprise the system, the composition, or the cell contacted with the system or contacted with the composition. The pharmaceutical composition may comprise a composition such as a protein or nucleic acid disclosed herein. The pharmaceutical composition may comprise a cell comprising a composition or system disclosed herein. 
     A pharmaceutical composition may include a mixture of a pharmaceutical composition, with other chemical components (i.e. pharmaceutically acceptable inactive ingredients), such as carriers, excipients, binders, filling agents, suspending agents, flavoring agents, sweetening agents, disintegrating agents, dispersing agents, surfactants, lubricants, colorants, diluents, solubilizers, moistening agents, plasticizers, stabilizers, penetration enhancers, wetting agents, anti-foaming agents, antioxidants, preservatives, or one or more combination thereof. In practicing the methods of treatment or use provided herein, therapeutically effective amounts of pharmaceutical compositions described herein are administered to a mammal having a disease, disorder, or condition to be treated. In some aspects, the mammal is a human. A therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the pharmaceutical composition used and other factors. The pharmaceutical compositions can be used singly or in combination with one or more pharmaceutical compositions as components of mixtures. 
     The pharmaceutical composition may be formulated for administering intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, intratumorally, pulmonarily, endotracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, by inhalation, by inhaled nebulized form, by intraluminal-GI route, or a combination thereof to a subject in need thereof. 
     The pharmaceutical formulations described herein are administered to a subject by appropriate administration routes, including but not limited to, intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal administration routes. The pharmaceutical formulations described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations. Pharmaceutical compositions including a pharmaceutical composition are manufactured in a conventional manner, such as, by way of example only, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes. 
     The pharmaceutical compositions may include at least a pharmaceutical composition as an active ingredient in free-acid or free-base form, or in a pharmaceutically acceptable salt form. In addition, the methods and pharmaceutical compositions described herein include the use of N-oxides (if appropriate), crystalline forms, amorphous phases, as well as active metabolites of these compounds having the same type of activity. In some aspects, pharmaceutical compositions exist in unsolvated form or in solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. The solvated forms of the pharmaceutical compositions are also considered to be disclosed herein. 
     In some aspects, a pharmaceutical composition exists as a tautomer. All tautomers are included within the scope of the agents presented herein. As such, it is to be understood that a pharmaceutical composition or a salt thereof may exhibit the phenomenon of tautomerism whereby two chemical compounds that are capable of facile interconversion by exchanging a hydrogen atom between two atoms, to either of which it forms a covalent bond. Since the tautomeric compounds exist in mobile equilibrium with each other they can be regarded as different isomeric forms of the same compound. 
     In some aspects, a pharmaceutical composition exists as an enantiomer, diastereomer, or other steroisomeric form. The agents disclosed herein include all enantiomeric, diastereomeric, and epimeric forms as well as mixtures thereof. 
     In some aspects, pharmaceutical compositions described herein can be prepared as prodrugs. A “prodrug” refers to an agent that is converted into the parent drug in vivo. Prodrugs are often useful because, in some situations, they can be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent is not. The prodrug may also have improved solubility in pharmaceutical compositions over the parent drug. In certain embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically active form of the pharmaceutical composition. In certain embodiments, a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically or therapeutically active form of the pharmaceutical composition. 
     Kits 
     Described herein, in some aspects, are kits for using the system, the composition, or the pharmaceutical composition described herein. In some aspects, the kits disclosed herein may be used to treat a disease or condition in a subject. In some aspects, the kit comprises an assemblage of materials or components apart from the system, the composition, or the pharmaceutical composition. In some aspects, the kit comprises the components for assaying and selecting for suitable guide nucleic acid or donor strand for treating a disease or a condition. In some aspects, the kit comprises components for performing assays such as enzyme-linked immunosorbent assay (ELISA), single-molecular array (Simoa), PCR, or qPCR. The exact nature of the components configured in the kit depends on its intended purpose. For example, some embodiments are configured for the purpose of treating a disease or condition disclosed herein in a subject. In some aspects, the kit is configured particularly for the purpose of treating mammalian subjects. In some aspects, the kit is configured particularly for the purpose of treating human subjects. 
     Instructions for use may be included in the kit. In some aspects, the kit comprises instructions for administering the composition to a subject in need thereof. In some aspects, the kit comprises instructions for further engineering the system described herein. In some aspects, the kit comprises instructions for thawing or otherwise restoring biological activity of at least one component of the system, which may have been cryopreserved or lyophilized during storage or transportation. In some aspects, the kit comprises instructions for measuring efficacy for its intended purpose (e.g., therapeutic efficacy if used for treating a subject). 
     The kit may comprise a system or composition disclosed herein, and a container. The composition may be a pharmaceutical composition. 
     Methods 
     Described herein are methods such as methods of modifying a target nucleic acid. Described herein are methods such as methods of gene editing or gene replacement. The method may include use of any aspect of composition described herein such as an endonuclease, ligase, guide nucleic acid, integrating nucleic acid, system, kit, or pharmaceutical composition. 
     Gene Editing or Replacement 
     Disclosed herein are editing methods such as gene editing methods or nucleic acid editing methods. The editing tools and methods disclosed herein may be useful for genetic enhancement, genetic correction, treatment of a disease, development of research tools, or for disease diagnosis. The methods may be performed for therapeutic, agricultural, industrial, and research purposes. The editing method may include contacting a target nucleic acid with an editing system and a ligase. The target nucleic acid may be double-stranded. The target nucleic acid may include a host or cell genome. The target nucleic acid may include a pathogen genome in a host. The target nucleic acid may be part of a gene, or may include a non-gene or intergenic sequence. The target nucleic acid may reside in a nucleus of a cell. The target nucleic acid may include chromatin, euchromatin, or heterochromatin. The target nucleic acid may comprise DNA. The methods referred to herein as gene editing methods or genome editing methods may be useful for nucleic acid editing without necessarily being limited to editing of a certain gene. The method may include replacing a target nucleic acid sequence with a sequence of an integrating nucleic acid. The method may be performed in vitro. The method may be performed in vivo. The method may be performed in a cell. The editing may be performed without homologous recombination. The editing may be performed without prior insertion into host genome. 
     Disclosed herein, in some aspects, are editing methods. The method may include editing a nucleic acid. The nucleic acid may be in a cell. The editing may be performed using a DNA ligase. The editing may be performed using a CRISPR protein. The editing may be performed using a CRISPR protein or DNA ligase without any significant chemical interaction with an endogenous factor. The editing may be performed using a CRISPR protein or DNA ligase without any significant chemical interaction with a polymerase such as a DNA polymerase. In some aspects, the editing may be performed using an endonuclease (e.g., a Cas endonuclease) described herein or DNA ligase, where the endonuclease and the DNA ligase are coupled. For example, the endonuclease and the DNA ligase can be covalently coupled as a fusion protein for performing the editing. The method may include editing a nucleic acid in a cell, wherein the editing is performed using a Cas endonuclease without any significant chemical interaction with an endogenous factor or polymerase. The method may include editing a nucleic acid in a cell, wherein the editing is performed using a Cas endonuclease without any significant chemical interaction with endogenous cellular components of NHEJ or HDR. The editing method may exclude polymerization or in-cell synthesis of a nucleic acid. For example, the method may exclude in-cell synthesis from a template on a guide nucleic acid. 
     The editing may be performed, in some aspects, solely by factors exogenous to the cell. The exogenous factors may be added to the cell or are encoded by a nucleic acid added to the cell. In some aspects, the exogenous factors are added to the cell. In some aspects, the exogenous factors encoded by a nucleic acid added to the cell. The factors may include a Cas endonuclease and a DNA ligase. The Cas endonuclease may be or include a DNA-binding protein. 
     The editing may include replacing a nucleotide or nucleotide sequence within a target nucleic acid. The editing may include replacing a nucleotide. The editing may include replacing a nucleotide sequence. The nucleotide or nucleotide sequence may be replaced with an integrating nucleic acid. The editing may include replacing a nucleotide or nucleotide sequence of the nucleic acid with an integrating nucleic acid. In some aspects, replacing the nucleotide comprises breaking a phosphodiester bond of the nucleic acid and forming a new phosphodiester bond with the integrating nucleic acid. In some aspects, the replacement is performed at a replacement site within the nucleic acid, without leaving a remaining nick or strand break in the nucleic acid at the replacement site. In some aspects, the editing generates an edited nucleic acid comprising an edited region flanked by phosphodiester bonds to unedited regions of the edited nucleic acid. 
     Described herein, in some aspects, is a method for correcting a gene or modifying gene expression in a cell. In some aspects, the method comprises contacting the cell with a system or composition described herein. In some aspects, the method comprises delivering a heterologous polynucleotide into the cell, where the heterologous polynucleotide encodes at least one component of system. In some aspects, the system described herein can introduce a donor strand into a genomic locus. In some aspects, the system can introduce the donor strand without the need of endogenous machinery of the cell. In some aspects, the system can introduce the donor strand without the need to synchronize cell cycling. In some aspects, the system can introduce the donor strand in non-dividing cell or slow dividing cell. Such technical aspect can be especially useful for correcting genetic mutation in non-dividing cell or slow dividing cell for treating a disease or condition. 
     The method may include editing a nucleic acid of a cell. In some embodiments, the cell is quiescent or senescent cell. The cell may be quiescent. The cell may be senescent. In some aspects, the cell is not actively dividing. The cell may have a low dNTP concentration relative to other cells or cell types. Some examples of cells may include a neuron, myocyte, cardiomyocyte, or osteocyte. The cell may include a neuron. The cell may include a myocyte. The cell may include a cardiomyocyte. The cell may include an osteocyte. The cell may include an eye cell. 
     The cell may include a stem cell such as an embryonic stem cell, or such as an adult stem cell. The cell may be a circulating cell such as a blood cell. The cell may include a bone marrow cell. The cell may be an immune cell. The cell may be an innate immune cell. 
     The cell may be an airway cell. The cell may be a lung cell. The cell may be a bronchial cell. The cell may be an endothelial cell. 
     Described herein, in some aspects, is an editing method, comprising: editing a nucleic acid in a cell, wherein the editing is performed using a CRISPR protein (e.g. an RNA-guided endonuclease such as a Cas endonuclease) without any significant chemical interaction with an endogenous factor or polymerase. In some embodiments, the editing is performed solely by factors exogenous to the cell. In some embodiments, the exogenous factors are added to the cell or are encoded by a nucleic acid added to the cell. 
     In some embodiments, the editing is performed using a DNA ligase. In some embodiments, the editing comprises replacing a nucleotide or nucleotide sequence of the nucleic acid with an integrating nucleic acid. In some embodiments, replacing the nucleotide comprises breaking a phosphodiester bond of the nucleic acid and forming a new phosphodiester bond with the integrating nucleic acid. In some embodiments, the replacement is performed at a replacement site within the nucleic acid, without leaving a nick or strand break in the nucleic acid at the replacement site. In some embodiments, the editing generates an edited nucleic acid comprising an edited region flanked by phosphodiester bonds to unedited regions of the edited nucleic acid. 
     Some aspects include a method for modifying a cell comprising contacting a cell with a system or composition such as a pharmaceutical composition disclosed herein. In some aspects, the cell is not a dividing cell. The integrating nucleic acid may be inserted into the genomic locus of the cell independent of endogenous non-homologous end joining (NHEJ) and independent of endogenous homology-directed repair (HDR). 
     In some aspects, described herein is a method for modifying or replacing a nucleotide or nucleotide sequence in a cell by contacting the cell with the system or composition described herein, where the system or composition comprises a guide nucleic acid comprising: a spacer complementary to a region of a genomic locus of a genomic strand; a scaffold for complexing with an endonuclease; an optional donor binding site that is at least partially complementary to an integrating nucleic acid; and a flap binding site that is at least partially identical or complementary to a genomic flap at or adjacent to the genomic locus. In some embodiments, the guide nucleic acid comprises the donor binding site is complexed with the integrating nucleic acid. The complexing between the guide nucleic acid and the integrating nucleic acid can occur in vivo or in vitro. In some embodiments, the flap binding site can be complexed with a genomic flap generated by the endonuclease cleaving the genomic strand. The complexing between the flap binding site and the genomic flap can bring the integrating nucleic acid to close proximity to the cleaved genomic strand. The decreased proximity between the donor nucleic and the cleaved genomic strand can increase editing efficiency, decease off-target effect, or decrease introduction of unwanted mutations such as indels. In such case, the integrating nucleic acid can replace one strand of the cleaved genomic strand, thus editing or correcting the cleaved genomic strand.  FIG.  1 A - FIG.  1 C  illustrate the complexing between the guide nucleic acid and the integrating nucleic acid described herein, where the complexing between the guide nucleic acid and the integrating nucleic acid brings the integrating nucleic acid to close proximity to the cleaved genomic strand. In some embodiments, the integrating nucleic acid comprises a 5′ end to be ligated to a 3′ terminus of a genomic strand generated by an endonuclease cleaving the genomic strand. In some embodiments, the integrating nucleic acid comprises a 3′ end to be ligated to a 5′ terminus of a genomic strand generated by an endonuclease cleaving the genomic strand. In some embodiments, the endonuclease can be a fusion protein described herein. For example, the endonuclease can be fused to a DNA ligase described herein, where the endonuclease and DNA ligase fusion can cleave the genomic strand and ligate the integrating nucleic acid to the cleaved genomic strand with increased efficiency. 
     In some embodiments, the integrating nucleic acid is double stranded or partially double stranded, where the integrating nucleic acid can replace both strands of the cleaved genomic strand. In such case, the integrating nucleic acid can comprise single stranded guide binding site to be complexed with a guide nucleic acid comprising the donor binding site. The guide binding site can locate at 5′ end of the integrating nucleic acid. The guide binding site can locate at 3′ end of the integrating nucleic acid. The guide binding site can locate at both 5′ end and 3′ end of the integrating nucleic acid.  FIG.  2 A - FIG.  2 C  illustrate a double stranded integrating nucleic acid comprising the guide binding site at both 5′ end and 3′ end of the integrating nucleic acid, where the integrating nucleic acid can edit and replace the cleaved genomic strand. 
     In some embodiments, the integrating nucleic acid is double stranded or partially double stranded, where the integrating nucleic acid comprises a flap binding site and a guide binding site. In such case, the guide binding site can complex with the donor binding site of the guide nucleic acid.  FIG.  3 A  illustrates such arrangement, where the integrating nucleic acid (and not the guide nucleic acid) can be complexed with the genomic flap to bring the integrating nucleic acid to close proximity to the cleaved genomic strand. In some embodiments, the donor nucleic comprises two flap binding sites to be complexed with two different genomic flaps.  FIG.  4 A  illustrates such arrangement, where the integrating nucleic acid (and not the guide nucleic acid) can be complexed with the two genomic flaps to bring the integrating nucleic acid to close proximity to the two cleaved genomic strand. 
     In some embodiments, the integrating nucleic acid comprises the guide binding site, where the guide binding site can be complexed with the donor binding site of the guide nucleic acid. The guide nucleic acid can comprise the flap binding site to be complexed with the genomic flap at the cleaved genomic strand. As shown in  FIG.  5 A , the guide nucleic acid brings the integrating nucleic acid to close proximity to the cleaved genomic strand for editing and replacing the cleaved genomic strand with the integrating nucleic acid. In some embodiments, the integrating nucleic acid can be double strand and comprises the two guide binding sites to be complexed with two different guide nucleic acids.  FIG.  6 A  illustrates such arrangement, where the two guide nucleic acids bring the integrating nucleic acid to close proximity to two cleaved genomic strands. 
     In some aspects, described herein is a method for modifying or replacing a nucleotide or nucleotide sequence in a cell by contacting the cell with the system or composition described herein, where the system or composition comprises a guide nucleic acid comprising a spacer complementary to a region of a genomic locus of a genomic strand; a scaffold for complexing with an endonuclease, and an optional donor binding site that is at least partially complementary to a splinting nucleic acid. In some embodiments, the system or composition comprises an integrating nucleic acid, where the integrating nucleic acid can be ligated into the cleaved or nicked genomic strand. In some embodiments, the integrating nucleic acid comprises a 5′ end to be ligated to a 3′ terminus of the genomic strand generated by an endonuclease. In some embodiments, the integrating nucleic acid comprises a 3′ end to be ligated to a 5′ terminus of the genomic strand generated by an endonuclease. In some embodiments, the system or composition comprises a splinting nucleic acid comprising a flap binding site that is at least partially identical or complementary to a genomic flap at or adjacent to the genomic locus, and comprising an optional guide binding site that is at least partially complementary to a guide nucleic acid. In some embodiments, the splinting nucleic acid may include a guide binding site. The guide binding site may be complementary to a guide nucleic acid. The guide binding site may be partially complementary to a guide nucleic acid. The splinting nucleic acid may include a donor binding site. The donor binding site may be complementary to an integrating nucleic acid. The donor binding site may be partially complementary to an integrating nucleic acid. The splinting strand may be or include DNA. The splinting strand may be or include RNA. The splinting nucleic acid may be included as part of an integrating nucleic acid. The splinting nucleic acid may be included as a strand of a double stranded integrating nucleic acid. 
     In some embodiments, the method described herein decreases proximity between the integrating nucleic acid and the cleaved or nicked site. In some embodiments, the decreased proximity between the integrating nucleic acid and the cleaved or nicked site increases gene editing rate by at least 0.1 fold, 0.2 fold, 0.5 fold, 1.0 fold, 2.0 fold, 5.0 fold, 10.0 fold, or more compared to a gene editing rate without using a composition or a replacer described herein. In some embodiments, the decreased proximity between the integrating nucleic acid and the cleaved or nicked site decreases introduction of unwanted mutation such as indel by at least 0.1 fold, 0.2 fold, 0.5 fold, 1.0 fold, 2.0 fold, 5.0 fold, 10.0 fold, or more compared to a introduction of unwanted mutation without using a composition or a replacer described herein. In some embodiments, the decreased proximity between the integrating nucleic acid and the cleaved or nicked site decreases off-target editing by at least 0.1 fold, 0.2 fold, 0.5 fold, 1.0 fold, 2.0 fold, 5.0 fold, 10.0 fold, or more compared to off-target editing without using a composition or a replacer described herein. 
     In some aspects, the method edits a gene. In some aspects, the method replaces a gene. In some aspects, the method removes a gene. In some aspects, the method introduces a methylated nucleotide into the target nucleic acid. In some aspects, the method introduces an unmethylated nucleotide into the target nucleic acid. 
     The method may be used to edit a nucleic acid in a plant cell. Some aspects include enhancing a plant. Some examples of plant enhancement may include editing of a disease susceptibility gene or introducing an herbicide resistance gene. An example of a disease susceptibility gene may include bacterial leaf streak disease susceptibility gene OsSULTR3;6 in rice. An example of introducing an herbicide resistance gene may include editing of acetolactate synthase in potato for herbicide resistance 
     Treatment 
     A method such as a gene editing method may be useful for treatment of a disease or disorder. The disease or disorder may be genetic. The treatment may be of a diseased or damaged cell. The disease may include a genetic disease, cancer, or an infection. The treatment may include administration of a composition disclosed herein to a subject in need thereof. The subject in need may include a subject identified as having a disease or disorder. 
     The methods described herein may be useful for treating a genetic disease. The genetic disease may be caused by a DNA mutation such as a point mutation, a deletion, an insertion, a duplication, or a repeat, relative to normal non-diseased DNA. The treatment may correct the mutation. Some examples of genetic diseases may include Angelman syndrome, Canavan disease, Charcot-Marie-Tooth disease, color blindness, cri du chat syndrome, cystic fibrosis, DiGeorge syndrome, Duchenne muscular dystrophy, familial hypercholesterolemia, haemochromatosis type 1, hemophilia, neurofibromatosis, phenylketonuria, polycystic kidney disease, Prader-Willi syndrome, sickle cell disease, spinal muscular atrophy, or Tay-Sachs disease. Some examples of diseases that may be treated using a method herein may include sickle cell disease, beta thalassemia, familial hypercholesterolemia (e.g. PCSK9 disruption), alpha I antitrypsin deficiency, phenylketonuria, cystic fibrosis, tyrosinemia, arginase I deficiency, Wilson&#39;s disease, a repeat expansion disorder, hemophilia (e.g. insertion of Factor IX at ALB in a hepatocyte), Duchenne muscular dystrophy. Some examples of repeat expansion disorders like Huntington&#39;s disease, Amyotrophic lateral sclerosis/frontotemporal dementia, Friedreich ataxia, Fragile X Syndrome. The method may be included in immuno-oncology, such as for T-cell engineering or in cancer treatment. 
     Two non-limiting examples of genetic diseases for which efficient and precise editing of slowly dividing and nondividing cells is beneficial for therapeutic gene therapy are sickle cell anemia (SCA) and alpha-1 antitrypsin deficiency (AATD). Sickle cell anemia is caused by the E6V missense mutation in the HBB gene resulting in aggregation of mutant beta-globin protein and ‘sickling’ of red blood cells. Autologous gene therapies using hematopoetic stem cells with corrected HBB alleles have been proposed as curative treatments for SCA. While expansion of ex vivo HSC cultures can be induced using cytokine cocktails, HSCs in the human body typically reside in niches within the bone marrow where they exist in a quiescent or slowly dividing state. AATD is most commonly caused by the E366K missense mutation in the SERPINA1 gene which encodes alpha-1 antitrypsin, a serine protease inhibitor secreted by hepatocytes. Mutant AAT is misfolded, forming aggregates in the endoplasmic reticulum of the hepatocytes rather than being secreted, ultimately leading to liver disease. Although hepatocytes possess the ability to rapidly proliferate in response to liver damage, their life cycles are typically spent in a state of quiescence. As such, high efficiency in vivo editing of these two disorders necessitates a novel gene therapy platform which can effectively perform precise edits in nondividing or slowly dividing cells. 
     Some aspects include a method for treating a disease or condition in subject in need thereof comprising: (a) contacting a cell of the subject with a system or composition such as a pharmaceutical composition disclosed herein; and (b) replacing a genomic locus in a cell with an integrating nucleic acid, thereby treating the disease or condition in the subject. In some aspects, the cell is not a dividing cell. In some aspects, the integrating nucleic acid is inserted into the genomic locus of the cell independent of endogenous non-homologous end joining (NHEJ) and independent of endogenous homology-directed repair (HDR). 
     In some embodiments, the method described herein decreases proximity between the integrating nucleic acid and the cleaved or nicked site, where the decreased proximity between the integrating nucleic acid and the cleaved or nicked site increases gene editing rate by at least 0.1 fold, 0.2 fold, 0.5 fold, 1.0 fold, 2.0 fold, 5.0 fold, 10.0 fold, or more compared to a gene editing rate without using a composition or a replacer described herein. In some embodiments, the decreased proximity between the integrating nucleic acid and the cleaved or nicked site increases therapeutic efficacy (e.g., by increasing gene editing rate) by at least 0.1 fold, 0.2 fold, 0.5 fold, 1.0 fold, 2.0 fold, 5.0 fold, 10.0 fold, or more compared to a therapeutic efficacy without using a composition or a replacer described herein. 
     Delivery 
     Described herein, in some aspects, are methods of delivering the system described herein to a cell. In some aspects, the method comprises delivering directly or indirectly at least one component of the system to the cell. In some aspects, the method comprises delivering the cell with at least one heterologous polynucleotide, where the cell can then express the at least one component of the system. In some aspects, the at least one heterologous polynucleotide can be delivered into the cell via any of the transfection methods described herein. In some aspects, the at least one heterologous polynucleotide can be delivered into the cell via the use of expression vectors such as viral vectors. In the context of an expression vector, the vector can be readily introduced into the cell described herein by any method in the art. For example, the expression vector can be transferred into the cell by physical, chemical, or biological means. 
     Physical methods for introducing the oligonucleotide or vector encoding the oligonucleotide into the cell can include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, gene gun, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are suitable for methods herein. One method for the introduction of oligonucleotide or vector encoding the oligonucleotide into a host cell is calcium phosphate transfection. 
     Chemical means for introducing the oligonucleotide or vector encoding the oligonucleotide into the cell can include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, spherical nucleic acid (SNA), liposomes, or lipid nanoparticles. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of oligonucleotide or vector encoding the oligonucleotide with targeted nanoparticles or other suitable sub-micron sized delivery system. 
     In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the oligonucleotide or vector encoding the oligonucleotide into a cell (in vitro, ex vivo or in vivo). In another aspect, the oligonucleotide or vector encoding the oligonucleotide can be associated with a lipid. The oligonucleotide or vector encoding the oligonucleotide associated with a lipid, In some aspects, is encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, In some aspects, they are present in a bilayer structure, as micelles, or with a “collapsed” structure. Alternately, they may be simply interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which are, In some aspects, naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. 
     Lipids suitable for use are obtained from commercial sources. Stock solutions of lipids in chloroform or chloroform/methanol are often stored at about −20° C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic tem) encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes are often characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers. However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids, In some aspects, assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes. 
     In some cases, non-viral delivery method comprises lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, exosomes, polycation or lipid:cargo conjugates (or aggregates), naked polypeptide (e.g., recombinant polypeptides), naked DNA, artificial virions, and agent-enhanced uptake of polypeptide or DNA. In some aspects, the delivery method comprises conjugating or encapsulating the compositions or the oligonucleotides described herein with at least one polymer such as natural polymer or synthetic materials. The polymer can be biocompatible or biodegradable. Non-limiting examples of suitable biocompatible, biodegradable synthetic polymers can include aliphatic polyesters, poly(amino acids), copoly(ether-esters), polyalkylenes oxalates, polyamides, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamidoesters, polyoxaesters containing amine groups, and poly(anhydrides). Such synthetic polymers can be homopolymers or copolymers (e.g., random, block, segmented, graft) of a plurality of different monomers, e.g., two or more of lactic acid, lactide, glycolic acid, glycolide, epsilon-caprolactone, trimethylene carbonate, p-dioxanone, etc. In an example, the scaffold can be comprised of a polymer comprising glycolic acid and lactic acid, such as those with a ratio of glycolic acid to lactic acid of 90/10 or 5/95. Non-limiting examples of naturally occurring biocompatible, biodegradable polymers can include glycoproteins, proteoglycans, polysaccharides, glycosamineoglycan (GAG) and fragment(s) derived from these components, elastin, laminins, decrorin, fibrinogen/fibrin, fibronectins, osteopontin, tenascins, hyaluronic acid, collagen, chondroitin sulfate, heparin, heparan sulfate, ORC, carboxymethyl cellulose, and chitin. 
     In some cases, the oligonucleotide or vector encoding the oligonucleotide described herein can be packaged and delivered to the cell via extracellular vesicles. The extracellular vesicles can be any membrane-bound particles. In some aspects, the extracellular vesicles can be any membrane-bound particles secreted by at least one cell. In some instances, the extracellular vesicles can be any membrane-bound particles synthesized in vitro. In some instances, the extracellular vesicles can be any membrane-bound particles synthesized without a cell. In some cases, the extracellular vesicles can be exosomes, microvesicles, retrovirus-like particles, apoptotic bodies, apoptosomes, oncosomes, exophers, enveloped viruses, exomeres, or other very large extracellular vesicles. 
     In aspects, the system described herein or the at least one heterologous polynucleotide encoding the system described herein can be delivered into a cell as a vector such as a viral vector. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors, in some embodiments, are derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. Exemplary viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors (AAVs), pox vectors, parvoviral vectors, baculovirus vectors, measles viral vectors, or herpes simplex virus vectors (HSVs). In some instances, the retroviral vectors include gamma-retroviral vectors such as vectors derived from the Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV) or the Murine Stem cell Virus (MSCV) genome. In some instances, the retroviral vectors also include lentiviral vectors such as those derived from the human immunodeficiency virus (HIV) genome. In some instances, AAV vectors include AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 serotype. In some instances, viral vector is a chimeric viral vector, comprising viral portions from two or more viruses. In additional instances, the viral vector is a recombinant viral vector. 
     In some cases, the at least one heterologous polynucleotide encoding the system described herein can be administered to the subject in need thereof via the use of the transgenic cells generated by introduction of the at least one heterologous polynucleotide first into allogeneic or autologous cells. In some cases, the cell can be isolated. In some aspects, the cell can be isolated from the subject. 
     Subjects and Cells 
     The methods described herein may involve cells. For example, a composition may be delivered to a cell to edit a nucleic acid in the cell. The aspects delivered to the cell may be heterologous to the cell. “Heterologous” may include anything that does not exist in the cell in its natural state. 
     Any cell or cell type may be used. Examples of cells or cell types may include stem cells, red blood cells, white blood cells, platelets, nerve cells, neuroglial cells, muscle cells, cartilage cells, bone cells, skin cells, endothelial cells, epithelial cells, fat cells, or sex cells. The cell may include a stem cell. The cell may include a bone cell. The cell may include a blood cell. The cell may include a sperm cell. The cell may include an egg cell. The cell may include a fat cell. The cell may include a nerve cell. The cell may include a muscle cell. The cell may include an endocrine cell. The cell may include an endothelial cell. The cell may include a pancreatic cell. 
     The cell may be eukaryotic. The cell may be a plant cell. The cell may be an animal cell. The cell may be protozoan. The cell may be a fungal cell. The cell may be prokaryotic. The cell may be a bacterial cell. The cell may be an archaeon cell. The cell may be from a cell line. The cell may be part of a subject. The cell may be separated from a subject. The cell may be an autologous cell of a subject. The cell may be an allogenic cell of a subject. 
     The cell may include a diseased cell. The cell may include a cancer cell. The cell may be infected. The cell may be damaged. The cell may be a pathogen such as a fungal pathogen. 
     The methods described herein may involve a subject. For example, a composition may be delivered to the subject. Some aspects of the methods described herein include treatment of the subject. Non-limiting examples of subjects include vertebrates, animals, mammals, dogs, cats, cattle, rodents, mice, rats, primates, monkeys, and humans. The subject may be an invertebrate. The subject may be a arthropod. The subject may be a vertebrate. The subject may be an animal. The subject may be a fish. The subject may be a reptile. The subject may be a mammal. The subject may be a dog. The subject may be a cat. The subject may be a cattle. The subject may be a rodent. The subject may be a mouse. The subject may be a rat. The subject may be a primate. The subject may be a non-human primate. The subject may be a monkey. The subject may be an animal, a mammal, a dog, a cat, cattle, a rodent, a mouse, a rat, a primate, or a monkey. The subject may be a human. 
     The subject may be a non-animal subject. For example, the subject may include a plant. Examples of plants may include trees, flowers, shrubs, or grasses. The subject may include a crop. Examples of crops may include almond, apricot, apple, artichoke, banana, barley, beet, blackberry, blueberry, broccoli, Brussels sprout, cabbage, cannabis, capsicum, carrot, celery, chard, cherry, citrus, corn, cucurbit, date, fig, garlic, grape, herb, spice, kale, lettuce, oil palm, olive, onion, pea, pear, peach, peanut, papaya, parsnip, pecan, persimmon, plum, pomegranate, potato, quince, radish, raspberry, rose, rice, sloe, sorghum, soybean, spinach, strawberry, sweet potato, tobacco, tomato, turnip greens, walnut, or wheat. 
     Definitions 
     Use of absolute or sequential terms, for example, “will,” “will not,” “shall,” “shall not,” “must,” “must not,” “first,” “initially,” “next,” “subsequently,” “before,” “after,” “lastly,” and “finally,” are not meant to limit scope of the present embodiments disclosed herein but as exemplary. 
     As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.” 
     As used herein, the phrases “at least one”, “one or more”, and “and/or” are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions “at least one of A, B and C”, “at least one of A, B”, or C, “one or more of A, B”, and C, “one or more of A, BC”, or and “A, B, and/or C” means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together. 
     As used herein, “or” may refer to “and”, “or,” or “and/or” and may be used both exclusively and inclusively. For example, the term “A or B” may refer to “A or B”, “A but not B”, “B but not A”, and “A and B”. In some cases, context may dictate a particular meaning. 
     Any systems, methods, software, and platforms described herein are modular. Accordingly, terms such as “first” and “second” do not necessarily imply priority, order of importance, or order of acts. 
     The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and the number or numerical range may vary from, for example, from 1% to 15% of the stated number or numerical range. In examples, the term “about” refers to ±10% of a stated number or value. 
     The terms “increased”, “increasing”, or “increase” are used herein to generally mean an increase by a statically significant amount. In some aspects, the terms “increased,” or “increase,” mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, standard, or control. Other examples of “increase” include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more as compared to a reference level. 
     The terms “decreased”, “decreasing”, or “decrease” are used herein generally to mean a decrease by a statistically significant amount. In some aspects, “decreased” or “decrease” means a reduction by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level or non-detectable level as compared to a reference level), or any decrease between 10-100% as compared to a reference level. In the context of a marker or symptom, by these terms is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without a given disease. 
     Where sequences are provided, nucleic acids containing phosphorothioate bonds between nucleotides are signified with an asterisk (*). 2′-O-methyl nucleotides are signified with a lowercase “m” in front of the nucleotide, for example mC instead of C. The code “/5Phos/” in front of a nucleotide sequence indicates that the sequence is phosphorylated at the 5′ end. Locked nucleic acid (LNA) nucleotides comprising a methylene bridge connecting the 2′ oxygen and 4′ carbon are signified with a “+” in front of the nucleotide, for example +C instead of C. 
     EMBODIMENTS 
     Some aspects include an embodiment as follows: 
     Embodiment 1. Described herein, in some aspects, is a composition, comprising: 
     a DNA-binding protein coupled to a DNA ligase. 
     Embodiment 2. The composition of Embodiment 1, wherein the coupling is covalent. 
     Embodiment 3. The composition of Embodiment 2, comprising a fusion protein comprising the DNA-binding protein and the DNA ligase. 
     Embodiment 4. The composition of Embodiment 3, wherein the DNA-binding protein is amino (N)-terminal relative to the DNA ligase within the fusion protein. 
     Embodiment 5. The composition of Embodiment 3, wherein the DNA-binding protein is carboxy (C)-terminal relative to the DNA ligase within the fusion protein. 
     Embodiment 6. The composition of any one of Embodiments 2-5, wherein the connection comprises a linker comprising 1-100 amino acids. 
     Embodiment 7. The composition of Embodiment 1, wherein the coupling is non-covalent. 
     Embodiment 8. The composition of Embodiment 7, wherein the composition comprises a first polypeptide comprising at least part of the DNA-binding protein, and a second polypeptide comprising at least part of the DNA ligase, wherein the first and second polypeptides are non-covalently coupled. 
     Embodiment 9. The composition of Embodiment 8, wherein the first polypeptide comprises a first heterodimerization domain that binds a second heterodimerization domain, and wherein the second polypeptide comprises the second heterodimerization domain. 
     Embodiment 10. The composition of Embodiment 9, wherein the heterodimer domains comprise a leucine zipper, PDZ domain, streptavidin, streptavidin binding protein, foldon domain, hydrophobic moiety, or a functional binding fragment thereof. 
     Embodiment 11. The composition of Embodiment 8, wherein the first polypeptide comprises a first intein that binds a second intein, and wherein the second polypeptide comprises the second intein. 
     Embodiment 12. The composition of Embodiment 1, wherein the ligase comprises a hairpin binding motif, and wherein the DNA-binding protein and the DNA ligase are coupled with a nucleic acid comprising a scaffold that binds to the DNA-binding protein and a hairpin that binds to the hairpin binding motif. 
     Embodiment 13. The composition of Embodiment 12, wherein the hairpin binding motif comprises an MS2 coat protein (MCP) peptide, and wherein the hairpin comprises an MS2 hairpin. 
     Embodiment 14. The composition of Embodiment 1, wherein the DNA-binding protein and the DNA ligase are coupled with a heterobifunctional molecule comprising an endonuclease binding domain and a DNA ligase binding domain. 
     Embodiment 15. The composition of Embodiment 14, wherein the heterobifunctional molecule comprises a small molecule. 
     Embodiment 16. Described herein, in some aspects, is a composition comprising a cell containing a DNA-binding protein and a DNA ligase, both of which are heterologous to the cell. 
     Embodiment 17. The composition of any one of Embodiments 1-16, wherein the DNA-binding protein comprises a class II CRISPR/Cas endonuclease. 
     Embodiment 18. The composition of any one of Embodiments 1-17, wherein the DNA-binding protein comprises a Cas9 endonuclease. 
     Embodiment 19. The composition of any one of Embodiments 1-18, wherein the DNA-binding protein comprises a nickase. 
     Embodiment 20. The composition of any one of Embodiments 1-19, wherein the DNA-binding protein comprises an amino acid sequence at least 80% identical to the amino acid sequence of any one of SEQ ID NOS: 1-13, or a functional fragment thereof. 
     Embodiment 21. The composition of any one of Embodiments 1-20, wherein the DNA ligase ligates DNA strands base paired to a DNA splint. 
     Embodiment 22. The composition of any one of Embodiments 1-20, wherein the DNA ligase ligates DNA strands base paired to an RNA splint. 
     Embodiment 23. The composition of any one of Embodiments 1-22, wherein the DNA ligase comprises an amino acid sequence at least 80% identical to the amino acid sequence of any one of SEQ ID NOS: 55-96, or a functional fragment thereof. 
     Embodiment 24. The composition of any one of Embodiments 1-23, wherein the DNA-binding protein or the DNA ligase comprises a nuclear localization signal, chromatin modifying domain, cell penetrating peptide, or tag polypeptide. 
     Embodiment 25. The composition of any one of Embodiments 1-24, further comprising a guide RNA and an integrating nucleic acid. 
     Embodiment 26. One or more nucleic acids encoding the composition of any one of Embodiments 1-25. 
     Embodiment 27. A cell comprising the composition of any one of Embodiments 1-25, or comprising the one or more nucleic acids of Embodiment 26. 
     Embodiment 28. A system of nucleic acids comprising:
         a. a guide nucleic acid comprising:
           i. a spacer complementary to a region of a genomic locus of a genomic strand,   ii. a scaffold for complexing with a DNA-binding protein,   iii. an optional donor binding site that is at least partially complementary to an integrating nucleic acid, and   iv. a flap binding site that is at least partially identical or complementary to a genomic flap at or adjacent to the genomic locus; and   
           b. an integrating nucleic acid comprising a 5′ end to be ligated to a 3′ terminus of the genomic strand generated by a DNA-binding protein.       

     Embodiment 29. A system of nucleic acids comprising:
         a. a guide nucleic acid comprising:
           i. a spacer complementary to a region of a genomic locus of a genomic strand,   ii. a scaffold for complexing with a DNA-binding protein, and   iii. an optional donor binding site that is at least partially complementary to a splinting nucleic acid;   
           b. an integrating nucleic acid comprising a 5′ end to be ligated to a 3′ terminus of the genomic strand generated by a DNA-binding protein; and   c. a splinting nucleic acid comprising a flap binding site that is at least partially identical or complementary to a genomic flap at or adjacent to the genomic locus, and comprising an optional guide binding site that is at least partially complementary to a guide nucleic acid.       

     Embodiment 30. The system of Embodiment 28 or 29, wherein the genomic strand is in a cell. 
     Embodiment 31. The system of any one of Embodiments 28-30, wherein the splinting nucleic acid further comprises a donor binding site that is at least partially identical or complementary to a portion of the integrating nucleic acid. 
     Embodiment 32. The system of any one of Embodiment 28-31, wherein the guide nucleic acid comprises a sequence of linking nucleic acids between the scaffold and the donor binding site. 
     Embodiment 33. The system of any one of Embodiment 28-32, wherein the guide nucleic acid, the integrating nucleic acid, or the splinting nucleic acid comprises a modified internucleoside linkage. 
     Embodiment 34. The system of Embodiment 33, wherein the modified internucleoside linkage comprises a phosphorothioate linkage. 
     Embodiment 35. The system of Embodiment 33 or 34, wherein the modified internucleoside linkage is between any of the 4 terminal nucleosides at a 5′ end or at a 3′ end of the guide nucleic acid or the integrating nucleic acid. 
     Embodiment 36. The system of any one of Embodiments 28-35, wherein the guide nucleic acid, the integrating nucleic acid, or the splinting nucleic acid comprises a modified nucleoside. 
     Embodiment 37. The system of Embodiment 36, wherein the modified nucleoside comprises a locked nucleic acid (LNA), a 2′ fluoro, a 2′ O-alkyl, or a combination thereof. 
     Embodiment 38. The system of Embodiment 36 or 37, wherein the modified nucleoside is any of the 3 terminal nucleosides at a 5′ end or at a 3′ end of the guide nucleic acid or the integrating nucleic acid. 
     EXAMPLES 
     Example 1. Editing to Convert BFP to GFP by Replacer 1 
     Components used to edit the blue fluorescent protein (BFP) gene stably integrated into HEK293 cells are co-delivered by lipid nanoparticle (LNP) transfection. The components include chemically synthesized guide RNAs (gRNAs), single-stranded DNA donors, and mRNA encoding protein effectors for Replacer 1 editing including nicking Cas9 (nCas9), a SplintR ligase and nuclear localization sequences (NLS). The gRNAs are synthesized by Agilent, the DNA donors are synthesized by IDT, and the mRNA is synthesized by TriLink or RiboPro. The gRNA, DNA donor, and mRNA are mixed and formulated into lipid nanoparticles prior to delivery to adherent cells in 96 well plates. After 48 hours, the cells are detached from the plate by trypsinization and green fluorescent protein (GFP) fluorescence is measured using an Attune NxT flow cytometer to assess the percentage of BFP-to-GFP editing. Following the Replacer 1 editing format, the gRNAs contain a spacer, scaffold, donor binding site (DBS), and flap binding site (FBS). The gRNAs are delivered individually (1-sided Replacer 1) or as pairs with spacers targeting opposite strands of the genomic locus (2-sided Replacer 1). Some of the DBSs contain a mutation in the spacer region or in the protospacer adjacent motif region (SpPAMmut). The gRNAs contain 2′-O-methyl 3′-phosphorothioate nucleotides at the first three and last three positions. The DNA donors are delivered individually (1-sided Replacer 1) or in pairs (2-sided Replacer 1). Some donors have mutations in the spacer or protospacer adjacent motif (PAM) regions (SpPAMmut). Some donors have phosphorothioate bonds at the first three and last three positions. Some donors are recoded with silent mutations that change the nucleotide sequence but retain the amino acid sequence. The DNA donors are phosphorylated on the 5′ end. In some conditions, the gRNAs and donor DNAs are annealed by a thermal cycler annealing reaction prior to LNP formulation. Plasmids can be used in the place of mRNA. Table 12 details this experiment. Sequences corresponding to the names in the table may be found herein. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 12 
               
               
                   
               
               
                   
                   
                   
                   
                   
                   
                 Anneal 
               
               
                 Condition 
                 Forward Guide 
                 Reverse Guide 
                 Top Donor 
                 Bottom Donor 
                 Ligase 
                 both sides? 
               
               
                   
               
             
            
               
                 1 
                 Rep1.BFP. 
                   
                 Rep1. 
                   
                 NLS-nCas9- 
                 N/A 
               
               
                   
                 FwdGuide 
                   
                 BFP2GFP. 
                   
                 linker- 
               
               
                   
                   
                   
                 TopDonor. 
                   
                 SplintR- 
               
               
                   
                   
                   
                 5P 
                   
                 bpNLS 
               
               
                 2 
                   
                 Rep1.BFP. 
                   
                 Rep1. 
                 NLS-nCas9- 
                 N/A 
               
               
                   
                   
                 RevGuide 
                   
                 BFP2GFP. 
                 linker- 
               
               
                   
                   
                   
                   
                 BotDonor. 
                 SplintR- 
               
               
                   
                   
                   
                   
                 5P 
                 bpNLS 
               
               
                 3 
                 Rep1.BFP. 
                 Rep1.BFP. 
                 Rep1. 
                 Rep1. 
                 NLS-nCas9- 
                 Yes 
               
               
                   
                 FwdGuide 
                 RevGuide 
                 BFP2GFP. 
                 BFP2GFP. 
                 linker- 
               
               
                   
                   
                   
                 TopDonor. 
                 BotDonor. 
                 SplintR- 
               
               
                   
                   
                   
                 5P 
                 5P 
                 bpNLS 
               
               
                 4 
                 Rep1.BFP. 
                 Rep1.BFP. 
                 Rep1. 
                 Rep1. 
                 NLS-nCas9- 
                 No 
               
               
                   
                 FwdGuide 
                 RevGuide 
                 BFP2GFP. 
                 BFP2GFP. 
                 linker- 
               
               
                   
                   
                   
                 TopDonor. 
                 BotDonor. 
                 SplintR- 
               
               
                   
                   
                   
                 5P 
                 5P 
                 bpNLS 
               
               
                 5 
                 Rep1.BFP. 
                 Rep1.BFP. 
                 Rep1. 
                 Rep1. 
                 NLS-nCas9- 
                 Yes 
               
               
                   
                 FwdGuide 
                 RevGuide 
                 BFP2GFP. 
                 BFP2GFP. 
                 linker- 
               
               
                   
                   
                   
                 TopDonor. 
                 BotDonor. 
                 SplintR- 
               
               
                   
                   
                   
                 Recoded.5P 
                 Recoded.5P 
                 bpNLS 
               
               
                 6 
                 Rep1.BFP. 
                 Rep1.BFP. 
                 Rep1. 
                 Rep1. 
                 NLS-nCas9- 
                 Yes 
               
               
                   
                 FwdGuide. 
                 RevGuide. 
                 BFP2GFP. 
                 BFP2GFP. 
                 linker- 
               
               
                   
                 SpPAMmut 
                 SpPAMmut 
                 TopDonor. 
                 BotDonor. 
                 SplintR- 
               
               
                   
                   
                   
                 SpPAMmut. 
                 SpPAMmut. 
                 bpNLS 
               
               
                   
                   
                   
                 Recoded.5P 
                 Recoded.5P 
               
               
                 7 
                 Rep1.BFP. 
                 Rep1.BFP. 
                 Rep1. 
                 Rep1. 
                 NLS-nCas9- 
                 Yes 
               
               
                   
                 FwdGuide 
                 RevGuide 
                 BFP2GFP. 
                 BFP2GFP. 
                 linker- 
               
               
                   
                   
                   
                 TopDonor. 
                 BotDonor. 
                 SplintR- 
               
               
                   
                   
                   
                 Recoded. 
                 Recoded. 
                 bpNLS 
               
               
                   
                   
                   
                 5P.endPhos 
                 5P.endPhos 
               
               
                   
               
            
           
         
       
     
     Example 2. Editing to Convert BFP to GFP by Replacer 2 
     An experiment can be performed similar to Example 1 but adjusted to fit a Replacer 2 format. The ligases used here are T4 ligase, hLIG1(233-919), and hLIG1(119-919). The Replacer 2 gRNA contains a spacer, scaffold, and DBS. The gRNAs are delivered individually (1-sided Replacer 2) or in pairs (2-sided Replacer 2), and the gRNAs contain 2′-O-methyl 3′-phosphorothioate nucleotides at the first three and last three positions. The DNA donors include a FBS and a guide binding site (GBS) that can hybridize to the DBS. Some DNA donors contain SpPAM mutations and some DNA donors have phosphorothioate bonds at the first three and last three positions. Some DNA donors are recoded. The DNA donors are phosphorylated on the 5′ end. The DNA donors are delivered as pairs in the Replacer 2 format. Some of the gRNAs and donor DNAs are annealed prior to LNP formulation. Table 13 details this experiment. Sequences corresponding to the names in the table may be found herein. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 13 
               
               
                   
               
               
                   
                   
                   
                   
                   
                   
                 Anneal 
               
               
                 Condition 
                 Forward Guide 
                 Reverse Guide 
                 Top Donor 
                 Bottom Donor 
                 Ligase 
                 both sides? 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 1 
                 Rep2.BFP. 
                   
                 Rep2. 
                 Rep2. 
                 NLS-nCas9- 
                 Yes 
               
               
                   
                 FwdGuide 
                   
                 BFP2GFP. 
                 BFP2GFP. 
                 linker- 
               
               
                   
                   
                   
                 TopDonor. 
                 BotDonor. 
                 hLIG1(233- 
               
               
                   
                   
                   
                 SpPAMmut. 
                 SpPAMmut. 
                 919)-bpNLS 
               
               
                   
                   
                   
                 5P 
                 5P 
               
               
                 2 
                   
                 Rep2.BFP. 
                 Rep2. 
                 Rep2. 
                 NLS-nCas9- 
                 Yes 
               
               
                   
                   
                 RevGuide 
                 BFP2GFP. 
                 BFP2GFP. 
                 linker- 
               
               
                   
                   
                   
                 TopDonor. 
                 BotDonor. 
                 hLIG1(233- 
               
               
                   
                   
                   
                 SpPAMmut. 
                 SpPAMmut. 
                 919)-bpNLS 
               
               
                   
                   
                   
                 5P 
                 5P 
               
               
                 3 
                 Rep2.BFP. 
                 Rep2.BFP. 
                 Rep2. 
                 Rep2. 
                 NLS-nCas9- 
                 Yes 
               
               
                   
                 FwdGuide 
                 RevGuide 
                 BFP2GFP. 
                 BFP2GFP. 
                 linker- 
               
               
                   
                   
                   
                 TopDonor. 
                 BotDonor. 
                 hLIG1(233- 
               
               
                   
                   
                   
                 SpPAMmut. 
                 SpPAMmut. 
                 919)-bpNLS 
               
               
                   
                   
                   
                 5P 
                 5P 
               
               
                 4 
                 Rep2.BFP. 
                 Rep2.BFP. 
                 Rep2. 
                 Rep2. 
                 NLS-nCas9- 
                 No 
               
               
                   
                 FwdGuide 
                 RevGuide 
                 BFP2GFP. 
                 BFP2GFP. 
                 linker- 
               
               
                   
                   
                   
                 TopDonor. 
                 BotDonor. 
                 hLIG1(233- 
               
               
                   
                   
                   
                 SpPAMmut. 
                 SpPAMmut. 
                 919)-bpNLS 
               
               
                   
                   
                   
                 5P 
                 5P 
               
               
                 5 
                 Rep2.BFP. 
                 Rep2.BFP. 
                 Rep2.BFP2GFP. 
                 Rep2. 
                 NLS-nCas9- 
                 Yes 
               
               
                   
                 FwdGuide 
                 RevGuide 
                 TopDonor. 
                 BFP2GFP. 
                 linker- 
               
               
                   
                   
                   
                 SpPAMmut. 
                 BotDonor. 
                 hLIG1(233- 
               
               
                   
                   
                   
                 Recoded.5P 
                 SpPAMmut. 
                 919)-bpNLS 
               
               
                   
                   
                   
                   
                 Recoded.5P 
               
               
                 6 
                 Rep2.BFP. 
                 Rep2.BFP. 
                 Rep2. 
                 Rep2. 
                 NLS-nCas9- 
                 Yes 
               
               
                   
                 FwdGuide 
                 RevGuide 
                 BFP2GFP. 
                 BFP2GFP. 
                 linker- 
               
               
                   
                   
                   
                 TopDonor. 
                 BotDonor. 
                 hLIG1(233- 
               
               
                   
                   
                   
                 5P 
                 5P 
                 919)-bpNLS 
               
               
                 7 
                 Rep2.BFP. 
                 Rep2.BFP. 
                 Rep2. 
                 Rep2. 
                 NLS-nCas9- 
                 Yes 
               
               
                   
                 FwdGuide 
                 RevGuide 
                 BFP2GFP. 
                 BFP2GFP. 
                 linker- 
               
               
                   
                   
                   
                 TopDonor. 
                 BotDonor. 
                 hLIG1(233- 
               
               
                   
                   
                   
                 SpPAMmut. 
                 SpPAMmut. 
                 919)-bpNLS 
               
               
                   
                   
                   
                 Recoded. 
                 Recoded. 
               
               
                   
                   
                   
                 5P.endPhos 
                 5P.endPhos 
               
               
                 8 
                 Rep2.BFP. 
                   
                 Rep2. 
                 Rep2. 
                 NLS-nCas9- 
                 Yes 
               
               
                   
                 FwdGuide 
                   
                 BFP2GFP. 
                 BFP2GFP. 
                 linker- 
               
               
                   
                   
                   
                 TopDonor. 
                 BotDonor. 
                 T4LIG- 
               
               
                   
                   
                   
                 SpPAMmut. 
                 SpPAMmut. 
                 bpNLS 
               
               
                   
                   
                   
                 5P 
                 5P 
               
               
                 9 
                   
                 Rep2.BFP. 
                 Rep2. 
                 Rep2. 
                 NLS-nCas9- 
                 Yes 
               
               
                   
                   
                 RevGuide 
                 BFP2GFP. 
                 BFP2GFP. 
                 linker- 
               
               
                   
                   
                   
                 TopDonor. 
                 BotDonor. 
                 T4LIG- 
               
               
                   
                   
                   
                 SpPAMmut. 
                 SpPAMmut. 
                 bpNLS 
               
               
                   
                   
                   
                 5P 
                 5P 
               
               
                 10 
                 Rep2.BFP. 
                 Rep2.BFP. 
                 Rep2. 
                 Rep2. 
                 NLS-nCas9- 
                 Yes 
               
               
                   
                 FwdGuide 
                 RevGuide 
                 BFP2GFP. 
                 BFP2GFP. 
                 linker- 
               
               
                   
                   
                   
                 TopDonor. 
                 BotDonor. 
                 T4LIG- 
               
               
                   
                   
                   
                 SpPAMmut. 
                 SpPAMmut. 
                 bpNLS 
               
               
                   
                   
                   
                 Recoded.5P 
                 Recoded.5P 
               
               
                 11 
                 Rep2.BFP. 
                   
                 Rep2. 
                 Rep2. 
                 NLS-nCas9- 
                 Yes 
               
               
                   
                 FwdGuide 
                   
                 BFP2GFP. 
                 BFP2GFP. 
                 linker- 
               
               
                   
                   
                   
                 TopDonor. 
                 BotDonor. 
                 hLIG1(119- 
               
               
                   
                   
                   
                 SpPAMmut. 
                 SpPAMmut. 
                 919)-bpNLS 
               
               
                   
                   
                   
                 5P 
                 5P 
               
               
                 12 
                   
                 Rep2.BFP. 
                 Rep2. 
                 Rep2. 
                 NLS-nCas9- 
                 Yes 
               
               
                   
                   
                 RevGuide 
                 BFP2GFP. 
                 BFP2GFP. 
                 linker- 
               
               
                   
                   
                   
                 TopDonor. 
                 BotDonor. 
                 hLIG1(119- 
               
               
                   
                   
                   
                 SpPAMmut. 
                 SpPAMmut. 
                 919)-bpNLS 
               
               
                   
                   
                   
                 5P 
                 5P 
               
               
                 13 
                 Rep2.BFP. 
                 Rep2.BFP. 
                 Rep2. 
                 Rep2. 
                 NLS-nCas9- 
                 Yes 
               
               
                   
                 FwdGuide 
                 RevGuide 
                 BFP2GFP. 
                 BFP2GFP. 
                 linker- 
               
               
                   
                   
                   
                 TopDonor. 
                 BotDonor. 
                 hLIG1(119- 
               
               
                   
                   
                   
                 SpPAMmut. 
                 SpPAMmut. 
                 919)-bpNLS 
               
               
                   
                   
                   
                 Recoded.5P 
                 Recoded.5P 
               
               
                   
               
            
           
         
       
     
     Example 3. Editing to Insert mGL in Front of CBX1 by Replacer 2 
     An editing experiment can be performed to insert monomeric Green Lantern (mGL) in the genome of HEK293T cells in front of the CBX1 gene such that a fusion protein is formed that exhibits green fluorescence. This fluorescence can be detected by flow cytometry as in Examples 1 and 2. The experiment is conducted in a similar way to Example 2 except that the sequences of the gRNAs and DNA donors are different and enable insertion of mGL into the genome rather than insertion of a sequence that changes blue fluorescent protein (BFP) to green fluorescent protein (GFP). The DNA donors in Example 3 are longer than in Example 2 and are synthesized by GenScript. The DNA donors are phosphorylated on the 5′ end. Table 14 details this experiment. Sequences corresponding to the names in the table may be found herein. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 14 
               
               
                   
               
               
                   
                   
                   
                   
                   
                   
                 Anneal 
               
               
                 Condition 
                 Forward Guide 
                 Reverse Guide 
                 Top Donor 
                 Bottom Donor 
                 Ligase 
                 both sides? 
               
               
                   
               
             
            
               
                 1 
                 Rep2.CBX1. 
                   
                 Rep2. 
                 Rep2. 
                 NLS-nCas9- 
                 Yes 
               
               
                   
                 FwdGuide 
                   
                 mGL-CBX1. 
                 mGL-CBX1. 
                 linker- 
               
               
                   
                   
                   
                 TopDonor. 
                 BotDonor. 
                 hLIG1(233- 
               
               
                   
                   
                   
                 SpPAMmut. 
                 SpPAMmut. 
                 919)-bpNLS 
               
               
                   
                   
                   
                 5P 
                 5P 
               
               
                 2 
                   
                 Rep2.CBX1. 
                 Rep2. 
                 Rep2. 
                 NLS-nCas9- 
                 Yes 
               
               
                   
                   
                 RevGuide 
                 mGL-CBX1. 
                 mGL-CBX1. 
                 linker- 
               
               
                   
                   
                   
                 TopDonor. 
                 BotDonor. 
                 hLIG1(233- 
               
               
                   
                   
                   
                 SpPAMmut. 
                 SpPAMmut. 
                 919)-bpNLS 
               
               
                   
                   
                   
                 5P 
                 5P 
               
               
                 3 
                 Rep2.CBX1. 
                 Rep2.CBX1. 
                 Rep2. 
                 Rep2. 
                 NLS-nCas9- 
                 Yes 
               
               
                   
                 FwdGuide 
                 RevGuide 
                 mGL-CBX1. 
                 mGL-CBX1. 
                 linker- 
               
               
                   
                   
                   
                 TopDonor. 
                 BotDonor. 
                 hLIG1(233- 
               
               
                   
                   
                   
                 SpPAMmut. 
                 SpPAMmut. 
                 919)-bpNLS 
               
               
                   
                   
                   
                 5P 
                 5P 
               
               
                 4 
                 Rep2.CBX1. 
                   
                 Rep2. 
                 Rep2. 
                 NLS-nCas9- 
                 Yes 
               
               
                   
                 FwdGuide 
                   
                 mGL-CBX1. 
                 mGL-CBX1. 
                 linker- 
               
               
                   
                   
                   
                 TopDonor. 
                 BotDonor. 
                 T4LIG- 
               
               
                   
                   
                   
                 SpPAMmut. 
                 SpPAMmut. 
                 bpNLS 
               
               
                   
                   
                   
                 5P 
                 5P 
               
               
                 5 
                   
                 Rep2.CBX1. 
                 Rep2. 
                 Rep2. 
                 NLS-nCas9- 
                 Yes 
               
               
                   
                   
                 RevGuide 
                 mGL-CBX1. 
                 mGL-CBX1. 
                 linker- 
               
               
                   
                   
                   
                 TopDonor. 
                 BotDonor. 
                 T4LIG- 
               
               
                   
                   
                   
                 SpPAMmut. 
                 SpPAMmut. 
                 bpNLS 
               
               
                   
                   
                   
                 5P 
                 5P 
               
               
                 6 
                 Rep2.CBX1. 
                 Rep2.CBX1. 
                 Rep2. 
                 Rep2. 
                 NLS-nCas9- 
                 Yes 
               
               
                   
                 FwdGuide 
                 RevGuide 
                 mGL-CBX1. 
                 mGL-CBX1. 
                 linker- 
               
               
                   
                   
                   
                 TopDonor. 
                 BotDonor. 
                 T4LIG- 
               
               
                   
                   
                   
                 SpPAMmut. 
                 SpPAMmut. 
                 bpNLS 
               
               
                   
                   
                   
                 5P 
                 5P 
               
               
                 7 
                 Rep2.CBX1. 
                   
                 Rep2. 
                 Rep2. 
                 NLS-nCas9- 
                 Yes 
               
               
                   
                 FwdGuide 
                   
                 mGL-CBX1. 
                 mGL-CBX1. 
                 linker- 
               
               
                   
                   
                   
                 TopDonor. 
                 BotDonor. 
                 hLIG1(119- 
               
               
                   
                   
                   
                 SpPAMmut. 
                 SpPAMmut. 
                 919)-bpNLS 
               
               
                   
                   
                   
                 5P 
                 5P 
               
               
                 8 
                   
                 Rep2.CBX1. 
                 Rep2. 
                 Rep2. 
                 NLS-nCas9- 
                 Yes 
               
               
                   
                   
                 RevGuide 
                 mGL-CBX1. 
                 mGL-CBX1. 
                 linker- 
               
               
                   
                   
                   
                 TopDonor. 
                 BotDonor. 
                 hLIG1(119- 
               
               
                   
                   
                   
                 SpPAMmut. 
                 SpPAMmut. 
                 919)-bpNLS 
               
               
                   
                   
                   
                 5P 
                 5P 
               
               
                 9 
                 Rep2.CBX1. 
                 Rep2.CBX1. 
                 Rep2. 
                 Rep2. 
                 NLS-nCas9- 
                 Yes 
               
               
                   
                 FwdGuide 
                 RevGuide 
                 mGL-CBX1. 
                 mGL-CBX1. 
                 linker- 
               
               
                   
                   
                   
                 TopDonor. 
                 BotDonor. 
                 hLIG1(119- 
               
               
                   
                   
                   
                 SpPAMmut. 
                 SpPAMmut. 
                 919)-bpNLS 
               
               
                   
                   
                   
                 5P 
                 5P 
               
               
                   
               
            
           
         
       
     
     Example 4. Treatment of a Genetic Disease in a Patient 
     A human patient with sickle cell disease comes to a physician for treatment. The patient is identified as having a hemoglobin gene mutation. Hematopoietic stem and progenitor cells are collected from the patient&#39;s peripheral blood. The cells are edited by contacting the cells&#39; genomes with a nCas9-DNA ligase fusion protein, a gRNA, and a donor DNA that includes a corrected hemoglobin gene. The gRNA recruits the fusion protein to the gene mutation, and the nCas9 nicks the patient&#39;s DNA on one side flanking the mutation. The gRNA binds to a genomic flap generated by the nick, and to the donor DNA, and forms an RNA splint for the ligase to ligate the genomic flap to the donor DNA. Another fusion protein nicks the opposite strand of the mutated hemoglobin gene using a second gRNA on the other side of the mutation, and ligates the other side of the donor DNA. The mutated DNA is thus replaced with the donor DNA, and the cell with the donor DNA is transfused back into the patient, thus treating the genetic disease in the patient. 
     Example 5. Enhancing a Crop 
     In a soybean plant, a germ cell is microinjected with an expression vector encoding an nCas9-DNA ligase fusion protein, and with a gRNA and donor DNA encoding an herbicide resistance gene. gRNA recruits the fusion protein to a suitable spot within the soybean genome which doesn&#39;t already include a gene. The nCas9 nicks the soybean&#39;s DNA on one side flanking the spot. The gRNA also recruits the donor DNA to bind to a genomic flap created by the nick, and the ligase seals the nick using the donor DNA itself as a splint. Another fusion protein nicks the opposite strand of the soybean&#39;s DNA on the other side flanking the spot, and ligates the other side of the donor DNA, thus integrating the herbicide resistance gene into the germ cell. The germ cell eventually produces a seed, and the seeds are harvested to grow herbicide resistant soybeans. 
     Example 6. In Vitro 1-Sided Replacer 2 Using T4 Ligase 
     To demonstrate the usefulness of the components and methods described herein for editing nucleic acids, in vitro experiments were performed. The experiments in this example specifically assessed the feasibility of 1-sided Replacer 2. The experiments used a 100 bp, 5′-Cy5-labeled double-stranded DNA (dsDNA) substrate (IDT) that corresponded to the blue fluorescent protein (BFP) target region (see examples 1 and 2), with the site of nicking located in the middle at base pair 50. 5′-phosphorylated dsDNA donors (IDT) containing a variable GBS, 13 nt flap binding site (FBS), and a protospacer adjacent motif (PAM) mutation were used in conjunction with gRNAs (Agilent) containing the corresponding variable DBS. 5′-Cy5-labeled dsDNA substrate and 5′-phosphorylated dsDNA donor were separately annealed using complementary oligonucleotides by heating to 95 C for 5 min followed by slowly cooling to room temperature. 
     In vitro 1-sided Replacer 2 reactions were performed by first incubating gRNA (30 nM final) and dsDNA donor (30 nM final) with recombinant  S. pyogenes  nicking Cas9 (Cas9n; IDT; 30 nM final) for 10 min at room temperature, followed by the addition of T4 ligase (NEB; 200U final), ATP (1 mM final), and 5′-Cy5-labeled dsDNA substrate (3 nM Final). Reactions were carried out in the presence of NEB Buffer 3.1 (lx final) at 37 C for 1 hr (final volume of 10 ul). Reactions were terminated by the addition of 0.5% SDS and 100 ug/ml Proteinase K, and incubated at 37 C for 30 min. Reaction products were then combined with 2× formamide gel loading buffer (90% formamide; 10% glycerol; 0.01% bromophenol blue), denatured at 95° C. for 10 min, and separated by denaturing urea PAGE gel (15% TBE-urea, 55° C., 200 V). DNA products were visualized by Cy5 fluorescence signal using a LI-COR Odyssey CLx imager. 
     In addition to the intact 100 bp 5′-Cy5-labeled dsDNA substrate, a nicked 5′-Cy5-labeled dsDNA substrate and a final ligation product were included as size controls. The nicked 5′-Cy5-labeled dsDNA control was annealed using two 50 mers corresponding to the top strand oligo of the 100 bp 5′-Cy5-labeled dsDNA substrate (a 5′-Cy5-labeled 50 mer and a 5′-phosphorylated 50 mer) and its complementary 100 mer bottom strand oligo. The final ligation product control was annealed and ligated using the 5′-Cy5-labeled 50 mer and the bottom 100 mer from the nicked control along with the 150 nt top strand donor oligo. 
       FIG.  8 A  illustrates an exemplary nicking and ligation pattern of an integrating nucleic acid.  FIG.  8 B  illustrates an exemplary nucleic acid gel showing pattern associated with In Vitro 1-Sided Replacer 2 using 30 nt GBS/DBS and Thermostable T4 Ligase. Using a 30 nt GBS/DBS combination, a donor containing a PAM mutation, and a thermostable T4 ligase (Hi-T4, NEB), we were able to produce a final Replacer product (Lane 3) corresponding to the size of our control product (Lane 1). Replacer products were not detected in the absence of nicking Cas9 (Cas9n) (Lane 2), or in the absence of the bottom donor which serves as the splint (Lanes 4 &amp; 5).  FIG.  8 C  illustrates an exemplary nucleic acid gel showing pattern associated with in vitro 1-Sided Replacer 2 using Variable Length GBS/DBS Combinations and T4 Ligase. Using regular T4 ligase (NEB), we were to produce a final Replacer product corresponding to the size of the control when using multiple GBS/DBS combinations, including No GBS/DBS, 20 nt GBS/DBS, and 30 nt GBS/DBS. 
     Additionally, in this experiment, recoded dsDNA donors containing PAM mutation were more efficient at producing final Replacer products compared to PAM mutant dsDNA donors that were not recoded. The results indicate that a DNA ligase may be used with an RNA-guided endonuclease to edit a target nucleic acid. 
     Example 7. Use of 1-Sided Replacer 2 with Nicking Cas9 and Multiple DNA Ligases in Various Coupling Architectures in Mammalian Cells 
     Components used to edit a blue fluorescent protein (BFP) gene stably integrated into HEK293T cells were co-delivered by lipofectamine 2000 transfection. The components included a chemically synthesized guide RNA (SEQ ID NO: 166, mG*mC*mU*GAAGCACUGCACGCCAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCU AGUCCGUUAUCGACUUGAAAAAGUCGGACCGAGUCGGUCCAGCUGCGGUAUUGUGGmC*mG* mU) with 2′-O-methyl and phosphorothioate chemical modifications on the 5′ and 3′ ends, an integrating nucleic acid with a 5′ phosphate end modification (SEQ ID NO: 167, /5Phos/cgtaTgtcagggtggtcacGAGgg), a splinting nucleic acid with locked nucleic acid and phosphorothioate modifications (SEQ ID NO: 169, +c*c*+CT+CG+TG+AC+CA+CC+CT+GA+CA+TA+CGGCGTGCAgtgcttACGCCA+CA+AT+AC+CG+C A+G*C*+T), and either a single mRNA encoding nicking Cas9 fused to a ligase, or a pair of mRNAs encoding nicking Cas9 and a ligase. 
     The integrating nucleic acid and splinting nucleic acid were synthesized by Integrated DNA Technologies (IDT). All mRNAs corresponding to Cas9n (H840A) and all ligases are generated via in vitro transcription (IVT) reactions using the HiScribe T7 High Yield RNA Synthesis Kit (NEB). Coding sequences are cloned into an IVT vector that contains a single copy of the 5′UTR and two copies of the 3′UTR from the human beta globin gene, in addition to a 152 nt polyA tail. Plasmid DNA containing coding sequences are linearized using an XbaI restriction site located immediately downstream of the polyA tail. Linearized plasmids are then purified via phenol:chloroform extraction followed by ethanol precipitation. mRNAs are produced via IVT reactions that contain N1-Methylpseudouridine-5′-Triphosphate (TriLink BioTech) in place of Uridine-Triphosphate, and capped co-transcriptionally with CleanCap Reagent AG (3′ OMe) (TriLink BioTech). IVT reactions are incubated at 37° C. for 2 hours, followed by DNAse I digestion of the template DNA. Finally, mRNA products are purified using LiCl precipitation, quantified (Qubit Fluorometric Quantification; ThermoFisher), and checked for integrity by denaturing gel electrophoresis. “Ligase in trans” refers to Cas9 H840A nickase combined with T4 ligase fused to leucine zipper on its C terminus (T4-LZ, SEQ ID NO: 145). “LZ; C terminal Ligase” refers to Cas9 H840A nickase fused to a leucine zipper on its C terminus (nCas9-LZ, SEQ ID NO: 133) combined with a ligase fused to a leucine zipper on its N terminus for T4 (LZ-T4, SEQ ID NO: 142), SplintR (LZ-SplintR, SEQ ID NO: 141), or hLIG4(1-620) (LZ-hLIG4(1-620), SEQ ID NO: 146). “LZ; N terminal Ligase” refers to Cas9 H840A nickase fused to a leucine zipper on its N terminus (LZ-nCas9, SEQ ID NO: 147) combined with a ligase fused to a leucine zipper on its C terminus for T4 (T4-LZ, SEQ ID NO: 145), SplintR (SplintR-LZ, SEQ ID NO: 148), or hLIG4(1-620) (hLIG4(1-620)-LZ, SEQ ID NO: 149). “Fusion; C terminal Ligase” refers to Cas9 H840A nickase fused to a ligase with the ligase on the C terminus for T4 (nCas9-T4, SEQ ID NO: 131), SplintR (nCas9-SplintR, SEQ ID NO: 129), or hLIG4(1-620) (nCas9-hLIG4(1-620) SEQ ID NO: 150). “Fusion; N terminal Ligase” refers to Cas9 H840A nickase fused to a ligase with the ligase on the N terminus for T4 (T4-nCas9, SEQ ID NO: 151), SplintR (SplintR-nCas9, SEQ ID NO: 152), or hLIG4(1-620) (hLIG4(1-620)-nCas9, (SEQ ID NO: 153). The gRNA contained a spacer, scaffold, and donor binding site. The splinting integrating nucleic acid contained a guide binding site and a flap binding site. The ligating integrating nucleic acid and splinting nucleic acid were partially complementary. 
     The integrating nucleic acid and splinting nucleic acid were hybridized using an annealing reaction, then mixed with the guide RNA and mRNA and formulated with lipofectamine 2000 in OptiMEM prior to delivery to the adherent HEK293 cells in 96-well plates. After 24-48 hours, the cells were detached with 0.05% Trypsin-EDTA and run through a flow cytometer to measure the percentage of cells expressing green fluorescent protein (GFP), indicating gene editing from BFP to GFP ( FIG.  9   ). Gene editing was observed with T4, SplintR, and hLIG4(1-620) ligases when fused to nCas9, interacting with nCas9 through leucine zippers, or delivered in trans with no leucine zipper interaction. 
     The results here demonstrate the usefulness of using a DNA ligase with an RNA-guided endonuclease to edit a target nucleic acid in a cell. The experiments in this example specifically demonstrated the feasibility of including 1-sided Replacer 2 components to edit a target nucleic acid in a mammalian cell. This example shows the effectiveness of including a DNA ligase coupled through a heterodimerization domain (here, leucine zippers) to an RNA guided endonuclease (e.g. a nicking Cas9) in nucleic acid editing such as gene editing. This also shows nucleic acid editing is possible in mammalian cells with a DNA ligase fused to an RNA guided endonuclease (e.g. T4 ligase fused to Cas9 H840A nickase), and that nucleic acid editing can be achieved by delivering the DNA ligase and RNA guided endonuclease as separate non-coupled components. 
     Example 8. Use of 1-Sided Replacer 2 with Nicking Cas9 and T4 DNA Ligase to Make a Variety of Edits at Multiple Genomic Targets 
     Components used to edit genomic targets in HEK293T cells were co-delivered by lipofectamine 2000 transfection. The components included a chemically synthesized guide with 2′-O-methyl and phosphorothioate chemical modifications on the 5′ and 3′ ends, an integrating nucleic acid with a 5′ phosphate end modification, a splinting nucleic acid with locked nucleic acid and phosphorothioate modifications, an mRNA encoding nicking Cas9 (LZ-nCas9, SEQ ID NO: 147), and an mRNA encoding a ligase (T4-LZ, SEQ ID NO: 145). Target-specific guides, splinting and integrating nucleic acids are listed in Table 15. The integrating nucleic acid and splinting nucleic acid were synthesized by Integrated DNA Technologies (IDT) and both mRNAs were generated via in vitro transcription reactions using the methods described in Example 7. The gRNA contained a spacer, scaffold, and donor binding site. The splinting integrating nucleic acid contained a guide binding site and a flap binding site. The ligating integrating nucleic acid and splinting nucleic acid were partially complementary. The integrating nucleic acid and splinting nucleic acid were hybridized using an annealing reaction, then mixed with the guide RNA and mRNA and formulated with lipofectamine 2000 in OptiMEM prior to delivery to the adherent HEK293 cells in 96-well plates. After 24-48 hours, genomic DNA was extracted from the cells using QuickExtract and genomic targets were amplified using Q5 DNA Polymerase. The PCR program ran at 98 C for 30 seconds, then 35 cycles of 98 C for 5 seconds, 67 C for 20 seconds, and 72 C for 20 seconds, then finally 72 C for 2 minutes. PCR primers are listed in Table 15. PCR products were cleaned up with ExoCIP treatment and submitted for next generation sequencing (NGS) by Azenta using their Amplicon-EZ service. Sequencing reads were merged and aligned to the amplicon of interest, and the percentage total reads that matched the intended edit was calculated ( FIG.  10   ). This example shows the effectiveness of gene editing with 1-sided Replacer 2 in mammalian cells at a variety of genomic targets. The types of edits here include making a single point mutation (HEK3 F+5 G to T), a pair of point mutations (VEGFA R+5 G to T and +2 A to T, VEGFA F+5 G to T and +2 G to C, and AAVS1 R+5 G to T), or a trinucleotide insertion (HEK3 F CAC insertion and AAVS1 R CAC insertion) using 1-sided Replacer 2. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 15 
               
               
                   
               
               
                 Condi- 
                   
                   
                   
                 PCR  
               
               
                 tion 
                 Guide 
                 Splint 
                 Donor 
                 Primers 
               
               
                   
               
             
            
               
                 VEGFA  
                 SEQ ID  
                 SEQ ID 
                 SEQ ID  
                 SEQ ID  
               
               
                 R +5 
                 NO: 170 
                 NO: 174 
                 NO: 180 
                 NO: 186 
               
               
                 G to T 
                 mC*mA*mC* 
                 +C*C*+TT+ 
                 /5Phos/ 
                 ACACTCTTT 
               
               
                 and +2 
                 CCCGGCUC 
                 TC+CA+AA 
                 ATGATG 
                 CCCTACAC 
               
               
                 A to T 
                 UGGCUAAA 
                 +GC+CC+A 
                 GAATGGG 
                 GACGCTCTT 
               
               
                   
                 GGUUUUAG 
                 T+TC+CA+T 
                 CTTT 
                 CCGATCTT 
               
               
                   
                 AGCUAGAA 
                 C+ATtagccag 
                 GGAAAGG 
                 GCCGCTCAC 
               
               
                   
                 AUAGCAAG 
                 agccggACGC 
                   
                 TTTGATGT 
               
               
                   
                 UUAAAAUA 
                 CA+CA+AT 
                   
                 CT; 
               
               
                   
                 AGGCUAGU 
                 +AC+CG+C 
                   
                 SEQ ID 
               
               
                   
                 CCGUUAUC 
                 A+G*C*+T 
                   
                 NO: 187 
               
               
                   
                 GACUUGAA 
                   
                   
                 GACTGGAGT 
               
               
                   
                 AAAGUCGG 
                   
                   
                 TCAGACG 
               
               
                   
                 ACCGAGUC 
                   
                   
                 TGTGCTCTT 
               
               
                   
                 GGUCCAGC 
                   
                   
                 CCGATCTG 
               
               
                   
                 UGCGGUAU 
                   
                   
                 GGGAGAGGG 
               
               
                   
                 UGUGGmC* 
                   
                   
                 ACACACA 
               
               
                   
                 mG*mU 
                   
                   
                 GA 
               
               
                   
               
               
                 VEGFA  
                 SEQ ID  
                 SEQ ID 
                 SEQ ID  
                 SEQ ID  
               
               
                 F +5  
                 NO: 171 
                 NO: 175 
                 NO: 181 
                 NO: 223 
               
               
                 G to T 
                 mG*mA*mU* 
                 +A*C*+AA+ 
                 /5Phos/ 
                 ACACTCTTT 
               
               
                 and +2 
                 GUCUGCAG 
                 TG+TG+CC 
                 TCAGT 
                 CCCTACAC 
               
               
                 G to C 
                 GCCAGAUG 
                 +AT+CT+G 
                 GCTCCA 
                 GACGCTCTT 
               
               
                   
                 AGUUUUAG 
                 G+AG+CA+ 
                 GATGGC 
                 CCGATCTT 
               
               
                   
                 AGCUAGAA 
                 CT+GAtctgg 
                 ACATTGT 
                 GCCGCTCA 
               
               
                   
                 AUAGCAAG 
                 cctgcagaTC 
                   
                 CTTTGATGT 
               
               
                   
                 UUAAAAUA 
                 ATGC+AG+ 
                   
                 CT; 
               
               
                   
                 AGGCUAGU 
                 CC+CG+GA 
                   
                 SEQ ID  
               
               
                   
                 CCGUUAUC 
                 +AC+C*A*+ 
                   
                 NO: 224 
               
               
                   
                 GACUUGAA 
                 C 
                   
                 GACTGGAGT 
               
               
                   
                 AAAGUCGG 
                   
                   
                 TCAGACG 
               
               
                   
                 ACCGAGUC 
                   
                   
                 TGTGCTCTT 
               
               
                   
                 GGUCCGUG 
                   
                   
                 CCGATCTG 
               
               
                   
                 GUUCCGG 
                   
                   
                 GGGAGAGGG 
               
               
                   
                 GCUGCAmU* 
                   
                   
                 ACACACA 
               
               
                   
                 mG*mA 
                   
                   
                 GA 
               
               
                   
               
               
                 HEK3  
                 SEQ ID  
                 SEQ ID 
                 SEQ ID  
                 SEQ ID  
               
               
                 F CAC 
                 NO: 172 
                 NO: 176 
                 NO: 182 
                 NO: 188 
               
               
                 inser- 
                 mG*mG*mC* 
                 +G*C*+TT+ 
                 /5Phos/ 
                 ACACTCTTT 
               
               
                 tion 
                 ccagacuga 
                 CC+TT+TC+ 
                 gtgTGAT 
                 CCCTACAC 
               
               
                   
                 gcacgugaG 
                 CT+CT+GC+ 
                 GGCAGAG 
                 GACGCTCTT 
               
               
                   
                 UUUUAGAGC 
                 CA+TC+Ac+ 
                 GAA 
                 CCGATCT 
               
               
                   
                 UAGAAAUAG 
                 accgtgctcag 
                 AGGAAGC 
                 ccctggcctg 
               
               
                   
                 CAAGUUAAA 
                 tctgTCATGC 
                   
                 ggtcaatcc; 
               
               
                   
                 AUAAGGCUA 
                 +AG+CC+CG 
                   
                 SEQ ID 
               
               
                   
                 GUCCGUUA 
                 +GA+AC+C* 
                   
                 NO: 189 
               
               
                   
                 UCGACUUGA 
                 A*+C 
                   
                 GACTGGAGTT 
               
               
                   
                 AAAAGUC 
                   
                   
                 CAGACG 
               
               
                   
                 GGACCGAGU 
                   
                   
                 TGTGCTCTT 
               
               
                   
                 CGGUCCG 
                   
                   
                 CCGATCTG 
               
               
                   
                 UGGUUCCGG 
                   
                   
                 tgaagggcca 
               
               
                   
                 GCUGCAm 
                   
                   
                 ggtccctc 
               
               
                   
                 U*mG*mA 
                   
                   
                   
               
               
                   
               
               
                 HEK3   
                 SEQ ID  
                 SEQ ID 
                 SEQ ID  
                 SEQ ID  
               
               
                 F +5 
                 NO: 221 
                 NO: 177 
                 NO: 183 
                 NO: 225 
               
               
                 G to T 
                 mG*mG*mC* 
                 +A*G*+GG+ 
                 /5Phos/ 
                 ACACTCTTT 
               
               
                   
                 ccagacuga 
                 CT+TC+CT+ 
                 TGATTG 
                 CCCTACAC 
               
               
                   
                 gcacgugaG 
                 TT+CC+TC+ 
                 CAGAGGA 
                 GACGCTCTT 
               
               
                   
                 UUUUAGAGC 
                 TG+CA+AT+ 
                 AAGGA 
                 CCGATCT 
               
               
                   
                 UAGAAAUAG 
                 CAcgtgctca 
                 AGCCCT 
                 ccctggcctg 
               
               
                   
                 CAAGUUAAA 
                 gtctgTCATG 
                   
                 ggtcaatcc; 
               
               
                   
                 AUAAGGCUA 
                 C+AG+CC+C 
                   
                 SEQ ID 
               
               
                   
                 GUCCGUUA 
                 G+GA+AC+ 
                   
                 NO: 226 
               
               
                   
                 UCGACUUGA 
                 C*A*+C 
                   
                 GACTGGAGTT 
               
               
                   
                 AAAAGUC 
                   
                   
                 CAGACG 
               
               
                   
                 GGACCGAGU 
                   
                   
                 TGTGCTCTTC 
               
               
                   
                 CGGUCCG 
                   
                   
                 CGATCTG 
               
               
                   
                 UGGUUCCGG 
                   
                   
                 tgaagggcca 
               
               
                   
                 GCUGCAm 
                   
                   
                 ggtccctc 
               
               
                   
                 U*mG*mA 
                   
                   
                   
               
               
                   
               
               
                 AAVS1  
                 SEQ ID  
                 SEQ ID 
                 SEQ ID  
                 SEQ ID  
               
               
                 R CAC  
                 NO: 173 
                 NO: 178 
                 NO: 184 
                 NO: 190 
               
               
                 inser- 
                 mG*mC*mG* 
                 +A*T*+TA+ 
                 /5Phos/ 
                 ACACTCTTT 
               
               
                 tion 
                 acuccugga 
                 GC+AG+AA 
                 gtgCCA 
                 CCCTACAC 
               
               
                   
                 aguggccaG 
                 +GT+GG+C 
                 AGGGCC 
                 GACGCTCTT 
               
               
                   
                 UUUUAGAGC 
                 C+CT+TG+ 
                 ACTTCT 
                 CCGATCT 
               
               
                   
                 UAGAAAUAG 
                 Gc+acccactt 
                 GCTAAT 
                 CGCCGGGAA 
               
               
                   
                 CAAGUUAAA 
                 ccaggACGC 
                   
                 CTGCCG 
               
               
                   
                 AUAAGGCUA 
                 CA+CA+AT 
                   
                 CTGGC; 
               
               
                   
                 GUCCGUUA 
                 +AC+CG+C 
                   
                 SEQ ID  
               
               
                   
                 UCGACUUGA 
                 A+G*C*+T 
                   
                 NO: 191 
               
               
                   
                 AAAAGUC 
                   
                   
                 GACTGGAGTT 
               
               
                   
                 GGACCGAGU 
                   
                   
                 CAGACG 
               
               
                   
                 CGGUCCA 
                   
                   
                 TGTGCTCTTC 
               
               
                   
                 GCUGCGGUA 
                   
                   
                 CGATCT 
               
               
                   
                 UUGUGG 
                   
                   
                 GAGGAGGCCC 
               
               
                   
                 mC*mG*mU 
                   
                   
                 TCATCT 
               
               
                   
                   
                   
                   
                 GGCG 
               
               
                   
               
               
                 AAVS1 
                 SEQ ID  
                 SEQ ID 
                 SEQ ID  
                 SEQ ID  
               
               
                 R +5 
                 NO: 222 
                 NO: 179 
                 NO: 185 
                 NO: 227 
               
               
                 G to T 
                 mG*mC*mG* 
                 +T*C*+CA+ 
                 /5Phos/ 
                 ACACTCTTT 
               
               
                   
                 acuccugga 
                 TT+AG+CA 
                 CCAAT 
                 CCCTACAC 
               
               
                   
                 aguggccaG 
                 +GA+AG+T 
                 GGCCACT 
                 GACGCTCTT 
               
               
                   
                 UUUUAGAGC 
                 G+GC+CA+ 
                 TCTGCT 
                 CCGATCT 
               
               
                   
                 UAGAAAUAG 
                 TT+GGccac 
                 AATGGA 
                 CGCCGGGAA 
               
               
                   
                 CAAGUUAAA 
                 ttccaggACG 
                   
                 CTGCCG 
               
               
                   
                 AUAAGGCUA 
                 CCA+CA+A 
                   
                 CTGGC; 
               
               
                   
                 GUCCGUUA 
                 T+AC+CG+ 
                   
                 SEQ ID  
               
               
                   
                 UCGACUUGA 
                 CA+G*C*+T 
                   
                 NO: 228 
               
               
                   
                 AAAAGUC 
                   
                   
                 GACTGGAGT 
               
               
                   
                 GGACCGAGU 
                   
                   
                 TCAGACG 
               
               
                   
                 CGGUCCA 
                   
                   
                 TGTGCTCTT 
               
               
                   
                 GCUGCGGUA 
                   
                   
                 CCGATCT 
               
               
                   
                 UUGUGG 
                   
                   
                 GAGGAGGCC 
               
               
                   
                 mC*mG*mU 
                   
                   
                 CTCATCT 
               
               
                   
                   
                   
                   
                 GGCG 
               
               
                   
               
            
           
         
       
     
     Example 9. Use of 2-Sided Replacer 2 with Nicking Cas9 and T4 DNA Ligase to Make Deletions and Sequence Replacements at Multiple Genomic Targets 
     Components used to edit genomic targets in HEK293T cells were co-delivered by lipofectamine 2000 transfection. The components included two chemically synthesized guides with 2′-O-methyl and phosphorothioate chemical modifications on the 5′ and 3′ ends, two integrating nucleic acids with a 5′ phosphate end modification, two splinting nucleic acids with locked nucleic acid and phosphorothioate modifications, an mRNA encoding nicking Cas9 (LZ-nCas9, SEQ ID NO: 147), and an mRNA encoding a ligase (T4-LZ, SEQ ID NO: 145). For both “VEGFA replacement of 175 nt with attB” and “VEGFA 175 nt deletion”, the two guide RNAs used were VEGFA_R (SEQ ID NO: 170) and VEGFA F (SEQ ID NO: 171). For both “AAVS1 replacement of 117 nt with attB” and “AAVS1 117 nt deletion”, the two guide RNAs used were AAVS1_R (SEQ ID NO: 173) and AAVS1_F (SEQ ID NO: 192, mG*mC*mU*ggccccccaccgccccaGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG UUAUCGACUUGAAAAAGUCGGACCGAGUCGGUCCGUGGUUCCGGGCUGCAmU*mG*mA). For “VEGFA replacement of 175 nt with attB”, the splinting nucleic acids used were SEQ ID NO: 193 (+g*g*+ag+ac+cg+cc+gt+cg+tc+ga+ca+ag+cctctggcctgcagaTCATGC+AG+CC+CG+GA+AC+C*A*+C) and SEQ ID NO: 194 (+g*g*+cg+gt+ct+cc+gt+cg+tc+ag+ga+tc+attagccagagccggACGCCA+CA+AT+AC+CG+CA+G*C*+T), and the integrating nucleic acids used were SEQ ID NO: 195 (/5Phos/ggcttgtcgacgacggcggtctcc) and SEQ ID NO: 196 (/5Phos/atgatcctgacgacggagaccgcc). For “VEGFA 175 nt deletion”, the splinting nucleic acids used were SEQ ID NO: 197 (+C* C*+GT+CT+GC+AC+AC+CC+CG+GC+TC+TG+GC+TAtctggcctgcagaTCATGC+AG+CC+CG+GA+AC+C*A*+C) and SEQ ID NO: 198 (+G*C*+TC+AC+TT+TG+AT+GT+CT+GC+AG+GC+CA+GAtagccagagccggACGCCA+CA+AT+AC+CG +CA+G*C*+T), and the integrating nucleic acids used were SEQ ID NO: 199 (/5Phos/TAGCCAGAGCCGGGGTGTGCAGACGG) and SEQ ID NO: 200 (/5Phos/TCTGGCCTGCAGACATCAAAGTGAGC). For “AAVS1 replacement of 117 nt with attB”, the splinting nucleic acids used were SEQ ID NO: 201 (+g*g*+ag+ac+cg+cc+gt+cg+tc+ga+ca+ag+ccggcggtgggTCATGC+AG+CC+CG+GA+AC+C*A*+C) and SEQ ID NO: 202 (+g*g*+cg+gt+ct+cc+gt+cg+tc+ag+ga+tc+atccacttccaggACGCCA+CA+AT+AC+CG+CA+G*C*+T), and the integrating nucleic acids used were SEQ ID NO: 195 and SEQ ID NO: 196. For “AAVS1 117 nt deletion”, the splinting nucleic acids used were SEQ ID NO: 203 (+C*G*+GG+GC+AC+AG+CG+AC+TC+CT+GG+AA+GT+GGggcggtgggTCATGC+AG+CC+CG+GA+A C+C*A*+C) and SEQ ID NO: 204 (+G* G*+AA+CT+GC+CG+CT+GG+CC+CC+CC+AC+CG+CCccacttccaggACGCCA+CA+AT+AC+CG+CA+G*C*+T), and the integrating nucleic acids used were SEQ ID NO: 205 (/5Phos/CCACTTCCAGGAGTCGCTGTGCCCCG) and SEQ ID NO: 206 (/5Phos/GGCGGTGGGGGGCCAGCGGCAGTTCC). The integrating nucleic acid and splinting nucleic acid were synthesized by Integrated DNA Technologies (IDT) and both mRNAs were generated via in vitro transcription reactions using the methods described in Example 7. The gRNA contained a spacer, scaffold, and donor binding site. The splinting integrating nucleic acid contained a guide binding site and a flap binding site. There were two pairs of ligating integrating nucleic acid and splinting nucleic acid, and each pair was partially complementary to each other. The integrating nucleic acid and splinting nucleic acid were hybridized using an annealing reaction, then mixed with the guide RNA and mRNA and formulated with lipofectamine 2000 in OptiMEM prior to delivery to the adherent HEK293 cells in 96-well plates. After 24-48 hours, genomic DNA was extracted from the cells using QuickExtract and genomic targets were amplified using Q5 DNA Polymerase. The PCR program ran at 98 C for 30 seconds, then 35 cycles of 98 C for 5 seconds, 67 C for 20 seconds, and 72 C for 20 seconds, then finally 72 C for 2 minutes. PCR primers used for both “VEGFA replacement of 175 nt with attB” and “VEGFA 175 nt deletion” are SEQ ID NO: 186 and SEQ ID NO: 187. PCR primers used for both “AAVS1 replacement of 117 nt with attB” and “AAVS1 117 nt deletion” are SEQ ID NO: 190 and SEQ ID NO: 191. PCR products were cleaned up with ExoCIP treatment and submitted for next generation sequencing (NGS). Sequencing reads were merged and aligned to the amplicon of interest, and the percentage total reads that matched the intended edit was calculated ( FIG.  11   ). This example shows that when Replacer 2 is delivered as 2 full sets of guide RNA, splint, and donor, it can delete an entire region of DNA between the nicking sites on each guide RNA, and optionally replace that region of DNA with a new DNA sequence. Since Replacer is making two separate flaps that can hybridize to each other here, this gene editing mechanism would not rely on the MMR pathway. After an attB sequence is inserted into a targeted site in the genome by Replacer, an entire synthetic gene could be inserted at that attB site if it is delivered with a Bxb1 integrase. Thus, the attB sequence replacement described here could be used for targeted insertion of large 1 kb+ DNA fragments into the genome without double strand break or mismatch repair mediated gene editing. 
     Example 10. Use of 1-Sided Replacer 2 with Nicking Cas9 and T4 DNA Ligase to Integrate Methylated DNA into a Genomic Target 
     Components used to edit genomic targets in HEK293T cells were co-delivered by lipofectamine 2000 transfection. The components included a chemically synthesized guide with 2′-O-methyl and phosphorothioate chemical modifications on the 5′ and 3′ ends (SEQ ID NO: 166), an integrating nucleic acid, a splinting nucleic acid, an mRNA encoding nicking Cas9 (LZ-nCas9, SEQ ID NO: 147), and an mRNA encoding a ligase (T4-LZ, SEQ ID NO: 145). Conditions with the “non-methylated donor” used an integrating nucleic acid with a 5′ phosphate end modification (SEQ ID NO: 207, /5Phos/CGTATGTCAGGGTGGTCACG). Conditions with the “donor with all cytosines methylated” used an integrating nucleic acid with a 5′ phosphate end modification and methylated cytosines (SEQ ID NO: 207, /5Phos//5Me-dC/gtaTgt/iMe-dC/agggtggt/iMe-dC/a/iMe-dC/G). Conditions under “Splint is LNA” used a splinting nucleic acid with locked nucleic acid and phosphorothioate modifications (SEQ ID NO: 208, +C*g*+tg+ac+ca+cc+ct+ga+cA+TA+CGGCGTGCAgtgcttACGCCA+CA+AT+AC+CG+CA+G*C*+T). Conditions under “Splint is OMe” used a splinting nucleic acid with locked nucleic acid, 2′-O-methyl, and phosphorothioate modifications (SEQ ID NO: 209, mC*g*mUgmacmcamccmctmgamcAmUAmCGGCGTGCAgtgcttACGCCA+CA+AT+AC+CG+CA+G*C*+T). The integrating nucleic acid and splinting nucleic acid were synthesized by Integrated DNA Technologies (IDT) and both mRNAs were generated via in vitro transcription reactions using the methods described in Example 7. The gRNA contained a spacer, scaffold, and donor binding site. The splinting integrating nucleic acid contained a guide binding site and a flap binding site. The ligating integrating nucleic acid and splinting nucleic acid were partially complementary. The integrating nucleic acid and splinting nucleic acid were hybridized using an annealing reaction, then mixed with the guide RNA and mRNA and formulated with lipofectamine 2000 in OptiMEM prior to delivery to the adherent HEK293 cells in 96-well plates. After 24-48 hours, the cells were detached with 0.05% Trypsin-EDTA and run through a flow cytometer to measure the percentage of cells expressing green fluorescent protein (GFP), indicating gene editing from BFP to GFP ( FIG.  12   ). This example shows that methylated DNA can be used in the integrating nucleic acid and does not negatively impact editing efficiency under ideal conditions, when the splint has LNA bases. When the splint has OMe bases instead of LNAs and thus lower affinity to the donor, methylated DNA in the donor boosts efficiency, showing that DNA methylation can improve the system by stabilizing the nucleic acid components. A methylated donor could also be used to specifically introduce DNA methylation into the genome at functional epigenetic sites such as promoters to regulate gene expression. A follow-up experiment could be conducted by performing bisulfate sequencing on the genomic region that Replacer is introducing methylated DNA into to confirm that epigenetic editing has occurred. If Replacer successfully introduces DNA methylation into this genomic region and it is believed that the region&#39;s methylation state controls gene expression, quantitative PCR could be conducted to confirm that a gene of interest has reduced mRNA expression after editing. 
     While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby. 
     While the foregoing disclosure has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the disclosure. For example, all the techniques and apparatus described above can be used in various combinations. All publications, patents, patent applications, and/or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and/or other document were individually and separately indicated to be incorporated by reference for all purposes.