Patent Publication Number: US-11661590-B2

Title: Programmable CAS9-recombinase fusion proteins and uses thereof

Description:
RELATED APPLICATIONS 
     This application is a national stage filing under 35 U.S.C. § 371 of international PCT application, PCT/US2017/046144, filed Aug. 9, 2017, which claims priority under 35 U.S.C. § 119(e) to U.S. provisional patent application Ser. No. 62/372,755, filed Aug. 9, 2016, and U.S. provisional patent application Ser. No. 62/456,048, filed Feb. 7, 2017, each of which is incorporated herein by reference. 
    
    
     GOVERNMENT FUNDING 
     This invention was made with government support under EB022376 and GM118062 awarded by National Institutes of Health (NIH). The government has certain rights in this invention. 
    
    
     BACKGROUND OF THE INVENTION 
     Efficient, programmable, and site-specific homologous recombination remains a longstanding goal of genetics and genome editing. Early attempts at directing recombination to loci of interest relied on the transfection of donor DNA with long flanking sequences that are homologous to a target locus. This strategy was hampered by very low efficiency and thus the need for a stringent selection to identify integrants. More recent efforts have exploited the ability of double-stranded DNA breaks (DSBs) to induce homology-directed repair (HDR). Homing endonucleases and later programmable endonucleases such as zinc finger nucleases, TALE nucleases, Cas9, and fCas9 have been used to introduce targeted DSBs and induce HDR in the presence of donor DNA. In most post-mitotic cells, however, DSB-induced HDR is strongly down regulated and generally inefficient. Moreover, repair of DSBs by error-prone repair pathways such as non-homologous end-joining (NHEJ) or single-strand annealing (SSA) causes random insertions or deletions (indels) of nucleotides at the DSB site at a higher frequency than HDR. The efficiency of HDR can be increased if cells are subjected to conditions forcing cell-cycle synchronization or if the enzymes involved in NHEJ are inhibited. However, such conditions can cause many random and unpredictable events, limiting potential applications. The instant disclosure provides a fusion protein that can recombine DNA sites containing a minimal recombinase core site flanked by guide RNA-specified sequences and represents a step toward programmable, scarless genome editing in unmodified cells that is independent of endogenous cellular machinery or cell state. 
     SUMMARY OF THE INVENTION 
     The instant disclosure describes the development of a fusion protein comprising a guide nucleotide sequence-programmable DNA binding protein domain, an optional linker, and a recombinase catalytic domain (e.g., a serine recombinase catalytic domain such as a Gin recombinase catalytic domain, a tyrosine recombinase catalytic domain, or any evolved recombinase catalytic domain). This fusion protein operates on a minimal gix core recombinase site (NNNNAAASSWWSSTTTNNNN, SEQ ID NO: 19) flanked by two guide RNA-specified DNA sequences. Recombination mediated by the described fusion protein is dependent on both guide RNAs, resulting in orthogonality among different guide nucleotide:fusion protein complexes, and functions efficiently in cultured human cells on DNA sequences matching those found in the human genome. The fusion protein of the disclosure can also operate directly on the genome of human cells (e.g., cultured human cells), catalyzing a deletion, insertion, inversion, translocation, or recombination between two recCas9 psuedosites located approximately 14 kilobases apart. This work provides engineered enzymes that can catalyze gene insertion, deletion, inversion, or chromosomal translocation with user-defined, single base-pair resolution in unmodified genomes. 
     In one aspect, the instant disclosure provides a fusion protein comprising: (i) a guide nucleotide sequence-programmable DNA binding protein domain; (ii) an optional linker; and (iii) a recombinase catalytic domain such as any serine recombinase catalytic domain (including but not limited to a Gin, Sin, Tn3, Hin, β, γδ, or PhiC31 recombinase catalytic domain), any tyrosine recombinase domain (including, but not limited to a Cre or FLP recombinase catalytic domain), or any evolved recombinase catalytic domain. 
     The guide nucleotide sequence-programmable DNA binding protein domain may be selected from the group consisting of nuclease inactive Cas9 (dCas9) domains, nuclease inactive Cpf1 domains, nuclease inactive Argonaute domains, and variants thereof. In certain embodiments, the guide nucleotide sequence-programmable DNA-binding protein domain is a nuclease inactive Cas9 (dCas9) domain. In certain embodiments, the amino acid sequence of the dCas9 domain comprises mutations corresponding to a D10A and/or H840A mutation in SEQ ID NO: 1. In another embodiment, the amino acid sequence of the dCas9 domain comprises a mutation corresponding to a D10A mutation in SEQ ID NO: 1 and a mutation corresponding to an H840A mutation in SEQ ID NO: 1. In another embodiment, the amino acid sequence of the dCas9 domain further does not include the N-terminal methionine shown in SEQ ID NO: 1. In a certain embodiment, the amino acid sequence of the dCas9 domain comprises SEQ ID NO: 712. In one embodiment, the amino acid sequence of the dCas9 domain has a greater than 95% sequence identity with SEQ ID NO: 712. In one embodiment, the amino acid sequence of the dCas9 domain has a greater than 96, 97, 98, 99% or greater sequence identity with SEQ ID NO: 712. In some embodiments, the recombinase catalytic domain is a serine recombinase catalytic domain or a tyrosine recombinase catalytic domain. 
     In one embodiment, the amino acid sequence of the recombinase catalytic domain is a Gin recombinase catalytic domain. In some embodiments, the Gin recombinase catalytic domain comprises a mutation corresponding to one or more of the mutations selected from: a H106Y, I127L, I136R and/or G137F mutation in SEQ ID NO: 713. In an embodiment, the amino acid sequence of the Gin recombinase catalytic domain comprises mutations corresponding to two or more of the mutations selected from: a I127L, I136R and/or G137F mutation in SEQ ID NO: 713. In an embodiment, the amino acid sequence of the Gin recombinase catalytic domain comprises mutations corresponding to a I127L, I136R and G137F mutation in SEQ ID NO: 713. In another embodiment, the amino acid sequence of the Gin recombinase has been further mutated. In a specific embodiment, the amino acid sequence of the Gin recombinase catalytic domain comprises SEQ ID NO: 713. 
     In another embodiment, the amino acid sequence of the recombinase catalytic domain is a Hin recombinase, β recombinase, Sin recombinase, Tn3 recombinase, γδ recombinase, Cre recombinase; FLP recombinase; or a phiC31 recombinase catalytic domain. 
     In one embodiment, the amino acid sequence of the Cre recombinase is truncated. In another embodiment, the tyrosine recombinase catalytic domain is the 25 kDa carboxy-terminal domain of the Cre recombinase. In another embodiment, the Cre recombinase begins with amino acid R118, A127, E138, or R154 (preceded in each case by methionine). In one embodiment, the amino acid sequence of the recombinase has been further mutated. In certain embodiments, the recombinase catalytic domain is an evolved recombinase catalytic domain. In some embodiments, the amino acid sequence of the recombinase has been further mutated. 
     In some embodiments, the linker (e.g., the first, second, or third linker) may have a length of about 0 angstroms to about 81 angstroms. The linker typically has a length of about 33 angstroms to about 81 angstroms. The linker may be peptidic, non-peptidic, or a combination of both types of linkers. In certain embodiments, the linker is a peptide linker. In certain embodiments, the peptide linker comprises an XTEN linker SGSETPGTSESATPES (SEQ ID NO: 7), SGSETPGTSESA (SEQ ID NO: 8), or SGSETPGTSESATPEGGSGGS (SEQ ID NO: 9), an amino acid sequence comprising one or more repeats of the tri-peptide GGS, or any of the following amino acid sequences: VPFLLEPDNINGKTC (SEQ ID NO: 10), GSAGSAAGSGEF (SEQ ID NO: 11), SIVAQLSRPDPA (SEQ ID NO: 12), MKIIEQLPSA (SEQ ID NO: 13), VRHKLKRVGS (SEQ ID NO: 14), GHGTGSTGSGSS (SEQ ID NO: 15), MSRPDPA (SEQ ID NO: 16), or GGSM (SEQ ID NO: 17). In another embodiment, the peptide linker comprises one or more repeats of the tri-peptide GGS. In one embodiment, the peptide linker comprises from one to five repeats of the tri-peptide GGS. In another embodiment, the peptide linker comprises from six to ten repeats of the tri-peptide GGS. In a specific embodiment, the peptide linker comprises eight repeats of the tri-peptide GGS. In another embodiment, the peptide linker is from 18 to 27 amino acids long. In certain embodiments, the peptide linker is 24 amino acids long. In certain embodiments, the peptide linker has the amino acid sequence GGSGGSGGSGGSGGSGGSGGSGGS (SEQ ID NO: 183). 
     In certain embodiments, the linker is a non-peptide linker. In certain embodiments, the non-peptide linker comprises polyethylene glycol (PEG), polypropylene glycol (PPG), co-poly(ethylene/propylene) glycol, polyoxyethylene (POE), polyurethane, polyphosphazene, polysaccharides, dextran, polyvinyl alcohol, polyvinylpyrrolidones, polyvinyl ethyl ether, polyacryl amide, polyacrylate, polycyanoacrylates, lipid polymers, chitins, hyaluronic acid, heparin, or an alkyl linker. In certain embodiments, the alkyl linker has the formula: —NH—(CH 2 ) s —C(O)—, wherein s is any integer between 1 and 100, inclusive. In certain embodiments, s is any integer from 1-20, inclusive. 
     In another embodiment, the fusion protein further comprises a nuclear localization signal (NLS) domain. In certain embodiments, the NLS domain is bound to the guide nucleotide sequence-programmable DNA binding protein domain or the recombinase catalytic domain via one or more second linkers. 
     In one embodiment, the fusion protein comprises the structure NH 2 -[recombinase catalytic domain]-[optional linker sequence]-[guide nucleotide sequence-programmable DNA binding protein domainHoptional, second linker sequence]-[NLS domain]-COOH. In certain embodiments, the fusion protein has greater than 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 719. In a specific embodiment, the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 719. In one embodiment, the fusion protein consists of the amino acid sequence shown in SEQ ID NO: 719. 
     In another embodiment, the fusion protein further comprises one or more affinity tags. In one embodiment, the affinity tag is selected from the group consisting of a FLAG tag, a polyhistidine (poly-His) tag, a polyarginine (poly-Arg) tag, a Myc tag, and an HA tag. In an embodiment, the affinity tag is a FLAG tag. In a specific embodiment, the FLAG tag has the sequence PKKKRKV (SEQ ID NO: 702). In another embodiment, the one or more affinity tags are bound to the guide nucleotide sequence-programmable DNA binding protein domain, the recombinase catalytic domain, or the NLS domain via one or more third linkers. In certain embodiments, the third linker is a peptide linker. 
     The elements of the fusion protein described herein may be in any order, without limitation. In some embodiments, the fusion protein has the structure NH 2 -[recombinase catalytic domain]-[linker sequence]-[guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[optional affinity tag]-COOH, NH 2 -[guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[optional affinity tag]-COOH, or NH 2 —[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[optional affinity tag]-COOH. 
     In some embodiments, the fusion protein has the structure NH 2 -[optional affinity tag]-[optional linker sequence]-[recombinase catalytic domain]-[linker sequence]-[guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[NLS domain]-COOH, NH 2 -[optional affinity tag]-[optional linker sequence]-[guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[NLS domain]-COOH, or NH 2 -[optional affinity tag]-[optional linker sequence]-[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[NLS domain]-COOH. 
     In a certain embodiment, the fusion protein has greater than 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 185. In a specific embodiment, the fusion protein has the amino acid sequence shown in SEQ ID NO: 185. In certain embodiments, the recombinase catalytic domain of the fusion protein has greater than 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence shown in amino acids 1-142 of SEQ ID NO: 185, which is identical to the sequence shown in SEQ ID NO: 713. In certain embodiments, the dCas9 domain has greater than 90%, 95%, or 99% sequence identity with the amino acid sequence shown in amino acids 167-1533 of SEQ ID NO: 185, which is identical to the sequence shown in SEQ ID NO: 712. In certain embodiments, the fusion protein of the instant disclosure has greater than 90%, 95%, or 99% sequence identity with the amino acid sequence shown in amino acids 1-1544 of SEQ ID NO: 185, which is identical to the sequence shown in SEQ ID NO: 719. In one embodiment, the fusion protein is bound to a guide RNA (gRNA). 
     In one aspect, the instant disclosure provides a dimer of the fusion protein described herein. In certain embodiments, the dimer is bound to a target DNA molecule. In certain embodiments, each fusion protein of the dimer is bound to the same strand of the target DNA molecule. In certain embodiments, each fusion protein of the dimer is bound to an opposite strand of the target DNA molecule. In certain embodiments, the gRNAs of the dimer hybridize to gRNA binding sites flanking a recombinase site of the target DNA molecule. In certain embodiments, the recombinase site comprises a res, gix, hix, six, resH, LoxP, FTR, or att core, or related core sequence. In certain embodiments, the recombinase site comprises a gix core or gix-related core sequence. In further embodiments, the distance between the gix core or gix-related core sequence and at least one gRNA binding site is from 3 to 7 base pairs. In certain embodiments, the distance between the gix core or gix-related core sequence and at least one gRNA binding site is from 5 to 6 base pairs. 
     In certain embodiments, a first dimer binds to a second dimer thereby forming a tetramer of the fusion protein. In one aspect, the instant disclosure provides a tetramer of the fusion protein described herein. In certain embodiments, the tetramer is bound to a target DNA molecule. In certain embodiments, each dimer is bound to an opposite strand of DNA. In other embodiments, each dimer is bound to the same strand of DNA. 
     In another aspect, the instant disclosure provides methods for site-specific recombination between two DNA molecules, comprising: (a) contacting a first DNA with a first fusion protein, wherein the guide nucleotide sequence-programmable DNA binding protein domain binds a first gRNA that hybridizes to a first region of the first DNA; (b) contacting the first DNA with a second fusion protein, wherein the guide nucleotide sequence-programmable DNA binding protein domain of the second fusion protein binds a second gRNA that hybridizes to a second region of the first DNA; (c) contacting a second DNA with a third fusion protein, wherein the guide nucleotide sequence-programmable DNA binding protein domain of the third fusion protein binds a third gRNA that hybridizes to a first region of the second DNA; and (d) contacting the second DNA with a fourth fusion protein, wherein the guide nucleotide sequence-programmable DNA binding protein domain of the fourth fusion protein binds a fourth gRNA that hybridizes to a second region of the second DNA; wherein the binding of the fusion proteins in steps (a)-(d) results in the tetramerization of the recombinase catalytic domains of the fusion proteins, under conditions such that the DNAs are recombined, and wherein the first, second, third, and/or fourth fusion protein is any of the fusion proteins described herein. 
     In one embodiment, the first and second DNA molecules have different sequences. In another embodiment, the gRNAs of steps (a) and (b) hybridize to opposing strands of the first DNA, and the gRNAs of steps (c) and (d) hybridize to opposing strands of the second DNA. In another embodiment, wherein the gRNAs of steps (a) and (b); and/or the gRNAs of steps (c) and (d) hybridize to regions of their respective DNAs that are no more than 10, no more than 15, no more than 20, no more than 25, no more than 30, no more than 40, no more than 50, no more than 60, no more than 70, no more than 80, no more than 90, or no more than 100 base pairs apart. In certain embodiments, the gRNAs of steps (a) and (b), and/or the gRNAs of steps (c) and (d) hybridize to regions of their respective DNAs at gRNA binding sites that flank a recombinase site (see, for example,  FIG.  1 D ). In certain embodiments, the recombinase site comprises a res, gix, hix, six, resH, LoxP, FTR, or att core, or related core sequence. In certain embodiments, the recombinase site comprises a gix core or gix-related core sequence. In certain embodiments, the distance between the gix core or gix-related core sequence and at least one gRNA binding site is from 3 to 7 base pairs. In certain embodiments, the distance between the gix core or gix-related core sequence and at least one gRNA binding site is from 5 to 6 base pairs. 
     The method for site-specific recombination provided herein may also be used with a single DNA molecule. In one aspect, the instant disclosure provides a method for site-specific recombination between two regions of a single DNA molecule, comprising: (a) contacting the DNA with a first fusion protein, wherein the guide nucleotide sequence-programmable DNA binding protein domain binds a first gRNA that hybridizes to a first region of the DNA; (b) contacting the DNA with a second fusion protein, wherein the guide nucleotide sequence-programmable DNA binding protein domain of the second fusion protein binds a second gRNA that hybridizes to a second region of the DNA; (c) contacting the DNA with a third fusion protein, wherein the guide nucleotide sequence-programmable DNA binding protein domain of the third fusion protein binds a third gRNA that hybridizes to a third region of the DNA; and (d) contacting the DNA with a fourth fusion protein, wherein the guide nucleotide sequence-programmable DNA binding protein domain of the fourth fusion protein binds a fourth gRNA that hybridizes to a fourth region of the DNA; wherein the binding of the fusion proteins in steps (a)-(d) results in the tetramerization of the recombinase catalytic domains of the fusion proteins, under conditions such that the DNA is recombined, and wherein the first, second, third, and/or fourth fusion protein is any of the fusion proteins described. 
     In certain embodiments, the two regions of the single DNA molecule that are recombined have different sequences. In another embodiment, the recombination results in the deletion of a region of the DNA molecule. In a specific embodiment, the region of the DNA molecule that is deleted is prone to cross-over events in meiosis. In one embodiment, the first and second gRNAs of steps (a)-(d) hybridize to the same strand of the DNA, and the third and fourth gRNAs of steps (a)-(d) hybridize to the opposing strand of the DNA. In another embodiment, the gRNAs of steps (a) and (b) hybridize to regions of the DNA that are no more than 50, no more than 60, no more than 70, no more than 80, no more than 90, or no more than 100 base pairs apart, and the gRNAs of steps (c) and (d) hybridize to regions of the DNA that are no more than 10, no more than 15, no more than 20, no more than 25, no more than 30, no more than 40, no more than 50, no more than 60, no more than 70, no more than 80, no more than 90, or no more than 100 base pairs apart. In certain embodiments, the gRNAs of steps (a) and (b); and/or the gRNAs of steps (c) and (d) hybridize to gRNA binding sites flanking a recombinase site. In certain embodiments, the recombinase site comprises a res, gix, hix, six, resH, LoxP, FTR, or att core or related core sequence. In one embodiment, the recombinase site comprises a gix core or gix-related core sequence. In certain embodiments, the distance between the gix core or gix-related core sequence and at least one gRNA binding site is from 3 to 7 base pairs. In certain embodiments, the distance between the gix core or gix-related core sequence and at least one gRNA binding site is from 5 to 6 base pairs. 
     The DNA described herein may be in a cell. In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the cell is a plant cell. In certain embodiments, the cell is a prokaryotic cell. In some embodiments, the cell may be a mammalian cell. In some embodiments, the cell may be a human cell. In certain embodiments, the cell is in a subject. In some embodiments, the subject may be a mammal. In certain embodiments, the subject is a human. In certain embodiments, the cell may be a plant cell. 
     In one aspect, the instant disclosure provides a polynucleotide encoding any of the fusion proteins disclosed herein. In certain embodiments, the instant disclosure provides a vector comprising the polynucleotide encoding any of the fusion proteins disclosed herein. 
     In another aspect, the instant disclosure provides a cell comprising a genetic construct for expressing any fusion protein disclosed herein. 
     In one aspect, the instant disclosure provides a kit comprising any fusion protein disclosed herein. In another aspect, the instant disclosure provides a kit comprising a polynucleotide encoding any fusion protein disclosed herein. In another aspect, the instant disclosure provides a kit comprising a vector for recombinant protein expression, wherein the vector comprises a polynucleotide encoding any fusion protein disclosed herein. In another aspect, the instant disclosure provides a kit comprising a cell that comprises a genetic construct for expressing any fusion protein disclosed herein. In one embodiment, the kit further comprises one or more gRNAs and/or vectors for expressing one or more gRNAs. 
     The details of certain embodiments of the invention are set forth in the Detailed Description of Certain Embodiments, as described below. Other features, objects, and advantages of the invention will be apparent from the Definitions, Examples, Figures, and Claims. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS.  1 A- 1 D . Overview of the experimental setup. Cells are transfected with ( FIG.  1 A ) guide RNA expression vector(s) under the control of an hU6 promoter, ( FIG.  1 B ) a recCas9 expression vector under the control of a CMV promoter, and ( FIG.  1 C ) a recCas9 reporter plasmid. Co-transfection of these components results in reassembly of guide RNA-programmed recCas9 at the target sites ( FIG.  1 D ). This will mediate deletion of the polyA terminator, allowing transcription of GFP. Guide RNA expression vectors and guide RNA sequences are abbreviated as gRNA. 
         FIGS.  2 A- 2 F . Optimization of fusion linker lengths and target site spacer variants. A single target guide RNA expression vector, pHU6-NT1, or non-target vector pHU6-BC74 was used in these experiments. The sequences can be found in Tables 6-9. ( FIG.  2 A ) A portion of the target site is shown with guide RNA target sites in black with dashed underline and a gix core sequence site in black. The 5′ and 3′ sequences on either side of the pseudo-gix sites are identical, but inverted, and are recognized by pHU6-NT1. The number of base pairs spacers separating the gix pseudo-site from the 5′ and 3′ binding sites is represented by an X and Y, respectively. This figure depicts SEQ ID NOs: 700 and 703, respectively. ( FIG.  2 B ) Z represents the number of GGS repeats connecting Ginβ to dCas9. recCas9 activity is assessed when X=Y for ( FIG.  2 C ) (GGS) 2  (SEQ ID NO: 182), ( FIG.  2 D ) (GGS) 5  (SEQ ID NO: 701), and ( FIG.  2 E ) (GGS) 8  (SEQ ID NO: 183) linkers connecting the Gin catalytic domain to the dCas9 domain. ( FIG.  2 F ) The activity of recCas9 on target sites composed of uneven base pair spacers (X≠Y) was determined; X=Y=6 is included for comparison. All experiments are performed in triplicate and background fluorescence is subtracted from these experiments. The percentage of eGFP-positive cells is of only those transfected (i.e., expressing a constitutively expressed iRFP gene) and at least 6,000 live events are recorded for each experiment. Guide RNA expression vectors and guide RNA sequences are abbreviated as “gRNA”. Values and error bars represent the mean and standard deviation, respectively, of three independent biological replicates. 
         FIGS.  3 A- 3 B . The dependence of forward and reverse guide RNAs on recCas9 activity. ( FIG.  3 A ) A sequence found within PCDH15 replaces the target site tested in  FIGS.  1 A- 1 D . Two offset sequences can be targeted by guide RNAs on both the 5′ and 3′ sides of a pseudo-gix core site. This figure depicts SEQ ID NOs: 704-705, respectively. ( FIG.  3 B ) recCas9 activity was measured by co-transfecting a recCas9 expression vector and reporter plasmid with all four guide RNA expression vector pairs and individual guide RNA vectors with off target (O.T.) guide RNA vectors. The off-target forward and reverse contained guide RNA sequences targeting CLTA and VEGF, respectively. Control experiments transfected with the reporter plasmid but without a target guide RNA are also shown. The results of reporter plasmid cotransfected with different guide RNA expression vectors, but without recCas9 expression vectors, are also shown. All experiments were performed in quadruplicate, and background fluorescence is not subtracted from these experiments. The percentage of eGFP-positive cells is of only those transfected (i.e., expressing a constitutively expressed iRFP gene), and at least 6,000 live events are recorded for each experiment. Guide RNA expression vectors and guide RNA sequences are abbreviated as gRNA. Values and error bars represent the mean and standard deviation, respectively, of four independent biological replicates. 
         FIGS.  4 A- 4 D . recCas9 can target multiple sequences identical to those in the human genome. ( FIG.  4 A ) The target sites shown in  FIGS.  1 A- 1 D  are replaced by sequences found within the human genome. See Table 6 for sequences. A recCas9 expression vector was cotransformed with all combinations of guide RNA vectors pairs and reporter plasmids. Off-target guide RNA vectors were also cotransformed with the recCas9 expression vector and reporter plasmids and contain guide RNA sequences targeting CLTA and VEGF (see, e.g., Guilinger et al., Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nature biotechnology, (2014), the entire contents of which is hereby incorporated by reference). The percentage of eGFP-positive cells reflects that of transfected (iRFP-positive) cells. At least 6,000 live events are recorded for each experiment. Values and error bars represent the mean and standard deviation, respectively, of at least three independent biological replicates. ( FIG.  4 B ) Transfection experiments were performed again, replacing the resistance marker in the recCas9 expression vector and pUC with SpecR. After cotransfection and incubation, episomal DNA was extracted, transformed into  E. coli  and selected for carbenicillin resistance. Colonies were then sequenced to determine ( FIG.  4 C ) the ratio of recombined to fully intact plasmids. ( FIG.  4 D ) Sequencing data from episomal extractions isolated from transfected cells. Columns and rows represent the transfection conditions. Each cell shows the percent of recombined plasmid and the ratio. The values shown reflect the mean and standard deviation of two independent biological replicates. The average difference between the mean and each replicate is shown as the error. Guide RNA expression vectors and guide RNA sequences are abbreviated as gRNA. 
         FIGS.  5 A- 5 D . recCas9 mediates guide RNA- and recCas9-dependent deletion of genomic DNA in cultured human cells. ( FIG.  5 A ) Schematic showing predicted recCas9 target sites located within an intronic region of the FAM19A2 locus of chromosome 12 and the positions of primers used for nested PCR. This figure depicts SEQ ID NOs: 706-709 from top to bottom and left to right, respectively. ( FIG.  5 B ) Representative results of nested genomic PCR of template from cells transfected with the indicated expression vectors (n=3 biological replicates; NTC=no template control). The asterisk indicates the position of the 1.3-kb predicted primary PCR product. Arrow indicates the predicted deletion product after the secondary PCR. Both panes are from the same gel but were cut to remove blank lanes. ( FIG.  5 C ) Sanger sequencing of PCR products resulting from nested genomic PCR of cells transfected with all four gRNA expression vectors, and the recCas9 expression vector matches the predicted post-recombination product. This figure depicts SEQ ID NOs: 710 and 711 from top to bottom, respectively. ( FIG.  5 D ) Estimated minimum deletion efficiency of FAM19A2 locus determined by limiting-dilution nested PCR. The values shown reflect the mean and standard deviation of three replicates. 
         FIG.  6   . Reporter plasmid construction. Golden Gate assembly was used to construct the reporter plasmids described in this work. All assemblies started with a common plasmid, pCALNL-EGFP-Esp3I, that was derived from pCALNL-EGFP and contained to Esp3I restriction sites. The fragments shown are flanked by Esp3I sites. Esp3I digestion creates a series of compatible, unique 4-base pair 5′ overhangs so that assembly occurs in the order shown. To assemble the target sites, Esp3I (ThermoFisher Scientific, Waltham, Mass.) and five fragments were added to a single reaction tube to allow for iterative cycles of Esp3I digestion and T7 ligation. Reactions were then digested with Plasmid-Safe-ATP-dependent DNAse (Epicentre, Madison, Wis.) to reduce background. Colonies were analyzed by colony PCR to identify PCR products that matched the expected full length 5 part assembly product; plasmid from these colonies was then sent for sanger sequencing. For the genomic reporters shown in  FIG.  4   , fragments 1 and 2 as well as fragments 4 and 5 were combined into two gBlocks (IDT, Coralville, Iowa) fragments encoding the entire target site (not shown in the figure). Assembly was then completed as described above. Details for construction can be found in the methods for the supporting material. Oligonucleotides and gBLOCKS for creation of fragments can be found in Table 2. 
         FIGS.  7 A and  7 B . A Cre recombinase evolved to target a site in the Rosa locus of the human genome called “36C6” was fused to dCas9. This fusion was then used to recombine a plasmid-based reporter containing the Rosa target site in a guide-RNA dependent fashion.  FIG.  7 A  demonstrates the results of linker optimization using wild-type Cre and 36C6. A GinB construct, targeting its cognate reporter, is shown for reference. The 1×2×, 5×, and 8× linkers shown are the number of GGS repeats in the linker.  FIG.  7 B  shows the results of a reversion analysis which demonstrated that making mutations to 36C6 fused to dCas9 could impact the relative guide dependence of the chimeric fusion. A GinB construct, targeting its cognate reporter, is shown for reference. GGS-36C6: 1×GGS linker; 2GGS-36C6 (using linker SEQ ID NO: 181): 2×GGS linker (using linker SEQ ID NO: 181). 
         FIG.  8   . PAMs were identified flanking the Rosa26 site in the human genome that could support dCas9 binding (see at top). Guide RNAs and a plasmid reporter were designed to test whether the endogenous protospacers could support dCas9-36C6 activity. A GinB construct, targeting the gix reporter, is shown for reference. Mix: equal parts mixture of all 5 linker variants between Cas9 and 36C6. The sequences correspond to SEQ ID NO: 769 (the nucleotide sequence) and 770, 776, and 777 (the amino acid sequences from left to right). 
         FIGS.  9 A- 9 B . Locations of various tested truncations of Cre recombinase are shown in  FIG.  9 A . Truncated variants of Cre recombinase fused to dCas9 show both appreciable recombinase activity as well as a strict reliance on the presence of guide RNA in a Lox plasmid reporter system ( FIG.  9 B ). Wild type Cre fused to dCas9 is shown as a positive control. 
     
    
    
     DEFINITIONS 
     As used herein, the singular forms “a,” “an,” and “the” include the singular and the plural reference unless the context clearly indicates otherwise. Thus, for example, a reference to “an agent” includes a single agent and a plurality of such agents. 
     Non-limiting, exemplary RNA-programmable DNA-binding proteins include Cas9 nucleases, Cas9 nickases, nuclease inactive Cas9 (dCas9), CasX, CasY, Cpf1, C2c1, C2c2, C2C3, and Argonaute. The term “Cas9” or “Cas9 nuclease” refers to an RNA-guided nuclease comprising a Cas9 protein, or a fragment thereof (e.g., a protein comprising an active or inactive DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9). Cas9 has two cleavage domains, which cut specific DNA strands (e.g., sense and antisense strands). Cas9 nickases can be generated that cut either strand (including, but not limited to D10A and H840A of spCas9). A Cas9 domain (e.g., nuclease active Cas9, nuclease inactive Cas9, or Cas9 nickases) may be used without limitation in the fusion proteins and methods described herein. Further, any of the guide nucleotide sequence-programmable DNA binding proteins described herein may be useful as nickases. 
     A Cas9 nuclease is also referred to sometimes as a casn1 nuclease or a CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves a linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3′-5′ exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNA sequences. However, single guide RNAs (“sgRNA”, or simply “gRNA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E.  Science  337:816-821(2012), the entire contents of which is hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., “Complete genome sequence of an M1 strain of  Streptococcus pyogenes .” Ferretti et al., J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S. W., Roe B. A., McLaughlin R. E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E.  Science  337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to,  S. pyogenes  and  S. thermophilus . Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase. As one example, the Cas9 nuclease (e.g., Cas9 nickase) may cleave the DNA strand that is bound to the gRNA. As another example, the Cas9 nuclease (e.g., Cas9 nickase) may cleave the DNA strand that is not bound to the gRNA. In another embodiment, any of the guide nucleotide sequence-programmable DNA binding proteins may have an inactive (e.g., an inactivated) DNA cleavage domain, that is, the guide nucleotide sequence-programmable DNA binding protein is a nickase. As one example, the guide nucleotide sequence-programmable DNA binding protein may cleave the DNA strand that is bound to the gRNA. As another example, the guide nucleotide sequence-programmable DNA binding protein may cleave the DNA strand that is not bound to the gRNA. 
     Additional exemplary Cas9 sequences may be found in International Publication No.: WO/2017/070633, published Apr. 27, 2017, and entitled “Evolved Cas9 Proteins for Gene Editing.” 
     A nuclease-inactivated Cas9 protein may interchangeably be referred to as a “dCas9” protein (for nuclease “dead” Cas9). In some embodiments, dCas9 corresponds to, or comprises in part or in whole, the amino acid set forth as SEQ ID NO: 1, below. In some embodiments, variants of dCas9 (e.g., variants of SEQ ID NO: 1) are provided. For example, in some embodiments, variants having mutations other than D10A and H840A are provided, which e.g., result in nuclease inactivated Cas9 (dCas9). Such mutations, by way of example, include other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domains of Cas9 (e.g., substitutions in the HNH nuclease subdomain and/or the RuvC1 subdomain). In some embodiments, variants or homologues of dCas9 (e.g., variants of SEQ ID NO: 1) are provided which are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% to SEQ ID NO: 1. In some embodiments, variants of dCas9 (e.g., variants of SEQ ID NO: 1) are provided having amino acid sequences which are shorter, or longer than SEQ ID NO: 1, by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids, or more. 
     
       
         
           
               
            
               
                 dCas9 (D10A and H840A): 
               
            
           
           
               
            
               
                 (SEQ ID NO: 1) 
               
            
           
           
               
            
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV 
               
               
                   
               
               
                 QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE 
               
               
                   
               
               
                 KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
               
               
                   
               
               
                 YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE 
               
               
                   
               
               
                 DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK 
               
               
                   
               
               
                 PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ 
               
               
                   
               
               
                 SITGLYETRIDLSQLGGD 
               
            
           
         
       
     
     Methods for generating a Cas9 protein (or a fragment thereof) having an inactive DNA cleavage domain are known (See, e.g., Jinek et al.,  Science.  337:816-821(2012); Qi et al., “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression” (2013)  Cell.  28; 152(5):1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of  S. pyogenes  Cas9 (See e.g., Jinek et al.,  Science.  337:816-821(2012); Qi et al.,  Cell.  28; 152(5):1173-83 (2013)). In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, a protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9, or fragments thereof, are referred to as “Cas9 variants.” A Cas9 variant shares homology to Cas9, or a fragment thereof. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% to wild type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% to the corresponding fragment of wild type Cas9. In some embodiments, wild type Cas9 corresponds to Cas9 from  Streptococcus pyogenes  (NCBI Reference Sequence: NC_017053.1, SEQ ID NO: 2 (nucleotide); SEQ ID NO: 3 (amino acid)). In some embodiments the Cas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to wild type Cas9. In some embodiments, the Cas9 domain comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more or more mutations compared to wild type Cas9. In some embodiments, the Cas9 domain comprises an amino acid sequence that has at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues as compared to wild type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9. In some embodiments, the fragment is 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% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9. 
     In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or 1300 amino acids in length. 
     
       
         
           
               
            
               
                 (SEQ ID NO: 2) 
               
            
           
           
               
            
               
                 ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGG 
               
               
                   
               
               
                 ATGGGCGGTGATCACTGATGATTATAAGGTTCCGTCTAAAAAGTTCAAGG 
               
               
                   
               
               
                 TTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCT 
               
               
                   
               
               
                 CTTTTATTTGGCAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGAC 
               
               
                   
               
               
                 AGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGG 
               
               
                   
               
               
                 AGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGA 
               
               
                   
               
               
                 CTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCC 
               
               
                   
               
               
                 TATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAA 
               
               
                   
               
               
                 CTATCTATCATCTGCGAAAAAAATTGGCAGATTCTACTGATAAAGCGGAT 
               
               
                   
               
               
                 TTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCA 
               
               
                   
               
               
                 TTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAAC 
               
               
                   
               
               
                 TATTTATCCAGTTGGTACAAATCTACAATCAATTATTTGAAGAAAACCCT 
               
               
                   
               
               
                 ATTAACGCAAGTAGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAG 
               
               
                   
               
               
                 TAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGA 
               
               
                   
               
               
                 GAAATGGCTTGTTTGGGAATCTCATTGCTTTGTCATTGGGATTGACCCCT 
               
               
                   
               
               
                 AATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTC 
               
               
                   
               
               
                 AAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAG 
               
               
                   
               
               
                 ATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATT 
               
               
                   
               
               
                 TTACTTTCAGATATCCTAAGAGTAAATAGTGAAATAACTAAGGCTCCCCT 
               
               
                   
               
               
                 ATCAGCTTCAATGATTAAGCGCTACGATGAACATCATCAAGACTTGACTC 
               
               
                   
               
               
                 TTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATC 
               
               
                   
               
               
                 TTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGC 
               
               
                   
               
               
                 TAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGG 
               
               
                   
               
               
                 ATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGC 
               
               
                   
               
               
                 AAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGG 
               
               
                   
               
               
                 TGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAA 
               
               
                   
               
               
                 AAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTAT 
               
               
                   
               
               
                 TATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCG 
               
               
                   
               
               
                 GAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATA 
               
               
                   
               
               
                 AAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAA 
               
               
                   
               
               
                 AATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTA 
               
               
                   
               
               
                 TTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAGGGAA 
               
               
                   
               
               
                 TGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGAT 
               
               
                   
               
               
                 TTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGA 
               
               
                   
               
               
                 TTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTG 
               
               
                   
               
               
                 AAGATAGATTTAATGCTTCATTAGGCGCCTACCATGATTTGCTAAAAATT 
               
               
                   
               
               
                 ATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGA 
               
               
                   
               
               
                 GGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGGGATGATTGAGG 
               
               
                   
               
               
                 AAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAG 
               
               
                   
               
               
                 CTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGAT 
               
               
                   
               
               
                 TAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGA 
               
               
                   
               
               
                 AATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGAT 
               
               
                   
               
               
                 AGTTTGACATTTAAAGAAGATATTCAAAAAGCACAGGTGTCTGGACAAGG 
               
               
                   
               
               
                 CCATAGTTTACATGAACAGATTGCTAACTTAGCTGGCAGTCCTGCTATTA 
               
               
                   
               
               
                 AAAAAGGTATTTTACAGACTGTAAAAATTGTTGATGAACTGGTCAAAGTA 
               
               
                   
               
               
                 ATGGGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCA 
               
               
                   
               
               
                 GACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCG 
               
               
                   
               
               
                 AAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTT 
               
               
                   
               
               
                 GAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTACAAAA 
               
               
                   
               
               
                 TGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTG 
               
               
                   
               
               
                 ATTATGATGTCGATCACATTGTTCCACAAAGTTTCATTAAAGACGATTCA 
               
               
                   
               
               
                 ATAGACAATAAGGTACTAACGCGTTCTGATAAAAATCGTGGTAAATCGGA 
               
               
                   
               
               
                 TAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGAC 
               
               
                   
               
               
                 AACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACG 
               
               
                   
               
               
                 AAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAA 
               
               
                   
               
               
                 ACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTT 
               
               
                   
               
               
                 TGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGA 
               
               
                   
               
               
                 GAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAA 
               
               
                   
               
               
                 AGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCC 
               
               
                   
               
               
                 ATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATAT 
               
               
                   
               
               
                 CCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGT 
               
               
                   
               
               
                 TCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAA 
               
               
                   
               
               
                 AATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACA 
               
               
                   
               
               
                 CTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGA 
               
               
                   
               
               
                 AACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCA 
               
               
                   
               
               
                 AAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAG 
               
               
                   
               
               
                 ACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAA 
               
               
                   
               
               
                 GCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTG 
               
               
                   
               
               
                 ATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAA 
               
               
                   
               
               
                 GGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAAT 
               
               
                   
               
               
                 TATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTA 
               
               
                   
               
               
                 AAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATAT 
               
               
                   
               
               
                 AGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGG 
               
               
                   
               
               
                 AGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATT 
               
               
                   
               
               
                 TTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGAT 
               
               
                   
               
               
                 AACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGA 
               
               
                   
               
               
                 GATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATG 
               
               
                   
               
               
                 CCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCA 
               
               
                   
               
               
                 ATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCT 
               
               
                   
               
               
                 TGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAAC 
               
               
                   
               
               
                 GATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCC 
               
               
                   
               
               
                 ATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGGTGA 
               
               
                   
               
               
                 CTGA 
               
               
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 3) 
               
            
           
           
               
            
               
                 MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFGSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENP 
               
               
                   
               
               
                 INASRVDAKAILSARLSKSRRLENLIAQLPGEKRNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNSEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGAYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGHSLHEQIANLAGSPAIKKGILQTVKIVDELVKV 
               
               
                   
               
               
                 MGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV 
               
               
                   
               
               
                 ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDS 
               
               
                   
               
               
                 IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT 
               
               
                   
               
               
                 KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR 
               
               
                   
               
               
                 EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY 
               
               
                   
               
               
                 PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT 
               
               
                   
               
               
                 LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ 
               
               
                   
               
               
                 TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK 
               
               
                   
               
               
                 GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY 
               
               
                   
               
               
                 SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED 
               
               
                   
               
               
                 NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP 
               
               
                   
               
               
                 IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS 
               
               
                   
               
               
                 ITGLYETRIDLSQLGGD 
               
            
           
         
       
     
     In some embodiments, wild type Cas9 corresponds to or comprises, SEQ ID NO: 4 (nucleotide) and/or SEQ ID NO: 5 (amino acid). 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 4) 
               
               
                   
                 ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCAC 
               
               
                   
                   
               
               
                   
                 TAATTCCGTTGGATGGGCTGTCATAACCGATGAATAC 
               
               
                   
                   
               
               
                   
                 AAAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACAC 
               
               
                   
                   
               
               
                   
                 AGACCGTCATTCGATTAAAAAGAATCTTATCGGTGCC 
               
               
                   
                   
               
               
                   
                 CTCCTATTCGATAGTGGCGAAACGGCAGAGGCGACTCG 
               
               
                   
                   
               
               
                   
                 CCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGC 
               
               
                   
                   
               
               
                   
                 AAGAACCGAATATGTTACTTACAAGAAATTTTTAGCAA 
               
               
                   
                   
               
               
                   
                 TGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGT 
               
               
                   
                   
               
               
                   
                 TTGGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAACA 
               
               
                   
                   
               
               
                   
                 TGAACGGCACCCCATCTTTGGAAACATAGTAGATGAG 
               
               
                   
                   
               
               
                   
                 GTGGCATATCATGAAAAGTACCCAACGATTTATCACCT 
               
               
                   
                   
               
               
                   
                 CAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGAC 
               
               
                   
                   
               
               
                   
                 CTGAGGTTAATCTACTTGGCTCTTGCCCATATGATAAA 
               
               
                   
                   
               
               
                   
                 GTTCCGTGGGCACTTTCTCATTGAGGGTGATCTAAAT 
               
               
                   
                   
               
               
                   
                 CCGGACAACTCGGATGTCGACAAACTGTTCATCCAGTT 
               
               
                   
                   
               
               
                   
                 AGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCT 
               
               
                   
                   
               
               
                   
                 ATAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAG 
               
               
                   
                   
               
               
                   
                 CGCCCGCCTCTCTAAATCCCGACGGCTAGAAAACCTG 
               
               
                   
                   
               
               
                   
                 ATCGCACAATTACCCGGAGAGAAGAAAAATGGGTTGTT 
               
               
                   
                   
               
               
                   
                 CGGTAACCTTATAGCGCTCTCACTAGGCCTGACACCA 
               
               
                   
                   
               
               
                   
                 AATTTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAA 
               
               
                   
                   
               
               
                   
                 ATTGCAGCTTAGTAAGGACACGTACGATGACGATCTC 
               
               
                   
                   
               
               
                   
                 GACAATCTACTGGCACAAATTGGAGATCAGTATGCGGA 
               
               
                   
                   
               
               
                   
                 CTTATTTTTGGCTGCCAAAAACCTTAGCGATGCAATC 
               
               
                   
                   
               
               
                   
                 CTCCTATCTGACATACTGAGAGTTAATACTGAGATTAC 
               
               
                   
                   
               
               
                   
                 CAAGGCGCCGTTATCCGCTTCAATGATCAAAAGGTAC 
               
               
                   
                   
               
               
                   
                 GATGAACATCACCAAGACTTGACACTTCTCAAGGCCCT 
               
               
                   
                   
               
               
                   
                 AGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATA 
               
               
                   
                   
               
               
                   
                 TTCTTTGATCAGTCGAAAAACGGGTACGCAGGTTATAT 
               
               
                   
                   
               
               
                   
                 TGACGGCGGAGCGAGTCAAGAGGAATTCTACAAGTTT 
               
               
                   
                   
               
               
                   
                 ATCAAACCCATATTAGAGAAGATGGATGGGACGGAAGA 
               
               
                   
                   
               
               
                   
                 GTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGA 
               
               
                   
                   
               
               
                   
                 AAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCA 
               
               
                   
                   
               
               
                   
                 AATCCACTTAGGCGAATTGCATGCTATACTTAGAAGG 
               
               
                   
                   
               
               
                   
                 CAGGAGGATTTTTATCCGTTCCTCAAAGACAATCGTGA 
               
               
                   
                   
               
               
                   
                 AAAGATTGAGAAAATCCTAACCTTTCGCATACCTTAC 
               
               
                   
                   
               
               
                   
                 TATGTGGGACCCCTGGCCCGAGGGAACTCTCGGTTCGC 
               
               
                   
                   
               
               
                   
                 ATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCA 
               
               
                   
                   
               
               
                   
                 TGGAATTTTGAGGAAGTTGTCGATAAAGGTGCGTCAGC 
               
               
                   
                   
               
               
                   
                 TCAATCGTTCATCGAGAGGATGACCAACTTTGACAAG 
               
               
                   
                   
               
               
                   
                 AATTTACCGAACGAAAAAGTATTGCCTAAGCACAGTTT 
               
               
                   
                   
               
               
                   
                 ACTTTACGAGTATTTCACAGTGTACAATGAACTCACG 
               
               
                   
                   
               
               
                   
                 AAAGTTAAGTATGTCACTGAGGGCATGCGTAAACCCGC 
               
               
                   
                   
               
               
                   
                 CTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGAT 
               
               
                   
                   
               
               
                   
                 CTGTTATTCAAGACCAACCGCAAAGTGACAGTTAAGCA 
               
               
                   
                   
               
               
                   
                 ATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTC 
               
               
                   
                   
               
               
                   
                 GATTCTGTCGAGATCTCCGGGGTAGAAGATCGATTTAA 
               
               
                   
                   
               
               
                   
                 TGCGTCACTTGGTACGTATCATGACCTCCTAAAGATA 
               
               
                   
                   
               
               
                   
                 ATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGA 
               
               
                   
                   
               
               
                   
                 AGATATCTTAGAAGATATAGTGTTGACTCTTACCCTC 
               
               
                   
                   
               
               
                   
                 TTTGAAGATCGGGAAATGATTGAGGAAAGACTAAAAAC 
               
               
                   
                   
               
               
                   
                 ATACGCTCACCTGTTCGACGATAAGGTTATGAAACAG 
               
               
                   
                   
               
               
                   
                 TTAAAGAGGCGTCGCTATACGGGCTGGGGACGATTGTC 
               
               
                   
                   
               
               
                   
                 GCGGAAACTTATCAACGGGATAAGAGACAAGCAAAGT 
               
               
                   
                   
               
               
                   
                 GGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTT 
               
               
                   
                   
               
               
                   
                 CGCCAATAGGAACTTTATGCAGCTGATCCATGATGAC 
               
               
                   
                   
               
               
                   
                 TCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGT 
               
               
                   
                   
               
               
                   
                 TTCCGGACAAGGGGACTCATTGCACGAACATATTGCG 
               
               
                   
                   
               
               
                   
                 AATCTTGCTGGTTCGCCAGCCATCAAAAAGGGCATACT 
               
               
                   
                   
               
               
                   
                 CCAGACAGTCAAAGTAGTGGATGAGCTAGTTAAGGTC 
               
               
                   
                   
               
               
                   
                 ATGGGACGTCACAAACCGGAAAACATTGTAATCGAGAT 
               
               
                   
                   
               
               
                   
                 GGCACGCGAAAATCAAACGACTCAGAAGGGGCAAAAA 
               
               
                   
                   
               
               
                   
                 AACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGGTAT 
               
               
                   
                   
               
               
                   
                 TAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCT 
               
               
                   
                   
               
               
                   
                 GTGGAAAATACCCAATTGCAGAACGAGAAACTTTACCT 
               
               
                   
                   
               
               
                   
                 CTATTACCTACAAAATGGAAGGGACATGTATGTTGAT 
               
               
                   
                   
               
               
                   
                 CAGGAACTGGACATAAACCGTTTATCTGATTACGACGT 
               
               
                   
                   
               
               
                   
                 CGATCACATTGTACCCCAATCCTTTTTGAAGGACGAT 
               
               
                   
                   
               
               
                   
                 TCAATCGACAATAAAGTGCTTACACGCTCGGATAAGAA 
               
               
                   
                   
               
               
                   
                 CCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGTC 
               
               
                   
                   
               
               
                   
                 GTAAAGAAAATGAAGAACTATTGGCGGCAGCTCCTAAA 
               
               
                   
                   
               
               
                   
                 TGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTA 
               
               
                   
                   
               
               
                   
                 ACTAAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAA 
               
               
                   
                   
               
               
                   
                 GGCCGGATTTATTAAACGTCAGCTCGTGGAAACCCGC 
               
               
                   
                   
               
               
                   
                 CAAATCACAAAGCATGTTGCACAGATACTAGATTCCCG 
               
               
                   
                   
               
               
                   
                 AATGAATACGAAATACGACGAGAACGATAAGCTGATT 
               
               
                   
                   
               
               
                   
                 CGGGAAGTCAAAGTAATCACTTTAAAGTCAAAATTGGT 
               
               
                   
                   
               
               
                   
                 GTCGGACTTCAGAAAGGATTTTCAATTCTATAAAGTT 
               
               
                   
                   
               
               
                   
                 AGGGAGATAAATAACTACCACCATGCGCACGACGCTTA 
               
               
                   
                   
               
               
                   
                 TCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAA 
               
               
                   
                   
               
               
                   
                 TACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGATTA 
               
               
                   
                   
               
               
                   
                 CAAAGTTTATGACGTCCGTAAGATGATCGCGAAAAGC 
               
               
                   
                   
               
               
                   
                 GAACAGGAGATAGGCAAGGCTACAGCCAAATACTTCTT 
               
               
                   
                   
               
               
                   
                 TTATTCTAACATTATGAATTTCTTTAAGACGGAAATC 
               
               
                   
                   
               
               
                   
                 ACTCTGGCAAACGGAGAGATACGCAAACGACCTTTAAT 
               
               
                   
                   
               
               
                   
                 TGAAACCAATGGGGAGACAGGTGAAATCGTATGGGAT 
               
               
                   
                   
               
               
                   
                 AAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTC 
               
               
                   
                   
               
               
                   
                 CATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTG 
               
               
                   
                   
               
               
                   
                 CAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAA 
               
               
                   
                   
               
               
                   
                 AAGGAATAGTGATAAGCTCATCGCTCGTAAAAAGGAC 
               
               
                   
                   
               
               
                   
                 TGGGACCCGAAAAAGTACGGTGGCTTCGATAGCCCTAC 
               
               
                   
                   
               
               
                   
                 AGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAG 
               
               
                   
                   
               
               
                   
                 AAGGGAAAATCCAAGAAACTGAAGTCAGTCAAAGAATT 
               
               
                   
                   
               
               
                   
                 ATTGGGGATAACGATTATGGAGCGCTCGTCTTTTGAA 
               
               
                   
                   
               
               
                   
                 AAGAACCCCATCGACTTCCTTGAGGCGAAAGGTTACAA 
               
               
                   
                   
               
               
                   
                 GGAAGTAAAAAAGGATCTCATAATTAAACTACCAAAG 
               
               
                   
                   
               
               
                   
                 TATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGAT 
               
               
                   
                   
               
               
                   
                 GTTGGCTAGCGCCGGAGAGCTTCAAAAGGGGAACGAA 
               
               
                   
                   
               
               
                   
                 CTCGCACTACCGTCTAAATACGTGAATTTCCTGTATTT 
               
               
                   
                   
               
               
                   
                 AGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAA 
               
               
                   
                   
               
               
                   
                 GATAACGAACAGAAGCAACTTTTTGTTGAGCAGCACAA 
               
               
                   
                   
               
               
                   
                 ACATTATCTCGACGAAATCATAGAGCAAATTTCGGAA 
               
               
                   
                   
               
               
                   
                 TTCAGTAAGAGAGTCATCCTAGCTGATGCCAATCTGGA 
               
               
                   
                   
               
               
                   
                 CAAAGTATTAAGCGCATACAACAAGCACAGGGATAAA 
               
               
                   
                   
               
               
                   
                 CCCATACGTGAGCAGGCGGAAAATATTATCCATTTGTT 
               
               
                   
                   
               
               
                   
                 TACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAG 
               
               
                   
                   
               
               
                   
                 TATTTTGACACAACGATAGATCGCAAACGATACACTTC 
               
               
                   
                   
               
               
                   
                 TACCAAGGAGGTGCTAGACGCGACACTGATTCACCAA 
               
               
                   
                   
               
               
                   
                 TCCATCACGGGATTATATGAAACTCGGATAGATTTGTC 
               
               
                   
                   
               
               
                   
                 ACAGCTTGGGGGTGACGGATCCCCCAAGAAGAAGAGG 
               
               
                   
                   
               
               
                   
                 AAAGTCTCGAGCGACTACAAAGACCATGACGGTGATTA 
               
               
                   
                   
               
               
                   
                 TAAAGATCATGACATCGATTACAAGGATGACGATGAC 
               
               
                   
                   
               
               
                   
                 AAGGCTGCAGGA 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 5) 
               
               
                   
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNT 
               
               
                   
                   
               
               
                   
                 DRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRR 
               
               
                   
                   
               
               
                   
                 KNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH 
               
               
                   
                   
               
               
                   
                 ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
                   
               
               
                   
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQL 
               
               
                   
                   
               
               
                   
                 VQTYNQLFEENPINASGVDAKAILSARLSKSRRLENL 
               
               
                   
                   
               
               
                   
                 IAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAK 
               
               
                   
                   
               
               
                   
                 LQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
                   
               
               
                   
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKAL 
               
               
                   
                   
               
               
                   
                 VRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKF 
               
               
                   
                   
               
               
                   
                 IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQ 
               
               
                   
                   
               
               
                   
                 IHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
                   
               
               
                   
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASA 
               
               
                   
                   
               
               
                   
                 QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELT 
               
               
                   
                   
               
               
                   
                 KVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQ 
               
               
                   
                   
               
               
                   
                 LKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
                   
               
               
                   
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT 
               
               
                   
                   
               
               
                   
                 YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQS 
               
               
                   
                   
               
               
                   
                 GKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQV 
               
               
                   
                   
               
               
                   
                 SGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
                   
               
               
                   
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGI 
               
               
                   
                   
               
               
                   
                 KELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVD 
               
               
                   
                   
               
               
                   
                 QELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKN 
               
               
                   
                   
               
               
                   
                 RGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
                   
               
               
                   
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSR 
               
               
                   
                   
               
               
                   
                 MNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV 
               
               
                   
                   
               
               
                   
                 REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDY 
               
               
                   
                   
               
               
                   
                 KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
                   
               
               
                   
                 TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS 
               
               
                   
                   
               
               
                   
                 MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKD 
               
               
                   
                   
               
               
                   
                 WDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKEL 
               
               
                   
                   
               
               
                   
                 LGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
               
               
                   
                   
               
               
                   
                 YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYL 
               
               
                   
                   
               
               
                   
                 ASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISE 
               
               
                   
                   
               
               
                   
                 FSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLF 
               
               
                   
                   
               
               
                   
                 TLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ 
               
               
                   
                   
               
               
                   
                 SITGLYETRIDLSQLGGD 
               
            
           
         
       
     
     In some embodiments, Cas9 refers to Cas9 from:  Corynebacterium ulcerans  (NCBI Refs: NC_015683.1, NC_017317.1);  Corynebacterium diphtheria  (NCBI Refs: NC_016782.1, NC_016786.1);  Spiroplasma syrphidicola  (NCBI Ref: NC_021284.1);  Prevotella intermedia  (NCBI Ref: NC_017861.1);  Spiroplasma taiwanense  (NCBI Ref: NC_021846.1);  Streptococcus iniae  (NCBI Ref: NC_021314.1);  Belliella baltica  (NCBI Ref: NC_018010.1);  Psychroflexus torquisl  (NCBI Ref: NC_018721.1);  Streptococcus  thermophiles (NCBI Ref: YP_820832.1),  Listeria innocua  (NCBI Ref: NP_472073.1);  Campylobacter jejuni  (NCBI Ref: YP_002344900.1); or  Neisseria meningitidis  (NCBI Ref: YP_002342100.1) or to a Cas9 from any other organism. 
     Cas9 recognizes a short motif (PAM motif) in the CRISPR repeat sequences in the target DNA sequence. A “PAM motif,” or “protospacer adjacent motif,” as used herein, refers a DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. PAM is a component of the invading virus or plasmid, but is not a component of the bacterial CRISPR locus. Naturally, Cas9 will not successfully bind to or cleave the target DNA sequence if it is not followed by the PAM sequence. PAM is a targeting component (not found in the bacterial genome) which distinguishes bacterial self from non-self DNA, thereby preventing the CRISPR locus from being targeted and destroyed by the Cas9 nuclease activity. 
     Wild-type  Streptococcus pyogenes  Cas9 recognizes a canonical PAM sequence (e.g., Cas9 from  Streptococcus thermophiles, Staphylococcus aureus, Neisseria meningitidis , or  Treponema denticolaor ) and Cas9 variants thereof have been described in the art to have different, or more relaxed PAM requirements. Typically, Cas9 proteins, such as Cas9 from  S. pyogenes  (spCas9), require a canonical NGG PAM sequence to bind a particular nucleic acid region, where the “N” in “NGG” is adenine (A), thymine (T), guanine (G), or cytosine (C), and the G is guanine. This may limit the ability to edit desired bases within a genome. In some embodiments, the base editing fusion proteins provided herein need to be positioned at a precise location, for example, where a target base is within a 4 base region (e.g., a “deamination window”), which is approximately 15 bases upstream of the PAM. See Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage”  Nature  533, 420-424 (2016), the entire contents of which are hereby incorporated by reference. In some embodiments, the deamination window is within a 2, 3, 4, 5, 6, 7, 8, 9, or 10 base region. In some embodiments, the deamination window is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases upstream of the PAM. Accordingly, in some embodiments, any of the fusion proteins provided herein may contain a Cas9 domain that is capable of binding a nucleotide sequence that does not contain a canonical (e.g., NGG) PAM sequence. Cas9 domains that bind to non-canonical PAM sequences have been described in the art and would be apparent to the skilled artisan. For example, Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver, B. P., et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities”  Nature  523, 481-485 (2015); and Kleinstiver, B. P., et al., “Broadening the targeting range of  Staphylococcus aureus  CRISPR-Cas9 by modifying PAM recognition”  Nature Biotechnology  33, 1293-1298 (2015); the entire contents of each are hereby incorporated by reference. See also: Klenstiver et al.,  Nature  529, 490-495, 2016; Ran et al.,  Nature , April 9; 520(7546): 186-191, 2015; Hou et al.,  Proc Natl Acad Sci USA,  110(39):15644-9, 2014; Prykhozhij et al.,  PLoS One,  10(3): e0119372, 2015; Zetsche et al.,  Cell  163, 759-771, 2015; Gao et al.,  Nature Biotechnology , doi:10.1038/nbt.3547, 2016; Want et al.,  Nature  461, 754-761, 2009; Chavez et al., doi: dx dot doi dot org/10.1101/058974; Fagerlund et al.,  Genome Biol.  2015; 16: 25, 2015; Zetsche et al.,  Cell,  163, 759-771, 2015; and Swarts et al.,  Nat Struct Mol Biol,  21(9):743-53, 2014, the entire contents of each of which is incorporated herein by reference. 
     Thus, the guide nucleotide sequence-programmable DNA-binding protein of the present disclosure may recognize a variety of PAM sequences including, without limitation: NGG, NGAN (SEQ ID NO: 741), NGNG (SEQ ID NO: 742), NGAG (SEQ ID NO: 743), NGCG (SEQ ID NO: 744), NNGRRT (SEQ ID NO: 745), NGRRN (SEQ ID NO: 746), NNNRRT (SEQ ID NO: 747), NNNGATT (SEQ ID NO: 748), NNAGAAW (SEQ ID NO: 749), NAAAC (SEQ ID NO: 750), TTN, TTTN (SEQ ID NO: 751), and YTN, wherein Y is a pyrimidine, and N is any nucleobase. 
     One example of an RNA-programmable DNA-binding protein that has different PAM specificity is Clustered Regularly Interspaced Short Palindromic Repeats from  Prevotella  and  Francisella  1 (Cpf1). Similar to Cas9, Cpf1 is also a class 2 CRISPR effector. It has been shown that Cpf1 mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif (TTN, TTTN (SEQ ID NO: 751), or YTN). Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, two enzymes from Acidaminococcus and Lachnospiraceae are shown to have efficient genome-editing activity in human cells. 
     Also provided herein are nuclease-inactive Cpf1 (dCpf1) variants that may be used as a RNA-programmable DNA-binding protein domain. The Cpf1 protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9 but does not have a HNH endonuclease domain, and the N-terminal of Cpf1 does not have the alpha-helical recognition lobe of Cas9. It was shown in Zetsche et al.,  Cell,  163, 759-771, 2015 (the entire contents of which is incorporated herein by reference) that the RuvC-like domain of Cpf1 is responsible for cleaving both DNA strands and inactivation of the RuvC-like domain inactivates Cpf1 nuclease activity. For example, mutations corresponding to D917A, E1006A, or D1255A in  Francisella novicida  Cpf1 (SEQ ID NO: 714) inactivates Cpf1 nuclease activity. In some embodiments, the dCpf1 of the present disclosure comprises mutations corresponding to D917A, E1006A, D1255A, D917A/E1006A, D917A/D1255A, E1006A/D1255A, or D917A/E1006A/D1255A in SEQ ID NO: 714. It is to be understood that any mutations, e.g., substitution mutations, deletions, or insertions that inactivates the RuvC domain of Cpf1 may be used in accordance with the present disclosure. 
     In some embodiments, the guide nucleotide sequence-programmable DNA-binding protein domain of the present disclosure has no requirements for a PAM sequence. One example of such a guide nucleotide sequence-programmable DNA-binding protein may be an Argonaute protein from  Natronobacterium gregoryi  (NgAgo). NgAgo is a ssDNA-guided endonuclease. NgAgo binds 5′ phosphorylated ssDNA of ˜24 nucleotides (gDNA) to guide it to its target site and will make DNA double-strand breaks at gDNA site. In contrast to Cas9, the NgAgo-gDNA system does not require a protospacer-adjacent motif (PAM). Using a nuclease inactive NgAgo (dNgAgo) can greatly expand the codons that may be targeted. The characterization and use of NgAgo have been described in Gao et al.,  Nat Biotechnol. Epub  2016 May 2. PubMed PMID: 27136078; Swarts et al.,  Nature.  507(7491) (2014):258-61; and Swarts et al.,  Nucleic Acids Res.  43(10) (2015):5120-9, the entire contents of each of which are incorporated herein by reference. The sequence of  Natronobacterium gregoryi  Argonaute is provided in SEQ ID NO: 718. 
     Also provided herein are Cas9 variants that have relaxed PAM requirements (PAMless Cas9). PAMless Cas9 exhibits an increased activity on a target sequence that does not comprise a canonical PAM (NGG) at its 3′-end as compared to  Streptococcus pyogenes  Cas9 as provided by SEQ ID NO: 1, e.g., increased activity by at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, or at least 1,000,000-fold. Thus, the dCas9 or Cas9 nickase of the present disclosure may further comprise mutations that relax the PAM requirements, e.g., mutations that correspond to A262T, K294R, S409I, E480K, E543D, M694I, or E1219V in SEQ ID NO: 1. 
     It should be appreciated that additional Cas9 proteins (e.g., a nuclease dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease active Cas9), including variants and homologs thereof, are within the scope of this disclosure. Exemplary Cas9 proteins include, without limitation, those provided below. In some embodiments, the Cas9 protein is a nuclease dead Cas9 (dCas9). In some embodiments, the dCas9 comprises the amino acid sequence shown below. In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9). In some embodiments, the nCas9 comprises the amino acid sequence shown below. In some embodiments, the Cas9 protein is a nuclease active Cas9. In some embodiments, the nuclease active Cas9 comprises the amino acid sequence shown below. 
     
       
         
           
               
               
            
               
                 Exemplary catalytically inactive Cas9 (dCas9): 
                   
               
            
           
           
               
               
            
               
                 (SEQ ID NO: 752) 
                   
               
            
           
           
               
               
            
               
                 DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA 
                   
               
               
                   
               
               
                 EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERH 
               
               
                   
               
               
                 PIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDL 
               
               
                   
               
               
                 NPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGE 
               
               
                   
               
               
                 KKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADL 
               
               
                   
               
               
                 FLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKY 
               
               
                   
               
               
                 KEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTF 
               
               
                   
               
               
                 DNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW 
               
               
                   
               
               
                 MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVY 
               
               
                   
               
               
                 NELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS 
               
               
                   
               
               
                 VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL 
               
               
                   
               
               
                 KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN 
               
               
                   
               
               
                 FMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVK 
               
               
                   
               
               
                 VMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL 
               
               
                   
               
               
                 QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDK 
               
               
                   
               
               
                 NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK 
               
               
                   
               
               
                 RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV 
               
               
                   
               
               
                 REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK 
               
               
                   
               
               
                 ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM 
               
               
                   
               
               
                 PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFE 
               
               
                   
               
               
                 LENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ 
               
               
                   
               
               
                 HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA 
               
               
                   
               
               
                 PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 Exemplary Cas9 nickase (nCas9): 
               
            
           
           
               
               
            
               
                 (SEQ ID NO: 753) 
                   
               
            
           
           
               
               
            
               
                 DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA 
                   
               
               
                   
               
               
                 EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERH 
               
               
                   
               
               
                 PIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDL 
               
               
                   
               
               
                 NPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGE 
               
               
                   
               
               
                 KKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADL 
               
               
                   
               
               
                 FLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKY 
               
               
                   
               
               
                 KEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTF 
               
               
                   
               
               
                 DNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW 
               
               
                   
               
               
                 MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVY 
               
               
                   
               
               
                 NELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS 
               
               
                   
               
               
                 VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL 
               
               
                   
               
               
                 KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN 
               
               
                   
               
               
                 FMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVK 
               
               
                   
               
               
                 VMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL 
               
               
                   
               
               
                 QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDK 
               
               
                   
               
               
                 NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK 
               
               
                   
               
               
                 RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV 
               
               
                   
               
               
                 REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK 
               
               
                   
               
               
                 ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM 
               
               
                   
               
               
                 PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFE 
               
               
                   
               
               
                 LENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ 
               
               
                   
               
               
                 HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA 
               
               
                   
               
               
                 PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 Exemplary catalytically active Cas9: 
               
            
           
           
               
               
            
               
                 (SEQ ID NO: 754) 
                   
               
            
           
           
               
               
            
               
                 DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA 
                   
               
               
                   
               
               
                 EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERH 
               
               
                   
               
               
                 PIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDL 
               
               
                   
               
               
                 NPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGE 
               
               
                   
               
               
                 KKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADL 
               
               
                   
               
               
                 FLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKY 
               
               
                   
               
               
                 KEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTF 
               
               
                   
               
               
                 DNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW 
               
               
                   
               
               
                 MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVY 
               
               
                   
               
               
                 NELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS 
               
               
                   
               
               
                 VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL 
               
               
                   
               
               
                 KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN 
               
               
                   
               
               
                 FMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVK 
               
               
                   
               
               
                 VMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL 
               
               
                   
               
               
                 QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDK 
               
               
                   
               
               
                 NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK 
               
               
                   
               
               
                 RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV 
               
               
                   
               
               
                 REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK 
               
               
                   
               
               
                 ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM 
               
               
                   
               
               
                 PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFE 
               
               
                   
               
               
                 LENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ 
               
               
                   
               
               
                 HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA 
               
               
                   
               
               
                 PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
            
           
         
       
     
     In some embodiments, Cas9 refers to a Cas9 from arehaea (e.g. nanoarchaea), which constitute a domain and kingdom of single-celled prokaryotic microbes. In some embodiments, Cas9 refers to CasX or CasY, which have been described in, for example, Burstein et al., “New CRISPR-Cas systems from uncultivated microbes.” Cell Res. 2017 Feb. 21. doi: 10.1038/cr.2017.21, the entire contents of which is hereby incorporated by reference. Using genome-resolved metagenomics, a number of CRISPR-Cas systems were identified, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems were discovered, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. In some embodiments, Cas9 refers to CasX, or a variant of CasX. In some embodiments, Cas9 refers to a CasY, or a variant of CasY. It should be appreciated that other RNA-guided DNA binding proteins may be used as a guide nucleotide sequence-programmable DNA-binding protein, and are within the scope of this disclosure. 
     In some embodiments, the guide nucleotide sequence-programmable DNA-binding protein domain of any of the fusion proteins provided herein may be a CasX or CasY protein. In some embodiments, guide nucleotide sequence-programmable DNA-binding protein domain is a CasX protein. In some embodiments, the guide nucleotide sequence-programmable DNA-binding protein domain is a CasY protein. In some embodiments, the guide nucleotide sequence-programmable DNA-binding protein domain comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at ease 99.5% identical to a naturally-occurring CasX or CasY protein. In some embodiments, the guide nucleotide sequence-programmable DNA-binding protein domain is a naturally-occurring CasX or CasY protein. In some embodiments, the guide nucleotide sequence-programmable DNA-binding protein domain comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of of the exemplary CasX or CasY proteins described herein. In some embodiments, the guide nucleotide sequence-programmable DNA-binding protein domain comprises an amino acid sequence of any one of of the exemplary CasX or CasY proteins described herein. It should be appreciated that CasX and CasY from other bacterial species may also be used in accordance with the present disclosure. 
     
       
         
           
               
            
               
                 CasX (uniprot.org/uniprot/F0NN87; uniprot.org/ 
               
               
                 uniprot/F0NH53) 
               
               
                 &gt;tr|F0NN87|F0NN87_SULIH CRISPR-associated 
               
               
                 Casx protein OS =  Sulfolobus islandicus   
               
               
                 (strain HVE10/4) 
               
               
                 GN = SiH_0402 PE = 4 SV = 1 
               
            
           
           
               
            
               
                 (SEQ ID NO: 755) 
               
            
           
           
               
            
               
                 MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAK 
               
               
                   
               
               
                 NNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFP 
               
               
                   
               
               
                 TTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLE 
               
               
                   
               
               
                 VEPHYLIIAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNG 
               
               
                   
               
               
                 IVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGG 
               
               
                   
               
               
                 FSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG 
               
               
                   
               
               
                 SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG 
               
               
                   
               
               
                 &gt;tr|F0NH53|F0NH53_SULIR CRISPR associated protein, 
               
               
                 Casx OS =  Sulfolobus islandicus  (strain REY15A) 
               
               
                 GN = SiRe_0771 PE = 4 SV = 1 
               
            
           
           
               
            
               
                 (SEQ ID NO: 756) 
               
            
           
           
               
            
               
                 MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAK 
               
               
                   
               
               
                 NNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFP 
               
               
                   
               
               
                 TTVALSEVFKNFSQVKECEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLE 
               
               
                   
               
               
                 VEPHYLIMAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNG 
               
               
                   
               
               
                 IVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGG 
               
               
                   
               
               
                 FSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG 
               
               
                   
               
               
                 SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG 
               
               
                   
               
               
                 CasY (ncbi.nlm.nih.gov/protein/APG80656.1) 
               
               
                   
               
               
                 &gt;APG80656.1 CRISPR-associated protein CasY 
               
               
                 [uncultured  Parcubacteria group bacterium ] 
               
            
           
           
               
            
               
                 (SEQ ID NO: 757) 
               
            
           
           
               
            
               
                 MSKRHPRISGVKGYRLHAQRLEYTGKSGAMRTIKYPLYSSPSGGRTVPRE 
               
               
                   
               
               
                 IVSAINDDYVGLYGLSNFDDLYNAEKRNEEKVYSVLDFWYDCVQYGAVFS 
               
               
                   
               
               
                 YTAPGLLKNVAEVRGGSYELTKTLKGSHLYDELQIDKVIKFLNKKEISRA 
               
               
                   
               
               
                 NGSLDKLKKDIIDCFKAEYRERHKDQCNKLADDIKNAKKDAGASLGERQK 
               
               
                   
               
               
                 KLFRDFFGISEQSENDKPSFTNPLNLTCCLLPFDTVNNNRNRGEVLFNKL 
               
               
                   
               
               
                 KEYAQKLDKNEGSLEMWEYIGIGNSGTAFSNFLGEGFLGRLRENKITELK 
               
               
                   
               
               
                 KAMMDITDAWRGQEQEEELEKRLRILAALTIKLREPKFDNHWGGYRSDIN 
               
               
                   
               
               
                 GKLSSWLQNYINQTVKIKEDLKGHKKDLKKAKEMINRFGESDTKEEAVVS 
               
               
                   
               
               
                 SLLESIEKIVPDDSADDEKPDIPAIAIYRRFLSDGRLTLNRFVQREDVQE 
               
               
                   
               
               
                 ALIKERLEAEKKKKPKKRKKKSDAEDEKETIDFKELFPHLAKPLKLVPNF 
               
               
                   
               
               
                 YGDSKRELYKKYKNAAIYTDALWKAVEKIYKSAFSSSLKNSFFDTDFDKD 
               
               
                   
               
               
                 FFIKRLQKIFSVYRRFNTDKWKPIVKNSFAPYCDIVSLAENEVLYKPKQS 
               
               
                   
               
               
                 RSRKSAAIDKNRVRLPSTENIAKAGIALARELSVAGFDWKDLLKKEEHEE 
               
               
                   
               
               
                 YIDLIELHKTALALLLAVTETQLDISALDFVENGTVKDFMKTRDGNLVLE 
               
               
                   
               
               
                 GRFLEMFSQSIVFSELRGLAGLMSRKEFITRSAIQTMNGKQAELLYIPHE 
               
               
                   
               
               
                 FQSAKITTPKEMSRAFLDLAPAEFATSLEPESLSEKSLLKLKQMRYYPHY 
               
               
                   
               
               
                 FGYELTRTGQGIDGGVAENALRLEKSPVKKREIKCKQYKTLGRGQNKIVL 
               
               
                   
               
               
                 YVRSSYYQTQFLEWFLHRPKNVQTDVAVSGSFLIDEKKVKTRWNYDALTV 
               
               
                   
               
               
                 ALEPVSGSERVFVSQPFTIFPEKSAEEEGQRYLGIDIGEYGIAYTALEIT 
               
               
                   
               
               
                 GDSAKILDQNFISDPQLKTLREEVKGLKLDQRRGTFAMPSTKIARIRESL 
               
               
                   
               
               
                 VHSLRNRIHHLALKHKAKIVYELEVSRFEEGKQKIKKVYATLKKADVYSE 
               
               
                   
               
               
                 IDADKNLQTTVWGKLAVASEISASYTSQFCGACKKLWRAEMQVDETITTQ 
               
               
                   
               
               
                 ELIGTVRVIKGGTLIDAIKDFMRPPIFDENDTPFPKYRDFCDKHHISKKM 
               
               
                   
               
               
                 RGNSCLFICPFCRANADADIQASQTIALLRYVKEEKKVEDYFERFRKLKN 
               
               
                   
               
               
                 IKVLGQMKKI 
               
            
           
         
       
     
     The terms “conjugating,” “conjugated,” and “conjugation” refer to an association of two entities, for example, of two molecules such as two proteins, two domains (e.g., a binding domain and a cleavage domain), or a protein and an agent, e.g., a protein binding domain and a small molecule. In some aspects, the association is between a protein (e.g., RNA-programmable nuclease) and a nucleic acid (e.g., a guide RNA). The association can be, for example, via a direct or indirect (e.g., via a linker) covalent linkage. In some embodiments, the association is covalent. In some embodiments, two molecules are conjugated via a linker connecting both molecules. For example, in some embodiments where two proteins are conjugated to each other, e.g., a binding domain and a cleavage domain of an engineered nuclease, to form a protein fusion, the two proteins may be conjugated via a polypeptide linker, e.g., an amino acid sequence connecting the C-terminus of one protein to the N-terminus of the other protein. 
     The term “consensus sequence,” as used herein in the context of nucleic acid sequences, refers to a calculated sequence representing the most frequent nucleotide residues found at each position in a plurality of similar sequences. Typically, a consensus sequence is determined by sequence alignment in which similar sequences are compared to each other and similar sequence motifs are calculated. In the context of recombinase target site sequences, a consensus sequence of a recombinase target site may, in some embodiments, be the sequence most frequently bound, or bound with the highest affinity, by a given recombinase. 
     The term “engineered,” as used herein refers to a protein molecule, a nucleic acid, complex, substance, or entity that has been designed, produced, prepared, synthesized, and/or manufactured by a human. Accordingly, an engineered product is a product that does not occur in nature. 
     The term “effective amount,” as used herein, refers to an amount of a biologically active agent that is sufficient to elicit a desired biological response. In some embodiments, an effective amount of a recombinase may refer to the amount of the recombinase that is sufficient to induce recombination at a target site specifically bound and recombined by the recombinase. As will be appreciated by the skilled artisan, the effective amount of an agent, e.g., a nuclease, a recombinase, a hybrid protein, a fusion protein, a protein dimer, a complex of a protein (or protein dimer) and a polynucleotide, or a polynucleotide, may vary depending on various factors as, for example, on the desired biological response, the specific allele, genome, target site, cell, or tissue being targeted, and the agent being used. 
     A “guide nucleotide sequence-programmable DNA-binding protein,” as used herein, refers to a protein, a polypeptide, or a domain that is able to bind DNA, and the binding to its target DNA sequence is mediated by a guide nucleotide sequence. The “guide nucleotide” may be an RNA or DNA molecule (e.g., a single-stranded DNA or ssDNA molecule) that is complementary to the target sequence and can guide the DNA binding protein to the target sequence. As such, a guide nucleotide sequence-programmable DNA-binding protein may be a RNA-programmable DNA-binding protein, or an ssDNA-programmable DNA-binding protein. “Programmable” means the DNA-binding protein may be programmed to bind any DNA sequence that the guide nucleotide targets. The guide nucleotide sequence-programmable DNA-binding protein referred to herein may be any guide nucleotide sequence-programmable DNA-binding protein known in the art without limitation including, but not limited to, a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA-binding protein. The term “circularly permuted” refers to proteins in which the order of the amino acids in a protein has been altered, resulting in a protein structure with altered connectivity but a similar (overall) three-dimensional shape. Circular permutations are formed when the original n and c terminal amino acids are connected via a peptide bond; the peptide sequence is then broken in another location within the peptide sequence, causing a new n and c-terminus. Circular permutations may occur through a number of processes including evolutionary events, post-translational modifications, or artificially engineered mutations. For example, circular permutations may be used to improve the catalytic activity or thermostability of proteins. A circularly permuted guide nucleotide sequence-programmable DNA-binding protein may be used with any of the embodiments described herein. The term “bifurcated” typically refers to a monomeric protein that is split into two parts. Typically both parts are required for the function of the monomeric protein. Bifurcated proteins may or may not dimerize on their own to reconstitute a functional protein. Bifurcations may occur through a number of processes including evolutionary events, post-translational modifications, or artificially engineered mutations. Other protein domains, when fused to bifurcated domains, can be used to force the reassembly of the bifurcated protein. In some cases, protein domains, whose interaction depends on a small molecule, can be fused to each bifurcated domain, resulting in the small-molecule regulated dimerization of the bifurcated protein. 
     The term “homologous,” as used herein, is an art-understood term that refers to nucleic acids or polypeptides that are highly related at the level of nucleotide and/or amino acid sequence. Nucleic acids or polypeptides that are homologous to each other are termed “homologues.” Homology between two sequences can be determined by sequence alignment methods known to those of skill in the art. In accordance with the invention, two sequences are considered to be homologous if they are at least about 50-60% identical, e.g., share identical residues (e.g., amino acid residues) in at least about 50-60% of all residues comprised in one or the other sequence, at least about 70% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical, for at least one stretch of at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 150, or at least 200 amino acids. 
     The term “sequence identity” or “percent sequence identity” as used herein, may refer to the percentage of nucleic acid or amino acid residues within a given DNA or protein, respectively, that are identical to the reference sequence. See, for example: Christopher M. Holman, Protein Similarity Score: A Simplified Version of the BLAST Score as a Superior Alternative to Percent Identity for Claiming Genuses of Related Protein Sequences, 21 SANTA CLARA COMPUTER &amp; HIGH TECH. L. J. 55, 60 (2004), which is herein incorporated by reference in its entirety. 
     The term “linker,” as used herein, refers to a bond (e.g., covalent bond), chemical group, or a molecule linking two molecules or moieties, e.g., two domains of a fusion protein, such as, for example, a nuclease-inactive Cas9 domain and a nucleic acid-editing domain (e.g., an adenosine deaminase). In some embodiments, a linker joins a gRNA binding domain of an RNA-programmable nuclease, including a Cas9 nuclease domain, and the catalytic domain of a nucleic-acid editing protein. In some embodiments, a linker joins a dCas9 and a nucleic-acid editing protein. Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-100 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, a linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 7), which may also be referred to as the XTEN linker. In some embodiments, a linker comprises the amino acid sequence SGGS (SEQ ID NO: 758). In some embodiments, a linker comprises (SGGS) n  (SEQ ID NO: 758), (GGGS) n  (SEQ ID NO: 759), (GGGGS) n  (SEQ ID NO: 722), (G) n , (EAAAK). (SEQ ID NO: 723), (GGS) n , or (XP) n  motif, or a combination of any of these, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. 
     The term “mutation,” as used herein, refers to a substitution of a residue within a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or a deletion or insertion of one or more residues within a sequence. Mutations are typically described herein by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art, and are provided by, for example, Green and Sambrook,  Molecular Cloning: A Laboratory Manual  (4 th  ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)). 
     The term “nuclear localization sequence” or “NLS” refers to an amino acid sequence that promotes import of a protein into the cell nucleus, for example, by nuclear transport. Nuclear localization sequences are known in the art and would be apparent to the skilled artisan. For example, NLS sequences are described in Plank et al., international PCT application, PCT/EP2000/011690, filed Nov. 23, 2000, published as WO/2001/038547 on May 31, 2001, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In some embodiments, a NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 702) or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 761). 
     The term “nuclease,” as used herein, refers to an agent, for example, a protein, capable of cleaving a phosphodiester bond connecting two nucleotide residues in a nucleic acid molecule. In some embodiments, “nuclease” refers to a protein having an inactive DNA cleavage domain, such that the nuclease is incapable of cleaving a phosphodiester bond. In some embodiments, a nuclease is a protein, e.g., an enzyme that can bind a nucleic acid molecule and cleave a phosphodiester bond connecting nucleotide residues within the nucleic acid molecule. A nuclease may be an endonuclease, cleaving a phosphodiester bonds within a polynucleotide chain, or an exonuclease, cleaving a phosphodiester bond at the end of the polynucleotide chain. In some embodiments, a nuclease is a site-specific nuclease, binding and/or cleaving a specific phosphodiester bond within a specific nucleotide sequence, which is also referred to herein as the “recognition sequence,” the “nuclease target site,” or the “target site.” In some embodiments, a nuclease is a RNA-guided (i.e., RNA-programmable) nuclease, which is associated with (e.g., binds to) an RNA (e.g., a guide RNA, “gRNA”) having a sequence that complements a target site, thereby providing the sequence specificity of the nuclease. In some embodiments, a nuclease recognizes a single stranded target site, while in other embodiments, a nuclease recognizes a double-stranded target site, for example, a double-stranded DNA target site. The target sites of many naturally occurring nucleases, for example, many naturally occurring DNA restriction nucleases, are well known to those of skill in the art. A nuclease protein typically comprises a “binding domain” that mediates the interaction of the protein with the nucleic acid substrate, and also, in some cases, specifically binds to a target site, and a “cleavage domain” that catalyzes the cleavage of the phosphodiester bond within the nucleic acid backbone. In some embodiments a nuclease protein can bind and cleave a nucleic acid molecule in a monomeric form, while, in other embodiments, a nuclease protein has to dimerize or multimerize in order to cleave a target nucleic acid molecule. Binding domains and cleavage domains of naturally occurring nucleases, as well as modular binding domains and cleavage domains that can be fused to create nucleases binding specific target sites, are well known to those of skill in the art. For example, the binding domain of a guide nucleotide sequence-programmable DNA binding protein such as an RNA-programmable nucleases (e.g., Cas9), or a Cas9 protein having an inactive DNA cleavage domain, can be used as a binding domain (e.g., that binds a gRNA to direct binding to a target site) to specifically bind a desired target site, and fused or conjugated to a cleavage domain. 
     The terms “nucleic acid” and “nucleic acid molecule,” as used herein, refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides). In some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms “oligonucleotide” and “polynucleotide” can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, “nucleic acid” encompasses RNA as well as single and/or double-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, gRNA, plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA/RNA hybrid, or including non-naturally occurring nucleotides or nucleosides. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and/or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5′ to 3′ direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages). 
     The term “orthogonal” refers to biological components that interact minimally, if at all. Recombinase target sites containing different gRNA binding sites are orthogonal if the gRNA-directed recCas9 proteins do not interact, or interact minimally, with other potential recombinase sites. The term “orthogonality” refers to the idea that system components can be varied independently without affecting the performance of the other components. The gRNA directed nature of the complex makes the set of gRNA molecules complexed to recCas9 proteins capable of directing recombinase activity at only the gRNA-directed site. Orthogonality of the system is demonstrated by the complete or near complete dependence of the set of gRNA molecules on the enzymatic activity on a targeted recombinase site. 
     The term “pharmaceutical composition,” as used herein, refers to a composition that can be administrated to a subject in the context of treatment and/or prevention of a disease or disorder. In some embodiments, a pharmaceutical composition comprises an active ingredient, e.g., a recombinase fused to a Cas9 protein, or fragment thereof (or a nucleic acid encoding a such a fusion), and optionally a pharmaceutically acceptable excipient. In some embodiments, a pharmaceutical composition comprises inventive Cas9 variant/fusion (e.g., fCas9) protein(s) and gRNA(s) suitable for targeting the Cas9 variant/fusion protein(s) to a target nucleic acid. In some embodiments, the target nucleic acid is a gene. In some embodiments, the target nucleic acid is an allele associated with a disease, wherein the allele is cleaved by the action of the Cas9 variant/fusion protein(s). In some embodiments, the allele is an allele of the CLTA gene, the VEGF gene, the PCDH15, gene or the FAM19A2 gene. See, e.g., the Examples. 
     The term “proliferative disease,” as used herein, refers to any disease in which cell or tissue homeostasis is disturbed in that a cell or cell population exhibits an abnormally elevated proliferation rate. Proliferative diseases include hyperproliferative diseases, such as pre-neoplastic hyperplastic conditions and neoplastic diseases. Neoplastic diseases are characterized by an abnormal proliferation of cells and include both benign and malignant neoplasms. Malignant neoplasia is also referred to as cancer. In some embodiments, the compositions and methods provided herein are useful for treating a proliferative disease. For example, in some embodiments, pharmaceutical compositions comprising Cas9 (e.g., fCas9) protein(s) and gRNA(s) suitable for targeting the Cas9 protein(s) to an VEGF allele, wherein the allele is inactivated by the action of the Cas9 protein(s). See, e.g., the Examples. 
     The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein, and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. The term “fusion protein” as used herein refers to a hybrid polypeptide that comprises protein domains from at least two different proteins. One protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) protein thus forming an “amino-terminal fusion protein” or a “carboxy-terminal fusion protein,” respectively. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook,  Molecular Cloning: A Laboratory Manual  (4 th  ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference. A specific fusion protein referred to herein is recCas9, an RNA programmed small serine recombinase capable of functioning in mammalian cells created by fusion a catalytically inactive dCas9 to the catalytic domain of recombinase. 
     A “pseudo-gix” site or a “gix pseudo-site” as discussed herein is a specific pseudo-palindromic core DNA sequence that resembles the Gix recombinases&#39; natural DNA recognition sequence. See, for example, N. D. F. Grindley, K. L. Whiteson, P. A. Rice, Mechanisms of site-specific recombination.  Annu Rev Biochem  75, 567-605 (2006), which is incorporated by reference herein in its entirety. Similarly, a “pseudo-hix” or “hix-pseudo-site;” a “pseudo-six” or “six-pseudo site;” a “pseudo-resH” or “resH-pseudo-site;” “pseudo-res”or “res-pseudo-site;” “pseudo-LoxP” or “LoxP-pseudo-site;” “pseudo-att” or “att-pseudo-site;” “pseudo-FTR” or “FTR-pseudo-site” is a specific pseudo-palindromic core DNA sequence that resembles the Hin recombinase&#39;s, β recombinase&#39;s, Sin recombinase&#39;s, Tn3 or γδ recombinase&#39;s, Cre recombinase&#39;s, λ phage integrase&#39;s, or FLP recombinase&#39;s natural DNA recognition sequence. 
     The terms “RNA-programmable nuclease” and “RNA-guided nuclease” are used interchangeably herein and refer to a nuclease that forms a complex with (e.g., binds or associates with) one or more RNA that is not a target for cleavage. In some embodiments, an RNA-programmable nuclease, when in a complex with an RNA, may be referred to as a nuclease:RNA complex. Typically, the bound RNA(s) is referred to as a guide RNA (gRNA). gRNAs can exist as a complex of two or more RNAs, or as a single RNA molecule. gRNAs that exist as a single RNA molecule may be referred to as single-guide RNAs (sgRNAs), though “gRNA” is used interchangeabley to refer to guide RNAs that exist as either single molecules or as a complex of two or more molecules. Typically, gRNAs that exist as single RNA species comprise two domains: (1) a domain that shares homology to a target nucleic acid (e.g., and directs binding of a Cas9 complex to the target); and (2) a domain that binds a Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as a tracrRNA, and comprises a stem-loop structure. For example, in some embodiments, domain (2) is homologous to a tracrRNA as depicted in  FIG.  1 E  of Jinek et al.,  Science  337:816-821(2012), the entire contents of which is incorporated herein by reference. Other examples of gRNAs (e.g., those including domain 2) can be found in U.S. Provisional Patent Application Ser. No. 61/874,682, filed Sep. 6, 2013, entitled “Switchable Cas9 Nucleases And Uses Thereof;” U.S. Provisional Patent Application Ser. No. 61/874,746, filed Sep. 6, 2013, entitled “Delivery System For Functional Nucleases;” PCT Application WO 2013/176722, filed Mar. 15, 2013, entitled “Methods and Compositions for RNA-Directed Target DNA Modification and for RNA-Directed Modulation of Transcription;” and PCT Application WO 2013/142578, filed Mar. 20, 2013, entitled “RNA-Directed DNA Cleavage by the Cas9-crRNA Complex;” the entire contents of each are hereby incorporated by reference in their entirety. Still other examples of gRNAs are provided herein. See e.g., the Examples. In some embodiments, a gRNA comprises two or more of domains (1) and (2), and may be referred to as an “extended gRNA.” For example, an extended gRNA will e.g., bind two or more Cas9 proteins and bind a target nucleic acid at two or more distinct regions, as described herein. The gRNA comprises a nucleotide sequence that complements a target site, which mediates binding of the nuclease/RNA complex to said target site, providing the sequence specificity of the nuclease:RNA complex. In some embodiments, the guide nucleotide sequence-programmable DNA binding protein is an RNA-programmable nuclease such as the (CRISPR-associated system) Cas9 endonuclease, for example, Cas9 (Csn1) from  Streptococcus pyogenes  (see, e.g., “Complete genome sequence of an M1 strain of  Streptococcus pyogenes .” Ferretti J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S. W., Roe B. A., McLaughlin R. E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference. 
     Because RNA-programmable nucleases (e.g., Cas9) use RNA:DNA hybridization to determine target DNA cleavage sites, these proteins are able to cleave, in principle, any sequence specified by the guide RNA. Methods of using RNA-programmable nucleases, such as Cas9, for site-specific cleavage (e.g., to modify a genome) are known in the art (see e.g., Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems.  Science  339, 819-823 (2013); Mali, P. et al. RNA-guided human genome engineering via Cas9 . Science  339, 823-826 (2013); Hwang, W. Y. et al. Efficient genome editing in zebrafish using a CRISPR-Cas system.  Nature biotechnology  31, 227-229 (2013); Jinek, M. et al. RNA-programmed genome editing in human cells.  eLife  2, e00471 (2013); Dicarlo, J. E. et al. Genome engineering in  Saccharomyces cerevisiae  using CRISPR-Cas systems.  Nucleic acids research  (2013); Jiang, W. et al. RNA-guided editing of bacterial genomes using CRISPR-Cas systems.  Nature biotechnology  31, 233-239 (2013); the entire contents of each of which are incorporated herein by reference). 
     The term “recombinase,” as used herein, refers to a site-specific enzyme that mediates the recombination of DNA between recombinase recognition sequences, which results in the excision, integration, inversion, or exchange (e.g., translocation) of DNA fragments between the recombinase recognition sequences. Recombinases can be classified into two distinct families: serine recombinases (e.g., resolvases and invertases) and tyrosine recombinases (e.g., integrases). Examples of serine recombinases include, without limitation, Hin, Gin, Tn3, β-six, CinH, ParA, γδ, Bxb1, ϕC31, TP901, TG1, ϕBT1, R4, ϕRV1, ϕFC1, MR11, A118, U153, and gp29. Examples of tyrosine recombinases include, without limitation, Cre, FLP, R, Lambda, HK101, HK022, and pSAM2. The Gin recombinase referred to herein may be any Gin recombinase known in the art including, but not limited to, the Gin recombinases presented in T. Gaj et al., A comprehensive approach to zinc-finger recombinase customization enables genomic targeting in human cells.  Nucleic Acids Research  41, 3937-3946 (2013), incorporated herein by reference in its entirety. In certain embodiments, the Gin recombinase catalytic domain has greater than 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 713. In another embodiment, the amino acid sequence of the Gin recombinase catalytic domain comprises a mutation corresponding to H106Y, and/or I127L, and/or I136R and/or G137F. In yet another embodiment, the amino acid sequence of the Gin recombinase catalytic domain comprises a mutation corresponding to H106Y, I127L, I136R, and G137F. In a further embodiment, the amino acid sequence of the Gin recombinase has been further mutated. In a specific embodiment, the amino acid sequence of the Gin recombinase catalytic domain comprises SEQ ID NO: 713. Gin recombinases bind to gix target sites (also referred to herein as “gix core,” “minimal gix core,” or “gix-related core” sequences). The minimal gix core recombinase site is NNNNAAASSWWSSTTTNNNN (SEQ ID NO: 19), wherein N is defined as any amino acid, W is an A or a T, and S is a G or a C. The gix target site may include any other mutations known in the art. In certain embodiments, the gix target site has greater than 90%, 95%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 19. The distance between the gix core or gix-related core sequence and at least one gRNA binding site may be from 1 to 10 base pairs, from 3 to 7 base pairs, from 5 to 7 base pairs, or from 5 to 6 base pairs. The distance between the gix core or gix-related core sequence and at least one gRNA binding site may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base pairs. 
     The serine and tyrosine recombinase names stem from the conserved nucleophilic amino acid residue that the recombinase uses to attack the DNA and which becomes covalently linked to the DNA during strand exchange. Recombinases have numerous applications, including the creation of gene knockouts/knock-ins and gene therapy applications. See, e.g., Brown et al., “Serine recombinases as tools for genome engineering.”  Methods.  2011; 53(4):372-9; Hirano et al., “Site-specific recombinases as tools for heterologous gene integration.”  Appl. Microbiol. Biotechnol.  2011; 92(2):227-39; Chavez and Calos, “Therapeutic applications of the ΦC31 integrase system.”  Curr. Gene Ther.  2011; 11(5):375-81; Turan and Bode, “Site-specific recombinases: from tag-and-target- to tag-and-exchange-based genomic modifications.”  FASEB J.  2011; 25(12):4088-107; Venken and Bellen, “Genome-wide manipulations of  Drosophila melanogaster  with transposons, Flp recombinase, and ΦC31 integrase.”  Methods Mol. Biol.  2012; 859:203-28; Murphy, “Phage recombinases and their applications.”  Adv. Virus Res.  2012; 83:367-414; Zhang et al., “Conditional gene manipulation: Creating a new biological era.”  J. Zhejiang Univ. Sci. B.  2012; 13(7):511-24; Karpenshif and Bernstein, “From yeast to mammals: recent advances in genetic control of homologous recombination.”  DNA Repair  ( Amst ). 2012; 1; 11(10):781-8; the entire contents of each are hereby incorporated by reference in their entirety. The recombinases provided herein are not meant to be exclusive examples of recombinases that can be used in embodiments of the invention. The methods and compositions of the invention can be expanded by mining databases for new orthogonal recombinases or designing synthetic recombinases with defined DNA specificities (See, e.g., Groth et al., “Phage integrases: biology and applications.”  J. Mol. Biol.  2004; 335, 667-678; Gordley et al., “Synthesis of programmable integrases.”  Proc. Natl. Acad. Sci. USA.  2009; 106, 5053-5058; the entire contents of each are hereby incorporated by reference in their entirety). 
     Other examples of recombinases that are useful in the methods and compositions described herein are known to those of skill in the art, and any new recombinase that is discovered or generated is expected to be able to be used in the different embodiments of the invention. In some embodiments, the catalytic domains of a recombinase are fused to a nuclease-inactivated RNA-programmable nuclease (e.g., dCas9, or a fragment thereof), such that the recombinase domain does not comprise a nucleic acid binding domain or is unable to bind to a target nucleic acid that subsequently results in enzymatic catalysis (e.g., the recombinase domain is engineered such that it does not have specific DNA binding activity). Recombinases lacking part of their DNA binding activity and those that act independently of accessory proteins and methods for engineering such are known, and include those described by Klippel et al., “Isolation and characterisation of unusual gin mutants.”  EMBO J.  1988; 7: 3983-3989: Burke et al., “Activating mutations of Tn3 resolvase marking interfaces important in recombination catalysis and its regulation.  Mol Microbiol.  2004; 51: 937-948; Olorunniji et al., “Synapsis and catalysis by activated Tn3 resolvase mutants.”  Nucleic Acids Res.  2008; 36: 7181-7191; Rowland et al., “Regulatory mutations in Sin recombinase support a structure-based model of the synaptosome.”  Mol Microbiol.  2009; 74: 282-298; Akopian et al., “Chimeric recombinases with designed DNA sequence recognition.”  Proc Natl Acad Sci USA.  2003; 100: 8688-8691; Gordley et al., “Evolution of programmable zinc finger-recombinases with activity in human cells.  J Mol Biol.  2007; 367: 802-813; Gordley et al., “Synthesis of programmable integrases.”  Proc Natl Acad Sci USA.  2009; 106: 5053-5058; Arnold et al., “Mutants of Tn3 resolvase which do not require accessory binding sites for recombination activity.”  EMBO J.  1999; 18: 1407-1414; Gaj et al., “Structure-guided reprogramming of serine recombinase DNA sequence specificity.”  Proc Natl Acad Sci USA.  2011; 108(2):498-503; and Proudfoot et al., “Zinc finger recombinases with adaptable DNA sequence specificity.”  PLoS One.  2011; 6(4):e19537; the entire contents of each are hereby incorporated by reference. For example, serine recombinases of the resolvase-invertase group, e.g., Tn3 and γδ resolvases and the Hin and Gin invertases, have modular structures with partly autonomous catalytic and DNA-binding domains (See, e.g., Grindley et al., “Mechanism of site-specific recombination.”  Ann Rev Biochem.  2006; 75: 567-605, the entire contents of which are incorporated by reference). The catalytic domains of these recombinases are therefore amenable to being recombined with nuclease-inactivated RNA-programmable nucleases (e.g., dCas9, or a fragment thereof) as described herein, e.g., following the isolation of ‘activated’ recombinase mutants which do not require any accessory factors (e.g., DNA binding activities) (See, e.g., Klippel et al., “Isolation and characterisation of unusual gin mutants.”  EMBO J.  1988; 7: 3983-3989: Burke et al., “Activating mutations of Tn3 resolvase marking interfaces important in recombination catalysis and its regulation.  Mol Microbiol.  2004; 51: 937-948; Olorunniji et al., “Synapsis and catalysis by activated Tn3 resolvase mutants.”  Nucleic Acids Res.  2008; 36: 7181-7191; Rowland et al., “Regulatory mutations in Sin recombinase support a structure-based model of the synaptosome.”  Mol Microbiol.  2009; 74: 282-298; Akopian et al., “Chimeric recombinases with designed DNA sequence recognition.”  Proc Natl Acad Sci USA.  2003; 100: 8688-8691). 
     Additionally, many other natural serine recombinases having an N-terminal catalytic domain and a C-terminal DNA binding domain are known (e.g., phiC31 integrase, TnpX transposase, IS607 transposase), and their catalytic domains can be co-opted to engineer programmable site-specific recombinases as described herein (See, e.g., Smith et al., “Diversity in the serine recombinases.”  Mol Microbiol.  2002; 44: 299-307, the entire contents of which are incorporated by reference). Similarly, the core catalytic domains of tyrosine recombinases (e.g., Cre, λ integrase) are known, and can be similarly co-opted to engineer programmable site-specific recombinases as described herein (See, e.g., Guo et al., “Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse.”  Nature.  1997; 389:40-46; Hartung et al., “Cre mutants with altered DNA binding properties.”  J Biol Chem  1998; 273:22884-22891; Shaikh et al., “Chimeras of the Flp and Cre recombinases: Tests of the mode of cleavage by Flp and Cre.  J Mol Biol.  2000; 302:27-48; Rongrong et al., “Effect of deletion mutation on the recombination activity of Cre recombinase.”  Acta Biochim Pol.  2005; 52:541-544; Kilbride et al., “Determinants of product topology in a hybrid Cre-Tn3 resolvase site-specific recombination system.”  J Mol Biol.  2006; 355:185-195; Warren et al., “A chimeric cre recombinase with regulated directionality.”  Proc Natl Acad Sci USA.  2008 105:18278-18283; Van Duyne, “Teaching Cre to follow directions.”  Proc Natl Acad Sci USA.  2009 Jan. 6; 106(1):4-5; Numrych et al., “A comparison of the effects of single-base and triple-base changes in the integrase arm-type binding sites on the site-specific recombination of bacteriophage λ.”  Nucleic Acids Res.  1990; 18:3953-3959; Tirumalai et al., “The recognition of core-type DNA sites by λ integrase.”  J Mol Biol.  1998; 279:513-527; Aihara et al., “A conformational switch controls the DNA cleavage activity of k integrase.”  Mol Cell.  2003; 12:187-198; Biswas et al., “A structural basis for allosteric control of DNA recombination by k integrase.”  Nature.  2005; 435:1059-1066; and Warren et al., “Mutations in the amino-terminal domain of λ-integrase have differential effects on integrative and excisive recombination.”  Mol Microbiol.  2005; 55:1104-1112; the entire contents of each are incorporated by reference). 
     The term “recombine” or “recombination,” in the context of a nucleic acid modification (e.g., a genomic modification), is used to refer to the process by which two or more nucleic acid molecules, or two or more regions of a single nucleic acid molecule, are modified by the action of a recombinase protein (e.g., an inventive recombinase fusion protein provided herein). Recombination can result in, inter alia, the insertion, inversion, excision, or translocation of nucleic acids, e.g., in or between one or more nucleic acid molecules. 
     The term “recombinant” as used herein in the context of proteins or nucleic acids refers to proteins or nucleic acids that do not occur in nature, but are the product of human engineering. For example, in some embodiments, a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence that comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations as compared to any naturally occurring sequence. 
     The term “subject,” as used herein, refers to an individual organism, for example, an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, a goat, a cattle, a cat, or a dog. In some embodiments, the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either sex and at any stage of development. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either sex and at any stage of development. 
     The terms “target nucleic acid,” and “target genome,” as used herein in the context of nucleases, refer to a nucleic acid molecule or a genome, respectively, that comprises at least one target site of a given nuclease. In the context of fusions comprising a (nuclease-inactivated) RNA-programmable nuclease and a recombinase domain, a “target nucleic acid” and a “target genome” refers to one or more nucleic acid molecule(s), or a genome, respectively, that comprises at least one target site. In some embodiments, the target nucleic acid(s) comprises at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight target sites. In some embodiments, the target nucleic acid(s) comprise four target sites. 
     The term “target site” refers to a sequence within a nucleic acid molecule that is bound and recombined (e.g., at or nearby the target site) by a recombinase (e.g., a dCas9-recombinase fusion protein provided herein). A target site may be single-stranded or double-stranded. For example, in some embodiments, four recombinase monomers are coordinated to recombine a target nucleic acid(s), each monomer being fused to a (nuclease-inactivated) Cas9 protein guided by a gRNA. In such an example, each Cas9 domain is guided by a distinct gRNA to bind a target nucleic acid(s), thus the target nucleic acid comprises four target sites, each site targeted by a separate dCas9-recombinase fusion (thereby coordinating four recombinase monomers which recombine the target nucleic acid(s)). For the RNA-guided nuclease-inactivated Cas9 (or gRNA-binding domain thereof) and inventive fusions of Cas9, the target site may be, in some embodiments, 17-20 base pairs plus a 3 base pair PAM (e.g., NNN, wherein N independently represents any nucleotide). Typically, the first nucleotide of a PAM can be any nucleotide, while the two downstream nucleotides are specified depending on the specific RNA-guided nuclease. Exemplary target sites (e.g., comprising a PAM) for RNA-guided nucleases, such as Cas9, are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, and NGG, wherein each N is independently any nucleotide. In addition, Cas9 nucleases from different species (e.g.,  S. thermophilus  instead of  S. pyogenes ) recognize a PAM that comprises the sequence NGGNG (SEQ ID NO: 763). Additional PAM sequences are known, including, but not limited to, NNAGAAW (SEQ ID NO: 749) and NAAR (SEQ ID NO: 771) (see, e.g., Esvelt and Wang, Molecular Systems Biology, 9:641 (2013), the entire contents of which are incorporated herein by reference). In some aspects, the target site of an RNA-guided nuclease, such as, e.g., Cas9, may comprise the structure [N Z ]-[PAM], where each N is independently any nucleotide, and z is an integer between 1 and 50, inclusive. In some embodiments, z is 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 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 25, at least 30, at least 35, at least 40, at least 45, or at least 50. In some embodiments, z is 5, 6, 7, 8, 9, 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, or 50. In some embodiments, z is 20. In certain embodiments, a “PAMless” RNA-guided nuclease (e.g., a Pamless Cas9) or an RNA-guided nuclease with relaxed PAM requirements as further described herein may be used. In some embodiments, “target site” may also refer to a sequence within a nucleic acid molecule that is bound but not cleaved by a nuclease. For example, certain embodiments described herein provide proteins comprising an inactive (or inactivated) Cas9 DNA cleavage domain. Such proteins (e.g., when also including a Cas9 RNA binding domain) are able to bind the target site specified by the gRNA; however, because the DNA cleavage site is inactivated, the target site is not cleaved by the particular protein. In some embodiments, such proteins are conjugated, fused, or bound to a recombinase (or a catalytic domain of a recombinase), which mediates recombination of the target nucleic acid. In some embodiments, the sequence actually cleaved or recombined will depend on the protein (e.g., recombinase) or molecule that mediates cleavage or recombination of the nucleic acid molecule, and in some cases, for example, will relate to the proximity or distance from which the inactivated Cas9 protein(s) is/are bound. 
     The term “Transcriptional Activator-Like Effector,” (TALE) as used herein, refers to bacterial proteins comprising a DNA binding domain, which contains a highly conserved 33-34 amino acid sequence comprising a highly variable two-amino acid motif (Repeat Variable Diresidue, RVD). The RVD motif determines binding specificity to a nucleic acid sequence and can be engineered according to methods known to those of skill in the art to specifically bind a desired DNA sequence (see, e.g., Miller, Jeffrey; et. al. (February 2011). “A TALE nuclease architecture for efficient genome editing”.  Nature Biotechnology  29 (2): 143-8; Zhang, Feng; et. al. (February 2011). “Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription”  Nature Biotechnology  29 (2): 149-53; Geiβler, R.; Scholze, H.; Hahn, S.; Streubel, J.; Bonas, U.; Behrens, S. E.; Boch, J. (2011), Shiu, Shin-Han. ed. “Transcriptional Activators of Human Genes with Programmable DNA-Specificity”.  PLoS ONE  6 (5): e19509; Boch, Jens (February 2011). “TALEs of genome targeting”.  Nature Biotechnology  29 (2): 135-6; Boch, Jens; et. al. (December 2009). “Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors”.  Science  326 (5959): 1509-12; and Moscou, Matthew J.; Adam J. Bogdanove (December 2009). “A Simple Cipher Governs DNA Recognition by TAL Effectors”  Science  326 (5959): 1501; the entire contents of each of which are incorporated herein by reference). The simple relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA binding domains by selecting a combination of repeat segments containing the appropriate RVDs. 
     The term “Transcriptional Activator-Like Element Nuclease,” (TALEN) as used herein, refers to an artificial nuclease comprising a transcriptional activator-like effector DNA binding domain to a DNA cleavage domain, for example, a FokI domain. A number of modular assembly schemes for generating engineered TALE constructs have been reported (see e.g., Zhang, Feng; et. al. (February 2011). “Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription”.  Nature Biotechnology  29 (2): 149-53; Geiβler, R.; Scholze, H.; Hahn, S.; Streubel, J.; Bonas, U.; Behrens, S. E.; Boch, J. (2011), Shiu, Shin-Han. ed. “Transcriptional Activators of Human Genes with Programmable DNA-Specificity”.  PLoS ONE  6 (5): e19509; Cermak, T.; Doyle, E. L.; Christian, M.; Wang, L.; Zhang, Y.; Schmidt, C.; Baller, J. A.; Somia, N. V. et al. (2011). “Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting”.  Nucleic Acids Research ; Morbitzer, R.; Elsaesser, J.; Hausner, J.; Lahaye, T. (2011). “Assembly of custom TALE-type DNA binding domains by modular cloning”.  Nucleic Acids Research ; Li, T.; Huang, S.; Zhao, X.; Wright, D. A.; Carpenter, S.; Spalding, M. H.; Weeks, D. P.; Yang, B. (2011). “Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes”.  Nucleic Acids Research .; Weber, E.; Gruetzner, R.; Werner, S.; Engler, C.; Marillonnet, S. (2011). Bendahmane, Mohammed. ed. “Assembly of Designer TAL Effectors by Golden Gate Cloning”.  PLoS ONE  6 (5): e19722; the entire contents of each of which are incorporated herein by reference). 
     The terms “treatment,” “treat,” and “treating,” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. As used herein, the terms “treatment,” “treat,” and “treating” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed and/or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence. 
     The term “vector” refers to a polynucleotide comprising one or more recombinant polynucleotides of the present invention, e.g., those encoding a Cas9 protein (or fusion thereof) and/or gRNA provided herein. Vectors include, but are not limited to, plasmids, viral vectors, cosmids, artificial chromosomes, and phagemids. The vector may be able to replicate in a host cell and may further be characterized by one or more endonuclease restriction sites at which the vector may be cut and into which a desired nucleic acid sequence may be inserted. Vectors may contain one or more marker sequences suitable for use in the identification and/or selection of cells which have or have not been transformed or genomically modified with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics (e.g., kanamycin, ampicillin) or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., β-galactosidase, alkaline phosphatase, or luciferase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies, or plaques. Any vector suitable for the transformation of a host cell (e.g.,  E. coli , mammalian cells such as CHO cell, insect cells, etc.) as embraced by the present invention, for example, vectors belonging to the pUC series, pGEM series, pET series, pBAD series, pTET series, or pGEX series. In some embodiments, the vector is suitable for transforming a host cell for recombinant protein production. Methods for selecting and engineering vectors and host cells for expressing proteins (e.g., those provided herein), transforming cells, and expressing/purifying recombinant proteins are well known in the art, and are provided by, for example, Green and Sambrook,  Molecular Cloning: A Laboratory Manual  (4 th  ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)). 
     The term “zinc finger,” as used herein, refers to a small nucleic acid-binding protein structural motif characterized by a fold and the coordination of one or more zinc ions that stabilize the fold. Zinc fingers encompass a wide variety of differing protein structures (see, e.g., Klug A, Rhodes D (1987). “Zinc fingers: a novel protein fold for nucleic acid recognition”.  Cold Spring Harb. Symp. Quant. Biol.  52: 473-82, the entire contents of which are incorporated herein by reference). Zinc fingers can be designed to bind a specific sequence of nucleotides, and zinc finger arrays comprising fusions of a series of zinc fingers, can be designed to bind virtually any desired target sequence. Such zinc finger arrays can form a binding domain of a protein, for example, of a nuclease, e.g., if conjugated to a nucleic acid cleavage domain. Different types of zinc finger motifs are known to those of skill in the art, including, but not limited to, Cys 2 His 2 , Gag knuckle, Treble clef, Zinc ribbon, Zn 2 /Cys 6 , and TAZ2 domain-like motifs (see, e.g., Krishna S S, Majumdar I, Grishin N V (January 2003). “Structural classification of zinc fingers: survey and summary”.  Nucleic Acids Res.  31 (2): 532-50). Typically, a single zinc finger motif binds 3 or 4 nucleotides of a nucleic acid molecule. Accordingly, a zinc finger domain comprising 2 zinc finger motifs may bind 6-8 nucleotides, a zinc finger domain comprising 3 zinc finger motifs may bind 9-12 nucleotides, a zinc finger domain comprising 4 zinc finger motifs may bind 12-16 nucleotides, and so forth. Any suitable protein engineering technique can be employed to alter the DNA-binding specificity of zinc fingers and/or design novel zinc finger fusions to bind virtually any desired target sequence from 3-30 nucleotides in length (see, e.g., Pabo C O, Peisach E, Grant R A (2001). “Design and selection of novel cys2His2 Zinc finger proteins”.  Annual Review of Biochemistry  70: 313-340; Jamieson A C, Miller J C, Pabo C O (2003). “Drug discovery with engineered zinc-finger proteins”.  Nature Reviews Drug Discovery  2 (5): 361-368; and Liu Q, Segal D J, Ghiara J B, Barbas C F (May 1997). “Design of polydactyl zinc-finger proteins for unique addressing within complex genomes”.  Proc. Natl. Acad. Sci. U.S.A.  94 (11); the entire contents of each of which are incorporated herein by reference). Fusions between engineered zinc finger arrays and protein domains that cleave a nucleic acid can be used to generate a “zinc finger nuclease.” A zinc finger nuclease typically comprises a zinc finger domain that binds a specific target site within a nucleic acid molecule, and a nucleic acid cleavage domain that cuts the nucleic acid molecule within or in proximity to the target site bound by the binding domain. Typical engineered zinc finger nucleases comprise a binding domain having between 3 and 6 individual zinc finger motifs and binding target sites ranging from 9 base pairs to 18 base pairs in length. Longer target sites are particularly attractive in situations where it is desired to bind and cleave a target site that is unique in a given genome. 
     The term “zinc finger nuclease,” as used herein, refers to a nuclease comprising a nucleic acid cleavage domain conjugated to a binding domain that comprises a zinc finger array. In some embodiments, the cleavage domain is the cleavage domain of the type II restriction endonuclease FokI. Zinc finger nucleases can be designed to target virtually any desired sequence in a given nucleic acid molecule for cleavage, and the possibility to design zinc finger binding domains to bind unique sites in the context of complex genomes allows for targeted cleavage of a single genomic site in living cells, for example, to achieve a targeted genomic alteration of therapeutic value. Targeting a double-strand break to a desired genomic locus can be used to introduce frame-shift mutations into the coding sequence of a gene due to the error-prone nature of the non-homologous DNA repair pathway. Zinc finger nucleases can be generated to target a site of interest by methods well known to those of skill in the art. For example, zinc finger binding domains with a desired specificity can be designed by combining individual zinc finger motifs of known specificity. The structure of the zinc finger protein Zif268 bound to DNA has informed much of the work in this field and the concept of obtaining zinc fingers for each of the 64 possible base pair triplets and then mixing and matching these modular zinc fingers to design proteins with any desired sequence specificity has been described (Pavletich N P, Pabo C O (May 1991). “Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A”.  Science  252 (5007): 809-17, the entire contents of which are incorporated herein). In some embodiments, separate zinc fingers that each recognizes a 3 base pair DNA sequence are combined to generate 3-, 4-, 5-, or 6-finger arrays that recognize target sites ranging from 9 base pairs to 18 base pairs in length. In some embodiments, longer arrays are contemplated. In other embodiments, 2-finger modules recognizing 6-8 nucleotides are combined to generate 4-, 6-, or 8-zinc finger arrays. In some embodiments, bacterial or phage display is employed to develop a zinc finger domain that recognizes a desired nucleic acid sequence, for example, a desired nuclease target site of 3-30 bp in length. Zinc finger nucleases, in some embodiments, comprise a zinc finger binding domain and a cleavage domain fused or otherwise conjugated to each other via a linker, for example, a polypeptide linker. The length of the linker determines the distance of the cut from the nucleic acid sequence bound by the zinc finger domain. If a shorter linker is used, the cleavage domain will cut the nucleic acid closer to the bound nucleic acid sequence, while a longer linker will result in a greater distance between the cut and the bound nucleic acid sequence. In some embodiments, the cleavage domain of a zinc finger nuclease has to dimerize in order to cut a bound nucleic acid. In some such embodiments, the dimer is a heterodimer of two monomers, each of which comprise a different zinc finger binding domain. For example, in some embodiments, the dimer may comprise one monomer comprising zinc finger domain A conjugated to a FokI cleavage domain, and one monomer comprising zinc finger domain B conjugated to a FokI cleavage domain. In this non-limiting example, zinc finger domain A binds a nucleic acid sequence on one side of the target site, zinc finger domain B binds a nucleic acid sequence on the other side of the target site, and the dimerize FokI domain cuts the nucleic acid in between the zinc finger domain binding sites. 
     DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION 
     The function and advantage of these and other embodiments of the present invention will be more fully understood from the Examples below. The following Examples are intended to illustrate the benefits of the present invention and to describe particular embodiments, but are not intended to exemplify the full scope of the invention. Accordingly, it will be understood that the Examples are not meant to limit the scope of the invention. 
     Guide Nucleotide Sequence-Programmable DNA Binding Protein 
     The fusion proteins and methods described herein may use any programmable DNA binding domain. 
     In some embodiments, the programmable DNA binding protein domain comprises the DNA binding domain of a zinc finger nuclease (ZFN) or a transcription activator-like effector domain (TALE). In some embodiments, the programmable DNA binding protein domain may be programmed by a guide nucleotide sequence and is thus referred as a “guide nucleotide sequence-programmable DNA binding-protein domain.” In some embodiments, the guide nucleotide sequence-programmable DNA binding protein is a nuclease inactive Cas9, or dCas9. A dCas9, as used herein, encompasses a Cas9 that is completely inactive in its nuclease activity, or partially inactive in its nuclease activity (e.g., a Cas9 nickase). Thus, in some embodiments, the guide nucleotide sequence-programmable DNA binding protein is a Cas9 nickase. In some embodiments, the guide nucleotide sequence-programmable DNA binding protein is a nuclease inactive Cpf1. In some embodiments, the guide nucleotide sequence-programmable DNA binding protein is a nuclease inactive Argonaute. 
     In some embodiments, the guide nucleotide sequence-programmable DNA binding protein is a dCas9 domain. In some embodiments, the guide nucleotide sequence-programmable DNA binding protein is a Cas9 nickase. In some embodiments, the dCas9 domain comprises an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the dCas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the Cas9 domains provided herein, and comprises mutations corresponding to D10X (X is any amino acid except for D) and/or H840X (X is any amino acid except for H) in SEQ ID NO: 1. In some embodiments, the dCas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the Cas9 domains provided herein, and comprises mutations corresponding to D10A and/or H840A in SEQ ID NO: 1. In some embodiments, the Cas9 nickase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the Cas9 domains provided herein, and comprises mutations corresponding to D10X (X is any amino acid except for D) in SEQ ID NO: 1 and a histidine at a position correspond to position 840 in SEQ ID NO: 1. In some embodiments, the Cas9 nickase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the Cas9 domains provided herein, and comprises mutations corresponding to D10A in SEQ ID NO: 1 and a histidine at a position correspond to position 840 in SEQ ID NO: 1. In some embodiments, variants or homologues of dCas9 or Cas9 nickase (e.g., variants of SEQ ID NO: 2 or SEQ ID NO: 3, respectively) are provided which are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to SEQ ID NO: 2 or SEQ ID NO: 3, respectively, and comprises mutations corresponding to D10A and/or H840A in SEQ ID NO: 1. In some embodiments, variants of Cas9 (e.g., variants of SEQ ID NO: 2) are provided having amino acid sequences which are shorter, or longer than SEQ ID NO: 2, by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids, or more, provided that the dCas9 variants comprise mutations corresponding to D10A and/or H840A in SEQ ID NO: 1. In some embodiments, variants of Cas9 nickase (e.g., variants of SEQ ID NO: 3) are provided having amino acid sequences which are shorter, or longer than SEQ ID NO: 3, by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids, or more, provided that the dCas9 variants comprise mutations corresponding to D10A and comprises a histidine at a position corresponding to position 840 in SEQ ID NO: 1. 
     Additional suitable nuclease-inactive dCas9 domains will be apparent to those of skill in the art based on this disclosure and knowledge in the field, and are within the scope of this disclosure. Such additional exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, D10A/H840A, D10A/D839A/H840A, D10A/D839A/H840A/N863A mutant domains in SEQ ID NO: 1 (See, e.g., Prashant et al., Nature Biotechnology. 2013; 31(9): 833-838, which is incorporated herein by reference), or K603R (See, e.g., Chavez et al., Nature Methods 12, 326-328, 2015, which is incorporated herein by reference). 
     In some embodiments, the nucleobase editors described herein comprise a Cas9 domain with decreased electrostatic interactions between the Cas9 domain and a sugar-phosphate backbone of a DNA, as compared to a wild-type Cas9 domain. In some embodiments, a Cas9 domain comprises one or more mutations that decreases the association between the Cas9 domain and a sugar-phosphate backbone of a DNA. In some embodiments, the nucleobase editors described herein comprises a dCas9 (e.g., with D10A and H840A mutations in SEQ ID NO: 1) or a Cas9 nickase (e.g., with D10A mutation in SEQ ID NO: 1), wherein the dCas9 or the Cas9 nickase further comprises one or more of a N497X, a R661X, a Q695X, and/or a Q926X mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, wherein X is any amino acid. In some embodiments, the nucleobase editors described herein comprises a dCas9 (e.g., with D10A and H840A mutations in SEQ ID NO: 1) or a Cas9 nickase (e.g., with D10A mutation in SEQ ID NO: 1), wherein the dCas9 or the Cas9 nickase further comprises one or more of a N497A, a R661A, a Q695A, and/or a Q926A mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the Cas9 domain (e.g., of any of the nucleobase editors provided herein) comprises the amino acid sequence as set forth in SEQ ID NO: 720. In some embodiments, the nucleobase editor comprises the amino acid sequence as set forth in SEQ ID NO: 721. Cas9 domains with high fidelity are known in the art and would be apparent to the skilled artisan. For example, Cas9 domains with high fidelity have been described in Kleinstiver, B. P., et al. “High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.”  Nature  529, 490-495 (2016); and Slaymaker, I. M., et al. “Rationally engineered Cas9 nucleases with improved specificity.”  Science  351, 84-88 (2015); the entire contents of each are incorporated herein by reference. 
     
       
         
           
               
               
            
               
                 Cas9 variant with decreased electrostatic interactions 
                   
               
               
                 between the Cas9 and DNA backbone 
               
            
           
           
               
               
            
               
                 (SEQ ID NO: 720) 
                   
               
            
           
           
               
               
            
               
                 DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA 
                   
               
               
                   
               
               
                 EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERH 
               
               
                   
               
               
                 PIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDL 
               
               
                   
               
               
                 NPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGE 
               
               
                   
               
               
                 KKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADL 
               
               
                   
               
               
                 FLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKY 
               
               
                   
               
               
                 KEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTF 
               
               
                   
               
               
                 DNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW 
               
               
                   
               
               
                 MTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEYFTVY 
               
               
                   
               
               
                 NELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS 
               
               
                   
               
               
                 VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL 
               
               
                   
               
               
                 KTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRN 
               
               
                   
               
               
                 FMALIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVK 
               
               
                   
               
               
                 VMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL 
               
               
                   
               
               
                 QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDK 
               
               
                   
               
               
                 NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK 
               
               
                   
               
               
                 RQLVETRAITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV 
               
               
                   
               
               
                 REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK 
               
               
                   
               
               
                 ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM 
               
               
                   
               
               
                 PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFE 
               
               
                   
               
               
                 LENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ 
               
               
                   
               
               
                 HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA 
               
               
                   
               
               
                 PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 High fidelity nucleobase editor 
               
            
           
           
               
               
            
               
                 (SEQ ID NO: 721) 
                   
               
            
           
           
               
               
            
               
                 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNT 
                   
               
               
                   
               
               
                 NKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIAR 
               
               
                   
               
               
                 LYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLW 
               
               
                   
               
               
                 VRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKSGSET 
               
               
                   
               
               
                 PGTSESATPESDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI 
               
               
                   
               
               
                 GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFL 
               
               
                   
               
               
                 VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK 
               
               
                   
               
               
                 FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRR 
               
               
                   
               
               
                 LENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNL 
               
               
                   
               
               
                 LAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLK 
               
               
                   
               
               
                 ALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLN 
               
               
                   
               
               
                 REDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGP 
               
               
                   
               
               
                 LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPK 
               
               
                   
               
               
                 HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKE 
               
               
                   
               
               
                 DYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLF 
               
               
                   
               
               
                 EDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFL 
               
               
                   
               
               
                 KSDGFANRNFMALIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTV 
               
               
                   
               
               
                 KVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKE 
               
               
                   
               
               
                 HPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN 
               
               
                   
               
               
                 KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLS 
               
               
                   
               
               
                 ELDKAGFIKRQLVETRAITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFR 
               
               
                   
               
               
                 KDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI 
               
               
                   
               
               
                 AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFA 
               
               
                   
               
               
                 TVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPT 
               
               
                   
               
               
                 VAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLII 
               
               
                   
               
               
                 KLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDN 
               
               
                   
               
               
                 EQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH 
               
               
                   
               
               
                 LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
            
           
         
       
     
     The Cas9 protein recognizes a short motif (PAM motif) within the target DNA sequence, which is required for the Cas9-DNA interaction but that is not determined by complementarity to the guide RNA nucleotide sequence. A “PAM motif” or “protospacer adjacent motif,” as used herein, refers to a DNA sequence adjacent to the 5′- or 3′-immediately following the DNA sequence that is complementary to the guide RNA oligonucleotide sequence. Cas9 will not successfully bind to, cleave, or nick the target DNA sequence if it is not followed by an appropriate PAM sequence. Without wishing to be bound by any particular theory, specific amino acid residues in the Cas9 enzyme are responsible for interacting with the bases of the PAM and determine the PAM specificity. Therefore, changes in these residues or nearby residues leads to a different or relaxed PAM specificity. Changing or relaxing the PAM specificity may shift the places where Cas9 can bind, as will be apparent to those of skill in the art based on the instant disclosure. 
     Wild-type  Streptococcus pyogenes  Cas9 recognizes a canonical PAM sequence (5′-NGG-3′). Other Cas9 nucleases (e.g., Cas9 from  Streptococcus thermophiles, Staphylococcus aureus, Neisseria meningitidis , or  Treponema denticolaor ) and Cas9 variants thereof have been described in the art to have different, or more relaxed PAM requirements. For example, in Kleinstiver et al., Nature 523, 481-485, 2015; Klenstiver et al., Nature 529, 490-495, 2016; Ran et al., Nature, April 9; 520(7546): 186-191, 2015; Kleinstiver et al., Nat Biotechnol, 33(12):1293-1298, 2015; Hou et al., Proc Natl Acad Sci USA, 110(39):15644-9, 2014; Prykhozhij et al., PLoS One, 10(3): e0119372, 2015; Zetsche et al., Cell 163, 759-771, 2015; Gao et al., Nature Biotechnology, doi:10.1038/nbt.3547, 2016; Want et al., Nature 461, 754-761, 2009; Chavez et al., doi: dx.doi dot org/10.1101/058974; Fagerlund et al., Genome Biol. 2015; 16: 25, 2015; Zetsche et al., Cell, 163, 759-771, 2015; and Swarts et al., Nat Struct Mol Biol, 21(9):743-53, 2014, each of which is incorporated herein by reference. 
     Thus, the guide nucleotide sequence-programmable DNA-binding protein of the present disclosure may recognize a variety of PAM sequences including, without limitation PAM sequences that are on the 3′ or the 5′ end of the DNA sequence determined by the guide RNA. For example, the sequence may be: NGG, NGAN (SEQ ID NO: 741), NGNG (SEQ ID NO: 742), NGAG (SEQ ID NO: 743), NGCG (SEQ ID NO: 744), NNGRRT (SEQ ID NO: 745), NGRRN (SEQ ID NO: 746), NNNRRT (SEQ ID NO: 747), NNNGATT (SEQ ID NO: 748), NNAGAAW (SEQ ID NO: 749), NAAAC (SEQ ID NO: 750), TTN, TTTN (SEQ ID NO: 751), and YTN, wherein Y is a pyrimidine, R is a purine, and N is any nucleobase. 
     Some aspects of the disclosure provide RNA-programmable DNA binding proteins, which may be used to guide a protein, such as a base editor, to a specific nucleic acid (e.g., DNA or RNA) sequence. Nucleic acid programmable DNA binding proteins include, without limitation, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1, C2c1, C2c2, C2C3, and Argonaute. One example of an RNA-programmable DNA-binding protein that has different PAM specificity is Clustered Regularly Interspaced Short Palindromic Repeats from  Prevotella  and  Francisella  1 (Cpf1). Similar to Cas9, Cpf1 is also a class 2 CRISPR effector. It has been shown that Cpf1 mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it may utilize a T-rich protospacer-adjacent motif (e.g., TTN, TTTN (SEQ ID NO: 751), or YTN), which is on the 5′-end of the DNA sequence determined by the guide RNA. Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, two enzymes from Acidaminococcus and Lachnospiraceae are shown to have efficient genome-editing activity in human cells. Cpf1 proteins are known in the art and have been described previously, for example Yamano et al., “Crystal structure of Cpf1 in complex with guide RNA and target DNA.”  Cell  (165) 2016, p. 949-962; the entire contents of which is hereby incorporated by reference. 
     Also useful in the present compositions and methods are nuclease-inactive Cpf1 (dCpf1) variants that may be used as a guide nucleotide sequence-programmable DNA-binding protein domain. The Cpf1 protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9 but does not have a HNH endonuclease domain, and the N-terminal of Cpf1 does not have the alfa-helical recognition lobe of Cas9. It was shown in Zetsche et al., Cell, 163, 759-771, 2015 (which is incorporated herein by reference) that, the RuvC-like domain of Cpf1 is responsible for cleaving both DNA strands and inactivation of the RuvC-like domain inactivates Cpf1 nuclease activity. For example, mutations corresponding to D917A, E1006A, or D1255A in  Francisella novicida  Cpf1 (SEQ ID NO: 714) inactivate Cpf1 nuclease activity. In some embodiments, the dCpf1 of the present disclosure may comprise mutations corresponding to D917A, E1006A, D1255A, D917A/E1006A, D917A/D1255A, E1006A/D1255A, or D917A/E1006A/D1255A in SEQ ID NO: 714. In other embodiments, the Cpf1 nickase of the present disclosure may comprise mutations corresponding to D917A, E1006A, D1255A, D917A/E1006A, D917A/D1255A, E1006A/D1255A, or D917A/E1006A/D1255A in SEQ ID NO: 714. A Cpf1 nickase useful for the embodiments of the instant disclosure may comprise other mutations and/or further mutations known in the field. It is to be understood that any mutations, e.g., substitution mutations, deletions, or insertions that fully or partially inactivates the RuvC domain of Cpf1 may be used in accordance with the present disclosure, and that these mutations of Cpf1 may result in, for example, a dCpf1 or Cpf1 nickase. 
     Thus, in some embodiments, the guide nucleotide sequence-programmable DNA binding protein is a nuclease inactive Cpf1 (dCpf1). In some embodiments, the dCpf1 comprises an amino acid sequence of any one SEQ ID NOs: 714-717. In some embodiments, the dCpf1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at ease 99.5% identical to any one of SEQ ID NOs: 714-717, and comprises mutations corresponding to D917A, E1006A, D1255A, D917A/E1006A, D917A/D1255A, E1006A/D1255A, or D917A/E1006A/D1255A in SEQ ID NO: 714. Cpf1 from other bacterial species may also be used in accordance with the present disclosure, as a dCpf1 or Cpf1 nickase. 
     
       
         
           
               
               
            
               
                 Wild type  Francisella novicida  Cpf1 (D917, E1006, and D1255 
                   
               
               
                 are bolded and underlined) 
               
            
           
           
               
               
            
               
                 (SEQ ID NO: 714) 
                   
               
            
           
           
               
               
            
               
                 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYH 
                   
               
               
                   
               
               
                 QFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSE 
               
               
                   
               
               
                 KFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWT 
               
               
                   
               
               
                 TYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIK 
               
               
                   
               
               
                 KDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGEN 
               
               
                   
               
               
                 TKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTM 
               
               
                   
               
               
                 QSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDY 
               
               
                   
               
               
                 SVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDI 
               
               
                   
               
               
                 DKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIK 
               
               
                   
               
               
                 DLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYI 
               
               
                   
               
               
                 TQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFD 
               
               
                   
               
               
                 DKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN 
               
               
                   
               
               
                 GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVE 
               
               
                   
               
               
                 NQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDER 
               
               
                   
               
               
                 NLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKR 
               
               
                   
               
               
                 FTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI   D   RGERHLAYYTLVDG 
               
               
                   
               
               
                 KGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQV 
               
               
                   
               
               
                 VHEIAKLVIEYNAIVVF   E   DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEF 
               
               
                   
               
               
                 DKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESV 
               
               
                   
               
               
                 SKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKN 
               
               
                   
               
               
                 HNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQM 
               
               
                   
               
               
                 RNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA   D   ANGAYHIGLKGLMLLGRI 
               
               
                   
               
               
                 KNNQEGKKLNLVIKNEEYFEFVQNRNN 
               
               
                   
               
               
                   Francisella novicida  Cpf1 D917A 
               
            
           
           
               
               
            
               
                 (SEQ ID NO: 715) 
                   
               
            
           
           
               
               
            
               
                 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYH 
                   
               
               
                   
               
               
                 QFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSE 
               
               
                   
               
               
                 KFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWT 
               
               
                   
               
               
                 TYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIK 
               
               
                   
               
               
                 KDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGEN 
               
               
                   
               
               
                 TKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTM 
               
               
                   
               
               
                 QSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDY 
               
               
                   
               
               
                 SVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDI 
               
               
                   
               
               
                 DKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIK 
               
               
                   
               
               
                 DLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYI 
               
               
                   
               
               
                 TQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFD 
               
               
                   
               
               
                 DKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN 
               
               
                   
               
               
                 GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVE 
               
               
                   
               
               
                 NQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDER 
               
               
                   
               
               
                 NLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKR 
               
               
                   
               
               
                 FTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI   A   RGERHLAYYTLVDG 
               
               
                   
               
               
                 KGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQV 
               
               
                   
               
               
                 VHEIAKLVIEYNAIVVF   E   DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEF 
               
               
                   
               
               
                 DKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESV 
               
               
                   
               
               
                 SKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKN 
               
               
                   
               
               
                 HNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQM 
               
               
                   
               
               
                 RNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA   D   ANGAYHIGLKGLMLLGRI 
               
               
                   
               
               
                 KNNQEGKKLNLVIKNEEYFEFVQNRNN 
               
               
                   
               
               
                   Francisella novicida  Cpf1 E1006A 
               
            
           
           
               
               
            
               
                 (SEQ ID NO: 716) 
                   
               
            
           
           
               
               
            
               
                 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYH 
                   
               
               
                   
               
               
                 QFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSE 
               
               
                   
               
               
                 KFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWT 
               
               
                   
               
               
                 TYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIK 
               
               
                   
               
               
                 KDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGEN 
               
               
                   
               
               
                 TKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTM 
               
               
                   
               
               
                 QSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDY 
               
               
                   
               
               
                 SVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDI 
               
               
                   
               
               
                 DKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIK 
               
               
                   
               
               
                 DLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYI 
               
               
                   
               
               
                 TQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFD 
               
               
                   
               
               
                 DKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN 
               
               
                   
               
               
                 GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVE 
               
               
                   
               
               
                 NQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDER 
               
               
                   
               
               
                 NLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKR 
               
               
                   
               
               
                 FTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI   D   RGERHLAYYTLVDG 
               
               
                   
               
               
                 KGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQV 
               
               
                   
               
               
                 VHEIAKLVIEYNAIVVF   A   DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEF 
               
               
                   
               
               
                 DKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESV 
               
               
                   
               
               
                 SKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKN 
               
               
                   
               
               
                 HNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQM 
               
               
                   
               
               
                 RNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA   D   ANGAYHIGLKGLMLLGRI 
               
               
                   
               
               
                 KNNQEGKKLNLVIKNEEYFEFVQNRNN 
               
               
                   
               
               
                   Francisella novicida  Cpf1 D1255A 
               
            
           
           
               
               
            
               
                 (SEQ ID NO: 717) 
                   
               
            
           
           
               
               
            
               
                 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYH 
                   
               
               
                   
               
               
                 QFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSE 
               
               
                   
               
               
                 KFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWT 
               
               
                   
               
               
                 TYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIK 
               
               
                   
               
               
                 KDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGEN 
               
               
                   
               
               
                 TKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTM 
               
               
                   
               
               
                 QSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDY 
               
               
                   
               
               
                 SVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDI 
               
               
                   
               
               
                 DKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIK 
               
               
                   
               
               
                 DLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYI 
               
               
                   
               
               
                 TQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFD 
               
               
                   
               
               
                 DKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN 
               
               
                   
               
               
                 GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVE 
               
               
                   
               
               
                 NQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDER 
               
               
                   
               
               
                 NLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKR 
               
               
                   
               
               
                 FTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI   D   RGERHLAYYTLVDG 
               
               
                   
               
               
                 KGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQV 
               
               
                   
               
               
                 VHEIAKLVIEYNAIVVF   E   DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEF 
               
               
                   
               
               
                 DKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESV 
               
               
                   
               
               
                 SKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKN 
               
               
                   
               
               
                 HNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQM 
               
               
                   
               
               
                 RNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDA   A   ANGAYHIGLKGLMLLGRI 
               
               
                   
               
               
                 KNNQEGKKLNLVIKNEEYFEFVQNRNN 
               
            
           
         
       
     
     In addition to Cas9 and Cpf1, three distinct Class 2 CRISPR-Cas systems (C2c1, C2c2, and C2c3) have been described by Shmakov et al., “Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems”, Mol. Cell, 2015 Nov. 5; 60(3): 385-397, the entire contents of which is hereby incorporated by reference. Effectors of two of the systems, C2c1 and C2c3, contain RuvC-like endonuclease domains related to Cpf1. A third system, C2c2 contains an effector with two predicated HEPN RNase domains. Production of mature CRISPR RNA is tracrRNA-independent, unlike production of CRISPR RNA by C2c1. C2c1 depends on both CRISPR RNA and tracrRNA for DNA cleavage. Bacterial C2c2 has been shown to possess a unique RNase activity for CRISPR RNA maturation distinct from its RNA-activated single-stranded RNA degradation activity. These RNase functions are different from each other and from the CRISPR RNA-processing behavior of Cpf1. See, e.g., East-Seletsky, et al., “Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection”, Nature, 2016 Oct. 13; 538(7624):270-273, the entire contents of which are hereby incorporated by reference. In vitro biochemical analysis of C2c2 in Leptotrichia shahii has shown that C2c2 is guided by a single CRISPR RNA and can be programmed to cleave ssRNA targets carrying complementary protospacers. Catalytic residues in the two conserved HEPN domains mediate cleavage. Mutations in the catalytic residues generate catalytically inactive RNA-binding proteins. See e.g., Abudayyeh et al., “C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector”,  Science,  2016 Aug. 5; 353(6299), the entire contents of which are hereby incorporated by reference. 
     The crystal structure of  Alicyclobaccillus acidoterrastris  C2c1 (AacC2c1) has been reported in complex with a chimeric single-molecule guide RNA (sgRNA). See, e.g., Liu et al., “C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism”,  Mol. Cell,  2017 Jan. 19; 65(2):310-322, the entire contents of which are hereby incorporated by reference. The crystal structure has also been reported in  Alicyclobacillus acidoterrestris  C2c1 bound to target DNAs as ternary complexes. See, e.g., Yang et al., “PAM-dependent Target DNA Recognition and Cleavage by C2C1 CRISPR-Cas endonuclease”,  Cell,  2016 Dec. 15; 167(7):1814-1828, the entire contents of which are hereby incorporated by reference. Catalytically competent conformations of AacC2c1, both with target and non-target DNA strands, have been captured independently positioned within a single RuvC catalytic pocket, with C2c1-mediated cleavage resulting in a staggered seven-nucleotide break of target DNA. Structural comparisons between C2c1 ternary complexes and previously identified Cas9 and Cpf1 counterparts demonstrate the diversity of mechanisms used by CRISPR-Cas9 systems. 
     In some embodiments, the guide nucleotide sequence-programmable DNA-binding protein of any of the fusion proteins provided herein may be a C2c1, a C2c2, or a C2c3 protein. In some embodiments, the guide nucleotide sequence-programmable DNA-binding protein is a C2c1 protein. In some embodiments, the guide nucleotide sequence-programmable DNA-binding protein is a C2c2 protein. In some embodiments, the guide nucleotide sequence-programmable DNA-binding protein is a C2c3 protein. In some embodiments, the guide nucleotide sequence-programmable DNA-binding protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring C2c1, C2c2, or C2c3 protein. In some embodiments, the guide nucleotide sequence-programmable DNA-binding protein is a naturally-occurring C2c1, C2c2, or C2c3 protein. In some embodiments, the guide nucleotide sequence-programmable DNA-binding protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any of the C2c1, C2c2, or C2c3 proteins described herein. In some embodiments, the guide nucleotide sequence-programmable DNA-binding protein comprises an amino acid sequence of any one of the C2c1, C2c2, or C2c3 proteins described herein. It should be appreciated that C2c1, C2c2, or C2c3 from other bacterial species may also be used in accordance with the present disclosure. 
     
       
         
           
               
            
               
                 C2c1 (uniprot.org/uniprot/T0D7A2#) 
               
               
                 sp|T0D7A2|C2C1_ALIAG CRISPR-associated 
               
               
                 endonuclease C2c1 OS =  Alicyclobacillus   
               
               
                   acidoterrestris  (strain ATCC 49025/DSM 3922/ 
               
               
                 CIP 6132/NCIMB 13137/GD3B) GN = c2c1 
               
               
                 PE = 1 SV = 1 
               
            
           
           
               
            
               
                 (SEQ ID NO: 762) 
               
            
           
           
               
            
               
                 MAVKSIKVKLRLDDMPEIRAGLWKLHKEVNAGVRYYTEWLSLLRQENLYR 
               
               
                   
               
               
                 RSPNGDGEQECDKTAEECKAELLERLRARQVENGHRGPAGSDDELLQLAR 
               
               
                   
               
               
                 QLYELLVPQAIGAKGDAQQIARKFLSPLADKDAVGGLGIAKAGNKPRWVR 
               
               
                   
               
               
                 MREAGEPGWEEEKEKAETRKSADRTADVLRALADFGLKPLMRVYTDSEMS 
               
               
                   
               
               
                 SVEWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGQEYAKLVEQKN 
               
               
                   
               
               
                 RFEQKNFVGQEHLVHLVNQLQQDMKEASPGLESKEQTAHYVTGRALRGSD 
               
               
                   
               
               
                 KVFEKWGKLAPDAPFDLYDAEIKNVQRRNTRRFGSHDLFAKLAEPEYQAL 
               
               
                   
               
               
                 WREDASFLTRYAVYNSILRKLNHAKMFATFTLPDATAHPIWTRFDKLGGN 
               
               
                   
               
               
                 LHQYTFLFNEFGERRHAIRFHKLLKVENGVAREVDDVTVPISMSEQLDNL 
               
               
                   
               
               
                 LPRDPNEPIALYFRDYGAEQHFTGEFGGAKIQCRRDQLAHMHRRRGARDV 
               
               
                   
               
               
                 YLNVSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHP 
               
               
                   
               
               
                 DDGKLGSEGLLSGLRVMSVDLGLRTSASISVFRVARKDELKPNSKGRVPF 
               
               
                   
               
               
                 FFPIKGNDNLVAVHERSQLLKLPGETESKDLRAIREERQRTLRQLRTQLA 
               
               
                   
               
               
                 YLRLLVRCGSEDVGRRERSWAKLIEQPVDAANHMTPDWREAFENELQKLK 
               
               
                   
               
               
                 SLHGICSDKEWMDAVYESVRRVWRHMGKQVRDWRKDVRSGERPKIRGYAK 
               
               
                   
               
               
                 DVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREH 
               
               
                   
               
               
                 IDHAKEDRLKKLADRIIMEALGYVYALDERGKGKWVAKYPPCQLILLEEL 
               
               
                   
               
               
                 SEYQFNNDRPPSENNQLMQWSHRGVFQELINQAQVHDLLVGTMYAAFSSR 
               
               
                   
               
               
                 FDARTGAPGIRCRRVPARCTQEHNPEPFPWWLNKFVVEHTLDACPLRADD 
               
               
                   
               
               
                 LIPTGEGEIFVSPFSAEEGDFHQIHADLNAAQNLQQRLWSDFDISQIRLR 
               
               
                   
               
               
                 CDWGEVDGELVLIPRLTGKRTADSYSNKVFYTNTGVTYYERERGKKRRKV 
               
               
                   
               
               
                 FAQEKLSEEEAELLVEADEAREKSVVLMRDPSGIINRGNWTRQKEFWSMV 
               
               
                   
               
               
                 NQRIEGYLVKQIRSRVPLQDSACENTGDI 
               
               
                   
               
               
                 C2c2 (uniprot.org/uniprot/P0DOC6) 
               
               
                   
               
               
                 &gt;sp|P0DOC6|C2C2_LEPSD CRISPR- 
               
               
                 associated endoribonuclease C2c2 OS = 
               
               
                   Leptotrichiashahii  (strain DSM 19757/CCUG 47503/ 
               
               
                 CIP 107916/JCM 16776/LB37) GN = c2c2 
               
               
                 PE = 1 SV = 1 
               
            
           
           
               
            
               
                 (SEQ ID NO: 764) 
               
            
           
           
               
            
               
                 MGNLFGHKRWYEVRDKKDFKIKRKVKVKRNYDGNKYILNINENNNKEKID 
               
               
                   
               
               
                 NNKFIRKYINYKKNDNILKEFTRKFHAGNILFKLKGKEGIIRIENNDDFL 
               
               
                   
               
               
                 ETEEVVLYIEAYGKSEKLKALGITKKKIIDEAIRQGITKDDKKIEIKRQE 
               
               
                   
               
               
                 NEEEIEIDIRDEYTNKTLNDCSIILRIIENDELETKKSIYEIFKNINMSL 
               
               
                   
               
               
                 YKIIEKIIENETEKVFENRYYEEHLREKLLKDDKIDVILTNFMEIREKIK 
               
               
                   
               
               
                 SNLEILGFVKFYLNVGGDKKKSKNKKMLVEKILNINVDLTVEDIADFVIK 
               
               
                   
               
               
                 ELEFWNITKRIEKVKKVNNEFLEKRRNRTYIKSYVLLDKHEKFKIERENK 
               
               
                   
               
               
                 KDKIVKFFVENIKNNSIKEKIEKILAEFKIDELIKKLEKELKKGNCDTEI 
               
               
                   
               
               
                 FGIFKKHYKVNFDSKKFSKKSDEEKELYKIIYRYLKGRIEKILVNEQKVR 
               
               
                   
               
               
                 LKKMEKIEIEKILNESILSEKILKRVKQYTLEHIMYLGKLRHNDIDMTTV 
               
               
                   
               
               
                 NTDDFSRLHAKEELDLELITFFASTNMELNKIFSRENINNDENIDFFGGD 
               
               
                   
               
               
                 REKNYVLDKKILNSKIKIIRDLDFIDNKNNITNNFIRKFTKIGTNERNRI 
               
               
                   
               
               
                 LHAISKERDLQGTQDDYNKVINIIQNLKISDEEVSKALNLDVVFKDKKNI 
               
               
                   
               
               
                 ITKINDIKISEENNNDIKYLPSFSKVLPEILNLYRNNPKNEPFDTIETEK 
               
               
                   
               
               
                 IVLNALIYVNKELYKKLILEDDLEENESKNIFLQELKKTLGNIDEIDENI 
               
               
                   
               
               
                 IENYYKNAQISASKGNNKAIKKYQKKVIECYIGYLRKNYEELFDFSDFKM 
               
               
                   
               
               
                 NIQEIKKQIKDINDNKTYERITVKTSDKTIVINDDFEYIISIFALLNSNA 
               
               
                   
               
               
                 VINKIRNRFFATSVWLNTSEYQNIIDILDEIMQLNTLRNECITENWNLNL 
               
               
                   
               
               
                 EEFIQKMKEIEKDFDDFKIQTKKEIFNNYYEDIKNNILTEFKDDINGCDV 
               
               
                   
               
               
                 LEKKLEKIVIFDDETKFEIDKKSNILQDEQRKLSNINKKDLKKKVDQYIK 
               
               
                   
               
               
                 DKDQEIKSKILCRIIFNSDFLKKYKKEIDNLIEDMESENENKFQEIYYPK 
               
               
                   
               
               
                 ERKNELYIYKKNLFLNIGNPNFDKIYGLISNDIKMADAKFLFNIDGKNIR 
               
               
                   
               
               
                 KNKISEIDAILKNLNDKLNGYSKEYKEKYIKKLKENDDFFAKNIQNKNYK 
               
               
                   
               
               
                 SFEKDYNRVSEYKKIRDLVEFNYLNKIESYLIDINWKLAIQMARFERDMH 
               
               
                   
               
               
                 YIVNGLRELGIIKLSGYNTGISRAYPKRNGSDGFYTTTAYYKFFDEESYK 
               
               
                   
               
               
                 KFEKICYGFGIDLSENSEINKPENESIRNYISHFYIVRNPFADYSIAEQI 
               
               
                   
               
               
                 DRVSNLLSYSTRYNNSTYASVFEVFKKDVNLDYDELKKKFKLIGNNDILE 
               
               
                   
               
               
                 RLMKPKKVSVLELESYNSDYIKNLIIELLTKIENTNDTL 
               
            
           
         
       
     
     In some embodiments, the guide nucleotide sequence-programmable DNA-binding protein domain of the present disclosure has no requirements for a PAM sequence. One example of such a guide nucleotide sequence-programmable DNA-binding protein may be an Argonaute protein from  Natronobacterium gregoryi  (NgAgo). NgAgo is a ssDNA-guided endonuclease. NgAgo binds 5′ phosphorylated ssDNA of ˜24 nucleotides (gDNA) to guide it to its target site and will make DNA double-strand breaks at the gDNA site. In contrast to Cas9, the NgAgo-gDNA system does not require a protospacer-adjacent motif (PAM). Using a nuclease inactive NgAgo (dNgAgo) can greatly expand the codons that may be targeted. The characterization and use of NgAgo have been described in Gao et al., Nat Biotechnol., 2016 July; 34(7):768-73. PubMed PMID: 27136078; Swarts et al., Nature. 507(7491) (2014):258-61; and Swarts et al., Nucleic Acids Res. 43(10) (2015):5120-9, each of which is incorporated herein by reference. The sequence of  Natronobacterium gregoryi  Argonaute is provided in SEQ ID NO: 718. 
     
       
         
           
               
            
               
                 Wild type  Natronobacterium gregoryi  Argonaute 
               
            
           
           
               
            
               
                 (SEQ ID NO: 718) 
               
            
           
           
               
            
               
                 MTVIDLDSTTTADELTSGHTYDISVTLTGVYDNTDEQHPRMSLAFEQDNG 
               
               
                   
               
               
                 ERRYITLWKNTTPKDVFTYDYATGSTYIFTNIDYEVKDGYENLTATYQTT 
               
               
                   
               
               
                 VENATAQEVGTTDEDETFAGGEPLDHHLDDALNETPDDAETESDSGHVMT 
               
               
                   
               
               
                 SFASRDQLPEWTLHTYTLTATDGAKTDTEYARRTLAYTVRQELYTDHDAA 
               
               
                   
               
               
                 PVATDGLMLLTPEPLGETPLDLDCGVRVEADETRTLDYTTAKDRLLAREL 
               
               
                   
               
               
                 VEEGLKRSLWDDYLVRGIDEVLSKEPVLTCDEFDLHERYDLSVEVGHSGR 
               
               
                   
               
               
                 AYLHINFRHRFVPKLTLADIDDDNIYPGLRVKTTYRPRRGHIVWGLRDEC 
               
               
                   
               
               
                 ATDSLNTLGNQSVVAYHRNNQTPINTDLLDAIEAADRRVVETRRQGHGDD 
               
               
                   
               
               
                 AVSFPQELLAVEPNTHQIKQFASDGFHQQARSKTRLSASRCSEKAQAFAE 
               
               
                   
               
               
                 RLDPVRLNGSTVEFSSEFFTGNNEQQLRLLYENGESVLTFRDGARGAHPD 
               
               
                   
               
               
                 ETFSKGIVNPPESFEVAVVLPEQQADTCKAQWDTMADLLNQAGAPPTRSE 
               
               
                   
               
               
                 TVQYDAFSSPESISLNVAGAIDPSEVDAAFVVLPPDQEGFADLASPTETY 
               
               
                   
               
               
                 DELKKALANMGIYSQMAYFDRFRDAKIFYTRNVALGLLAAAGGVAFTTEH 
               
               
                   
               
               
                 AMPGDADMFIGIDVSRSYPEDGASGQINIAATATAVYKDGTILGHSSTRP 
               
               
                   
               
               
                 QLGEKLQSTDVRDIMKNAILGYQQVTGESPTHIVIHRDGFMNEDLDPATE 
               
               
                   
               
               
                 FLNEQGVEYDIVEIRKQPQTRLLAVSDVQYDTPVKSIAAINQNEPRATVA 
               
               
                   
               
               
                 TFGAPEYLATRDGGGLPRPIQIERVAGETDIETLTRQVYLLSQSHIQVHN 
               
               
                   
               
               
                 STARLPITTAYADQASTHATKGYLVQTGAFESNVGFL 
               
            
           
         
       
     
     Also provided herein are Cas9 variants that have relaxed PAM requirements (PAMless Cas9). PAMless Cas9 exhibits an increased activity on a target sequence that does not include a canonical PAM (e.g., NGG) sequence at its 3′-end as compared to  Streptococcus pyogenes  Cas9 as provided by SEQ ID NO: 1, e.g., increased activity by at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, or at least 1,000,000-fold. Such Cas9 variants that have relaxed PAM requirements are described in US Provisional Application Ser. No. 62/245,828, filed Oct. 23, 2015; 62/279,346, filed Jan. 15, 2016; 62/311,763, filed Mar. 22, 2016; 62/322,178, filed Apr. 13, 2016; and 62/357,332, filed Jun. 30, 2016, each of which is incorporated herein by reference. In some embodiments, the dCas9 or Cas9 nickase useful in the present disclosure may further comprise mutations that relax the PAM requirements, e.g., mutations that correspond to A262T, K294R, S409I, E480K, E543D, M694I, or E1219V in SEQ ID NO: 1. 
     The “-” used in the general architecture discussed herein may indicate the presence of an optional linker. The term “linker,” as used herein, refers to a chemical group or a molecule linking two molecules or moieties, e.g., two domains of a fusion protein, such as, for example, a guide nucleotide sequence-programmable DNA binding protein domain and a recombinase catalytic domain. Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-100 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated. Linkers may be of any form known in the art. The linkers may also be unstructured, structured, helical, or extended. 
     In some embodiments, the guide nucleotide sequence-programmable DNA binding protein domain and the recombinase catalytic domain are fused to each other via a linker. Various linker lengths and flexibilities between the guide nucleotide sequence-programmable DNA binding protein domain and the recombinase catalytic domain can be employed (e.g., ranging from flexible linkers of the form (GGGS)n (SEQ ID NO: 759), (GGGGS)n (SEQ ID NO: 722), (GGS)n, and (G)n to more rigid linkers of the form (EAAAK)n (SEQ ID NO: 723), SGSETPGTSESATPES (SEQ ID NO: 724) (see, e.g., Guilinger et al., Nat. Biotechnol. 2014; 32(6): 577-82; the entire contents of which is incorporated herein by reference), (XP)n, or a combination of any of these, wherein X is any amino acid, and n is independently an integer between 1 and 30, in order to achieve the optimal length for activity for the specific application. In some embodiments, n is independently 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, 26, 27, 28, 29, or 30, or, if more than one linker or more than one linker motif is present, any combination thereof. In some embodiments, the linker comprises a (GGS)n motif, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In some embodiments, the linker comprises a (GGS)n motif, wherein n is 1, 3, or 7. In some embodiments, the linker comprises an XTEN linker. The XTEN linker may have the sequence SGSETPGTSESATPES (SEQ ID NO: 7), SGSETPGTSESA (SEQ ID NO: 8), or SGSETPGTSESATPEGGSGGS (SEQ ID NO: 9). In some embodiments, the linker comprises an amino acid sequence chosen from the group including, but not limited to, AGVF (SEQ ID NO: 772), GFLG (SEQ ID NO: 773), FK, AL, ALAL (SEQ ID NO: 774), and ALALA (SEQ ID NO: 775). In some embodiments, suitable linker motifs and configurations include those described in Chen et al., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013; 65(10):1357-69, which is incorporated herein by reference. In some embodiments, the linker may comprise any of the following amino acid sequences: VPFLLEPDNINGKTC (SEQ ID NO: 10), GSAGSAAGSGEF (SEQ ID NO: 11), SIVAQLSRPDPA (SEQ ID NO: 12), MKIIEQLPSA (SEQ ID NO: 13), VRHKLKRVGS (SEQ ID NO: 14), GHGTGSTGSGSS (SEQ ID NO: 15), MSRPDPA (SEQ ID NO: 16), GSAGSAAGSGEF (SEQ ID NO: 7), SGSETPGTSESA (SEQ ID NO: 8), SGSETPGTSESATPEGGSGGS (SEQ ID NO: 9), and GGSM (SEQ ID NO: 17). 
     Additional suitable linker sequences will be apparent to those of skill in the art based on the instant disclosure. In certain embodiments, the linker may have a length of about 33 angstroms to about 81 angstroms. In another embodiment, the linker may have a length of about 54 angstroms to about 81 angstroms. In a further embodiment, the linker may have a length of about 63 to about 81 angstroms. In another embodiment, the linker may have a length of about 65 angstroms to about 75 angstroms. In some embodiments, the linker may have a weight of about 1.20 kDa to about 1.85 kDa. In certain embodiments, the linker may have a weight of about 1.40 kDa to about 1.85 kDa. In certain embodiments, the linker may have a weight of about 1.60 kDa to about 1.7 kDa. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is any stretch of amino acids having 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 15, at least 20, at least 25, at least 30, at least 40, at least 50, or more amino acids. In certain embodiments, the peptide linker is from 18 to 27 amino acids long. In a specific embodiment, the peptide linker is 24 amino acids long. In some embodiments, the peptide linker comprises repeats of the tri-peptide Gly-Gly-Ser, e.g., comprising the sequence (GGS)., wherein n represents at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more repeats. In some embodiments, the linker comprises the sequence (GGS) 6  (SEQ ID NO: 6). In some embodiments, the peptide linker is the 16 residue “XTEN” linker, or a variant thereof (See, e.g., the Examples; and Schellenberger et al. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat. Biotechnol. 27, 1186-1190 (2009)). In some embodiments, the XTEN linker comprises the sequence SGSETPGTSESATPES (SEQ ID NO: 7), SGSETPGTSESA (SEQ ID NO: 8), or SGSETPGTSESATPEGGSGGS (SEQ ID NO: 9). In some embodiments, the peptide linker is selected from VPFLLEPDNINGKTC (SEQ ID NO: 10), GSAGSAAGSGEF (SEQ ID NO: 11), SIVAQLSRPDPA (SEQ ID NO: 12), MKIIEQLPSA (SEQ ID NO: 13), VRHKLKRVGS (SEQ ID NO: 14), GHGTGSTGSGSS (SEQ ID NO: 15), MSRPDPA (SEQ ID NO: 16); or GGSM (SEQ ID NO: 17). In some embodiments, the linker is a non-peptide linker. In certain embodiments, the non-peptide linker comprises one or more of polyethylene glycol (PEG), polypropylene glycol (PPG), co-poly(ethylene/propylene) glycol, polyoxyethylene (POE), polyurethane, polyphosphazene, polysaccharides, dextran, polyvinyl alcohol, polyvinylpyrrolidones, polyvinyl ethyl ether, polyacryl amide, polyacrylate, polycyanoacrylates, lipid polymers, chitins, hyaluronic acid, heparin, or an alkyl linker. In one embodiment, the alkyl linker has the formula —NH—(CH 2 ) s —C(O)—, wherein s may be any integer. In a further embodiment, s may be any integer from 1-20. 
     Recombinase Catalytic Domain 
     The recombinase catalytic domain for use in the compositions and methods of the instant disclosure may be from any recombinase. Suitable recombinases catalytic domains for use in the disclosed methods and compositions may be obtained from, for example, and without limitation, tyrosine recombinases and serine recombinases. Some exemplary suitable recombinases provided herein include, for example, and without limitation, Gin recombinase (acting on gix sites), Hin recombinase (acting on hix sites), β recombinase (acting on six sites), Sin recombinase (acting on resH sites), Tn3 recombinase (acting on res sites), γδ recombinase (acting on res sites), Cre recombinase from bacteriophage P1 (acting on LoxP sites); FLP recombinases of fungal origin (acting on FTR sites); and phiC31 integrase (acting on att sites). Non-limiting sequences of exemplary suitable recombinases may be found below. 
     
       
         
           
               
            
               
                 Cre recombinase sequence 
               
            
           
           
               
            
               
                 (SEQ ID NO: 725) 
               
            
           
           
               
            
               
                 MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCR 
               
               
                   
               
               
                 SWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHR 
               
               
                   
               
               
                 RSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLME 
               
               
                   
               
               
                 NSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRT 
               
               
                   
               
               
                 KTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAP 
               
               
                   
               
               
                 SATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMA 
               
               
                   
               
               
                 RAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLEDGD 
               
               
                   
               
               
                 FLP recombinase 
               
            
           
           
               
            
               
                 (SEQ ID NO: 726) 
               
            
           
           
               
            
               
                 MPQFGILCKTPPKVLVRQFVERFERPSGEKIALCAAELTYLCWMITHNGT 
               
               
                   
               
               
                 AIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAW 
               
               
                   
               
               
                 EFTIIPYYGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSE 
               
               
                   
               
               
                 GESIWEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDP 
               
               
                   
               
               
                 KSFKLVQNKYLGVIIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLR 
               
               
                   
               
               
                 NSEPVLKRVNRTGNSSSNKQEYQLLKDNLVRSYNKALKKNAPYSIFAIKN 
               
               
                   
               
               
                 GPKSHIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVARTTYTHQITAI 
               
               
                   
               
               
                 PDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIRY 
               
               
                   
               
               
                 PAWNGIISQEVLDYLSSYINRRI 
               
               
                   
               
               
                 γδ recombinase (Gamma Delta resolvase) 
               
            
           
           
               
            
               
                 (SEQ ID NO: 727) 
               
            
           
           
               
            
               
                 MRLFGYARVSTSQQSLDIQVRALKDAGVKANRIFTDKASGSSSDRKGLDL 
               
               
                   
               
               
                 LRMKVEEGDVILVKKLDRLGRDTADMIQLIKEFDAQGVSIRFIDDGISTD 
               
               
                   
               
               
                 GEMGKMVVTILSAVAQAERQRILERTNEGRQEAMAKGVVFGRKR 
               
               
                   
               
               
                 γδ recombinase (E124Q mutation) 
               
            
           
           
               
            
               
                 (SEQ ID NO: 728) 
               
            
           
           
               
            
               
                 MRLFGYARVSTSQQSLDIQVRALKDAGVKANRIFTDKASGSSSDRKGLDL 
               
               
                   
               
               
                 LRMKVEEGDVILVKKLDRLGRDTADMIQLIKEFDAQGVSIRFIDDGISTD 
               
               
                   
               
               
                 GEMGKMVVTILSAVAQAERQRILQRTNEGRQEAMAKGVVFGRKR 
               
               
                   
               
               
                 γδ recombinase (E102Y/E124Q mutation) 
               
            
           
           
               
            
               
                 (SEQ ID NO: 729) 
               
            
           
           
               
            
               
                 MRLFGYARVSTSQQSLDIQVRALKDAGVKANRIFTDKASGSSSDRKGLDL 
               
               
                   
               
               
                 LRMKVEEGDVILVKKLDRLGRDTADMIQLIKEFDAQGVSIRFIDDGISTD 
               
               
                   
               
               
                 GYMGKMVVTILSAVAQAERQRILQRTNEGRQEAMAKGVVFGRKR 
               
               
                   
               
               
                 β recombinase 
               
            
           
           
               
            
               
                 (SEQ ID NO: 730) 
               
            
           
           
               
            
               
                 MAKIGYARVSSKEQNLDRQLQALQGVSKVFSDKLSGQSVERPQLQAMLNY 
               
               
                   
               
               
                 IREGDIVVVTELDRLGRNNKELTELMNAIQQKGATLEVLDLPSMNGIEDE 
               
               
                   
               
               
                 NLRRLINNLVIELYKYQAESERKRIKERQAQGIEIAKSKGKFKGRQH 
               
               
                   
               
               
                 β recombinase (N95D mutation) 
               
            
           
           
               
            
               
                 (SEQ ID NO: 731) 
               
            
           
           
               
            
               
                 MAKIGYARVSSKEQNLDRQLQALQGVSKVFSDKLSGQSVERPQLQAMLNY 
               
               
                   
               
               
                 IREGDIVVVTELDRLGRNNKELTELMNAIQQKGATLEVLDLPSMDGIEDE 
               
               
                   
               
               
                 NLRRLINNLVIELYKYQAESERKRIKERQAQGIEIAKSKGKFKGRQH 
               
               
                   
               
               
                 Sin recombinase 
               
            
           
           
               
            
               
                 (SEQ ID NO: 732) 
               
            
           
           
               
            
               
                 MIIGYARVSSLDQNLERQLENLKTFGAEKIFTEKQSGKSIENRPILQKAL 
               
               
                   
               
               
                 NFVRMGDRFIVESIDRLGRNYNEVIHTVNYLKDKEVQLMITSLPMMNEVI 
               
               
                   
               
               
                 GNPLLDKFMKDLIIQILAMVSEQERNESKRRQAQGIQVAKEKGVYKGRPL 
               
               
                   
               
               
                 Sin recombinase (Q87R/Q115R mutations) 
               
            
           
           
               
            
               
                 (SEQ ID NO: 733) 
               
            
           
           
               
            
               
                 MIIGYARVSSLDQNLERQLENLKTFGAEKIFTEKQSGKSIENRPILQKAL 
               
               
                   
               
               
                 NFVRMGDRFIVESIDRLGRNYNEVIHTVNYLKDKEVRLMITSLPMMNEVI 
               
               
                   
               
               
                 GNPLLDKFMKDLIIRILAMVSEQERNESKRRQAQGIQVAKEKGVYKGRPL 
               
               
                   
               
               
                 Tn3 recombinase 
               
            
           
           
               
            
               
                 (SEQ ID NO: 734) 
               
            
           
           
               
            
               
                 MRLFGYARVSTSQQSLDLQVRALKDAGVKANRIFTDKASGSST 
               
               
                   
               
               
                 DREGLDLLRMKVKEGDVILVKKLDRLGRDTADMLQLIKEFDAQGVAV 
               
               
                   
               
               
                 RFIDDGISTDGDMGQMVVTILSAVAQAERRRILERTNEGRQEAK 
               
               
                   
               
               
                 LKGIKFGRRR 
               
               
                   
               
               
                 Tn3 recombinase (G70S/D102Y, E124Q mutations) 
               
            
           
           
               
            
               
                 (SEQ ID NO: 735) 
               
            
           
           
               
            
               
                 MRLFGYARVSTSQQSLDLQVRALKDAGVKANRIFTDKASGSSTDREGLDL 
               
               
                   
               
               
                 LRMKVKEGDVILVKKLDRLSRDTADMLQLIKEFDAQGVAVRFIDDGISTD 
               
               
                   
               
               
                 GYMGQMVVTILSAVAQAERRRILQRTNEGRQEAKLKGIKFGRRR 
               
               
                   
               
               
                 Hin recombinase 
               
            
           
           
               
            
               
                 (SEQ ID NO: 736) 
               
            
           
           
               
            
               
                 MATIGYIRVSTIDQNIDLQRNALTSANCDRIFED 
               
               
                   
               
               
                 RISGKIANRPGLKRALKYVNKGDTLVVWKLDRLGRSVKNLVALISELHER 
               
               
                   
               
               
                 GAHFHSLTDSIDTSSAMGRFFFHVMSALAEMERELIVERTLAGLAAARAQ 
               
               
                   
               
               
                 GRLGGRPV 
               
               
                   
               
               
                 Hin recombinase (H107Y mutation) 
               
            
           
           
               
            
               
                 (SEQ ID NO: 737) 
               
            
           
           
               
            
               
                 MATIGYIRVSTIDQNIDLQRNALTSANCDRIFEDRISGKIANRPGLKRAL 
               
               
                   
               
               
                 KYVNKGDTLVVWKLDRLGRSVKNLVALISELHERGAHFHSLTDSIDTSSA 
               
               
                   
               
               
                 MGRFFFYVMSALAEMERELIVERTLAGLAAARAQGRLGGRPV 
               
               
                   
               
               
                 PhiC31 recombinase 
               
            
           
           
               
            
               
                 (SEQ ID NO: 738) 
               
            
           
           
               
            
               
                 MDTYAGAYDRQSRERENSSAASPATQRSANEDKAADLQREVERDGGRFRF 
               
               
                   
               
               
                 VGHFSEAPGTSAFGTAERPEFERILNECRAGRLNMIIVYDVSRFSRLKVM 
               
               
                   
               
               
                 DAIPIVSELLALGVTIVSTQEGVFRQGNVMDLIHLIMRLDASHKESSLKS 
               
               
                   
               
               
                 AKILDTKNLQRELGGYVGGKAPYGFELVSETKEITRNGRMVNVVINKLAH 
               
               
                   
               
               
                 STTPLTGPFEFEPDVIRWWWREIKTHKHLPFKPGSQAAIHPGSITGLCKR 
               
               
                   
               
               
                 MDADAVPTRGETIGKKTASSAWDPATVMRILRDPRIAGFAAEVIYKKKPD 
               
               
                   
               
               
                 GTPTTKIEGYRIQRDPITLRPVELDCGPIIEPAEWYELQAWLDGRGRGKG 
               
               
                   
               
               
                 LSRGQAILSAMDKLYCECGAVMTSKRGEESIKDSYRCRRRKVVDPSAPGQ 
               
               
                   
               
               
                 HEGTCNVSMAALDKFVAERIFNKIRHAEGDEETLALLWEAARRFGKLTEA 
               
               
                   
               
               
                 PEKSGERANLVAERADALNALEELYEDRAAGAYDGPVGRKHFRKQQAALT 
               
               
                   
               
               
                 LRQQGAEERLAELEAAEAPKLPLDQWFPEDADADPTGPKSWWGRASVDDK 
               
               
                   
               
               
                 RVFVGLFVDKIVVTKSTTGRGQGTPIEKRASITWAKPPTDDDEDDAQDGT 
               
               
                   
               
               
                 EDVAATGA 
               
            
           
         
       
     
     Recombinases for use with the disclosed compositions and methods may also include further mutations. Some aspects of this disclosure provide recombinases comprising an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% identical to the sequence of the recombinase sequence discussed herein, wherein the amino acid sequence of the recombinase comprises at least one mutation as compared to the sequence of the recombinase sequence discussed herein. In some embodiments, the amino acid sequence of the recombinase comprises 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 11, at least 12, at least 13, at least 14, or at least 15 mutations as compared to the sequence of the recombinase sequence discussed herein. 
     For example, the γδ recombinase may comprise one or more mutations from the list: R2A, E56K, G1015, E102Y, M1031, or E124Q. In one embodiment, the γδ recombinase may comprise an E102Y mutation, an E124Q mutation, or both an E102Y and E124Q mutation. In another embodiment, the β recombinase may comprise one or more mutations including, but not limited to N95D. See, for example, Sirk et al., “Expanding the zinc-finger recombinase repertoire: directed evolution and mutational analysis of serine recombinase specificity determinants” Nucl Acids Res (2014) 42 (7): 4755-4766. In another embodiment, the Sin recombinase may have one or more mutations including, but not limited to: Q87R, Q115R, or Q87R and Q115R. In another embodiment, the Tn3 recombinase may have one or more mutations including, but not limited to: G705, D102Y, E124Q, and any combination thereof. In another embodiment, the Hin recombinase may have one or more mutations including, but not limited to: H107Y. In another embodiment, the Sin recombinase may have one or more mutations including, but not limited to: H107Y. Any of the recombinase catalytic domains for use with the disclosed compositions and methods may have greater than 85%, 90%, 95%, 98%, or 99% sequence identity with the native (or wild type) amino acid sequence. For example, in certain embodiments, the Gin recombinase catalytic domain has greater than 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 713. In another embodiment, the amino acid sequence of the Gin recombinase catalytic domain comprises a mutation corresponding to H106Y, and/or I127L, and/or I136R and/or G137F. In yet another embodiment, the amino acid sequence of the Gin recombinase catalytic domain comprises a mutation corresponding to H106Y, I127L, I136R, and G137F. In a further embodiment, the amino acid sequence of the Gin recombinase has been further mutated. In a specific embodiment, the amino acid sequence of the Gin recombinase catalytic domain comprises SEQ ID NO: 713. 
     The recombinase catalytic domain for use in the compositions and methods of the instant disclosure may be from an evolved recombinase. As used herein, the term “evolved recombinase” refers to a recombinase that has been altered (e.g., through mutation) to recognize non-native DNA target sequences. 
     Suitable recombinases that can be evolved include, for example, and without limitation, tyrosine recombinases and serine recombinases (e.g., any of the recombinases discussed herein). Some exemplary suitable recombinases that can be evolved by the methods and strategies provided herein include, for example, and without limitation, Gin recombinase (acting on gix sites), Hin recombinase (acting on hix sites), β recombinase (acting on six sites), Sin recombinase (acting on resH sites), Tn3 recombinase (acting on res sites), γδ recombinase (acting on res sites), Cre recombinase from bacteriophage P1 (acting on LoxP sites); λ phage integrase (acting on att sites); FLP recombinases of fungal origin (acting on FTR sites); phiC31 integrase; Dre recombinase, BxB 1; and prokaryotic β-recombinase. 
     For example, the evolved recombinase for use with the compositions and methods of the instant disclosure may have been altered to interact with (e.g., bind and recombine) a non-canonical recombinase target sequence. As a non-limiting example, the non-canonical recombinase target sequence may be naturally occurring, such as, for example, sequences within a “safe harbor” genomic locus in a mammalian genome, e.g., a genomic locus that is known to be tolerant to genetic modification without any undesired effects. Recombinases targeting such sequences allow, e.g., for the targeted insertion of nucleic acid constructs at a specific genomic location without the need for conventional time- and labor-intensive gene targeting procedures, e.g., via homologous recombination technology. In addition, the directed evolution strategies provided herein can be used to evolve recombinases with an altered activity profile, e.g., recombinases that favor integration of a nucleic acid sequence over excision of that sequence or vice versa. 
     Evolved recombinases exhibit altered target sequence preferences as compared to their wild type counterparts, can be used to target virtually any target sequence for recombinase activity. Accordingly, the evolved recombinases can be used to modify, for example, any sequence within the genome of a cell or subject. Because recombinases can effect an insertion of a heterologous nucleic acid molecule into a target nucleic acid molecule, an excision of a nucleic acid sequence from a nucleic acid molecule, an inversion, or a replacement of nucleic acid sequences, the technology provided herein enables the efficient modification of genomic targets in a variety of ways (e.g., integration, deletion, inversion, exchange of nucleic acid sequences). 
     Catalytic domains from evolved recombinases for use with the methods and compositions of the instant disclosure comprise an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% identical to the sequence of a wild-type recombinase, wherein the amino acid sequence of the evolved recombinase comprises at least one mutation as compared to the sequence of the wild-type recombinase, and wherein the evolved recombinase recognizes a DNA recombinase target sequence that differs from the canonical recombinase target sequence by at least one nucleotide. In some embodiments, the evolved recombinase recognizes a DNA recombinase target sequence that differs from the canonical recombinase target sequence (e.g., a res, gix, hix, six, resH, LoxP, FTR, or att core or related core sequence) by 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 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 25, or at least 30 nucleotides. In some embodiments, the evolved recombinase recognizes a DNA recombinase target sequence that differs from the canonical recombinase target sequence by 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, 26, 27, 28, 29, or 30 nucleotides. 
     In some embodiments, only a portion of the recombinase is used in the fusion proteins and methods described herein. As a non-limiting embodiment, only the C-terminal portion of the recombinase may be used in the fusion proteins and methods described herein. In a specific embodiment, the 25 kDa carboxy-terminal domain of Cre recombinase may be used in the compositions and methods. See, for example, Hoess et al, “DNA Specificity of the Cre Recombinase Resides in the 25 kDa Carboxyl Domain of the Protein,” J. Mol. Bio. 1990 Dec. 20, 216(4):873-82, which is incorporated by reference herein for all purposes. The 25 kDa carboxy-terminal domain of Cre recombinase is the portion stretching from R118 to the carboxy terminus of the protein. In some embodiments, the 25 kDa carboxy-terminal domain of Cre recombinase for use in the instant fusion proteins and methods may differ from the canonical 25 kDa carboxy-terminal domain of Cre recombinase by 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 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, or at least 20 amino acids. In some embodiments, the 25 kDa carboxy-terminal domain of Cre recombinase for use in the instant fusion proteins and methods may differ from the canonical 25 kDa carboxy-terminal domain of Cre recombinase by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In certain embodiments, only a portion of the 25 kDa carboxy-terminal domain of Cre recombinase may be used in the fusion proteins and methods described herein. For example, the portion of Cre recombinase used may be R130 to the carboxy terminus of the protein, T140 to the carboxy terminus of the protein, E150 to the carboxy terminus of the protein, N160 to the carboxy terminus of the protein, T170 to the carboxy terminus of the protein, 1180 to the carboxy terminus of the protein, G190 to the carboxy terminus of the protein, T200 to the carboxy terminus of the protein, E210 to the carboxy terminus of the protein, L220 to the carboxy terminus of the protein, V230 to the carboxy terminus of the protein, C240 to the carboxy terminus of the protein, P250 to the carboxy terminus of the protein, A260 to the carboxy terminus of the protein, R270 to the carboxy terminus of the protein, G280 to the carboxy terminus of the protein, S290 to the carboxy terminus of the protein, A300 to the carboxy terminus of the protein, or M310 to the carboxy terminus of the protein. As another set of non-limiting examples, the portion of Cre recombinase used may be R118-E340, R118-5330, R1184320, R118-M310, R118-A300, R118-S290, R118-G280, R118-R270, R118-A260, R118-P250, R118-C240, R118-V230, R118-L220, or R118-E210. As a further set of non-limiting examples, the portion of Cre recombinase used may be R118-E210, G190-R270, E210-5290, P250-M310, or R270 to the carboxy terminus of the protein. 
     In some embodiments, the Cre recombinase used in the fusion proteins and methods described herein may be truncated at any position. In a specific embodiment, the Cre recombinase used in the fusion proteins and methods described herein may be truncated such that it begins with amino acid R118, A127, E138, or R154) (preceded in each case by methionine). In another set of non-limiting embodiments, the Cre recombinase used in the fusion proteins and methods described herein may be truncated within 10 amino acids, 9 amino acids, 8 amino acids, 7 amino acids, 6 amino acids, 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, or 1 amino acid of R118, A127, E138, or R154. 
     In some embodiments, the recombinase target sequence is between 10-50 nucleotides long. In some embodiments, the recombinase is a Cre recombinase, a Hin recombinase, or a FLP recombinase. In some embodiments, the canonical recombinase target sequence is a LoxP site (5′-ATAACTTCGTATA GCATACAT TATACGAAGTTAT-3′ (SEQ ID NO: 739). In some embodiments, the canonical recombinase target sequence is an FRT site (5′-GAAGTTCCTATTCTCTAGAAA GTATAGGAACTTC-3′) (SEQ ID NO: 740). In some embodiments, the amino acid sequence of the evolved recombinase comprises 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 11, at least 12, at least 13, at least 14, or at least 15 mutations as compared to the sequence of the wild-type recombinase. In some embodiments, the evolved recombinase recognizes a DNA recombinase target sequence that comprises a left half-site, a spacer sequence, and a right half-site, and wherein the left half-site is not a palindrome of the right half-site. 
     In some embodiments, the evolved recombinase recognizes a DNA recombinase target sequence that comprises a naturally occurring sequence. In some embodiments, the evolved recombinase recognizes a DNA recombinase target sequence that is comprised in the genome of a mammal. In some embodiments, the evolved recombinase recognizes a DNA recombinase target sequence comprised in the genome of a human. In some embodiments, the evolved recombinase recognizes a DNA recombinase target sequence that occurs only once in the genome of a mammal. In some embodiments, the evolved recombinase recognizes a DNA recombinase target sequence in the genome of a mammal that differs from any other site in the genome by 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 11, at least 12, at least 13, at least 14, or at least 15 nucleotide(s). In some embodiments, the evolved recombinase recognizes a DNA recombinase target sequence located in a safe harbor genomic locus. In some embodiments, the safe harbor genomic locus is a Rosa26 locus. In some embodiments, the evolved recombinase recognizes a DNA recombinase target sequence located in a genomic locus associated with a disease or disorder. 
     In certain embodiments, the evolved recombinase may target a site in the Rosa locus of the human genome (e.g., 36C6). A non-limiting set of such recombinases may be found, for example, in International PCT Publication, WO 2017/015545A1, published Jan. 26, 2017, entitled “Evolution of Site Specific Recombinases,” which is incorporated by reference herein for this purpose. In some embodiments, the amino acid sequence of the evolved recombinase comprises 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 11, at least 12, at least 13, at least 14, or at least 15 mutations as compared to the sequence of the wild-type recombinase. The nucleotide sequence encoding 36C6 is shown below in bold; those encoding GGS linkers are shown in italics; those encoding dCas9 linkers are black; those encoding the FLAG tag and NLS are underlined and in lowercase, respectively. 
     
       
         
           
               
               
            
               
                 dCas9-36C6 (nucleotide) 
                   
               
            
           
           
               
               
            
               
                 (SEQ ID NO: 765) 
                   
               
            
           
           
               
               
            
               
                 
                   ATGTCCAACCTCCTTACCGTCCACCAGAATCTCCCTGCCCTTCCGGTGGATGCCACCTCTGATGAAGTGCGAAAA 
                 
                   
               
               
                   
               
               
                 
                   AACCTGATGGATATGTTTCGCGATAGGCAAGCTTTTTCTGAACACACGTGGAAGATGCTCCTGTCAGTGTGTAGA 
                 
               
               
                   
               
               
                 
                   AGCTGGGCAGCTTGGTGCAAGTTGAACAACCGAAAATGGTTTCCTGCCGAACCCGAAGATGTGAGAGACTACCTC 
                 
               
               
                   
               
               
                 
                   CTCTACCTGCAGGCTCGAGGGCTCGCCGTGAAAACAATCCAACAACACTTGGGTCAGCTCAACATGCTGCACAGG 
                 
               
               
                   
               
               
                 
                   AGATCTGGGCTGCCCCGGCCGAGTGACTCTAATGCCGTTAGTCTCGTAATGCGGCGCATTCGCAAAGAGAATGTG 
                 
               
               
                   
               
               
                 
                   GATGCTGGAGAACGGGCGAAACAGGCACTGGCTTTTGAACGGACCGACTTCGATCAGGTGCGGAGTCTTATGGAG 
                 
               
               
                   
               
               
                 
                   AATAGTGACAGATGCCAGGACATTCGGAACCTTGCATTCCTGGGTATCGCGTATAATACCCTGCTGAGAATCGCT 
                 
               
               
                   
               
               
                 
                   GAGATCGCCAGAATCAGGGTAAAGGATATTTCTCGAACGGACGGGGGACGGATGTTGATTCATATCGGTCGCACT 
                 
               
               
                   
               
               
                 
                   AAAACACTTGTGAGTACCGCCGGGGTAGAGAAAGCCCTGAGCCTTGGAGTTACTAAACTGGTGGAGCGGTGGATT 
                 
               
               
                   
               
               
                 
                   AGCGTGTCCGGCGTGGCGGATGACCCAAACAATTACTTGTTTTGTAGGGTGCGGAAAAATGGTGTAGCCGCTCCA 
                 
               
               
                   
               
               
                 
                   TCCGCTACCTCACAGTTGAGTACACGCGCGTTGGAGGGGATTTTCGAAGCCACACATCGCTTGATCTACGGCGCC 
                 
               
               
                   
               
               
                 
                   AAGGACGATTCAGGCCAGCGATATCTTGCCTGGAGCGGGCATAGTGCCCGGGTGGGTGCCGCCCGAGACATGGCA 
                 
               
               
                   
               
               
                 
                   AGGGCTGGCGTGTCAATTCCTGAAATCATGCAGGCCGGCGGGTGGACCAACGTGAACATTGTGATGAACTATATC 
                 
               
               
                   
               
               
                 
                   CGGAACCTGGATAGCGAGACCGGAGCAATGGTCAGACTGCTTGAGGATGGCGAC 
                   GGTGGATCCGGAGGGTCCGGA 
                 
               
               
                   
               
               
                   GGTAGTGGCGGCAGCGGTGGTTCAGGTGGCAGCGGAGGGTCAGGAGGCTCTG ATAAAAAGTATTCTATTGGTTTA 
               
               
                   
               
               
                 GCTATCGGCACTAATTCCGTTGGATGGGCTGTCATAACCGATGAATACAAAGTACCTTCAAAGAAATTTAAGGTG 
               
               
                   
               
               
                 TTGGGGAACACAGACCGTCATTCGATTAAAAAGAATCTTATCGGTGCCCTCCTATTCGATAGTGGCGAAACGGCA 
               
               
                   
               
               
                 GAGGCGACTCGCCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACAAGAA 
               
               
                   
               
               
                 ATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAAGAGTCCTTCCTTGTCGAAGAG 
               
               
                   
               
               
                 GACAAGAAACATGAACGGCACCCCATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAACG 
               
               
                   
               
               
                 ATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTCTTGCC 
               
               
                   
               
               
                 CATATGATAAAGTTCCGTGGGCACTTTCTCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTG 
               
               
                   
               
               
                 TTCATCCAGTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGTGGCGTGGATGCGAAG 
               
               
                   
               
               
                 GCTATTCTTAGCGCCCGCCTCTCTAAATCCCGACGGCTAGAAAACCTGATCGCACAATTACCCGGAGAGAAGAAA 
               
               
                   
               
               
                 AATGGGTTGTTCGGTAACCTTATAGCGCTCTCACTAGGCCTGACACCAAATTTTAAGTCGAACTTCGACTTAGCT 
               
               
                   
               
               
                 GAAGATGCCAAATTGCAGCTTAGTAAGGACACGTACGATGACGATCTCGACAATCTACTGGCACAAATTGGAGAT 
               
               
                   
               
               
                 CAGTATGCGGACTTATTTTTGGCTGCCAAAAACCTTAGCGATGCAATCCTCCTATCTGACATACTGAGAGTTAAT 
               
               
                   
               
               
                 ACTGAGATTACCAAGGCGCCGTTATCCGCTTCAATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTT 
               
               
                   
               
               
                 CTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATATTCTTTGATCAGTCGAAAAACGGGTAC 
               
               
                   
               
               
                 GCAGGTTATATTGACGGCGGAGCGAGTCAAGAGGAATTCTACAAGTTTATCAAACCCATATTAGAGAAGATGGAT 
               
               
                   
               
               
                 GGGACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGAAAGCAGCGGACTTTCGACAACGGTAGC 
               
               
                   
               
               
                 ATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTTCCTCAAA 
               
               
                   
               
               
                 GACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTTACTATGTGGGACCCCTGGCCCGAGGGAAC 
               
               
                   
               
               
                 TCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCATGGAATTTTGAGGAAGTTGTCGATAAA 
               
               
                   
               
               
                 GGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCAACTTTGACAAGAATTTACCGAACGAAAAAGTATTGCCT 
               
               
                   
               
               
                 AAGCACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCATG 
               
               
                   
               
               
                 CGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAAGTG 
               
               
                   
               
               
                 ACAGTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGGTAGAA 
               
               
                   
               
               
                 GATCGATTTAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGATAATTAAAGATAAGGACTTCCTGGATAAC 
               
               
                   
               
               
                 GAAGAGAATGAAGATATCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAA 
               
               
                   
               
               
                 AGACTAAAAACATACGCTCACCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGG 
               
               
                   
               
               
                 GGACGATTGTCGCGGAAACTTATCAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAG 
               
               
                   
               
               
                 AGCGACGGCTTCGCCAATAGGAACTTTATGCAGCTGATCCATGATGACTCTTTAACCTTCAAAGAGGATATACAA 
               
               
                   
               
               
                 AAGGCACAGGTTTCCGGACAAGGGGACTCATTGCACGAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCAAA 
               
               
                   
               
               
                 AAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTTAAGGTCATGGGACGTCACAAACCGGAAAACATT 
               
               
                   
               
               
                 GTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGAATA 
               
               
                   
               
               
                 GAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCAATTGCAGAACGAG 
               
               
                   
               
               
                 AAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGAACTGGACATAAACCGTTTATCT 
               
               
                   
               
               
                 GATTACGACGTCGATGCCATTGTACCCCAATCCTTTTTGAAGGACGATTCAATCGACAATAAAGTGCTTACACGC 
               
               
                   
               
               
                 TCGGATAAGAACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAGAACTATTGGCGG 
               
               
                   
               
               
                 CAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTAACTAAAGCTGAGAGGGGTGGCTTGTCT 
               
               
                   
               
               
                 GAACTTGACAAGGCCGGATTTATTAAACGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGATA 
               
               
                   
               
               
                 CTAGATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAG 
               
               
                   
               
               
                 TCAAAATTGGTGTCGGACTTCAGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCG 
               
               
                   
               
               
                 CACGACGCTTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTG 
               
               
                   
               
               
                 TATGGTGATTACAAAGTTTATGACGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAGCC 
               
               
                   
               
               
                 AAATACTTCTTTTATTCTAACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGCAAA 
               
               
                   
               
               
                 CGACCTTTAATTGAAACCAATGGGGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGA 
               
               
                   
               
               
                 AAAGTTTTGTCCATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAATCG 
               
               
                   
               
               
                 ATTCTTCCAAAAAGGAATAGTGATAAGCTCATCGCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTC 
               
               
                   
               
               
                 GATAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGGAAAATCCAAGAAACTGAAGTCA 
               
               
                   
               
               
                 GTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTTTTGAAAAGAACCCCATCGACTTCCTTGAGGCG 
               
               
                   
               
               
                 AAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAAAGTATAGTCTGTTTGAGTTAGAAAATGGC 
               
               
                   
               
               
                 CGAAAACGGATGTTGGCTAGCGCCGGAGAGCTTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGAAT 
               
               
                   
               
               
                 TTCCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTTTTGTT 
               
               
                   
               
               
                 GAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTCATCCTAGCTGAT 
               
               
                   
               
               
                 GCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGGAAAATATT 
               
               
                   
               
               
                 ATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCAAA 
               
               
                   
               
               
                 CGATACACTTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGG 
               
               
                   
               
               
                 ATAGATTTGTCACAGCTTGGGGGTGAC GGTGGCTCC   GATTATAAGGATGATGACGACAAG   GGAGGTTCC ccaaag 
               
               
                   
               
               
                 aagaaaaggaaggtcTGA 
               
               
                   
               
               
                 dCas9-36C6 (amino acid) 
               
            
           
           
               
               
            
               
                 (SEQ ID NO: 766) 
                   
               
            
           
           
               
               
            
               
                 
                   MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYL 
                 
                   
               
               
                   
               
               
                 
                   LYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLME 
                 
               
               
                   
               
               
                 
                   NSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWI 
                 
               
               
                   
               
               
                 
                   SVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMA 
                 
               
               
                   
               
               
                   RAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLEDGD   GGSGGSGGSGGSGGSGGSGGSGGS DKKYSIGL 
               
               
                   
               
               
                 AIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQE 
               
               
                   
               
               
                 IFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALA 
               
               
                   
               
               
                 HMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKK 
               
               
                   
               
               
                 NGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVN 
               
               
                   
               
               
                 TEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD 
               
               
                   
               
               
                 GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN 
               
               
                   
               
               
                 SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGM 
               
               
                   
               
               
                 RKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDN 
               
               
                   
               
               
                 EENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLK 
               
               
                   
               
               
                 SDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENI 
               
               
                   
               
               
                 VIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLS 
               
               
                   
               
               
                 DYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLS 
               
               
                   
               
               
                 ELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHA 
               
               
                   
               
               
                 HDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRK 
               
               
                   
               
               
                 RPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGF 
               
               
                   
               
               
                 DSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENG 
               
               
                   
               
               
                 RKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD 
               
               
                   
               
               
                 ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETR 
               
               
                   
               
               
                 IDLSQLGGD GGS   DYKDDDDK   GGS pkkkrkv Stop 
               
            
           
         
       
     
     Some aspects of this disclosure provide evolved recombinases (e.g., a Cre recombinase) comprising an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% identical to the sequence of the recombinase sequence (e.g., a Cre recombinase) discussed herein, wherein the amino acid sequence of the recombinase (e.g., a Cre recombinase) comprises at least one mutation as compared to the sequence of the recombinase (e.g., a Cre recombinase) sequence discussed herein, and wherein the recombinase (e.g., a Cre recombinase) recognizes a DNA recombinase target sequence that differs from the canonical LoxP site 5′-ATAACTTCGTATA GCATACAT TATACGAAGTTAT-3′ (SEQ ID NO: 739) in at least one nucleotide. 
     In some embodiments, the amino acid sequence of the evolved recombinase (e.g., a Cre recombinase) comprises 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 11, at least 12, at least 13, at least 14, or at least 15 mutations as compared to the sequence of the recombinase (e.g., a Cre recombinase) sequence discussed herein and recognizes a DNA recombinase target sequence that differs from the canonical target site (e.g., a LoxP site) in 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 11, at least 12, at least 13, at least 14, or at least 15 nucleotides. 
     In some embodiments, the evolved Cre recombinase recognizes a DNA recombinase target sequence that comprises a left half-site, a spacer sequence, and a right half-site, wherein the left half-site is not a palindrome of the right half-site. In some embodiments, the evolved Cre recombinase recognizes a DNA recombinase target sequence that comprises a naturally occurring sequence. In some embodiments, the evolved Cre recombinase recognizes a DNA recombinase target sequence that is comprised in the genome of a mammal. 
     In some embodiments, the evolved Cre recombinase recognizes a DNA recombinase target sequence that is comprised in the genome of a human. In some embodiments, the evolved Cre recombinase recognizes a DNA recombinase target sequence that is comprised only once in the genome of a mammal. In some embodiments, the evolved Cre recombinase recognizes a DNA recombinase target sequence in the genome of a mammal that differs from any other site in the genome by 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 11, at least 12, at least 13, at least 14, or at least 15 nucleotide(s). In some embodiments, the evolved Cre recombinase recognizes a DNA recombinase target sequence located in a safe harbor genomic locus. In some embodiments, the safe harbor genomic locus is a Rosa26 locus. In some embodiments, the evolved Cre recombinase recognizes a DNA recombinase target sequence located in a genomic locus associated with a disease or disorder. 
     Additional evolved recombinases (and methods for making the same) for use with the instant methods and compositions may be found in, for example, U.S. patent application Ser. No. 15/216,844, which is incorporated herein by reference. 
     Additional suitable recombinases will be apparent to those of skill in the art for both providing recombinase catalytic domains or evolved recombinase catalytic domains, and such suitable recombinases include, without limitation, those disclosed in Hirano et al., Site-specific recombinases as tools for heterologous gene integration. Appl Microbiol Biotechnol. 2011 October; 92(2):227-39; Fogg et al., New applications for phage integrases. J Mol Biol. 2014 Jul. 29; 426(15):2703; Brown et al., Serine recombinases as tools for genome engineering. Methods. 2011 April; 53(4):372-9; Smith et al., Site-specific recombination by phiC31 integrase and other large serine recombinases. Biochem Soc Trans. 2010 April; 38(2):388-94; Grindley et al., Mechanisms of site-specific recombination. Annu Rev Biochem. 2006; 75:567-605; Smith et al., Diversity in the serine recombinases. Mol Microbiol. 2002 April; 44(2):299-307; Grainge et al., The integrase family of recombinase: organization and function of the active site. Mol Microbiol. 1999 August; 33(3):449-56; Gopaul et al., Structure and mechanism in site-specific recombination. Curr Opin Struct Biol. 1999 February; 9(1):14-20; Cox et al., Conditional gene expression in the mouse inner ear using Cre-loxP. J Assoc Res Otolaryngol. 2012 June; 13(3):295-322; Birling et al., Site-specific recombinases for manipulation of the mouse genome. Methods Mol Biol. 2009; 561:245-63; and Mishina M, Sakimura K. Conditional gene targeting on the pure C57BL/6 genetic background. Neurosci Res. 2007 June; 58(2):105-12; the entire contents of each of which are incorporated herein by reference. 
     Structure of the Fusion Protein 
     The fusion protein of the instant instant disclosure may be any combination and order of the elements described herein. Exemplary fusion proteins include, but are not limited to, any of the following structures: NH 2 -[recombinase catalytic domain]-[linker sequence]-[guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[optional NLS domain]-[optional linker sequence]-[optional affinity tag]-COOH. In another embodiment, the fusion protein has the structure NH 2 -[recombinase catalytic domain]-[linker sequence]-[guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[optional affinity tag]-COOH. In another embodiment, the fusion protein has the structure NH 2 -[recombinase catalytic domain]-[linker sequence]-[guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[affinity tag]-COOH. In another embodiment, the fusion protein has the structure NH 2 -[recombinase catalytic domain]-[linker sequence]-[guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[NLS domain]-[linker sequence]-[affinity tag]-COOH. 
     In another embodiment, the fusion protein has the structure NH 2 -[recombinase catalytic domain]-[optional linker sequence]-[guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[affinity tag]-COOH, NH 2 -[guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[optional affinity tag]-COOH, NH 2 —[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[optional affinity tag]-COOH, NH 2 -[affinity tag]-[optional linker sequence]-[recombinase catalytic domain]-[linker sequence]-[guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[NLS domain]-COOH, NH 2 -[affinity tag]-[optional linker sequence]-[guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[NLS domain]-COOH, or NH 2 -[affinity tag]-[optional linker sequence]-[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[NLS domain]-COOH. 
     In another embodiment, the fusion protein has the structure: NH 2 -[guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[optional NLS domain]-[optional linker sequence]-[optional affinity tag]-COOH. In one embodiment, the fusion protein comprises the structure NH 2 -[guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[optional affinity tag]-COOH. In one embodiment, the fusion protein comprises the structure NH 2 -[guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[affinity tag]-COOH. In one embodiment, the fusion protein comprises the structure NH 2 -[guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-[linker sequence]-[NLS domain]-[linker sequence]-[affinity tag]-COOH. 
     In another embodiment, the fusion protein has the structure NH 2 —[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[optional NLS domain]-[optional linker sequence]-[optional affinity tag]-COOH. In another embodiment, the fusion protein comprises the structure NH 2 —[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[optional affinity tag]-COOH. In another embodiment, the fusion protein comprises the structure NH 2 —[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[affinity tag]-COOH. In another embodiment, the fusion protein comprises the structure NH 2 —[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[NLS domain]-[linker sequence]-[affinity tag]-COOH. 
     In another embodiment, the fusion protein has the structure NH 2 —[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[optional NLS domain]-[optional linker sequence]-[optional affinity tag]-COOH. In another embodiment, the fusion protein comprises the structure NH 2 —[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[optional affinity tag]-COOH. In another embodiment, the fusion protein comprises the structure NH 2 —[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[affinity tag]-COOH. In another embodiment, the fusion protein comprises the structure NH 2 —[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[NLS domain]-[linker sequence]-[affinity tag]-COOH. 
     In another embodiment, the fusion protein has the structure NH 2 —[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[recombinase catalytic domain]-[linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[optional NLS domain]-[optional linker sequence]-[optional affinity tag]-COOH. In another embodiment, the fusion protein comprises the structure NH 2 —[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[recombinase catalytic domain]-[linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[optional affinity tag]-COOH. In another embodiment, the fusion protein comprises the structure NH 2 —[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[recombinase catalytic domain]-[linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[affinity tag]-COOH. In another embodiment, the fusion protein comprises the structure NH 2 —[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[recombinase catalytic domain]-[linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[NLS domain]-[linker sequence]-[affinity tag]-COOH. 
     In another embodiment, the fusion protein has the structure NH 2 —[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-[linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[optional NLS domain]-[optional linker sequence]-[optional affinity tag]-COOH. In another embodiment, the fusion protein comprises the structure NH 2 —[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-[linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[optional affinity tag]-COOH. In another embodiment, the fusion protein comprises the structure NH 2 —[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-[linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[affinity tag]-COOH. In another embodiment, the fusion protein comprises the structure NH 2 —[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-[linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[NLS domain]-[linker sequence]-[affinity tag]-COOH. 
     In one embodiment, the fusion protein has the structure NH 2 -[optional affinity tag]-[optional linker sequence]-[optional NLS domain]-[optional linker sequence]-[recombinase catalytic domain]-[linker sequence]-[guide nucleotide sequence-programmable DNA binding protein domain]-COOH. In one embodiment, the fusion protein comprises the structure NH 2 -[optional affinity tag]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[recombinase catalytic domain]-[linker sequence]-[guide nucleotide sequence-programmable DNA binding protein domain]-COOH. In one embodiment, the fusion protein comprises the structure NH 2 -[affinity tag]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[recombinase catalytic domain]-[linker sequence]-[guide nucleotide sequence-programmable DNA binding protein domain]-COOH. In one embodiment, the fusion protein comprises the structure NH 2 -[affinity tag]-[linker sequence]-[NLS domain]-[linker sequence]-[recombinase catalytic domain]-[linker sequence]-[guide nucleotide sequence-programmable DNA binding protein domain]-COOH. 
     In one embodiment, the fusion protein has the structure NH 2 -[optional affinity tag]-[optional linker sequence]-[optional NLS domain]-[optional linker sequence]-[guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-COOH. In one embodiment, the fusion protein comprises the structure NH 2 -[optional affinity tag]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-COOH. In one embodiment, the fusion protein comprises the structure NH 2 -[affinity tag]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-COOH. In one embodiment, the fusion protein comprises the structure NH 2 -[affinity tag]-[linker sequence]-[NLS domain]-[linker sequence]-[guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-COOH. 
     In another embodiment, the fusion protein has the structure NH 2 -[optional affinity tag]-[optional linker sequence]-[optional NLS domain]-[optional linker sequence]-[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-COOH. In another embodiment, the fusion protein comprises the structure NH 2 -[optional affinity tag]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-COOH. In another embodiment, the fusion protein comprises the structure NH 2 -[affinity tag]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-COOH. In another embodiment, the fusion protein comprises the structure NH 2 -[affinity tag]-[linker sequence]-[NLS domain]-[linker sequence]-[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-COOH. 
     In another embodiment, the fusion protein has the structure NH 2 -[optional affinity tag]-[optional linker sequence]-[optional NLS domain]-[optional linker sequence]-[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-COOH. In another embodiment, the fusion protein comprises the structure NH 2 -[optional affinity tag]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-COOH. In another embodiment, the fusion protein comprises the structure NH 2 -[affinity tag]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-COOH. In another embodiment, the fusion protein comprises the structure NH 2 -[affinity tag]-[linker sequence]-[NLS domain]-[linker sequence]-[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-[optional linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-COOH. 
     In another embodiment, the fusion protein has the structure NH 2 -[optional affinity tag]-[optional linker sequence]-[optional NLS domain]-[optional linker sequence]-[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[recombinase catalytic domain]-[linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-COOH. In another embodiment, the fusion protein comprises the structure NH 2 -[optional affinity tag]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[recombinase catalytic domain]-[linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-COOH. In another embodiment, the fusion protein comprises the structure NH 2 -[affinity tag]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[recombinase catalytic domain]-[linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-COOH. In another embodiment, the fusion protein comprises the structure NH 2 -[affinity tag]-[linker sequence]-[NLS domain]-[linker sequence]-[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[optional linker sequence]-[recombinase catalytic domain]-[linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-COOH. 
     In another embodiment, the fusion protein has the structure NH 2 -[optional affinity tag]-[optional linker sequence]-[optional NLS domain]-[optional linker sequence]-[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-[linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-COOH. In another embodiment, the fusion protein comprises the structure NH 2 -[optional affinity tag]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-[linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-COOH. In another embodiment, the fusion protein comprises the structure NH 2 -[affinity tag]-[optional linker sequence]-[NLS domain]-[optional linker sequence]-[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-[linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-COOH. In another embodiment, the fusion protein comprises the structure NH 2 -[affinity tag]-[linker sequence]-[NLS domain]-[linker sequence]-[N-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-[linker sequence]-[recombinase catalytic domain]-[linker sequence]-[C-terminal portion of a bifurcated or circularly permuted guide nucleotide sequence-programmable DNA binding protein domain]-COOH. 
     The fusion protein may further comprise one or more affinity tags. Suitable affinity tags provided herein include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc-tags, calmodulin-tags, FLAG-tags, hemagglutinin (HA)-tags, polyhistidine tags, also referred to as histidine tags or His-tags, polyarginine (poly-Arg) tags, maltose binding protein (MBP)-tags, nus-tags, glutathione-S-transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g., Softag 1, Softag 3), strep-tags, biotin ligase tags, FlAsH tags, V5 tags, and SBP-tags. Additional suitable sequences will be apparent to those of skill in the art. The FLAG tag may have the sequence PKKKRKV (SEQ ID NO: 702). The one or more affinity tags are bound to the guide nucleotide sequence-programmable DNA binding protein domain, the recombinase catalytic domain, or the NLS domain via one or more third linkers. The third linker may be any peptide linker described herein. For example, the third linker may be a peptide linker. 
     As a non-limiting set of examples, the third linker may comprise an XTEN linker SGSETPGTSESATPES (SEQ ID NO: 7), SGSETPGTSESA (SEQ ID NO: 8), or SGSETPGTSESATPEGGSGGS (SEQ ID NO: 9), an amino acid sequence comprising one or more repeats of the tri-peptide GGS, or any of the following amino acid sequences: VPFLLEPDNINGKTC (SEQ ID NO: 10), GSAGSAAGSGEF (SEQ ID NO: 11), SIVAQLSRPDPA (SEQ ID NO: 12), MKIIEQLPSA (SEQ ID NO: 13), VRHKLKRVGS (SEQ ID NO: 14), GHGTGSTGSGSS (SEQ ID NO: 15), MSRPDPA (SEQ ID NO; 16), or GGSM (SEQ ID NO: 17). In certain embodiments, the third linker comprises one or more repeats of the tri-peptide GGS. In an embodiment, the third linker comprises from one to five repeats of the tri-peptide GGS. In another embodiment, the third linker comprises one repeat of the tri-peptide GGS. In a specific embodiment, the third linker has the sequence GGS. 
     The third linker may also be a non-peptide linker. In certain embodiments, the non-peptide linker comprises polyethylene glycol (PEG), polypropylene glycol (PPG), co-poly(ethylene/propylene) glycol, polyoxyethylene (POE), polyurethane, polyphosphazene, polysaccharides, dextran, polyvinyl alcohol, polyvinylpyrrolidones, polyvinyl ethyl ether, polyacryl amide, polyacrylate, polycyanoacrylates, lipid polymers, chitins, hyaluronic acid, heparin, or an alkyl linker. In other embodiments, the alkyl linker has the formula: —NH—(CH 2 ) s —C(O)—, wherein s may be any integer between 1 and 100, inclusive. In a specific embodiment, s is any integer between 1 and 20, inclusive. 
     The fusion protein of the instant disclosure has greater than 90%, 95%, or 99% sequence identity with the amino acid sequence shown in amino acids 1-1544 of SEQ ID NO: 185, which is identical to the sequence shown in SEQ ID NO: 719. 
     
       
         
           
               
            
               
                 (SEQ ID NO: 719) 
               
               
                 
                   MLIGYVRVSTNDQNTDLQRNALVCAGCEQIFEDKLSGTRTDRPGLKRALK 
                 
               
               
                   
               
               
                 
                   RLQKGDTLVVWKLDRLGRSMKHLISLVGELRERGINFRSLTDSIDTSSPM 
                 
               
               
                   
               
               
                 
                   GRFFFYVMGALAEMERELIIERTMAGLAAARNKGRRFGRPPK 
                   GGSGGSGG 
                 
               
               
                   
               
               
                   SGGSGGSGGSGGSGGS DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVL 
               
               
                   
               
               
                 GNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEI 
               
               
                   
               
               
                 FSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTI 
               
               
                   
               
               
                 YHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLF 
               
               
                   
               
               
                 IQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKN 
               
               
                   
               
               
                 GLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQ 
               
               
                   
               
               
                 YADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLL 
               
               
                   
               
               
                 KALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG 
               
               
                   
               
               
                 TEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD 
               
               
                   
               
               
                 NREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKG 
               
               
                   
               
               
                 ASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMR 
               
               
                   
               
               
                 KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVED 
               
               
                   
               
               
                 RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER 
               
               
                   
               
               
                 LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKS 
               
               
                   
               
               
                 DGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKK 
               
               
                   
               
               
                 GILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIE 
               
               
                   
               
               
                 EGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSD 
               
               
                   
               
               
                 YDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQ 
               
               
                   
               
               
                 LLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQIL 
               
               
                   
               
               
                 DSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAH 
               
               
                   
               
               
                 DAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAK 
               
               
                   
               
               
                 YFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRK 
               
               
                   
               
               
                 VLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFD 
               
               
                   
               
               
                 SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK 
               
               
                   
               
               
                 GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNF 
               
               
                   
               
               
                 LYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA 
               
               
                   
               
               
                 NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKR 
               
               
                   
               
               
                 YTSTKEVLDATLIHQSITGLYETRIDLSQLGGD GGS   DYKDDDDK  Stop 
               
            
           
         
       
     
     In the context of proteins that dimerize (or multimerize) such as, for example, fusions between a nuclease-inactivated Cas9 (or a Cas9 gRNA binding domain) and a recombinase (or catalytic domain of a recombinase), a target site typically comprises a left-half site (bound by one protein), a right-half site (bound by the second protein), and a spacer sequence between the half sites in which the recombination is made. In some embodiments, either the left-half site or the right half-site (and not the spacer sequence) is recombined. In other embodiments, the spacer sequence is recombined. This structure ([left-half site]-[spacer sequence]-[right-half site]) is referred to herein as an LSR structure. In some embodiments, the left-half site and/or the right-half site correspond to an RNA-guided target site (e.g., a Cas9 target site). In some embodiments, either or both half-sites are shorter or longer than e.g., a typical region targeted by Cas9, for example shorter or longer than 20 nucleotides. In some embodiments, the left and right half sites comprise different nucleic acid sequences. In some embodiments, the spacer sequence is at least 5, at least 6, at least 7, at least 8, at least 9, at least 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 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, or at least 250 bp long. In some embodiments, the spacer sequence is between approximately 15 bp and approximately 25 bp long. In some embodiments, the spacer sequence is approximately 15 bp long. In some embodiments, the spacer sequence is approximately 25 bp long. 
     EXAMPLES 
     Example 1: A Programmable Cas9-Serine Recombinase Fusion Protein that Operates on DNA Sequences in Mammalian Cells 
     Materials and Methods 
     Oligonucleotides and PCR 
     All oligonucleotides were purchased from Integrated DNA Technologies (IDT, Coralville, Calif.) and are listed in Tables 1-5. Enzymes, unless otherwise noted, were purchased from New England Biolabs (Ipswich, Mass.). Plasmid Safe ATP-dependent DNAse was purchased from Epicentre (Madison, Wis.). All assembled vectors were transformed into One Shot Mach1-T1 phage-resistant chemically competent cells (Fisher Scientific, Waltham, Mass.). Unless otherwise noted, all PCR reactions were performed with Q5 Hot Start High-Fidelity 2× Master Mix. Phusion polymerse was used for circular polymerase extension cloning (CPEC) assemblies. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Oligonucleotides for gRNA construction 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                 Oligonucleotide Name 
                 Sequence 
                 NO: 
               
               
                   
               
               
                 R.pHU6.TSS(−1).univ 
                 GGTGTTTCGTCCTTTCCACAAG 
                 20 
               
               
                   
               
               
                 F.non-target 
                 GCACACTAGTTAGGGATAACAGTTTTAG 
                 21 
               
               
                   
                 AGCTAGAAATAGC 
                   
               
               
                   
               
               
                 F.Chr10-1 
                 GCCCATGACCCTTCTCCTCTGTTTTAGAG 
                 22 
               
               
                   
                 CTAGAAATAGC 
                   
               
               
                   
               
               
                 F.Chr10-1-rev 
                 GCTCAGGGCCTGTGATGGGAGGTTTTAG 
                 23 
               
               
                   
                 AGCTAGAAATAGC 
                   
               
               
                   
               
               
                 F.Chr10-2 
                 GGCCCATGACCCTTCTCCTCGTTTTAGAG 
                 24 
               
               
                   
                 CTAGAAATAGC 
                   
               
               
                   
               
               
                 F.Chr10-2rev 
                 GCCTCAGGGCCTGTGATGGGAGTTTTAG 
                 25 
               
               
                   
                 AGCTAGAAATAGC 
                   
               
               
                   
               
               
                 F.Centromere_Chr_1_5_19- 
                 GACTTGAAACACTCTTTTTCGTTTTAGAG 
                 26 
               
               
                 gRNA-for 
                 CTAGAAATAGC 
                   
               
               
                   
               
               
                 F.Centromere_Chr_1_5_19- 
                 GAGTTGAAGACACACAACACAGTTTTAG 
                 27 
               
               
                 gRNA-rev 
                 AGCTAGAAATAGC 
                   
               
               
                   
               
               
                 F.Ch5_155183064-gRNA-for 
                 GGAACTCATGTGATTAACTGGTTTTAGA 
                 28 
               
               
                   
                 GCTAGAAATAGC 
                   
               
               
                   
               
               
                 F.Ch5_155183064-gRNA-rev-1 
                 GTCTACCTCTCATGAGCCGGTGTTTTAGA 
                 29 
               
               
                   
                 GCTAGAAATAGC 
                   
               
               
                   
               
               
                 F.Ch5_169395198-gRNA-for 
                 GTTTCCCGCAGGATGTGGGATGTTTTAG 
                 30 
               
               
                   
                 AGCTAGAAATAGC 
                   
               
               
                   
               
               
                 F.Ch5_169395198-gRNA-rev 
                 GCCTGGGGATTTATGTTCTTAGTTTTAGA 
                 31 
               
               
                   
                 GCTAGAAATAGC 
                   
               
               
                   
               
               
                 F.Ch12_62418577-gRNA-for 
                 GAAATAGCACAATGAATGGAAGTTTTAG 
                 32 
               
               
                   
                 AGCTAGAAATAGC 
                   
               
               
                   
               
               
                 F.Ch12_62418577-gRNA-rev 
                 GACTTTTTGGGGGAGAGGGAGGTTTTAG 
                 33 
               
               
                   
                 AGCTAGAAATAGC 
                   
               
               
                   
               
               
                 F.Ch13_102010574-gRNA-for 
                 GGAGACTTAAGTCCAAAACCGTTTTAGA 
                 34 
               
               
                   
                 GCTAGAAATAGC 
                   
               
               
                   
               
               
                 F.Ch13_102010574-gRNA- 
                 GTCAGCTATGATCACTTCCCTGTTTTAGA 
                 35 
               
               
                 rev 
                 GCTAGAAATAGC 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Oligonucleotides and gBlocks for reporter construction 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                 Construct Name 
                 Sequence 
                 NO: 
               
               
                   
               
            
           
           
               
               
               
            
               
                 1-0bp-for 
                 TCGTCTCGGCGTCCCCAATTTTCCCAAACAGAG 
                 36 
               
               
                   
                 GTCTGTAAACCGAGGTGAGACGG 
                   
               
               
                   
               
               
                 1-0bp-rev 
                 CCGTCTCACCTCGGTTTACAGACCTCTGTTTGG 
                 37 
               
               
                   
                 GAAAATTGGGGACGCCGAGACGA 
                   
               
               
                   
               
               
                 1-1bp-for 
                 TCGTCTCGGCGTCCCCAATTTTCCCAAACAGAG 
                 38 
               
               
                   
                 GTtCTGTAAACCGAGGTGAGACGG 
                   
               
               
                   
               
               
                 1-1bp-rev 
                 CCGTCTCACCTCGGTTTACAGaACCTCTGTTTGG 
                 39 
               
               
                   
                 GAAAATTGGGGACGCCGAGACGA 
                   
               
               
                   
               
               
                 1-2bp-for 
                 TCGTCTCGGCGTCCCCAATTTTCCCAAACAGAG 
                 40 
               
               
                   
                 GTatCTGTAAACCGAGGTGAGACGG 
                   
               
               
                   
               
               
                 1-2bp-rev 
                 CCGTCTCACCTCGGTTTACAGatACCTCTGTTTG 
                 41 
               
               
                   
                 GGAAAATTGGGGACGCCGAGACGA 
                   
               
               
                   
               
               
                 1-3bp-for 
                 TCGTCTCGGCGTCCCCAATTTTCCCAAACAGAG 
                 42 
               
               
                   
                 GTaatCTGTAAACCGAGGTGAGACGG 
                   
               
               
                   
               
               
                 1-3bp-rev 
                 CCGTCTCACCTCGGTTTACAGattACCTCTGTTTG 
                 43 
               
               
                   
                 GGAAAATTGGGGACGCCGAGACGA 
                   
               
               
                   
               
               
                 1-4bp-for 
                 TCGTCTCGGCGTCCCCAATTTTCCCAAACAGAG 
                 44 
               
               
                   
                 GTaaatCTGTAAACCGAGGTGAGACGG 
                   
               
               
                   
               
               
                 1-4bp-rev 
                 CCGTCTCACCTCGGTTTACAGatttACCTCTGTTT 
                 45 
               
               
                   
                 GGGAAAATTGGGGACGCCGAGACGA 
                   
               
               
                   
               
               
                 1-5bp-for 
                 TCGTCTCGGCGTCCCCAATTTTCCCAAACAGAG 
                 46 
               
               
                   
                 GTgaaatCTGTAAACCGAGGTGAGACGG 
                   
               
               
                   
               
               
                 1-5bp-rev 
                 CCGTCTCACCTCGGTTTACAGatttcACCTCTGTTT 
                 47 
               
               
                   
                 GGGAAAATTGGGGACGCCGAGACGA 
                   
               
               
                   
               
               
                 1-6bp-for 
                 TCGTCTCGGCGTCCCCAATTTTCCCAAACAGAG 
                 48 
               
               
                   
                 GTcgaaatCTGTAAACCGAGGTGAGACGG 
                   
               
               
                   
               
               
                 1-6bp-rev 
                 CCGTCTCACCTCGGTTTACAGatttcgACCTCTGTT 
                 49 
               
               
                   
                 TGGGAAAATTGGGGACGCCGAGACGA 
                   
               
               
                   
               
               
                 1-7bp-for 
                 TCGTCTCGGCGTCCCCAATTTTCCCAAACAGAG 
                 50 
               
               
                   
                 GTtcgaaatCTGTAAACCGAGGTGAGACGG 
                   
               
               
                   
               
               
                 1-7bp-rev 
                 CCGTCTCACCTCGGTTTACAGatttcgaACCTCTGT 
                 51 
               
               
                   
                 TTGGGAAAATTGGGGACGCCGAGACGA 
                   
               
               
                   
               
               
                 2-0bp-for 
                 TCGTCTCGGAGGTTTTGGAACCTCTGTTTGGGA 
                 52 
               
               
                   
                 AAATTGGGGAGTCTGAGACGG 
                   
               
               
                   
               
               
                 2-0bp-rev 
                 CCGTCTCAGACTCCCCAATTTTCCCAAACAGAG 
                 53 
               
               
                   
                 GTTCCAAAACCTCCGAGACGA 
                   
               
               
                   
               
               
                 2-1bp-for 
                 TCGTCTCGGAGGTTTTGGACACCTCTGTTTGGG 
                 54 
               
               
                   
                 AAAATTGGGGAGTCTGAGACGG 
                   
               
               
                   
               
               
                 2-1bp-rev 
                 CCGTCTCAGACTCCCCAATTTTCCCAAACAGAG 
                 55 
               
               
                   
                 GTGTCCAAAACCTCCGAGACGA 
                   
               
               
                   
               
               
                 2-2bp-for 
                 TCGTCTCGGAGGTTTTGGACTACCTCTGTTTGG 
                 56 
               
               
                   
                 GAAAATTGGGGAGTCTGAGACGG 
                   
               
               
                   
               
               
                 2-2bp-rev 
                 CCGTCTCAGACTCCCCAATTTTCCCAAACAGAG 
                 57 
               
               
                   
                 GTAGTCCAAAACCTCCGAGACGA 
                   
               
               
                   
               
               
                 2-3bp-for 
                 TCGTCTCGGAGGTTTTGGACTTACCTCTGTTTG 
                 58 
               
               
                   
                 GGAAAATTGGGGAGTCTGAGACGG 
                   
               
               
                   
               
               
                 2-3bp-rev 
                 CCGTCTCAGACTCCCCAATTTTCCCAAACAGAG 
                 59 
               
               
                   
                 GTAAGTCCAAAACCTCCGAGACGA 
                   
               
               
                   
               
               
                 2-4bp-for 
                 TCGTCTCGGAGGTTTTGGACTTAACCTCTGTTT 
                 60 
               
               
                   
                 GGGAAAATTGGGGAGTCTGAGACGG 
                   
               
               
                   
               
               
                 2-4bp-rev 
                 CCGTCTCAGACTCCCCAATTTTCCCAAACAGAG 
                 61 
               
               
                   
                 GTTAAGTCCAAAACCTCCGAGACGA 
                   
               
               
                   
               
               
                 2-5bp-for 
                 TCGTCTCGGAGGTTTTGGACTTAGACCTCTGTT 
                 62 
               
               
                   
                 TGGGAAAATTGGGGAGTCTGAGACGG 
                   
               
               
                   
               
               
                 2-5bp-rev 
                 CCGTCTCAGACTCCCCAATTTTCCCAAACAGAG 
                 63 
               
               
                   
                 GTCTAAGTCCAAAACCTCCGAGACGA 
                   
               
               
                   
               
               
                 2-6bp-for 
                 TCGTCTCGGAGGTTTTGGACTTAGCACCTCTGT 
                 64 
               
               
                   
                 TTGGGAAAATTGGGGAGTCTGAGACGG 
                   
               
               
                   
               
               
                 2-6bp-rev 
                 CCGTCTCAGACTCCCCAATTTTCCCAAACAGAG 
                 65 
               
               
                   
                 GTGCTAAGTCCAAAACCTCCGAGACGA 
                   
               
               
                   
               
               
                 2-7bp-for 
                 TCGTCTCGGAGGTTTTGGACTTAGCTACCTCTG 
                 66 
               
               
                   
                 TTTGGGAAAATTGGGGAGTCTGAGACGG 
                   
               
               
                   
               
               
                 2-7bp-rev 
                 CCGTCTCAGACTCCCCAATTTTCCCAAACAGAG 
                 67 
               
               
                   
                 GTAGCTAAGTCCAAAACCTCCGAGACGA 
                   
               
               
                   
               
               
                 4-0bp-for 
                 TCGTCTCTGCACCCCCAATTTTCCCAAACAGAG 
                 68 
               
               
                   
                 GTCTGTAAACCGATGAGACGG 
                   
               
               
                   
               
               
                 4-0bp-rev 
                 CCGTCTCATCGGTTTACAGACCTCTGTTTGGGA 
                 69 
               
               
                   
                 AAATTGGGGGTGCAGAGACGA 
                   
               
               
                   
               
               
                 4-1bp-for 
                 TCGTCTCTGCACCCCCAATTTTCCCAAACAGAG 
                 70 
               
               
                   
                 GTtCTGTAAACCGATGAGACGG 
                   
               
               
                   
               
               
                 4-1bp-rev 
                 CCGTCTCATCGGTTTACAGaACCTCTGTTTGGG 
                 71 
               
               
                   
                 AAAATTGGGGGTGCAGAGACGA 
                   
               
               
                   
               
               
                 4-2bp-for 
                 TCGTCTCTGCACCCCCAATTTTCCCAAACAGAG 
                 72 
               
               
                   
                 GTatCTGTAAACCGATGAGACGG 
                   
               
               
                   
               
               
                 4-2bp-rev 
                 CCGTCTCATCGGTTTACAGatACCTCTGTTTGGG 
                 73 
               
               
                   
                 AAAATTGGGGGTGCAGAGACGA 
                   
               
               
                   
               
               
                 4-3bp-for 
                 TCGTCTCTGCACCCCCAATTTTCCCAAACAGAG 
                 74 
               
               
                   
                 GTaatCTGTAAACCGATGAGACGG 
                   
               
               
                   
               
               
                 4-3bp-rev 
                 CCGTCTCATCGGTTTACAGattACCTCTGTTTGGG 
                 75 
               
               
                   
                 AAAATTGGGGGTGCAGAGACGA 
                   
               
               
                   
               
               
                 4-4bp-for 
                 TCGTCTCTGCACCCCCAATTTTCCCAAACAGAG 
                 76 
               
               
                   
                 GTaaatCTGTAAACCGATGAGACGG 
                   
               
               
                   
               
               
                 4-4bp-rev 
                 CCGTCTCATCGGTTTACAGatttACCTCTGTTTGG 
                 77 
               
               
                   
                 GAAAATTGGGGGTGCAGAGACGA 
                   
               
               
                   
               
               
                 4-5bp-for 
                 TCGTCTCTGCACCCCCAATTTTCCCAAACAGAG 
                 78 
               
               
                   
                 GTgaaatCTGTAAACCGATGAGACGG 
                   
               
               
                   
               
               
                 4-5bp-rev 
                 CCGTCTCATCGGTTTACAGatttcACCTCTGTTTGG 
                 79 
               
               
                   
                 GAAAATTGGGGGTGCAGAGACGA 
                   
               
               
                   
               
               
                 4-6bp-for 
                 TCGTCTCTGCACCCCCAATTTTCCCAAACAGAG 
                 80 
               
               
                   
                 GTcgaaatCTGTAAACCGATGAGACGG 
                   
               
               
                   
               
               
                 4-6bp-rev 
                 CCGTCTCATCGGTTTACAGatttcgACCTCTGTTTG 
                 81 
               
               
                   
                 GGAAAATTGGGGGTGCAGAGACGA 
                   
               
               
                   
               
               
                 4-7bp-for 
                 TCGTCTCTGCACCCCCAATTTTCCCAAACAGAG 
                 82 
               
               
                   
                 GTtcgaaatCTGTAAACCGATGAGACGG 
                   
               
               
                   
               
               
                 4-7bp-rev 
                 CCGTCTCATCGGTTTACAGatttcgaACCTCTGTTT 
                 83 
               
               
                   
                 GGGAAAATTGGGGGTGCAGAGACGA 
                   
               
               
                   
               
               
                 5-0bp-for 
                 TCGTCTCGCCGAGGTTTTGGAACCTCTGTTTGG 
                 84 
               
               
                   
                 GAAAATTGGGGCTCGTGAGACGG 
                   
               
               
                   
               
               
                 5-0bp-rev 
                 CCGTCTCACGAGCCCCAATTTTCCCAAACAGAG 
                 85 
               
               
                   
                 GTTCCAAAACCTCGGCGAGACGA 
                   
               
               
                   
               
               
                 5-1bp-for 
                 TCGTCTCGCCGAGGTTTTGGACACCTCTGTTTG 
                 86 
               
               
                   
                 GGAAAATTGGGGCTCGTGAGACGG 
                   
               
               
                   
               
               
                 5-1bp-rev 
                 CCGTCTCACGAGCCCCAATTTTCCCAAACAGAG 
                 87 
               
               
                   
                 GTGTCCAAAACCTCGGCGAGACGA 
                   
               
               
                   
               
               
                 5-2bp-for 
                 TCGTCTCGCCGAGGTTTTGGACTACCTCTGTTT 
                 88 
               
               
                   
                 GGGAAAATTGGGGCTCGTGAGACGG 
                   
               
               
                   
               
               
                 5-2bp-rev 
                 CCGTCTCACGAGCCCCAATTTTCCCAAACAGAG 
                 89 
               
               
                   
                 GTAGTCCAAAACCTCGGCGAGACGA 
                   
               
               
                   
               
               
                 5-3bp-for 
                 TCGTCTCGCCGAGGTTTTGGACTTACCTCTGTT 
                 90 
               
               
                   
                 TGGGAAAATTGGGGCTCGTGAGACGG 
                   
               
               
                   
               
               
                 5-3bp-rev 
                 CCGTCTCACGAGCCCCAATTTTCCCAAACAGAG 
                 91 
               
               
                   
                 GTAAGTCCAAAACCTCGGCGAGACGA 
                   
               
               
                   
               
               
                 5-4bp-for 
                 TCGTCTCGCCGAGGTTTTGGACTTAACCTCTGT 
                 92 
               
               
                   
                 TTGGGAAAATTGGGGCTCGTGAGACGG 
                   
               
               
                   
               
               
                 5-4bp-rev 
                 CCGTCTCACGAGCCCCAATTTTCCCAAACAGAG 
                 93 
               
               
                   
                 GTTAAGTCCAAAACCTCGGCGAGACGA 
                   
               
               
                   
               
               
                 5-5bp-for 
                 TCGTCTCGCCGAGGTTTTGGACTTAGACCTCTG 
                 94 
               
               
                   
                 TTTGGGAAAATTGGGGCTCGTGAGACGG 
                   
               
               
                   
               
               
                 5-5bp-rev 
                 CCGTCTCACGAGCCCCAATTTTCCCAAACAGAG 
                 95 
               
               
                   
                 GTCTAAGTCCAAAACCTCGGCGAGACGA 
                   
               
               
                   
               
               
                 5-6bp-for 
                 TCGTCTCGCCGAGGTTTTGGACTTAGCACCTCT 
                 96 
               
               
                   
                 GTTTGGGAAAATTGGGGCTCGTGAGACGG 
                   
               
               
                   
               
               
                 5-6bp-rev 
                 CCGTCTCACGAGCCCCAATTTTCCCAAACAGAG 
                 97 
               
               
                   
                 GTGCTAAGTCCAAAACCTCGGCGAGACGA 
                   
               
               
                   
               
               
                 5-7bp-for 
                 TCGTCTCGCCGAGGTTTTGGACTTAGCTACCTC 
                 98 
               
               
                   
                 TGTTTGGGAAAATTGGGGCTCGTGAGACGG 
                   
               
               
                   
               
               
                 5-7bp-rev 
                 CCGTCTCACGAGCCCCAATTTTCCCAAACAGAG 
                 99 
               
               
                   
                 GTAGCTAAGTCCAAAACCTCGGCGAGACGA 
                   
               
               
                   
               
               
                 1-Chr10--54913298- 
                 TCGTCTCGGCGTCCCCTCCCATCACAGGCCCTG 
                 100 
               
               
                 54913376-for 
                 AGGTTTAAGAGAAAACCTGAGACGG 
                   
               
               
                   
               
               
                 1-Chr10-54913298- 
                 CCGTCTCAGGTTTTCTCTTAAACCTCAGGGCCT 
                 101 
               
               
                 54913376-rev 
                 GTGATGGGAGGGGACGCCGAGACGA 
                   
               
               
                   
               
               
                 2-Chr10--54913298- 
                 TCGTCTCGAACCATGGTTTTGTGGGCCAGGCCC 
                 102 
               
               
                 54913376-for 
                 ATGACCCTTCTCCTCTGGGAGTCTGAGACGG 
                   
               
               
                   
               
               
                 2-Chr10--54913298- 
                 CCGTCTCAGACTCCCAGAGGAGAAGGGTCATG 
                 103 
               
               
                 54913376-rev 
                 GGCCTGGCCCACAAAACCATGGTTCGAGACGA 
                   
               
               
                   
               
               
                 4-Chr10-54913298- 
                 TCGTCTCTGCACCCCCTCCCATCACAGGCCCTG 
                 104 
               
               
                 54913376-for 
                 AGGTTTAAGAGAAAACCATTGAGACGG 
                   
               
               
                   
               
               
                 4-Chr10-54913298- 
                 CCGTCTCAATGGTTTTCTCTTAAACCTCAGGGC 
                 105 
               
               
                 54913376-rev 
                 CTGTGATGGGAGGGGGTGCAGAGACGA 
                   
               
               
                   
               
               
                 5-Chr10-54913298- 
                 TCGTCTCGCCATGGTTTTGTGGGCCAGGCCCAT 
                 106 
               
               
                 54913376-for 
                 GACCCTTCTCCTCTGGGCTCGTGAGACGG 
                   
               
               
                   
               
               
                 5-Chr10-54913298- 
                 CCGTCTCACGAGCCCAGAGGAGAAGGGTCATG 
                 107 
               
               
                 54913376-rev 
                 GGCCTGGCCCACAAAACCATGGCGAGACGA 
                   
               
               
                   
               
               
                 3-for 
                 ATCCGTCTCCAGTCGAGTCGGATTTGATCTGAT 
                 108 
               
               
                   
                 CAAGAGACAG 
                   
               
               
                   
               
               
                 3-rev 
                 AACCGTCTCGGTGCGTTCGGATTTGATCCAGAC 
                 109 
               
               
                   
                 ATGATAAGATAC 
                   
               
               
                   
               
               
                 Esp3I-insert-for 
                 /Phos/CGCGTTGAGACGCTGCCATCCGTCTCGC 
                 110 
               
               
                   
               
               
                 Esp3I-insert-rev 
                 /Phos/TCGAGCGAGACGGATGGCAGCGTCTCAA 
                 111 
               
               
                   
               
               
                 Centromere_Chr_1_5_19- 
                 GTTGTTCGTCTCGGCGTCCTTGTGTTGTGTGTCT 
                 112 
               
               
                 1_2* 
                 TCAACTCACAGAGTTAAACGATGCTTTACACA 
                   
               
               
                   
                 GAGTAGACTTGAAACACTCTTTTTCTGGAGTCT 
                   
               
               
                   
                 GAGACGGTTCTGTTTTGGTGTGATTAGTTAT 
                   
               
               
                   
               
               
                 Centromere_Chr_1_5_19- 
                 GTTGGTCGTCTCTGCACCCTTGTGTTGTGTGTCT 
                 113 
               
               
                 4_5* 
                 TCAACTCACAGAGTTAAACGATGCTTTACACA 
                   
               
               
                   
                 GAGTAGACTTGAAACACTCTTTTTCTGGCTCGT 
                   
               
               
                   
                 GAGACGGTTCTGTTTTGGTGTGATTAGTTAT 
                   
               
               
                   
               
               
                 Ch5_155183064- 
                 GTTGTTCGTCTCGGCGTCCCACCGGCTCATGAG 
                 114 
               
               
                 155183141-1_2* 
                 AGGTAGAGCTAAGGTCCAAACCTAGGTTTATC 
                   
               
               
                   
                 TGAGACCGGAACTCATGTGATTAACTGTGGAG 
                   
               
               
                   
                 TCTGAGACGGTTCTGTTTTGGTGTGATTAGTTAT 
                   
               
               
                   
               
               
                 Ch5_155183064- 
                 GTTGGTCGTCTCTGCACCCCACCGGCTCATGAG 
                 115 
               
               
                 155183141-4_5* 
                 AGGTAGAGCTAAGGTCCAAACCTAGGTTTATC 
                   
               
               
                   
                 TGAGACCGGAACTCATGTGATTAACTGTGGCTC 
                   
               
               
                   
                 GTGAGACGGTTCTGTTTTGGTGTGATTAGTTAT 
                   
               
               
                   
               
               
                 Ch5_169395198- 
                 GTTGTTCGTCTCGGCGTCCTTAAGAACATAAAT 
                 116 
               
               
                 169395274-1_2* 
                 CCCCAGGAATTCACAGAAACCTTGGTTTGAGCT 
                   
               
               
                   
                 TTGGATTTCCCGCAGGATGTGGGATAGGAGTCT 
                   
               
               
                   
                 GAGACGGTTCTGTTTTGGTGTGATTAGTTAT 
                   
               
               
                   
               
               
                 Ch5_169395198- 
                 GTTGGTCGTCTCTGCACCCTTAAGAACATAAAT 
                 117 
               
               
                 169395274-4_5* 
                 CCCCAGGAATTCACAGAAACCTTGGTTTGAGCT 
                   
               
               
                   
                 TTGGATTTCCCGCAGGATGTGGGATAGGCTCGT 
                   
               
               
                   
                 GAGACGGTTCTGTTTTGGTGTGATTAGTTAT 
                   
               
               
                   
               
               
                 Ch12_62418577- 
                 GTTGTTCGTCTCGGCGTCCACTCCCTCTCCCCC 
                 118 
               
               
                 62418652-1_2* 
                 AAAAAGTAAAGGTAGAAAACCAAGGTTTACAG 
                   
               
               
                   
                 GCAACAAATAGCACAATGAATGGAATGGAGTC 
                   
               
               
                   
                 TGAGACGGTTCTGTTTTGGTGTGATTAGTTAT 
                   
               
               
                   
               
               
                 Ch12_62418577- 
                 GTTGGTCGTCTCTGCACCCACTCCCTCTCCCCC 
                 119 
               
               
                 62418652-4_5* 
                 AAAAAGTAAAGGTAGAAAACCAAGGTTTACAG 
                   
               
               
                   
                 GCAACAAATAGCACAATGAATGGAATGGCTCG 
                   
               
               
                   
                 TGAGACGGTTCTGTTTTGGTGTGATTAGTTAT 
                   
               
               
                   
               
               
                 chr13_102010574- 
                 GTTGTTCGTCTCGGCGTCCTAGGGAAGTGATCA 
                 120 
               
               
                 102010650-1_2* 
                 TAGCTGAGTTTCTGGAAAAACCTAGGTTTTAAA 
                   
               
               
                   
                 GTTGAGGAGACTTAAGTCCAAAACCTGGAGTC 
                   
               
               
                   
                 TGAGACGGTTCTGTTTTGGTGTGATTAGTTAT 
                   
               
               
                   
               
               
                 chr13_102010574- 
                 GTTGGTCGTCTCTGCACCCTAGGGAAGTGATCA 
                 121 
               
               
                 102010650-4_5* 
                 TAGCTGAGTTTCTGGAAAAACCTAGGTTTTAAA 
                   
               
               
                   
                 GTTGAGGAGACTTAAGTCCAAAACCTGGCTCG 
                   
               
               
                   
                 TGAGACGGTTCTGTTTTGGTGTGATTAGTTAT 
               
               
                   
               
               
                 Oligonucleotide sequences were annealed to create the fragments shown in FIG. 1. The names correspond to the fragment number (1, 2, 4, or 5) and then to the number of base pair spacer nucleotides separating the Cas9 binding site from the gix core site. 
               
               
                 *Double stranded gBlocks as described in the methods within the supporting material document. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Oligonucleotides for recCas9 construction 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                 Oligonucleotide Name 
                 Sequence 
                 NO: 
               
               
                   
               
               
                 1GGS-link-for_BamHI 
                 TTCATCGGATCCGATAAAAAGTATTCTATTG 
                 122 
               
               
                   
                 GTTTAGCTATCGGCAC 
                   
               
               
                   
               
               
                 5GGS-link-for_BamHI 
                 TTCATCGGATCCGGTGGTTCAGGTGGCAGC 
                 123 
               
               
                   
                 GGAG 
                   
               
               
                   
               
               
                 8GGS-link-for_BamHI 
                 TTCATCGGATCCGGAGGGTCCGGAGGTAGT 
                 124 
               
               
                   
                 GGCGGCAGCGGTGGTTCAGGTGGCAGCGGAG 
                   
               
               
                   
               
               
                 Cas9-rev-FLAG-NLS- 
                 AATAACCGGTTCAGACCTTCCTTTTCTTCTT 
                 125 
               
               
                 AgeI 
                 TGGGGAACCTCCCTTGTCGTCATCATCCTTA 
                   
               
               
                   
                 TAATCGGAGCCACCGTCACCCCCAAGCTGT 
                   
               
               
                   
                 GACAAATC 
                   
               
               
                   
               
               
                 1GGS-rev-BamHI 
                 TGATAAGGATCCACCCTTTGGTGGTCTTCCA 
                 126 
               
               
                   
                 AACCGCC 
                   
               
               
                   
               
               
                 2GGS-rev-BamH 
                 TGATAAGGATCCACCGCTACCACCCTTTGG 
                 127 
               
               
                   
                 TGGTCTTC 
                   
               
               
                   
               
               
                 Gin-for_NotI 
                 AGATCCGCGGCCGCTAATAC 
                 128 
               
               
                   
               
               
                 Esp3I-for-plasmid 
                 TTGAGTcgtctcTATACTCTTCCTTTTTCAATAT 
                 129 
               
               
                   
                 TATTGAAGCATTTATCAGGG 
                   
               
               
                   
               
               
                 Esp3I-rev-plasmid 
                 CTGGAAcgtctcACTGTCAGACCAAGTTTACTC 
                 130 
               
               
                   
                 ATATATACTTTAGATTG 
                   
               
               
                   
               
               
                 spec-Esp3I-for 
                 GGTGTGcgtctcTACAGTTATTTGCCGACTACC 
                 131 
               
               
                   
                 TTGGTGATCTCGC 
                   
               
               
                   
               
               
                 spec-Esp3I-rev 
                 ACACCAcgtctcTGTATGAGGGAAGCGGTGAT 
                 132 
               
               
                   
                 CGCC 
                   
               
               
                   
               
               
                 cpec assembly-for- 
                 CATACTCTTCCTTTTTCAATATTATTGAAGC 
                 133 
               
               
                 plasmid 
                 ATTTATCAGGG 
                   
               
               
                   
               
               
                 cpec assembly-rev- 
                 CTGTCAGACCAAGTTTACTCATATATACTTT 
                 134 
               
               
                 plasmid 
                 AGATTG 
                   
               
               
                   
               
               
                 cpec assembly-for-spec 
                 CAATCTAAAGTATATATGAGTAAACTTGGT 
                 135 
               
               
                   
                 CTGACAGTTTGCCGACTACCTTGGTGATCTCG 
                   
               
               
                   
               
               
                 cpec assembly-for-spec2 
                 CAATCTAAAGTATATATGAGTAAACTTGGT 
                 136 
               
               
                   
                 CTGACAGTTATTTGCCGACTACCTTGGTGAT 
                   
               
               
                   
                 CTCG 
                   
               
               
                   
               
               
                 cpec assembly-rev-spec 
                 CCCTGATAAATGCTTCAATAATATTGAAAA 
                 137 
               
               
                   
                 AGGAAGAGTATG 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Custom sequencing oligonucleotides 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                 Oligonucleotide Name 
                 Sequence 
                 NO: 
               
               
                   
               
               
                 Fwd CMV 
                 CGCAAATGGGCGGTAGGCGTG 
                 138 
               
               
                   
               
               
                 Cas9coRevE1 
                 CCGTGATGGATTGGTGAATC 
                 139 
               
               
                   
               
               
                 Cas9coRevE2 
                 CCCATACGATTTCACCTGTC 
                 140 
               
               
                   
               
               
                 Cas9coRevE3 
                 GGGTATTTTCCACAGGATGC 
                 141 
               
               
                   
               
               
                 Cas9coRevE4 
                 CTTAGAAAGGCGGGTTTACG 
                 142 
               
               
                   
               
               
                 Cas9coRevE5 
                 CTTACTAAGCTGCAATTTGG 
                 143 
               
               
                   
               
               
                 Cas9coRevE6 
                 TGTATTCATCGGTTATGACAG 
                 144 
               
               
                   
               
               
                 bGH_PArev seq1 
                 CAGGGTCAAGGAAGGCACG 
                 145 
               
               
                   
               
               
                 pHU6-gRNA_for 
                 GTTCCGCGCACATTTCC 
                 146 
               
               
                   
               
               
                 pHU6-gRNA_rev 
                 GCGGAGCCTATGGAAAAAC 
                 147 
               
               
                   
               
               
                 pCALNL-for1 
                 GCCTTCTTCTTTTTCCTACAGC 
                 148 
               
               
                   
               
               
                 pCALNL-for2 
                 CGCATCGAGCGAGCAC 
                 149 
               
               
                   
               
            
           
         
       
     
                     TABLE 5                  Genomic PCR primers                                 SEQ       Oligonucleotide       ID       Name   Sequence   NO:               FAM19A2-F1   TCAAGTAGCAAAAGAAGTAGGAGTCAG   150               FAM19A2-F2   TTAGATGCATTCGTGCTTGAAG   151               FAM19A2-C1   TTAATTTCTGCTGCTAGAACTAAATCTGG   152               FAM19A2-R1   GGGAAGAAAACTGGATGGAGAATG   153               FAM19A2-R2   CATAAATGACCTAGTGGAGCTG   154               FAM19A2-C2   TGGTTATTTTGCCCATTAGTTGATGC   155                    
Reporter Construction
 
     A five-piece Golden Gate assembly was used to construct reporters described below. Fragments 1-5 were flanked by Esp3I sites; Esp3I digestion created complementary 5′ overhangs specifying the order of fragment assembly ( FIG.  6   ). Fragments 1, 2, 4, and 5 were created by annealing forward and reverse complementary oligonucleotides listed in Table 5. Fragments were annealed by mixing 10 μl of each oligonucleotide (100 μM) in 20 μl of molecular grade water, incubating at 95° C. for 3 minutes and reducing the temperature to 16° C. at a rate of −0.1° C./sec. Fragment 3 was created by PCR amplifying the region containing kanR and a PolyA stop codon with primers 3-for and 3-rev. These primers also appended Esp3I on the 5′ and 3′ ends of this sequence. 
     Annealed fragments 1, 2, 4 and 5 were diluted 12,000 fold and 0.625 μl of each fragment were added to a mixture containing the following:
         1) 40-50 ng fragment 3   2) 100 ng pCALNL EGFP-Esp3I   3) 1 μL Tango Buffer (10×)   4) 1 μL DTT (10 mM)   5) 1 μL ATP (10 mM)   6) 0.25 uL T7 ligase (3,000 U/μL)   7) 0.75 uL Esp3I (10 U/μL)   8) H 2 O up to 10 μL       

     Reactions were incubated in thermal cycler programmed for 20 cycles (37° C. for 5 min, 20° C.). 
     After completion of the Golden Gate reactions, 7 μL of each reaction was mixed with 1 μL of ATP (10 mM), 1 μL of 10× Plasmid Safe ATP-dependent DNAse buffer (10×), and 1 μL of Plasmid Safe ATP-dependent DNAse (10 U/μL) (Epicentre, Madison, Wis.) to remove linear DNA and reduce background. DNAse digestions were incubated at 37° C. for 30 min and heat killed at 70° C. for 30 min. Half (5 μL) of each reaction was transformed into Mach1-T1 cells. Colonies were analyzed by colony PCR and sequenced. 
     The protocol was modified for reporters used in  FIG.  4   . Two gBlocks, encoding target sites to the 5′ or 3′ of the PolyA terminator were used instead of fragments 1, 2, 4 and 5. These gBlocks (10 ng) were added to the MMX, which was cycled 10 times (37° C. for 5 min, 20° C.) and carried forward as described above. 
     Plasmids 
     Unless otherwise stated, DNA fragments were isolated from agarose gels using QIAquick Gel Extraction Kit (Qiagen, Valencia, Calif.) and further purified using DNA Clean &amp; Concentrator-5 (Zymo Research, Irvine, Calif.) or Qiaquick PCR purification kit (Qiagen, Valencia, Calif.). PCR fragments not requiring gel purification were isolated using one of the kits listed above. 
     The pCALNL-GFP subcloning vector, pCALNL-EGFP-Esp3I, was used to clone all recCas9 reporter plasmids and was based on the previously described pCALNL-GFP vector (Matsuda and Cepko, Controlled expression of transgenes introduced by in vivo electroporation. Proceedings of the National Academy of Sciences of the United States of America 104, 1027-1032 (2007), which is incorporated herein by reference). To create pCALNL-EGFP-Esp3I, pCALNL-GFP vectors were digested with XhoI and MluI and gel purified to remove the loxP sites, the kanamycin resistance marker, and the poly-A terminator. Annealed oligonucleotides formed an EspI-Insert, that contained inverted Esp3I sites as well as XhoI and MluI compatible overhangs; this insert was ligated into the XhoI and MluI digested plasmid and transformed. 
     pCALNL-GFP recCas9 reporter plasmids were created by Golden Gate assembly with annealed oligos and PCR products containing compatible Esp3I overhangs. Golden Gate reactions were set up and performed as described previously with Esp3I (ThermoFisher Scientific, Waltham, Mass.) (Sanjana et al., A transcription activator-like effector toolbox for genome engineering. Nature protocols 7, 171-192 (2012), the entire contents of which is hereby incorporated by reference).  FIG.  6    outlines the general assembly scheme and relevant primers for reporter assembly as well as sequences for all recCas9 target sites are listed in Tables 2 and 6, respectively. A representative DNA sequence containing KanR (bold and underlined) and PolyA terminator (in italics and underlined) flanked by two recCas9 target sites is shown below. The target sites shown are both PAM_NT1-0 bp-gix_core-0bp-NT1_PAM (see Table 6). Protoadjacent spacer motifs (PAMs) are in bold. Base pair spacers are lower case. Gix site or gix-related sites are in italics and dCas9 binding sites are underlined. For the genomic reporter plasmids used in the assays of  FIG.  4   , a G to T transversion was observed in the kanamycin resistance marker, denoted by a G/T in the sequence below. This was present in all the reporters used in this figure, and it is not expected to affect the results, as it is far removed from the PolyA terminator and recCas9 target sites. 
     
       
         
           
               
            
               
                 (SEQ ID NO: 156) 
               
               
                 ACGCGT CCC   CAATTTTCCCAAACAGAGGT   CTGTAAACCGAGGTTTTGGA   A   
               
               
                   
               
               
                   CCTCTGTTTGGGAAAATTG   GGG AGTCGAGTCGGATTTGATCTGATCAAGA 
               
               
                   
               
               
                 GACAGGATGAGGATCGTTTCGC   ATGATTGAACAAGATGGATTGCACGCAG     
               
               
                   
               
               
                 
                   
                     GTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAA 
                   
                 
               
               
                   
               
               
                 
                   
                     CAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGTCGCAGG 
                   
                 
               
               
                   
               
               
                 
                   
                     GGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAA 
                   
                 
               
               
                   
               
               
                 
                   
                     CTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCC 
                   
                 
               
               
                   
               
               
                 
                   
                     TTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGC 
                   
                 
               
               
                   
               
               
                 
                   
                     TATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCT 
                   
                 
               
               
                   
               
               
                 
                   
                     GCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCT 
                   
                 
               
               
                   
               
               
                 
                   
                     TGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGC 
                   
                 
               
               
                   
               
               
                 
                   
                     GAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGAC 
                   
                 
               
               
                   
               
               
                 
                   
                     GAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGC 
                   
                 
               
               
                   
               
               
                 
                   
                     GCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCT 
                   
                 
               
               
                   
               
               
                 
                   
                     TGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGT 
                   
                 
               
               
                   
               
               
                 
                   
                     GGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCG 
                   
                 
               
               
                   
               
               
                 
                   
                     TGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGC 
                   
                 
               
               
                   
               
               
                 
                   
                     TTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTT 
                   
                 
               
               
                   
               
               
                     CTTGACGAGTTCTTCTGA   GCGGGACTCTGGGGTTCGAAATGACCGACCAA 
               
               
                   
               
               
                 GCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTA 
               
               
                   
               
               
                 TGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCC 
               
               
                   
               
               
                 TCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCATCGATAAC 
               
               
                   
               
               
                 
                   
                     TTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAA 
                   
                 
               
               
                   
               
               
                 
                   
                     TTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCA 
                   
                 
               
               
                   
               
               
                     AACTCATCAATGTATCTTATC   ATGTCTGGATCAAATCCGAACGCAC CCC   C   
               
               
                   
               
               
                 
                   AATTTTCCCAAACAGAGGT 
                   CTGTAAACCGAGGTTTTGGA 
                   ACCTCTGTTTG 
                 
               
               
                   
               
               
                   GGAAAATTG   GGG CTCGAG 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 List of target site sequences used in reporter assays 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                 Target site name 
                 Sequence 
                 NO: 
               
               
                   
               
               
                 PAM_NT1-0bp- 
                   CCC   CAATTTTCCCAAACAGAGGT t CTGTAAACCGAG   
                 157 
               
               
                 gix_core-0bp- 
                 
                   GTTTTGG 
                   AACCTCTGTTTGGGAAAATTG 
                   GGG 
                 
                   
               
               
                 NT1_PAM 
                   
                   
               
               
                   
               
               
                 PAM_NT1-1bp- 
                   CCC   CAATTTTCCCAAACAGAGGT t CTGTAAACCGAG   
                 158 
               
               
                 gix_core-1bp- 
                   GTTTTGG c AACCTCTGTTTGGGAAAATTG   GGG   
                   
               
               
                 NT1_PAM 
                   
                   
               
               
                   
               
               
                 PAM_NT1-2bp- 
                   CCC   CAATTTTCCCAAACAGAGGT at CTGTAAACCGA   
                 159 
               
               
                 gix_core-2bp- 
                   GGTTTTGG ct AACCTCTGTTTGGGAAAATTG   GGG   
                   
               
               
                 NT1_PAM 
                   
                   
               
               
                   
               
               
                 PAM_NT1-3bp- 
                   CCC   CAATTTTCCCAAACAGAGGT aat CTGTAAACCG   
                 160 
               
               
                 gix_core-3bp- 
                   AGGTTTTGG ctt AACCTCTGTTTGGGAAAATTG   GGG   
                   
               
               
                 NT1_PAM 
                   
                   
               
               
                   
               
               
                 PAM_NT1-4bp- 
                   CCC   CAATTTTCCCAAACAGAGGT aaat CTGTAAACCG   
                 161 
               
               
                 gix_core-4bp- 
                   AGGTTTTGG ctta AACCTCTGTTTGGGAAAATTG   GGG   
                   
               
               
                 NT1_PAM 
                   
                   
               
               
                   
               
               
                 PAM_NT1-5bp- 
                   CCC   CAATTTTCCCAAACAGAGGT gaaat CTGTAAACC   
                 162 
               
               
                 gix_core-5bp- 
                   GAGGTTTTGG cttag AACCTCTGTTTGGGAAAATTG   G   
                   
               
               
                 NT1_PAM 
                 
                   GG 
                 
                   
               
               
                   
               
               
                 PAM_NT1-6bp- 
                   CCC   CAATTTTCCCAAACAGAGGT cgaaat CTGTAAAC   
                 163 
               
               
                 gix_core-6bp- 
                   CGAGGTTTTGG cttagc AACCTCTGTTTGGGAAAATTG   
                   
               
               
                 NT1_PAM 
                 
                   GGG 
                 
                   
               
               
                   
               
               
                 PAM_NT1-7bp- 
                   CCC   CAATTTTCCCAAACAGAGGT tcgaaat CTGTAAAC   
                 164 
               
               
                 gix_core-7bp- 
                   CGAGGTTTTGG cttagct AACCTCTGTTTGGGAAAATT   
                   
               
               
                 NT1_PAM 
                 
                   G 
                   GGG 
                 
                   
               
               
                   
               
               
                 PAM_NT1-6bp- 
                   CCC   CAATTTTCCCAAACAGAGGT tcgaaat CTGTAAAC   
                 165 
               
               
                 gix_core-0bp- 
                 
                   CGAGGTTTTGG 
                   AACCTCTGTTTGGGAAAATTG 
                   GGG 
                 
                   
               
               
                 NT1_PAM 
                   
                   
               
               
                   
               
               
                 PAM_NT1-6bp- 
                   CCC   CAATTTTCCCAAACAGAGGT tcgaaat CTGTAAAC   
                 166 
               
               
                 gix_core-1bp- 
                   CGAGGTTTTGG c AACCTCTGTTTGGGAAAATTG   GGG   
                   
               
               
                 NT1_PAM 
                   
                   
               
               
                   
               
               
                 PAM_NT1-6bp- 
                   CCC   CAATTTTCCCAAACAGAGGT cgaaat CTGTAAAC   
                 167 
               
               
                 gix_core-2bp- 
                   CGAGGTTTTGG ct AACCTCTGTTTGGGAAAATTG   GGG   
                   
               
               
                 NT1_PAM 
                   
                   
               
               
                   
               
               
                 PAM_NT1-6bp- 
                   CCC   CAATTTTCCCAAACAGAGGT cgaaat CTGTAAAC   
                 168 
               
               
                 gix_core-4bp- 
                   CGAGGTTTTGG ctta AACCTCTGTTTGGGAAAATTG   G   
                   
               
               
                 NT1_PAM 
                 
                   GG 
                 
                   
               
               
                   
               
               
                 PAM_NT1-6bp- 
                   CCC   CAATTTTCCCAAACAGAGGT cgaaat CTGTAAAC   
                 169 
               
               
                 gix_core-5bp- 
                   CGAGGTTTTGG cttag AACCTCTGTTTGGGAAAATTG   
                   
               
               
                 NT1_PAM 
                 
                   GGG 
                 
                   
               
               
                   
               
               
                 PAM_NT1-0bp- 
                 
                   CCC 
                   CAATTTTCCCAAACAGAGGT 
                   CTGTAAACCGAG 
                 
                 170 
               
               
                 gix_core-6bp- 
                   GTTTTGG cttagc AACCTCTGTTTGGGAAAATTG   GGG   
                   
               
               
                 NT1_PAM 
                   
                   
               
               
                   
               
               
                 PAM_NT1-1bp- 
                   CCC   CAATTTTCCCAAACAGAGGT t CTGTAAACCGAG   
                 171 
               
               
                 gix_core-6bp- 
                   GTTTTGG cttagc AACCTCTGTTTGGGAAAATTG   GGG   
                   
               
               
                 NT1_PAM 
                   
                   
               
               
                   
               
               
                 PAM_NT1-2bp- 
                   CCC   CAATTTTCCCAAACAGAGGT at CTGTAAACCGA   
                 172 
               
               
                 gix_core-6bp- 
                   GGTTTTGG cttagc AACCTCTGTTTGGGAAAATTG   GGG   
                   
               
               
                 NT1_PAM 
                   
                   
               
               
                   
               
               
                 PAM_NT1-3bp- 
                   CCC   CAATTTTCCCAAACAGAGGT aat CTGTAAACCG   
                 173 
               
               
                 gix_core-6bp- 
                   AGGTTTTGG cttagc AACCTCTGTTTGGGAAAATTG   G   
                   
               
               
                 NT1_PAM 
                 
                   GG 
                 
                   
               
               
                   
               
               
                 PAM_NT1-4bp- 
                   CCC   CAATTTTCCCAAACAGAGGT aaat CTGTAAACCG   
                 174 
               
               
                 gix_core-6bp- 
                   AGGTTTTGG cttagc AACCTCTGTTTGGGAAAATTG   G   
                   
               
               
                 NT1_PAM 
                 
                   GG 
                 
                   
               
               
                   
               
               
                 PAM_NT1-5bp- 
                   CCC   CAATTTTCCCAAACAGAGGT gaaat CTGTAAACC   
                 175 
               
               
                 gix_core-6bp- 
                   GAGGTTTTGG cttagc AACCTCTGTTTGGGAAAATTG   G   
                   
               
               
                 NT1_PAM 
                 
                   GG 
                 
                   
               
               
                   
               
               
                 Chromosome_10- 
                   CCC   CTCCCATCACAGGCCCTGAG gtttaa GAGAAAAC   
                 176 
               
               
                 54913298-54913376* 
                   CATGGTTTTGTG ggccag GCCCATGACCCTTCTCCTCT   
                   
               
               
                   
                 
                   GGG 
                 
                   
               
               
                   
               
               
                 Centromere_Chromosomes_1_5_19 
                   CCT   TGTGTTGTGTGTCTTCAACT cacag AGTTAAACGA   
                 177 
               
               
                   
                   TGCTTTACAC agagta GACTTGAAACACTCTTTTTC   TGG   
                   
               
               
                   
               
               
                 Chromosome_5_155183064- 
                   CCA   CCGGCTCATGAGAGGTAGAG ctaag GTCCAAAC   
                 178 
               
               
                 155183141 
                   CTAGGTTTATCT gagacc GGAACTCATGTGATTAACTG   
                   
               
               
                 (site 1) 
                 
                   TGG 
                 
                   
               
               
                   
               
               
                 Chromosome_5_169395198- 
                   CCT   TAAGAACATAAATCCCCAGG aattc ACAGAAACC   
                 179 
               
               
                 169395274 
                   TTGGTTTGAGC tttgga TTTCCCGCAGGATGTGGGAT   A   
                   
               
               
                 (site 2) 
                 
                   GG 
                 
                   
               
               
                   
               
               
                 Chromosome_12_62418577- 
                   CCA   CTCCCTCTCCCCCAAAAAGT aaagg TAGAAAACC   
                 180 
               
               
                 62418652 
                   AAGGTTTACAG gcaac AAATAGCACAATGAATGGAA   
                   
               
               
                   
                 
                   TGG 
                 
                   
               
               
                   
               
               
                 Chromosome_13_102010574- 
                   CCT   AGGGAAGTGATCATAGCTGA gtttct GGAAAAAC   
                 181 
               
               
                 102010650 
                   CTAGGTTTTAAA gttga GGAGACTTAAGTCCAAAACC   T   
                   
               
               
                 (FGF14) 
                 
                   GG 
                 
               
               
                   
               
               
                 Protoadjacent spacer motifs (PAMs) are in bold. Base pair spacers are lower case. Gix site or gix-related sites are in italics and dCas9 binding sites are underlined. 
               
               
                 *Chromosome_10 reporter contains two overlapping PAM sites and dCas9 binding sites on the 5′ and 3′ ends of the gix sites. 
               
            
           
         
       
     
     Plasmids containing the recCas9 gene were constructed by PCR amplification of a gBlock encoding an evolved, hyperactivated Gin variant (Ginβ) (Gaj et al., A comprehensive approach to zinc-finger recombinase customization enables genomic targeting in human cells. Nucleic acids research 41, 3937-3946 (2013), the entire contents of which is hereby incorporated by reference) with the oligonucleotides 1GGS-rev-BamHI or 2GGS-rev-BamHI (using linker SEQ ID NO: 182) and Gin-for-NotI. PCR fragments were digested with BamHI and NotI, purified and ligated into a previously described expression vector (Addgene plasmid 43861) (see, e.g., Fu et al., High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature biotechnology 31, 822-826 (2013), the entire contents of which is hereby incorporated by reference) to produce subcloning vectors pGin-1GGS and pGIN-2GGS (using linker SEQ ID NO: 182). Oligonucleotides 1GGS-link-for-BamHI, 5GGS-link-for-BamHI (using linker SEQ ID NO: 701), or 8GGS-link-for-BamHI (using linker SEQ ID NO: 183) were used with Cas9-rev-FLAG-NLS-AgeI to construct PCR fragments encoding Cas9-FLAG-NLS with a 1, 5, or 8 GGS linker (see Table 3). For DNA sequences encoding the GGS amino acid linkers, see Table 7. PCR fragments and subcloning plasmids were digested with BamHI and AgeI and ligated to create plasmids pGinβ-2×GGS-dCas9-FLAG-NLS (using linker SEQ ID NO: 182), pGinβ-5×GGS-dCas9-FLAG-NLS (using linker SEQ ID NO: 701), and pGinβ-8×GGS-dCas9-FLAG-NLS (using linker SEQ ID NO: 183). For the DNA and amino acid sequence of the pGinβ-8×GGS-dCas9-FLAG-NLS (i.e., recCas9), see below. The sequence encoding Ginβ is shown in bold; those encoding GGS linkers are shown in italics; those encoding dCas9 linkers are black; those encoding the FLAG tag and NLS are underlined and in lowercase, respectively. 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 184) 
                   
               
               
                 
                   ATGCTCATTGGCTACGTGCGCGTCTCAACTAACGACCAGAATACCGATCTTC 
                 
                   
               
               
                   
               
               
                 
                   AGAGGAACGCACTGGTTTGTGCAGGCTGCGAACAGATTTTCGAGGACAAAC 
                 
               
               
                   
               
               
                 
                   TCAGCGGGACACGGACGGACAGACCTGGCCTCAAGCGAGCACTCAAGAGGC 
                 
               
               
                   
               
               
                 
                   TGCAGAAAGGAGACACTCTGGTGGTCTGGAAATTGGACCGCCTGGGTCGAA 
                 
               
               
                   
               
               
                 
                   GCATGAAGCATCTCATTTCTCTGGTTGGCGAACTGCGAGAAAGGGGGATCA 
                 
               
               
                   
               
               
                 
                   ACTTTCGAAGTCTGACGGATTCCATAGATACAAGCAGCCCCATGGGCCGGT 
                 
               
               
                   
               
               
                 
                   TCTTCTTCTACGTGATGGGTGCACTGGCTGAAATGGAAAGAGAACTCATTAT 
                 
               
               
                   
               
               
                 
                   AGAGCGAACCATGGCAGGGCTTGCGGCTGCCAGGAATAAAGGCAGGCGGTT 
                 
               
               
                   
               
               
                 
                   TGGAAGACCACCAAAG 
                   GGTGGATCCGGAGGGTCCGGAGGTAGTGGCGGCAGCGG 
                 
               
               
                   
               
               
                   TGGTTCAGGTGGCAGCGGAGGGTCAGGAGGCTCT GATAAAAAGTATTCTATTGGTT 
               
               
                   
               
               
                 TAGCTATCGGCACTAATTCCGTTGGATGGGCTGTCATAACCGATGAATACAAAGT 
               
               
                   
               
               
                 ACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGATTAAAAA 
               
               
                   
               
               
                 GAATCTTATCGGTGCCCTCCTATTCGATAGTGGCGAAACGGCAGAGGCGACTCGC 
               
               
                   
               
               
                 CTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTAC 
               
               
                   
               
               
                 TTACAAGAAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACC 
               
               
                   
               
               
                 GTTTGGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAACATGAACGGCACCCCA 
               
               
                   
               
               
                 TCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAACGATTTA 
               
               
                   
               
               
                 TCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAAT 
               
               
                   
               
               
                 CTACTTGGCTCTTGCCCATATGATAAAGTTCCGTGGGCACTTTCTCATTGAGGGTG 
               
               
                   
               
               
                 ATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCAGTTAGTACAAAC 
               
               
                   
               
               
                 CTATAATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGTGGCGTGGATGCGAA 
               
               
                   
               
               
                 GGCTATTCTTAGCGCCCGCCTCTCTAAATCCCGACGGCTAGAAAACCTGATCGCA 
               
               
                   
               
               
                 CAATTACCCGGAGAGAAGAAAAATGGGTTGTTCGGTAACCTTATAGCGCTCTCAC 
               
               
                   
               
               
                 TAGGCCTGACACCAAATTTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATT 
               
               
                   
               
               
                 GCAGCTTAGTAAGGACACGTACGATGACGATCTCGACAATCTACTGGCACAAAT 
               
               
                   
               
               
                 TGGAGATCAGTATGCGGACTTATTTTTGGCTGCCAAAAACCTTAGCGATGCAATC 
               
               
                   
               
               
                 CTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCG 
               
               
                   
               
               
                 CTTCAATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGC 
               
               
                   
               
               
                 CCTAGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATATTCTTTGATCAGTCG 
               
               
                   
               
               
                 AAAAACGGGTACGCAGGTTATATTGACGGCGGAGCGAGTCAAGAGGAATTCTAC 
               
               
                   
               
               
                 AAGTTTATCAAACCCATATTAGAGAAGATGGATGGGACGGAAGAGTTGCTTGTA 
               
               
                   
               
               
                 AAACTCAATCGCGAAGATCTACTGCGAAAGCAGCGGACTTTCGACAACGGTAGC 
               
               
                   
               
               
                 ATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGAGG 
               
               
                   
               
               
                 ATTTTTATCCGTTCCTCAAAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTT 
               
               
                   
               
               
                 TCGCATACCTTACTATGTGGGACCCCTGGCCCGAGGGAACTCTCGGTTCGCATGG 
               
               
                   
               
               
                 ATGACAAGAAAGTCCGAAGAAACGATTACTCCATGGAATTTTGAGGAAGTTGTC 
               
               
                   
               
               
                 GATAAAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCAACTTTGACAAG 
               
               
                   
               
               
                 AATTTACCGAACGAAAAAGTATTGCCTAAGCACAGTTTACTTTACGAGTATTTCA 
               
               
                   
               
               
                 CAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCATGCGTAAAC 
               
               
                   
               
               
                 CCGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGATCTGTTATTCAAGA 
               
               
                   
               
               
                 CCAACCGCAAAGTGACAGTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTG 
               
               
                   
               
               
                 AATGCTTCGATTCTGTCGAGATCTCCGGGGTAGAAGATCGATTTAATGCGTCACT 
               
               
                   
               
               
                 TGGTACGTATCATGACCTCCTAAAGATAATTAAAGATAAGGACTTCCTGGATAAC 
               
               
                   
               
               
                 GAAGAGAATGAAGATATCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAG 
               
               
                   
               
               
                 ATCGGGAAATGATTGAGGAAAGACTAAAAACATACGCTCACCTGTTCGACGATA 
               
               
                   
               
               
                 AGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGACGATTGTCGC 
               
               
                   
               
               
                 GGAAACTTATCAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATT 
               
               
                   
               
               
                 TTCTAAAGAGCGACGGCTTCGCCAATAGGAACTTTATGCAGCTGATCCATGATGA 
               
               
                   
               
               
                 CTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGA 
               
               
                   
               
               
                 CTCATTGCACGAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGC 
               
               
                   
               
               
                 ATACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTTAAGGTCATGGGACGTCAC 
               
               
                   
               
               
                 AAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAG 
               
               
                   
               
               
                 GGGCAAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGGTATTAAAGA 
               
               
                   
               
               
                 ACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCAATTGCAGAA 
               
               
                   
               
               
                 CGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAG 
               
               
                   
               
               
                 GAACTGGACATAAACCGTTTATCTGATTACGACGTCGATGCCATTGTACCCCAAT 
               
               
                   
               
               
                 CCTTTTTGAAGGACGATTCAATCGACAATAAAGTGCTTACACGCTCGGATAAGAA 
               
               
                   
               
               
                 CCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAGA 
               
               
                   
               
               
                 ACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATA 
               
               
                   
               
               
                 ACTTAACTAAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTAT 
               
               
                   
               
               
                 TAAACGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGATACT 
               
               
                   
               
               
                 AGATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCGGGAAGT 
               
               
                   
               
               
                 CAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTCAGAAAGGATTTTCAA 
               
               
                   
               
               
                 TTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGCTTATCTTA 
               
               
                   
               
               
                 ATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGT 
               
               
                   
               
               
                 TTGTGTATGGTGATTACAAAGTTTATGACGTCCGTAAGATGATCGCGAAAAGCGA 
               
               
                   
               
               
                 ACAGGAGATAGGCAAGGCTACAGCCAAATACTTCTTTTATTCTAACATTATGAAT 
               
               
                   
               
               
                 TTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGCAAACGACCTTTA 
               
               
                   
               
               
                 ATTGAAACCAATGGGGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTC 
               
               
                   
               
               
                 GCGACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACATAGTAAAGAAAACT 
               
               
                   
               
               
                 GAGGTGCAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGT 
               
               
                   
               
               
                 GATAAGCTCATCGCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTC 
               
               
                   
               
               
                 GATAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGGAA 
               
               
                   
               
               
                 AATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGC 
               
               
                   
               
               
                 GCTCGTCTTTTGAAAAGAACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGA 
               
               
                   
               
               
                 AGTAAAAAAGGATCTCATAATTAAACTACCAAAGTATAGTCTGTTTGAGTTAGAA 
               
               
                   
               
               
                 AATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGCTTCAAAAGGGGAACGA 
               
               
                   
               
               
                 ACTCGCACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCATTACGAG 
               
               
                   
               
               
                 AAGTTGAAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTTTTGTTGAGCAG 
               
               
                   
               
               
                 CACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGA 
               
               
                   
               
               
                 GTCATCCTAGCTGATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCAC 
               
               
                   
               
               
                 AGGGATAAACCCATACGTGAGCAGGCGGAAAATATTATCCATTTGTTTACTCTTA 
               
               
                   
               
               
                 CCAACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCAA 
               
               
                   
               
               
                 ACGATACACTTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCATC 
               
               
                   
               
               
                 ACGGGATTATATGAAACTCGGATAGATTTGTCACAGCTTGGGGGTGAC GGTGGCT   
               
               
                   
               
               
                   CC   GATTATAAGGATGATGACGACAAG   GGAGGTTCC ccaaagaagaaaaggaaggtcTGA 
               
               
                   
               
               
                 (SEQ ID NO: 185) 
                   
               
               
                 
                   MLIGYVRVSTNDQNTDLQRNALVCAGCEQIFEDKLSGTRTDRPGLKRALKRLQ 
                 
                   
               
               
                   
               
               
                 
                   KGDTLVVWKLDRLGRSMKHLISLVGELRERGINFRSLTDSIDTSSPMGRFFFYV 
                 
               
               
                   
               
               
                 
                   MGALAEMERELIIERTMAGLAAARNKGRRFGRPPK 
                   GGSGGSGGSGGSGGSGGSG 
                 
               
               
                   
               
               
                   GSGGS DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD 
               
               
                   
               
               
                 SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDK 
               
               
                   
               
               
                 KHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFL 
               
               
                   
               
               
                 IEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQ 
               
               
                   
               
               
                 LPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQ 
               
               
                   
               
               
                 YADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL 
               
               
                   
               
               
                 PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK 
               
               
                   
               
               
                 QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNS 
               
               
                   
               
               
                 RFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY 
               
               
                   
               
               
                 FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE 
               
               
                   
               
               
                 CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIE 
               
               
                   
               
               
                 ERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFA 
               
               
                   
               
               
                 NRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDE 
               
               
                   
               
               
                 LVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVEN 
               
               
                   
               
               
                 TQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTR 
               
               
                   
               
               
                 SDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKA 
               
               
                   
               
               
                 GFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF 
               
               
                   
               
               
                 YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ 
               
               
                   
               
               
                 EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRK 
               
               
                   
               
               
                 VLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYS 
               
               
                   
               
               
                 VLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
               
               
                   
               
               
                 YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ 
               
               
                   
               
               
                 LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLT 
               
               
                   
               
               
                 NLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD GGS   DYK   
               
               
                   
               
               
                   DDDDK   GGS pkkkrkv Stop 
               
            
           
         
       
     
     The Gin recombinase catalytic domain, which is amino acids 1-142 of SEQ ID NO: 185, is identical to the sequence of SEQ ID NO: 713. The dCas9 domain, in which is amino acids 167-1533 of SEQ ID NO: 185 is identical to the sequence of SEQ ID NO: 712. 
     
       
         
           
               
            
               
                 (SEQ ID NO: 713) 
               
               
                 MLIGYVRVSTNDQNTDLQRNALVCAGCEQIFEDKLSGTRTDRPGLKRALK 
               
               
                   
               
               
                 RLQKGDTLVVWKLDRLGRSMKHLISLVGELRERGINFRSLTDSIDTSSPM 
               
               
                   
               
               
                 GRFFFYVMGALAEMERELIIERTMAGLAAARNKGRRFGRPPK 
               
               
                   
               
               
                 (SEQ ID NO: 712) 
               
               
                 DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL 
               
               
                   
               
               
                 LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL 
               
               
                   
               
               
                 EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL 
               
               
                   
               
               
                 RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI 
               
               
                   
               
               
                 NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN 
               
               
                   
               
               
                 FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL 
               
               
                   
               
               
                 LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF 
               
               
                   
               
               
                 FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK 
               
               
                   
               
               
                 QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY 
               
               
                   
               
               
                 VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN 
               
               
                   
               
               
                 LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL 
               
               
                   
               
               
                 LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII 
               
               
                   
               
               
                 KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL 
               
               
                   
               
               
                 KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS 
               
               
                   
               
               
                 LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM 
               
               
                   
               
               
                 GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV 
               
               
                   
               
               
                 ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDS 
               
               
                   
               
               
                 IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT 
               
               
                   
               
               
                 KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR 
               
               
                   
               
               
                 EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY 
               
               
                   
               
               
                 PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT 
               
               
                   
               
               
                 LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ 
               
               
                   
               
               
                 TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK 
               
               
                   
               
               
                 GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY 
               
               
                   
               
               
                 SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED 
               
               
                   
               
               
                 NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP 
               
               
                   
               
               
                 IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS 
               
               
                   
               
               
                 ITGLYETRIDLSQLGGD 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 DNA sequences encoding GGS linkers 
               
            
           
           
               
               
               
               
            
               
                 GGS 
                 SEQ ID 
                   
                 SEQ ID 
               
               
                 linkers 
                 NO: 
                 DNA sequences for GGS linkers 
                 NO: 
               
               
                   
               
               
                 2XGGS 
                 182 
                 GGTGGTAGCGGTGGATCC 
                 186 
               
               
                   
               
               
                 5XGGS 
                 701 
                 GGTGGATCCGGTGGTTCAGGTGGCAGCGGAGGGTCAG 
                 187 
               
               
                   
                   
                 GAGGCTCT 
                   
               
               
                   
               
               
                 8XGGS 
                 183 
                 GGTGGATCCGGAGGGTCCGGAGGTAGTGGCGGCAGC 
                 188 
               
               
                   
                   
                 GGTGGTTCAGGTGGCAGCGGAGGGTCAGGAGGCTCT 
               
               
                   
               
            
           
         
       
     
     For plasmid sequencing experiments, the AmpR gene in pGinβ-8×GGS-dCas9-FLAG-NLS (using linker SEQ ID NO: 183) was replaced with SpecR by golden gate cloning with PCR fragments. Esp3I sites were introduced into the pGinβ-8×GGS-dCas9-FLAG-NLS (using linker SEQ ID NO: 183) plasmid at sites flanking the AmpR gene by PCR with Esp3I-for-plasmid and Esp3I-rev-plasmid. The primers spec-Esp3I-for and spec-Esp3I-rev were used to amplify the SpecR marker as well as introduce Esp3I sites and Esp3I generated overhangs compatible with those generated by the Esp3I-cleaved plasmid PCR product. Golden gate assembly was performed on the two fragments following the protocol used to generate the reporter plasmids as described herein. 
     The pHU6-NT1 guide RNA expression vector was based on the previously described pFYF1328 (Fu et al., High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature biotechnology 31, 822-826 (2013), the entire contents of which is hereby incorporated by reference) altered to target a region within the bacterial luciferase gene LuxAB. Guide RNA expression vectors were created by PCR amplification of the entire vector with a universal primer R.pHU6.TSS(-1).univ and primers encoding unique guide RNA sequences (Table 1). A list of the guide RNA sequences is given in Table 8. These primers were phosphorylated with T4 polynucleotide kinase. The PCR reaction products and linear guide RNA expression vectors were blunt-end ligated and transformed. Guide RNA expression vectors used in initial optimizations, off target control guide RNA sequences and those targeting Chromosome 10 locus contained AmpR. All other plasmids described in this study contained specR to facilitate sequencing experiments. Spectinomycin resistance was initially introduced into guide RNA expression vectors via CPEC essentially as described (Quan et al., Circular polymerase extension cloning of complex gene libraries and pathways. PloS one 4, e6441 (2009); and Hillson (2010), vol. 2015, pp. CPEC protocol; each of which is incorporated herein by reference) and guide RNA plasmids were then constructed by PCR amplification of the vector, as described above. Reactions were incubated overnight at 37° C. with 40 U of DpnI, purified and transformed. Fragments for CPEC were generated by PCR amplification of a guide RNA expression vector with oligonucleotides cpec-assembly-for-spec2 and cpec assembly-rev. The specR fragment was generated by PCR amplification of the SpecR gene via the oligonucleotides cpec-assembly-for-spec and cpec-assembly-rev-spec. pUC19 (ThermoFisher Scientific, Waltham, Mass.) was similarly modified. 
                     TABLE 8                  List of gRNA sequences                                 SEQ ID       gRNA name   gRNA-sequence   NO:               on-target_gRNA   ACCTCTGTTTGGGAAAATTG   189               non-target_gRNA   gCACACTAGTTAGGGATAACA   190               Chromosome_10-54913298-   gCCTCAGGGCCTGTGATGGGA   191       54913376_gRNA-rev-5                       Chromosome_10-54913298-   gCTCAGGGCCTGTGATGGGAG   192       54913376_gRNA-rev-6                       Chromosome_10-54913298-   GGCCCATGACCCTTCTCCTC   193       54913376_gRNA-for-5                       Chromosome_10-54913298-   GCCCATGACCCTTCTCCTCT   194       54913376_gRNA-for-6                       Centromere_Chromosomes_1_5_19-   GACTTGAAACACTCTTTTTC   195       gRNA-for                       Centromere_Chromosomes_1_5_19-   gAGTTGAAGACACACAACACA   196       gRNA-rev                       Chromosome_5_155183064-   GGAACTCATGTGATTAACTG   197       155183141_(site 1)_gRNA-for                       Chromosome_5_155183064-   gTCTACCTCTCATGAGCCGGT   198       155183141_(site 1)_gRNA-rev                       Chromosome_5_169395198-   gTTTCCCGCAGGATGTGGGAT   199       169395274_(site 2)_gRNA-for                       Chromosome_5_169395198-   gCCTGGGGATTTATGTTCTTA   200       169395274_(site 2)_gRNA-rev                       Chromosome_12_62418577-   gAAATAGCACAATGAATGGAA   201       62418652_gRNA-for                       Chromosome_12_62418577-   gACTTTTTGGGGGAGAGGGAG   202       62418652_gRNA-rev                       Chromosome_13_102010574-   GGAGACTTAAGTCCAAAACC   203       102010650_(FGF14)_gRNA-for                       Chromosome_13_102010574-   gTCAGCTATGATCACTTCCCT   204       102010650_(FGF14)_gRNA-rev                       Off target-for (CLTA)   GCAGATGTAGTGTTTCCACA   205               Off target-rev(VEGF)   GGGTGGGGGGAGTTTGCTCC   206               Chromosome_12_62098359-   gATATCCGTTTATCAGTGTCA   207       62098434_(FAM19A2)_gRNA-rev                       Chromosome_12_62098359-   gTTCCTAAGCTTGGGCTGCAG   208       62098434_(FAM19A2)_gRNA-for                       Chromosome_12_62112591-   gCCTAAAAGTGACTGGGAGAA   209       62112668_(FAM19A2)_gRNA-rev                       Chromosome_12_62112591-   gCACAGTCCCATATTTCTTGG   210       62112668_(FAM19A2)_gRNA-for                    
Cell Culture and Transfection
 
     HEK293T cells were purchased from the American Type Culture Collection (ATCC, Manassas, Va.). Cells were cultured in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM)+GlutaMAX-I (4.5 g/L D glucose+110 mg/mL sodium pyruvate) supplemented with 10% fetal bovine serum (FBS, Life Technologies, Carlsbad, Calif.). Cells were cultured at 37° C. at 5% CO 2  in a humidified incubator. 
     Plasmid used for transfections were isolated from PureYield Plasmid Miniprep System (Promega, Madison, Wis.). The night before transfections, HEK293T cells were seeded at a density of 3×10 5  cells per well in 48 well collagen-treated plates (Corning, Corning, N.Y.). Transfections reactions were prepared in 25 μL of Opti-MEM (ThermoFisher Scientific, Waltham, Mass.). For each transfection, 45 ng of each guide RNA expression vector, 9 ng of reporter plasmid, 9 ng of piRFP670-N1 (Addgene Plasmid 45457), and 160 ng of recCas9 expression vector were mixed, combined with 0.8 μL lipofectamine 2000 in Opti-MEM (ThermoFisher Scientific, Waltham, Mass.) and added to individual wells. 
     Flow Cytometry 
     After 60-72 hours post-transfection, cells were washed with phosphate buffered saline and harvested with 50 μL of 0.05% trypsin-EDTA (Life Technologies, Carlsbad, Calif.) at 37° C. for 5-10 minutes. Cells were diluted in 250 μL culture media and run on a BD Fortessa analyzer. iRFP fluorescence was excited using a 635 nm laser and emission was collected using a 670/30 band pass filter. EGFP was excited using a 488 nM laser and emission fluorescence acquired with a 505 long pass and 530/30 band pass filters. Data was analyzed on FlowJo Software, gated for live and transfected events (expressing iRFP). Positive GFP-expressing cells were measured as a percentage of transfected cells gated from at least 6,000 live events. For optimization experiments, assay background was determined by measuring the percentage of transfected cells producing eGFP upon cotransfection with reporter plasmid and pUC, without recCas9 or guide RNA expression vectors. This background was then subtracted from percentage of eGFP-positive cells observed when the reporter plasmid was cotransfected with recCas9 and the on-target or non-target guide RNA expression vectors. 
     Identification of Genomic Target Sites 
     Searching for appropriate target sites was done using Bioconductor, an open-source bioinformatics package using the R statistical programming (Fu et al., High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature biotechnology 31, 822-826 (2013), the entire contents of which is hereby incorporated by reference). The latest release (GRCh38) of the human reference genome published by the Genome Reference Consortium was used to search for sites that matched both the PAM requirement of Cas9 and the evolved gix sequence as described in the text. With the genome loaded into R, each search pattern was represented as a Biostring, a container in R that allowed for string matching and manipulation Scanning both strands of DNA for the entire genome, using the stated parameters, reveals approximately 450 potential targets in the human genome when searching using the GRCh38 reference assembly (Table 9). 
                     TABLE 9                  recCas9 genomic targets identified in silico                                                     Pattern   SEQ ID       Chr.   Start   End   Sequence   ID   NO:                                             chr1   34169027   34169103   CCTTTAGTGAAAAGTAGACAGCTCTGAATAT   2   211                   GAAAGGTAGGTTTTCATTTCTGGGAAAGAGA                   CGCCAAGTGATGTGG               chr1   51006703   51006780   CCTCCAATAAATATGGGACTATGTGGAAAG   1   212                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGACGGGAAGAATGG               chr1   89229373   89229450   CCATTCTGCCCGTCACTTTCAGGTACACCAA   1   213                   TCAAACGTAGGTTTAGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr1   115638077   115638154   CCATTCTCCCCGTCACTTTCAGGTACAACAA   1   214                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr1   122552402   122552478   CCTTGTAGTGTGTGTATTCAACTCACAGAGT   2   215                   TAAACGATCCTTTACACAGAGCAGACTTGAA                   ACACTCTTGTTGTGG               chr1   122609874   122609950   CCTTGTAGTGTGTGTATTCAACTCACAGAGT   2   216                   TAAACGATCCTTTACACAGAGCATACTTGAA                   ACACTCTTTTTGTGG               chr1   122668677   122668753   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   217                   TAAACGATCCTTTACACAGAGCAGACTTGAA                   ACACTCTTTTTGTGG               chr1   123422419   123422495   CCTTGTGTTGTGTTTATTCAACTCACAGAGTT   2   218                   AAACGATCCTTTACACAGAGCAGACTTGAA                   ATACTCTTTTTGTGG               chr1   123648614   123648690   CCTTGTAGTGTGTGTATTCAACTCACAGAGT   2   219                   TAAACGATCCTTTACACAGAGCATACTTGAA                   ACACTCTTTTTGTGG               chr1   123806335   123806411   CCTTGTATTGTGAGTATTCAACTCACAGAGT   2   220                   TAAACGATCCTTTACACAGAGCAGACTTGAA                   ACACTCTTTTTGTGG               chr1   124078228   124078304   CCTTGTGTTGTGTGTCTTCAACTCACAGAGTT   2   221                   AAACGATGCTTTACACAGAGTAGACTTGAA                   ACACTCTTTTTCTGG               chr1   124231074   124231150   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   222                   TAAACGATCCTTTACACAGAGCAGACTTGTA                   ACACTCTTTTTGTGG               chr1   124232435   124232511   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   223                   TAAACGATCCTTTACACAGAGCAGACGTGA                   AACACTCTTTTTGTGG               chr1   124344781   124344857   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   224                   TAAACGATCCTTTACACAGAGCAGACTTGAA                   ACACTCTTTTTGTGG               chr1   124435716   124435792   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   225                   TAAACGATCCTTTACACAGAGGAGACTTGTA                   ACACTCTTTTTGTGG               chr1   158677186   158677262   CCTGAGGTTTTCCAGGTTTTAAAAGGAAACC   2   226                   TAAAGGTAGGTTTAGCATTAAGTGTCTTGAA                   GTTTATTTTAAAAGG               chr1   167629479   167629554   CCAAAATTCCCACAAAACCGAATGCATCAGT   4   227                   CAAAGCAAGGTTTGAAGAAAAGATTTACCA                   CTTCAGGGAGCTTGG               chr1   167783428   167783504   CCTTTTCTGGATATCGTTGATGCTCTGTATGC   3   228                   AAAAGGTAGGTTTTTGGGTTATGTTGTTAAA                   CAGTGATTGAATGG               chr1   169409367   169409444   CCTCCAAGAAATATGGAACTATGTGAAAAG   1   229                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGACAGAGAGAATGG               chr1   174145346   174145423   CCTCCAAGAAATATGGGACTATGTGAGAAG   1   230                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGATGGGGAGAATGG               chr1   183750168   183750245   CCATTCTCCCCATCGCTTTCAGGTACACCAA   1   231                   TCAAACGTAGGTTTGGTCTTTTCACATAGTT                   CCATATTCTTTGGAGG               chr1   200801540   200801617   CCATTCTCCCCATCACTTTCAGGTGTACCGA   1   232                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr1   207589936   207590013   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   233                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGACGGGGAGAATGG               chr1   209768370   209768445   CCTTCAGGGCAGAAACAGCTCTACTAGCAG   4   234                   AGAAAGCAAGCTTTCAATATTGTGCAATACA                   AAAACGAGAGCAGGG               chr1   218652378   218652455   CCATTCTCCTCATCTCCTTCTGGTACTCCAAT   1   235                   CAAACGTAGGTTTGGTCTTTTCTCATAGTCTC                   ATATTTCTTGGAGG               chr1   222147250   222147327   CCTCCAAGACATATAGGACTATGTGAAAATA   1   236                   CCAAACCTACGTTTGATTGGTGTACCTGAAA                   GTGACAGGGAGTATGG               chr1   245870710   245870785   CCTGCCAGATACCAGTAGTCACTGTGAATTA   4   237                   CAAAGCTACGTTTCTTCCATAGGGAAAGTTT                   GGAGTCCAGCCAGG               chr2   2376037   2376114   CCATTCTCCCTGTCACTTTCAGGTACACCAA   1   238                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr2   4119629   4119706   CCATTCTCCCCACCACTTTCAGGTACACCAA   1   239                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGTAGG               chr2   4909047   4909124   CCTAACCAGAAACTAACTAATAGATATGGG   1   240                   CAGAAAGCATCCTTTCACTTTTGTTCTGGGA                   GAGGGAAGAAGCAAAGG               chr2   28984877   28984953   CCATTTTGGGGAGGCCTTGATGGGAAGCTGG   2   241                   AAAAGGAAGCTTTCCTCCCAGTCCTGCTGAA                   GGCCTTGCCAGCTGG               chr2   31755833   31755910   CCTCCAAGAAACACAGGACTATGTGAAAAG   1   242                   ATCAAACCTACGTTTGATTGGTGTTCCTGAA                   AGTGATGGGGAGAATGG               chr2   39829583   39829660   CCATTCTCTTCATGACTTTCAGGTACACCATT   1   243                   GAAACGTAGGTTTGGTCTTTTCACATTGTCC                   CATATTTCTTGGAGG               chr2   60205947   60206024   CCATTCTCCCCATCACTTTCAGGTACACCAA   1   244                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCGTATTTCTTGGTGG               chr2   79082362   79082439   CCATTCTCCCTGTCACTTTCAGGTACACCAA   1   245                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGGGG               chr2   79082362   79082438   CCATTCTCCCTGTCACTTTCAGGTACACCAA   3   246                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGGG               chr2   108430915   108430992   CCTCCAAGAAATATGAGATTATATGAAAAG   1   247                   ACCAAACCTACGTTTGATTGGTGTACTTTAA                   AGTGACGGGGAGAATGG               chr2   115893685   115893762   CCATTCTCCCCGTCATTTTCAGGTACACCAA   1   248                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCAAATTTCTTGGAGG               chr2   119620068   119620145   CCCCCAAGAAATGTGGGACTATATGAAAAG   1   249                   ACCAAACCTACGTTTGACTGGTGTACCTAAA                   AGTGATGGGGAGAATGG               chr2   119620069   119620145   CCCCAAGAAATGTGGGACTATATGAAAAGA   2   250                   CCAAACCTACGTTTGACTGGTGTACCTAAAA                   GTGATGGGGAGAATGG               chr2   128495068   128495144   CCCATTGGTGCTGACCAGATGGTGAAGGAG   2   251                   GCAAAGGTTGCTTTGAATGACTGTGCTCTGG                   GGTGAGCCAGGCCTGG               chr2   133133559   133133634   CCCTTTACAGAGGTGAGCTTTGTTATTAGTA   4   252                   AAAAGGTAGGTTTCCCTGTTTTTCTGAAGAA                   AAGCTGTGAGTGGG               chr2   134174983   134175060   CCACTGCCCATTGACAGAGTGGCGAGGTGG   1   253                   GTGAAACCTTGCTTTCCTCCTGGCCCATGGG                   CAGGGTGGGGCTGTGGG               chr2   134174983   134175059   CCACTGCCCATTGACAGAGTGGCGAGGTGG   3   254                   GTGAAACCTTGCTTTCCTCCTGGCCCATGGG                   CAGGGTGGGGCTGTGG               chr2   138069945   138070022   CCATTCTCCCTGTCACTTTTAGATACACCAAT   1   255                   CAAACGTAGGTTTGGTCTTTTCACATAGTCC                   CATGTTTCTTGGAGG               chr2   138797420   138797496   CCTCCAAGAAATATCAACTGTGTGAAAAGA   2   256                   CGAAACCTACGTTTGATTAATGTACCTGAAA                   GTGACAGGGAGAATGG               chr2   145212434   145212511   CCATTCTCCCATTAACTTTCAAGTACACCAA   1   257                   TCAAAGGTAGGTTTGGTGTTTTCCCATAGTC                   CCGTATTTCTTGGAGG               chr2   147837842   147837919   CCTTTTCATCATGCCCCTTTCACTTTAAGGTG   1   258                   AAAACCTTGCTTTACATGTCAGAGAAAAGA                   AGAGCCCTCAGCTGGG               chr2   147837842   147837918   CCTTTTCATCATGCCCCTTTCACTTTAAGGTG   3   259                   AAAACCTTGCTTTACATGTCAGAGAAAAGA                   AGAGCCCTCAGCTGG               chr2   154152540   154152617   CCATTCACCCCGTCACTTTCAGGTACACCAA   1   260                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr2   157705943   157706019   CCTCCAAGAAATATGGGACTATGTGAAAAG   3   261                   ACCAAACCTACGTTTGATGGTGTACCCGAAA                   GTGACAGGGAGAATGG               chr2   158361152   158361229   CCACCAAGAAATATGGGACTATGTGAAAAG   1   262                   ACCAAACCTACGTTTGATAGGTATACCTGAA                   AGTGACAGGGAGAATGG               chr2   161461006   161461083   CCATTCTCCCCATCACTTTCAGGTGCACCAA   1   263                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr2   179077376   179077453   CCCTCAAGAAATATGAGACTATGTGAAAAG   1   264                   ACCAAACCTACGTTTGACTGGTATACCTGAA                   AGTGACAGGGAGAATGG               chr2   179077377   179077453   CCTCAAGAAATATGAGACTATGTGAAAAGA   2   265                   CCAAACCTACGTTTGACTGGTATACCTGAAA                   GTGACAGGGAGAATGG               chr2   181090699   181090776   CCTCCAACAAATATGGGACTATGTGAAAAG   1   266                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGACGGGGATAATGG               chr2   182331957   182332034   CCATTCTCTCCCTCACTTTCAAGTACACCAAT   1   267                   CAAACGTAGGTTTGGTCTTTTCACATAGTCT                   TATATTTCTTGGCGG               chr2   183620562   183620638   CCATTCTCCCTGTCACTGTCAGTACACCAAT   2   268                   CAAACGTAGGTTTGGTCTCTTCACATAGTCC                   CATATTTCTTGGAGG               chr2   207345927   207346003   CCTCCAAGAAATATGGGACTATGTGAACAG   3   269                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGATGGCAGAATGG               chr2   216652047   216652123   CCACCATGCCTGGCCACCACACATTTTTTTCT   2   270                   AAAGCTTGGTTTTGGCCACAGTGAGAGTTTC                   TTGGGCTGTCAGGG               chr2   216652047   216652122   CCACCATGCCTGGCCACCACACATTTTTTTCT   4   271                   AAAGCTTGGTTTTGGCCACAGTGAGAGTTTC                   TTGGGCTGTCAGG               chr2   223780040   223780116   CCCACTAGGTGGCGATATCTGAGGGTCCAAT   2   272                   GAAACCATGCTTTTTACTCAGATCTTCCACT                   AACCACCTCCCCCGG               chr2   224486595   224486672   CCTCTAAGAAATATGGGACTATGTGAAAAG   1   273                   ACCAAACCTACGTTTGACTGGTGTACCTGAA                   AGTGACGGGGAGAATGG               chr2   230526902   230526979   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   274                   ACCAAACCTACGTTTGATTAGTGTACCTGAA                   AGTGACGGGGAGAATGG               chr2   232036127   232036204   CCATTCTCCCTGTCACTTTCAGGTACATCAAT   1   275                   CAAACGTAGGTTTGGTCTTTTCACATAGTCC                   CATATTTCTTGGAGG               chr3   4072812   4072889   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   276                   ACCAAACCTACGTTTGACTGGTGTACCTGAA                   AGGGATGGGGAGAATGG               chr3   9261677   9261754   CCCCCAAGAAATATGAGACTATGTGAAAAG   1   277                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGACAGGGAGAATGG               chr3   9261678   9261754   CCCCAAGAAATATGAGACTATGTGAAAAGA   2   278                   CCAAACCTACGTTTGATTGGTGTACCTGAAA                   GTGACAGGGAGAATGG               chr3   16732146   16732223   CCTCTAAGAAATATGGGACTATGTGAAAAG   1   279                   ACCAAACCTACGTTTGATTGGTGTAACTGAA                   AGTGACAGGGAGAATGG               chr3   17450712   17450789   CCTCCAAGAAATATGCGCCTATGTGAAAAG   1   280                   ACCAAACCTACGTTTGATTGGTATACCTGAA                   AGTGATGGAGAGAATGG               chr3   21559769   21559846   CCATTCTCCCTGTCACTTTGAGGTACACCAA   1   281                   TCAAACGTAGGTTTGGTCTTTTCACATATTC                   GCATATTTCTTGGAGG               chr3   23416658   23416735   CCATTCTCCCCGTCACTTTCAGGTACACCAA   1   282                   CCAAACGTTGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr3   29984019   29984096   CCATTCTCCCTGTCACTTTCCAGTACACCAGT   1   283                   CAAACGTAGGTTTGGTCTTTTCACATACTCC                   CATATTTCTTGGAGG               chr3   38269551   38269627   CCTGGCCTAATTTTTAATTCTTAGTTTGACTT   2   284                   AAACCTTGCTTTTAGTGTGATGGCGACAAAA                   GCTGAGCTGAAAGG               chr3   40515213   40515288   CCAGTGCTTTTTGGTTTTAAAGGCAAGCCTC   4   285                   CAAACCTTCCTTTCTCCTGGATGCTGTGGTG                   GTTGCCATGCATGG               chr3   49233612   49233687   CCCAACTCCTGCGAGAAGTAGCTCACCATGA   4   286                   CAAAGCTACCTTTGCTTTTATCGTTTTGCAAA                   ACAAAAAAGGGGG               chr3   66292894   66292971   CCATTCTCCCCGTCACTTTGAGGTGTGCCAA   1   287                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CTATATTTCTTGGAGG               chr3   67541493   67541570   CCTCCAAAAAATATGGGACTACGTAAAAAG   1   288                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   ACTGACAGGGAGAATGG               chr3   82273011   82273088   CCATTCTCCCCGTCACTTTCAGGTACACCAA   1   289                   TCAAACGTAGGTTTGGTCTTTTCACATAGTT                   CCATATTTCTTGGAGG               chr3   98683349   98683426   CCTACAAGATATATGGGACTATGTGAAAAG   1   290                   ACCAAACCTACGTTTTACTGGTGTGCCTGAA                   ACTGACGGGGAGAATGG               chr3   101923653   101923730   CCATTCTCTCTGTCACTTTCAGGTACACCAAT   1   291                   CAAACGTAGGTTTGGTCTTTTCACATAGTCC                   CATATTTCTTGGAGG               chr3   114533467   114533544   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   292                   ACCAAACCTACGTTTCATTGGTGTACCTGAA                   AGTGATAGGGAGAATGG               chr3   132607602   132607679   CCTCCAAAAAATATGGGATGATGTGAAAAG   1   293                   ACCAAACCTAGGTTTGACTGGTGTACCTGAA                   AATGATGGGGAGAATGG               chr3   137545176   137545253   CCTCCAAGAAATATGAGACTATGTGAAAAG   1   294                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGACAGGGAGAATGG               chr3   137655679   137655756   CCTCCAAGAAATATGGGACTACGTGAAAAG   1   295                   ATCAAACCTACGTTTGATTGTTGTACCTGAA                   AGTGATGGGGAGAATGG               chr3   137662040   137662117   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   296                   ACCAAACCTACGTTTGATTGTTGTACCTGAA                   AGTGATGGGGAGAATGG               chr3   142133796   142133873   CCTCAAAAGTGTTCTGGTTTTGTTTTGTTTTT   1   297                   TAAACCATGGTTTTACCTCTGGCTTAGTGGG                   ACTAAAAATAGGAGG               chr3   146726949   146727026   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   298                   ACCAAACCTACGTTTGACTGGTGTACCTGAA                   AGTGATGGGGAAAATGG               chr3   152421096   152421173   CCTCCAAGAAATATGGGACTGTGTGTAAAG   1   299                   ACCAAACCTACGTTTGATTGGTGTACCTCAA                   AGTGATGGGGAGAATGG               chr3   170620247   170620324   CCATTCTCCCCATCACATTCAGGTACACCAA   1   300                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr3   181166873   181166949   CCCCTGGAAAAGTTGGAGCATCACAGGAAA   3   301                   AGCAAACCAACCTTTTTTCTCCCCTAGGTAA                   ACTGGGGAGCCAGGGG               chr3   181166874   181166949   CCCTGGAAAAGTTGGAGCATCACAGGAAAA   4   302                   GCAAACCAACCTTTTTTCTCCCCTAGGTAAA                   CTGGGGAGCCAGGGG               chr4   6604233   6604309   CCTTCCCCAGTTGCAGCAGACAAGAGTCTCG   2   303                   AAAAGCTTGCTTTGGTTGCTGCAGTGGATGG                   GTTGGTAGGCACAGG               chr4   6626269   6626344   CCCCCACCTCCCAAGCTGCTGGCTTCTCGAA   4   304                   TAAAGCTACCTTTCCTTTTACCAAAACTTGTC                   TCTCGAATGTCGG               chr4   8155396   8155472   CCTTGGCCCTGGACAGCTGCTTTTCCTTCCCT   2   305                   AAACCTTGGTTTCCCCCTTTGTGCAGGTGGG                   TGGGTTTGGGCTGG               chr4   10386803   10386880   CCTCTTCTAGTGAACCCATGGGGTTACCAAG   1   306                   GGAAAGCAACCTTTTGATAAATATTCCCATC                   TTTTTATGTTGTCTGG               chr4   20701579   20701656   CCACTTGAAAGGGTTACCAAGGATAAGATTT   1   307                   TTAAAGCTTGCTTTCACAAACAACTCATGCT                   CCAGGCTTGTCAGTGG               chr4   29594286   29594363   CCTTTCTCCCCATCACTTTCAGGTACACCAAT   1   308                   CAAACGTAGGTTTGATCTTTTCACATAGTCC                   CATATTTCTTGGAGG               chr4   53668422   53668499   CCATTCTCCCCATCAATTTCAGTTACACCAA   1   309                   TGAAACGTAGGTTTGGCCTTTTCACATAGTC                   CCATATTTCTTAGAGG               chr4   74914802   74914879   CCATTCTCCCTGTCACTCTCAGGTACACCAA   1   310                   TCAAACGTAGGTTTGGTCTTTTCATATAGTC                   CCATATTTCTTGGAGG               chr4   75332783   75332859   CCTCCAAGAAAATTGGGACTATGTGAAAAA   3   311                   ACCAAACCTACGTTTGATTGATGTACCTGAA                   AGTGACAGGAGAATGG               chr4   88123643   88123720   CCTTCAAGAAATATGGGACTATGTGAAAGG   1   312                   ACAAAACCTACGTTTTATTGGTGTACCTGAA                   AGTGACAGGGAGAATGG               chr4   89567192   89567269   CCATTCTCCCCATCACTTTCAGGTACGCTAA   1   313                   TCAAACGTAGGTTTGATCTTTTCACATAGTC                   TTATATTTCTTGGAGG               chr4   93556577   93556654   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   314                   ACCAAACCTACGTTTGACTGGTGTACCTCAA                   TGTGACAGGGAGAATGG               chr4   100266379   100266456   CCATTCTCCCTGTCACTTTTAGGTACACCAAT   1   315                   CAAACGTACGTTTGGTCTTTTCACATAGACC                   CATATTTCTTGGAGG               chr4   103486234   103486311   CCTTCAAGAAATATGGGACTGTGTGAAAAG   1   316                   ACCAAAGCTAGGTTTGATTGGTGTACCTGAA                   AGTGATGGGGAGAATGG               chr4   105923129   105923204   CCTACTATTCACAGAGTAATGCAGTTTGCTG   4   317                   AAAAGGTTGGTTTTTGCTGACCTCTGAGAGC                   TCACATTACAGTGG               chr4   106874711   106874788   CCATTCTCTCTGTCACTTTCTGGTACACCAAT   1   318                   CAAACGTAGGTTTGCTCTTTTCACATAATCC                   CATATTTATTGAAGG               chr4   115805791   115805867   CCATAACATGTATTTGCTGGTGCTAGACTCT   3   319                   CCAAAGCTAGGTTTCTTTCTACAACAATGGC                   TGGAAGTCTTCTTGG               chr4   122033277   122033354   CCATTCTCCCCATCACTTTCAGGTACACCAA   1   320                   TCAAACGTAGGTTTGGTCTTCTCACACAGTC                   CCATATTTCTTGGAGG               chr4   129125132   129125209   CCATTCTTCCCATTACTTTCAGGTACACCAAT   1   321                   CAAACGTAGGTTTGGTCTTTTCACATAGTCC                   CACATTTCTTGGAGG               chr4   135472562   135472639   CCATTCTCCCCCTCACTTTCAGGTACACCAA   1   322                   TCAAACGTAGGTTTGGTCTTTTCACATTGTCC                   CATATTTCTTGGAGG               chr4   138507099   138507176   CCATTCTCCCCAGCACTTACAGGTACACCAA   1   323                   TCAAACGTAGGTTTGGTCATTTCACATAGTC                   CCATATTTCTTGGAGG               chr4   144249093   144249170   CCATTCTCCCTGTCACTTTCAGGTACAGCAA   1   324                   TCAAACGTAGGTTTGGTCTTTTCACATGGTC                   CCATATTTCTTGGAGG               chr4   144436406   144436483   CCTCCAAGAAATATGAGACTATGTGAAAAG   1   325                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGACGGGGAAGATGG               chr4   154110259   154110336   CCTCCAAGAAATATGAGACTATGTGAAAAG   1   326                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGACAGGGAGAATGG               chr4   154893438   154893515   CCTCCAAGAGATATGAGACTATGTAAATAG   1   327                   ACCAAACCTACCTTTGATTGGTGTACGTGAA                   AGTGACAGGAAGAATGG               chr4   161116854   161116931   CCATTCTCCCCATCACTTTCAGGTACACCAA   1   328                   CCAAACGTAGGTTTGGTCTTTTCACATAGTC                   TCATATTTCTTGGAGG               chr4   165140748   165140823   CCTCCATTGACTACTCCTTATCATTGGCTAG   4   329                   AAAACCTACCTTTCAACCAGTTTCTAAGGCC                   AAGAAACTTGGAGG               chr4   181928508   181928585   CCACCAAGAAATATGGGACTACGTGAAAAG   1   330                   ACCAAACCTACGTTTGATGGGTGTGCCTGAA                   AGTGACGGGAAGAATGG               chr4   187521958   187522035   CCTCCAAGAAATAAGGGACTATGTGAAAAG   1   331                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   GGTGACAGGGAGAATGG               chr5   12675639   12675715   CCAAAGGGCCTTTGTGATTCTACTTTGTAAT   3   332                   ATAAAGGATGGTTTCTTACTACGGTTGGTGT                   CCTTGCAGGAGTGGG               chr5   29271804   29271881   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   333                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGATGGGGAGAATGG               chr5   35352660   35352737   CCATTCTCCCCGTTACTTTCAGGTACACCAA   1   334                   TAAAACCTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr5   38723235   38723310   CCCATATCTCTGGCAAGGGCAGCTCTCTGGC   4   335                   TAAACCAAGCTTTCCTGTAGAGCTTGAGTTC                   CAAGGCAGCGTTGG               chr5   47358339   47358415   CCTTGTAGTGTGTGTATTCAACTCACAGAGT   2   336                   TAAACGATCCTTTACACAGAGCAGACTTGAA                   ACACTCTTGTTGTGG               chr5   47415811   47415887   CCTTGTAGTGTGTGTATTCAACTCACAGAGT   2   337                   TAAACGATCCTTTACACAGAGCATACTTGAA                   ACACTCTTTTTGTGG               chr5   47474614   47474690   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   338                   TAAACGATCCTTTACACAGAGCAGACTTGAA                   ACACTCTTTTTGTGG               chr5   48228356   48228432   CCTTGTGTTGTGTTTATTCAACTCACAGAGTT   2   339                   AAACGATCCTTTACACAGAGCAGACTTGAA                   ATACTCTTTTTGTGG               chr5   48454551   48454627   CCTTGTAGTGTGTGTATTCAACTCACAGAGT   2   340                   TAAACGATCCTTTACACAGAGCATACTTGAA                   ACACTCTTTTTGTGG               chr5   48612272   48612348   CCTTGTATTGTGAGTATTCAACTCACAGAGT   2   341                   TAAACGATCCTTTACACAGAGCAGACTTGAA                   ACACTCTTTTTGTGG               chr5   48884165   48884241   CCTTGTGTTGTGTGTCTTCAACTCACAGAGTT   2   342                   AAACGATGCTTTACACAGAGTAGACTTGAA                   ACACTCTTTTTCTGG               chr5   49037011   49037087   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   343                   TAAACGATCCTTTACACAGAGCAGACTTGTA                   ACACTCTTTTTGTGG               chr5   49038372   49038448   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   344                   TAAACGATCCTTTACACAGAGCAGACGTGA                   AACACTCTTTTTGTGG               chr5   49150718   49150794   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   345                   TAAACGATCCTTTACACAGAGCAGACTTGAA                   ACACTCTTTTTGTGG               chr5   49241653   49241729   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   346                   TAAACGATCCTTTACACAGAGGAGACTTGTA                   ACACTCTTTTTGTGG               chr5   88582714   88582790   CCTTTTCATAAGAAGAAAATCGACTCATCAT   3   347                   TGAAACCAAGCTTTGGTACAATTTCATTGAT                   GTTTCCAGAAGCAGG               chr5   93497156   93497231   CCCATAGACTATGATAGAAACAAAATAACC   4   348                   CAAAAGCTAGCTTTCTGATTGAGTTTCCATA                   AATGCAATGTGAAGG               chr5   94295029   94295105   CCATTCACTTGTCACTTTCTGGTACACCAATC   2   349                   AAACGTAGGTTTGGTCTTTTCACATAGTCTC                   ATATTTCTTGGAGG               chr5   94956746   94956823   CCTCCAAGAAATATGGGACTCTGTAAAGAG   1   350                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGAAGGGGAGAATGG               chr5   106003488   106003565   CCATTCTCCCCGTCATTTTCAGGTACACCAA   1   351                   TCAAACCTAGGTTTGGTCTTTTTACATAGTCC                   CATATTTCTTGGAGG               chr5   118727905   118727982   CCTCCACGAAACATGGGACTATGTGAAAAG   1   352                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGACAGGGAGAATGG               chr5   132156032   132156109   CCAATTTCCCCCTCACTTTCAGATACACCAA   1   353                   TCAAACGTAGGTTTGGTCTTTTCACATAGTT                   CCATATTTCCTGGAGG               chr5   152037951   152038028   CCATTCTCCCCATCACTTTCAGGTACACCAA   1   354                   TCAAACGTAGGTTTGGTCTTTTCACATATTCC                   CATATGTCTTGGAGG               chr5   155183064   155183141   CCCACCGGCTCATGAGAGGTAGAGCTAAGG   1   355                   TCCAAACCTAGGTTTATCTGAGACCGGAACT                   CATGTGATTAACTGTGG               chr5   155183065   155183141   CCACCGGCTCATGAGAGGTAGAGCTAAGGT   2   356                   CCAAACCTAGGTTTATCTGAGACCGGAACTC                   ATGTGATTAACTGTGG               chr5   163148211   163148288   CCTTCAAGAAATATGGGACTATGTGAAGAG   1   357                   ACCAAACCTACGTTTGATTGGTGTAGCCAAA                   AGTGATGGGGAAAATGG               chr5   165889537   165889614   CCTCAGATTAGATTTACTTGCAAAGAGACAT   1   358                   TTAAAGGATCGTTTTGATACTATTTTGAAAG                   TACTATACAAAGATGG               chr5   169395198   169395274   CCTTAAGAACATAAATCCCCAGGAATTCACA   2   359                   GAAACCTTGGTTTGAGCTTTGGATTTCCCGC                   AGGATGTGGGATAGG               chr5   171021380   171021457   CCATTCTCTCTGTCACTTTCAGGTACACCAAT   1   360                   CAAACGTAGGTTTGGTCTTTTCTCATAGTCC                   CATATTTCTTGGAGG               chr5   173059898   173059973   CCATTTACCATCATTCTCTGTCATGGCAGGT   4   361                   GAAAGCAAGCTTTTATATAGACAATGTTCTA                   CTTAGTTTACAGGG               chr5   174102359   174102435   CCCAAAGTTAATTTTACTCTTTTTCTGAATCA   2   362                   AAAGGAACCTTTCCTCCATGAGAAGAATCCT                   GCCATATTTCTAGG               chr5   180927811   180927888   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   363                   ACCAAACCTACGTTTGATTGCTATACATGAA                   AGTGACGGGGAGAATGG               chr6   1752363   1752440   CCTTCAAGAAATATGGGACTATGTGAAAAG   1   364                   ACCAAACCTACCTTTGATTGGTGTACCTGAA                   AGTGATGGGAAGAATGG               chr6   20595279   20595356   CCATTCTCCCCATCACTTTCAGGTACACCAA   1   365                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATAGTTCTTGGAGG               chr6   23431370   23431447   CCATTCTCCCCGTCACTTTCAGGGACAACAA   1   366                   TCAAACGTAGGTTTGGCCTTTGCACATAGTC                   TTATATTTCTTGGAGG               chr6   29190624   29190701   CCATTCTCCCCATCACTTTCAGGTACACCAA   1   367                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr6   61533266   61533343   CCTCCAAAAAATATGGGACTATGTGAGAAG   1   368                   ACCAAACCTACGTTTTATTAGTGTACCTCAA                   AGTGACAGGGAGGATGG               chr6   101052764   101052841   CCATTCTCCCCATCACTTTCAGGTACACCAA   1   369                   TGAAACGTAGGTTTGGCCTTTTCACATAGTT                   TCATATTTCTTGGAGG               chr6   117176355   117176432   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   370                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGATGGGGAGAATGG               chr6   117747073   117747149   CCTACAAGAAATATGGAACTTGTAAAAAGA   2   371                   CCAAACCTACGTTTGATTGGTGTACCTGAAA                   GTGACGGGGAGAATGG               chr6   118422508   118422585   CCTCCAAGAAATATGGGACAATGTGAAAAG   1   372                   GCCAAAGCTACGTTTGATTGGTGTACCTGAA                   AGTGACAGGGAGAATGG               chr6   122035019   122035096   CCTTTCAAACTTAGAGGTAAACAAAAGTCCT   1   373                   GAAAACCTAGGTTTGACCATAAGTTGGGACC                   ATACGAGCATAGAAGG               chr6   134445210   134445287   CCAAAAATAAAAAAAAATTGACTTATAAGT   1   374                   AAGAAAGGTTCGTTTTCTCACATTCAGAAAG                   AGAACCCACATGTTGGG               chr6   134445210   134445286   CCAAAAATAAAAAAAAATTGACTTATAAGT   3   375                   AAGAAAGGTTCGTTTTCTCACATTCAGAAAG                   AGAACCCACATGTTGG               chr6   135154944   135155021   CCATTCTCCCCATCACTTTCAGGTACACCAA   1   376                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr6   137889995   137890072   CCATTCTCCCCGTCACTTTCAGGTACACCAA   1   377                   TCAAACGTTGGTTTAGTCTATTCACATAGTC                   CCATATTTCTTGGAGG               chr6   143993904   143993981   CCGAAAAGAATAAGACTATCAGCTGAAGTC   1   378                   TTAAAACGATCCTTTGGCCCCCAGTACTCTA                   TATGCAGGATAGAAAGG               chr6   152610473   152610549   CCTACAAAAATAGGGGACTATGTGATAAGA   2   379                   CCAAACCTACGTTTGATTGGTGTACCTGAAA                   GTGATGGGGAGAATGG               chr6   160372604   160372681   CCATTCTACCCATCACTTTCAGGTACACCAA   1   380                   TCAAACGTAGGTTTGGCCTTTTCATATAGTC                   TCATATTTCTTGGAGG               chr6   169352478   169352555   CCATTCTCCCCATCACTTTCTGGTATACCAAT   1   381                   CAAACGTAGGTTTGGTCTTTTCACATAGTCC                   CATATTTCTTAGAGG               chr6_GL000251v2_alt   677196   677273   CCATTCTCCCCATCACTTTCAGGTACACCAA   1   382                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr6_GL000252v2_alt   456242   456319   CCATTCTCCCCATCACTTTCAGGTACACCAA   1   383                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr6_GL000253v2_alt   456279   456202   CCATTCTCCCCATCACTTTCAGGTACACCAA   1   384                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr6_GL000254v2_alt   456371   456448   CCATTCTCCCCATCACTTTCAGGTACACCAA   1   385                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr6_GL000255v2_alt   456225   456302   CCATTCTCCCCATCACTTTCAGGTACACCAA   1   386                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr6_GL000256v2_alt   500011   500088   CCATTCTCCCCATCACTTTCAGGTACACCAA   1   387                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr7   5256551   5256627   CCACCACACCCAGCCTTATGGGATGGTTTTC   2   388                   AAAAGCATCCTTTTTTAGAAGTGGATTCTGA                   TATATAATCGGATGG               chr7   7392583   7392660   CCATTCTCAATGTCACTTTCAGGTACACCAA   1   389                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr7   8737741   8737818   CCATTCTCTCTGTCACTTTCAGGTACACCAGT   1   390                   CAAAGGTAGGTTTGTTTTATTCACACGTTCA                   CATATTTCTTGGAGG               chr7   11352226   11352303   CCATTCGCCCCATCACTTTCAGGTACACTAG   1   391                   TAAAACGTAGGTTTGGTCTTTTCACATAGTT                   CCATATTTCTTGGAGG               chr7   15519145   15519222   CCTCCAAGAAATATGGGACTATGTGAAGAG   1   392                   ATCAAACCTAGGTTTGATTGTTGTACCTGAA                   AGTGATAAGAAGAATGG               chr7   19228341   19228418   CCTCCAATAAATATGGGGCTATGTGAAAAG   1   393                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGACAGGGAGAATGG               chr7   23778445   23778522   CCCTTTTCCCTGTCACTTTCAGGTACACCAGT   1   394                   CAAACGTAGGTTTGGTCTTTTCACATAGTCG                   AATATTTCTTCAAGG               chr7   23778446   23778522   CCTTTTCCCTGTCACTTTCAGGTACACCAGTC   2   395                   AAACGTAGGTTTGGTCTTTTCACATAGTCGA                   ATATTTCTTCAAGG               chr7   26769065   26769142   CCATTCTCCCTGTCACTTTCAGGTACACTAAT   1   396                   CAAACGTAGGTTTGGTGTATTCACACAGTCC                   CATATTTCTTGGAGG               chr7   42864035   42864112   CCATTCTTCCTGTCACTTTCAGGTATACCAAT   1   397                   CAAACGTAGGTTTGGTCTTTTCACATAGTCC                   CATGTTTCTTGGAGG               chr7   46498923   46499000   CCTCCAAGAAATATGAGACTATATGAAAAT   1   398                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   AGAGACAGGGAGAATGG               chr7   51535360   51535437   CCATTCTCCCTATCACTTTCAGGTACACCAA   1   399                   TCAAACGTAGGTTTGGTCTTTTCATGTAGTC                   CCATATTTCTTGGAGG               chr7   51927106   51927183   CCATTCTGCCCGTCACTTTCAGGTACACCAA   1   400                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr7   56976942   56977018   CCGTCCGATTATATATCAGAATCTACTTCTA   3   401                   AAAAAGGATGCTTTTGAAAACCATCCCATAA                   GGCTGGGTGTGGTGG               chr7   80021598   80021675   CCTACAAGGAATATAGGACTATGTGAAAAT   1   402                   ACCAAACCTACGTTTCACTGCTGTACCTGAA                   GGTGACAGGGAGAATGG               chr7   89673853   89673930   CCATTCTCCCCATCATTTCCAGGTAAACCAA   1   403                   TCAAAGGTAGGTTTGGTCATTTCACATAGTC                   CCATATTTCTTGGAGG               chr7   103404790   103404867   CCATTCTCCCCGTCACTTTCAGGTACACCAG   1   404                   TCAAACGTAGGTTTGGTCTTTTCACACAGTC                   CCATATTTCCTGGAGG               chr7   113053651   113053728   CCATTCTCCCCATCACTTTCAGGTACAGCAA   1   405                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr7   125765204   125765279   CCACTACAGATTCTTGGGTCAAGATGTGTGC   4   406                   AAAAGGATGCTTTAGGGTGATGGATATGAG                   TGGGATGAAATGAGG               chr7   128042158   128042234   CCTGAAAAAAAACCCTGCCAGCCAGCAACT   3   407                   CTGAAAGGATGCTTTGTGTGAGTGAGCAGTG                   TCTGAGATGGACAGGG               chr7   130637332   130637409   CCATTCTCCCCATCACTTTCAGGTACGCCAA   1   408                   TCAAACGTAGGTTTGGTCTTTTGACATAGTC                   CCATATTTCTTGGAGG               chr7   136983050   136983127   CCGTTCTCCCCATCACTTTTAGGTACACCAA   1   409                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   TCATATTTCTTGGAGG               chr7   143579507   143579584   CCATTCTCCTGGTCACTTTCAGGTATACCAA   1   410                   TCAAACGTAGGTTTGGTCTTTTCATGTAGTC                   CCATATTTCTTGGAGG               chr7   143749881   143749958   CCTCCAAGAAATATGGGACTACATGAAAAG   1   411                   ACCAAACCTACGTTTGATTGGTATACCTGAA                   AGTGACCAGGAGAATGG               chr8   2338364   2338441   CCTCCAAGAACTATGGGACTATGTGAAAAG   1   412                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGACGGGGAGAATGG               chr8   2383289   2383366   CCATTCTCCCCGTCACTTTCAGGTACACCAA   1   413                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATAGTTCTTGGAGG               chr8   8414568   8414645   CCATTCTCCCCGTCACTTTCAGGTACACCAA   1   414                   TCAAACGTAGGTTTGGTCTTTTCACAGAGTC                   CCATATTTCTTGGAGG               chr8   24163142   24163219   CCATTCTCCCCGTCACTTTCATGTACACCAA   1   415                   GCAAACGTAGGTTTGATCTTTCCACATAGTC                   CCGTGTTTCTTGGAGG               chr8   34299051   34299128   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   416                   ACCAAACCTACGTTTGATTGGTGTACTTGAA                   AGTGACAGGGAGAATGG               chr8   40965485   40965562   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   417                   ACAAAACCTACGTTTCACTGGTGTACCTGAA                   AGTGACAGGGAGGATGG               chr8   48371659   48371735   CCCCCACCTTTTAAAAACATGCATACATACG   2   418                   GAAACGTTGCTTTCTGCACGATTTCATTTTA                   ATGGAACAGAACAGG               chr8   82534960   82535037   CCATTTCCCCTGTCACTTTCAGGTACACCAA   1   419                   TCAAACGTAGGTTTGGTCTTTTCACATAGTA                   TCATATTTCTTGGAGG               chr8   109217624   109217700   CCATTCTCCCCGTCACTTTCAGGTACACCAA   3   420                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTGGAGG               chr8   134790285   134790361   CCTTTTGTTAAAGTAATAGAATTCTGCTTCTT   2   421                   AAAGGAACCTTTCAGGCAAGATGGTGGTTA                   GAGCACCTAAATGGG               chr8   134790285   134790360   CCTTTTGTTAAAGTAATAGAATTCTGCTTCTT   4   422                   AAAGGAACCTTTCAGGCAAGATGGTGGTTA                   GAGCACCTAAATGG               chr8_KI270821v1_alt   519635   519712   CCTCCAAGAACTATGGGACTATGTGAAAAG   1   423                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGACGGGGAGAATGG               chr8_KI270821v1_alt   564557   564634   CCATTCTCCCCGTCACTTTCAGGTACACCAA   1   424                   TCAAACGTAGGTTTGGCCTTTTCACATAGTC                   CCATAGTTCTTGGAGG               chr9   14951207   14951283   CCTCCAAGAAATATGGGACTGGTGAAAAGA   2   425                   CCAAACCTACGTTTGACTGGTGTACCTGAAA                   GTGACGGGGAGACTGG               chr9   23249218   23249295   CCTCCAAGAAACATGGGAATGTGTGAAAAG   1   426                   ACCAAACCTACGTTTGATTGGCGTACCTGAA                   AGTGACGGGGAGTATGG               chr9   26278896   26278973   CCTCCAAGAAATATGGGACTGTGTGAAAAG   1   427                   ACCAAACCTACGTTTGATTGGTATACCTGAA                   AGTGACAGAGAGAATGG               chr9   27323237   27323314   CCATTCTCCCCTTCACTATCAGGTACACCAA   1   428                   TCAAACGTAGGTTTAGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr9   31517993   31518070   CCATTCTCCCCGTCACTTTCAGATACACCAG   1   429                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr9   39694860   39694937   CCATCTTACTTTGTACTACACTGTTCTTTAGA   1   430                   GAAAGCTTCCTTTTGGAGACCAACCAGGACT                   CCTTAGAAGCAGAGG               chr9   42451132   42451209   CCATCTTACTTTGTACTACACTGTTCTTTAGA   1   431                   GAAAGCTTCCTTTTGGAGACCAACCAGGACT                   CCTTAGAAGCAGAGG               chr9   60776573   60776650   CCTCTGCTTCTAAGGAGTCCTGGTTGGTCTC   1   432                   CAAAAGGAAGCTTTCTCTAAAGAACAGTGT                   AGTACAAAGTAAGATGG               chr9   62647482   62647559   CCTCTGCTTCTAAGGAGTCCTGGTTGGTCTC   1   433                   CAAAAGGAAGCTTTCTCTAAAGAACAGTGT                   AGTACAAAGTAAGATGG               chr9   66682030   66682107   CCTCTGCTTCTAAGGAGTCCTGGTTGGTCTC   1   434                   CAAAAGGAAGCTTTCTCTAAAGAACAGTGT                   AGTACAAAGTAAGATGG               chr9   82264427   82264503   CCACCACTGTGCCTGGCCATTTTCACTATTCT   3   435                   TAAAGGAAGCTTTGGTTTACAAAGGTTTGCT                   ACTGTACTTCCAGG               chr9   84042684   84042761   CCATTCTCCCTGTCACTTTCAGGTACACCATT   1   436                   CAAACGTAGGTTTGGTCTTTTCTCATAGTCC                   CATATTTCTTGGAGG               chr9   95256012   95256089   CCTCCAAGAAATTCGGGACTATGTGAAAAG   1   437                   ACAAAACCTACGTTTAATTGGTGTGTGGTGT                   ACCTGAAAGTGACAAGG               chr9   101816988   101817065   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   438                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGACCAGAAGAATGG               chr9   135842327   135842403   CCTCCAAGAAATATGGGACTATGTGAAAAG   3   439                   CCCAAACCTACGTTTGACTGATGTACCTAAA                   GTGACGGGGAGAATGG               chr9   136910865   136910940   CCCGCACTGTGAGCTTGGCCGAGTGCTGTCT   4   440                   GAAAGCATCCTTTCCCTTCACCTGGAGACTG                   GAGCGCCATAGAGG               chr10   13710312   13710389   CCTGTCTCCCCCATTCCATGCAAAATAAAAC   1   441                   ACAAACCAAGCTTTGCTTTAAGTGCTCCCTG                   ATGCAGTTCAGCGTGG               chr10   18938129   18938206   CCATTCTTCCCGTCACATTCAGGTACACCAA   1   442                   TCAAACGTAGGTTTGGTCTTTTCCCATAGTC                   CCATATTTCTTAGAGG               chr10   22712838   22712914   CCCCCTGCTCAGCTTGGGGAAGAAAAATAC   2   443                   AAAAACGATGCTTTTAGGCATTTTAAACAAC                   TTCACTACATTGAGGG               chr10   22712838   22712913   CCCCCTGCTCAGCTTGGGGAAGAAAAATAC   4   444                   AAAAACGATGCTTTTAGGCATTTTAAACAAC                   TTCACTACATTGAGG               chr10   40160932   40161009   CCTTTGTGTTGTGTGTATTCAACTCACAGAG   1   445                   TGAAACCTTCCTTTATTCAGAGCAGTTTTGA                   AACACTCTTTTTGTGG               chr10   40390136   40390213   CCTTTGTGTTGTGTGTATTCAACTCACAGAG   1   446                   TGAAACCTTCCTTTATTCAGAGCAGTTTTGA                   AAAACACTTTTTGTGG               chr10   40409152   40409229   CCTTTGTGTTGTGTGTATTCAACTCACAGAG   1   447                   TGAAACCTTCCTTTATTCAGAGCAGTTTTGA                   AAAACTCTTTTTGTGG               chr10   40433940   40434017   CCTTTGTGTTGTGTGTATTCAACTCACAGAG   1   448                   TGAAACCTTCCTTTATTCAGAGCAGTTTTGA                   AACACTCTTTTTGTGG               chr10   40588155   40588232   CCTTTGTGTTGTGTGTATTCAACTCACAGAG   1   449                   TGAAACCTTCCTTTATTCAGAGCAGTTTTGA                   AATACTCTTTTTGTGG               chr10   41146207   41146284   CCTTTGTGTTGTGTGTATTCAACTCACAGAG   1   450                   TGAAACCTTCCTTTATTCAGAGCAGTTTTGA                   AACACTCTTTTTGTGG               chr10   43835183   43835260   CCATTCTCCCTGTCACTTTCAAGTACACCAA   1   451                   TCAAACCTAGGTTTGGTCTTTTCACATAGTTC                   CATATTTCTTGGAGG               chr10   54913222   54913299   CCCCTCCCATCACAGGCCCTGAGGTTTAAGA   1   452                   GAAAACCATGGTTTTGTGGGCCAGGCCCATG                   ACCCTTCTCCTCTGGG               chr10   54913222   54913298   CCCCTCCCATCACAGGCCCTGAGGTTTAAGA   3   453                   GAAAACCATGGTTTTGTGGGCCAGGCCCATG                   ACCCTTCTCCTCTGG               chr10   54913223   54913299   CCCTCCCATCACAGGCCCTGAGGTTTAAGAG   2   454                   AAAACCATGGTTTTGTGGGCCAGGCCCATGA                   CCCTTCTCCTCTGGG               chr10   54913223   54913298   CCCTCCCATCACAGGCCCTGAGGTTTAAGAG   4   455                   AAAACCATGGTTTTGTGGGCCAGGCCCATGA                   CCCTTCTCCTCTGG               chr10   58035951   58036028   CCATTCTCCCCATCACTTTCAGGTACACCAA   1   456                   TCAAACGTAGGTTTCATCTTTTCACATAGTC                   CCACGGTTTTTGGAGG               chr10   58677525   58677602   CCTCCAAGATATATGGGACTATGTGAAAAG   1   457                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   ATTGATGGGGAGAATGG               chr10   84021390   84021467   CCTCCAAGAAATATGGGACTGTGTGAAAAG   1   458                   AACAAACCTACGTTTGATTGGTGTACGTGAA                   AGTGATGGGGAGAATGG               chr10   91442692   91442769   CCATTCCTCCCGTCACTTTCAGATACACCAA   1   459                   AAAAACGTAGGTTTGGTCTCTTCACATAGTC                   CCACATTTCTTGGAGG               chr10   91446848   91446925   CCTCCAAGAAATGTGGGACTATGTGAAGAG   1   460                   ACCAAACCTACGTTTTTTTGGTGTATCTGAA                   AGTGACGGGAGGAATGG               chr10   116928784   116928860   CCTCCAAGGGGAATCTGAGTTCTCTGAAGAC   3   461                   AAAAAGCATGGTTTCTTTTCTTCTGTATTTCT                   TATTGTTTCCTAGG               chr10   116937771   116937848   CCATTCTCCCTATCACTTTCCAGTACACCAAT   1   462                   CAAACGTAGGTTTGGTCTTTTCACATAGTCC                   CATATTTCTTGGAGG               chr11   31182070   31182147   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   463                   ACCAAACCTACGTTTGATTGGTATACTTGAA                   ATTGACAAGGAGAATGG               chr11   34739273   34739350   CCTCCAAGAAATATGGGACTATGTGGAAAG   1   464                   ACCAAACCTACGTTTGACTGGTGTACCTGAA                   AGTGATGGGGAGAATGG               chr11   86646529   86646606   CCTCTAAGAAATATGGGACTATGTGAAGAG   1   465                   ATGAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGACGAGGAGAATGG               chr11   90469791   90469867   CCCTCGTATACTACATGCTATAGTCAAAGCA   3   466                   GTAAACCTTCCTTTCCTTAAGCAGACCACAC                   TCTTTCATGCCTGGG               chr11   90469792   90469867   CCTCGTATACTACATGCTATAGTCAAAGCAG   4   467                   TAAACCTTCCTTTCCTTAAGCAGACCACACT                   CTTTCATGCCTGGG               chr11   92429985   92430062   CCATTCTCCCCATCACTTTCAGGTATACTAAT   1   468                   CAAAGGTAGGTTTGGTCTTTTCACATAGTCC                   CATATTTCATGGAGG               chr11   102818498   102818574   CCATTCCCCCGTCACTTTCAGGTACACCAAT   2   469                   CAAACGTAGGTTTGGTCTTTTCACATAGTCC                   CATATTTCTTGGAGG               chr11   120765065   120765142   CCATTCTCCCCGTCACTTTCAGGTACACCAA   1   470                   TCAAACGTAGGTTTTGTCTTTTCTTATAGTCC                   CATATTTCTTGGAGG               chr11   123131901   123131978   CCACTGCACCTGACCAAGATCCTTAATTTTT   1   471                   CTAAACCTACGTTTATCATCTATAAAATGAG                   CCATCTTTTCACATGG               chr11   129468520   129468597   CCTCCGAGAAATATGGGACTATGTGAAAAG   1   472                   ACCAAACCTACGTTTGATTGTTGTACCTGAA                   AGTGACAGGGAGAATGG               chr11   131272361   131272438   CCATTCTCCCCATCACTTTTAGGTACACCAA   1   473                   TCAAACGTAGGTTTGGTCCTTTTGCATAGAC                   CCATATTTCTTGGAGG               chr11   132761415   132761492   CCATTTTCCCCGTCAGTTTCATATACACCTAT   1   474                   CAAACGTAGGTTTACTGTTTTCACATAGTCC                   CTTATTTCTTGGAGG               chr12   22367416   22367493   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   475                   ACCAAACCTACCTTTGATTGGTGTACCTGAA                   AGTGACGGGCAGGATGG               chr12   33146384   33146461   CCATTCTTCTCGTCATTTTCAAGTACACCAAT   1   476                   CAAACGTAGGTTTGGTCTTTTCGCATAGTCC                   CATATTTCTTGGAGG               chr12   33198476   33198553   CCATTCTTCTCGTCACTTTCAAGTACACCAAT   1   477                   CAAACGTAGGTTTGGTCTTTTCACATAGTCC                   CATATTTCTTGGAGG               chr12   46038332   46038409   CCTCCAAGAAATATAGGACTATGTGAAAAG   1   478                   ACCAAACCTACGTTTGATTGGTGTACTTGAA                   AGTGACAGGGAGAATGG               chr12   60236126   60236203   CCTCCAAGAAATGTGGAACTATGTGAAAAG   1   479                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGACAGGGAGAATGG               chr12   62098359   62098434   CCCTGACACTGATAAACGGATATGAAGAGA   4   480                   AAAAAGCTAGGTTTTCGCTGGAATTCCTAAG                   CTTGGGCTGCAGTGG               chr12   62112591   62112668   CCCTTCTCCCAGTCACTTTTAGGTACACCAA   1   481                   TGAAACGTAGGTTTGGTCTTTTCACACAGTC                   CCATATTTCTTGGAGG               chr12   62112592   62112668   CCTTCTCCCAGTCACTTTTAGGTACACCAAT   2   482                   GAAACGTAGGTTTGGTCTTTTCACACAGTCC                   CATATTTCTTGGAGG               chr12   62418577   62418652   CCACTCCCTCTCCCCCAAAAAGTAAAGGTAG   4   483                   AAAACCAAGGTTTACAGGCAACAAATAGCA                   CAATGAATGGAATGG               chr12   71732311   71732388   CCAAACCCGCATCGCACACCCTGTGAGGGG   1   484                   GACAAAGGAACCTTTCCGTTCCAACATCAAG                   GTTGTTTTGACCCAAGG               chr12   78047816   78047893   CCATTCTTTCTGTCACTTTCAGGTATACCAGT   1   485                   CAAACCTAGGTTTGGTCTTTTCACATAGTCC                   CATATTTCTTGGAGG               chr12   81480016   81480093   CCATTCTCCCCATCACTTTCAGGTACACCAA   1   486                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr12   96840231   96840307   CCACACGGTAGAGGATAAACTAGGTGGATT   3   487                   CTCAAAGCAACCTTTGAAATAATCTATGCAG                   TTTTTCTGGGTACTGG               chr12   99187165   99187242   CCACCAAGAAACATGGGACTATGTGAAAAG   1   488                   ACCAAACCTACGTTTGGTTGGTGTACCTGGA                   AGTGACGGGGAGAGTGG               chr12   107860841   107860918   CCTCCAAGAAATATGGGACCATGTGAAAAG   1   489                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGACAGGGAGAATGG               chr12   110882809   110882885   CCTGTAAAAAGGTCACATGGTCAGGTGTGCC   2   490                   TAAACGATCCTTTTATTTATTTATTTATTTAT                   TTTTAAGAAACAGG               chr12   119063321   119063397   CCAGCCCCAAAATGTCAGGGGCTTAGAACA   2   491                   ACAAAGGTTCCTTTTCATGTTTATACTACAT                   GTTTGTCATGGGCTGG               chr13   35320704   35320781   CCGTTTTCCCCATCACTTTCAGGTACACCAG   1   492                   TCAAACGTAGGTTTGGTCTTTTCACATGGTC                   CCACATTTCTTGGAGG               chr13   53133477   53133554   CCTGGAATAGCTTTCCTGACTGTCTGACTTC   1   493                   AAAAACCTTGGTTTGACCACTTCGTCTATAT                   CATGAGGAAGGACTGG               chr13   53184880   53184956   CCCTACTCTGAACCTACCTTGATAAAGCCTA   3   494                   GAAAACCAAGCTTTGACAAGATTTGACAAG                   AGATGGAATTTGGAGG               chr13   53184881   53184956   CCTACTCTGAACCTACCTTGATAAAGCCTAG   4   495                   AAAACCAAGCTTTGACAAGATTTGACAAGA                   GATGGAATTTGGAGG               chr13   57896962   57897038   CCCTTATAAAACTGAAAACTTTAACCTTTTTT   2   496                   AAAGCATGCTTTTGAATAAATTCTTTTATTA                   CAAAAAAGACCAGG               chr13   62610100   62610177   CCATTCTCCCTGTCACTTTCAGGTACACCAA   1   497                   TCAAACGTAGGTTTGGTCTTTTCACGTAGTC                   CCATATTTCTTGGAGG               chr13   77004382   77004458   CCCTTTATTATCCAAGTGGTTTCCTGCTCTTC   2   498                   AAACCTTCCTTTCAAAATTTTGTCTCCTACTT                   AAAACAAGTTAGG               chr13   81646075   81646151   CCTTCTGTTGAGACCTACTGCTAAGAAAACA   3   499                   AAAAAGGTTCCTTTCAAATATTATTGTGAAT                   CAATAATGTACCTGG               chr13   83755854   83755931   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   500                   ACCAAACCTACGTTTCATTGATGGACCTGAA                   AGTGATGGGGAGAATGG               chr13   89719199   89719275   CCATTCTCCCTTCACTTTCAGTTACACCAATC   2   501                   AAACGTAGGTTTGGTCTTTTCACATAGTCCC                   ATATTTCTTGGAGG               chr13   102010574   102010650   CCTAGGGAAGTGATCATAGCTGAGTTTCTGG   3   502                   AAAAACCTAGGTTTTAAAGTTGAGGAGACTT                   AAGTCCAAAACCTGG               chr13_KI270841v1_alt   124240   124316   CCATTCTCCCTTCACTTTCAGTTACACCAATC   2   503                   AAACGTAGGTTTGGTCTTTTCACATAGTCCC                   ATATTTCTTGGAGG               chr14   25980646   25980723   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   504                   ACTAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGACAGGGAGAATGG               chr14   35842786   35842863   CCATTCTCCCTGTCACTTTCAGGTATGCCAGT   1   505                   CAAACGTAGGTTTGGTCTTTTCACATAGTCC                   CATATTCCTTGGAGG               chr14   42646400   42646477   CCTCCAAGAAATATGGGACTATGTAAAAAG   1   506                   ACGAAACCTACGTTTGATTGGTGTACTTAAA                   AGTGACGAGGAGAATGG               chr14   49063242   49063319   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   507                   ACCAAACCTACGTTTGATTTGTGTACCTGAA                   AGTGATGGGGAGAATGG               chr14   49130379   49130456   CCATTCTCCCCGTCACTTTCAGGCACACCAA   1   508                   TCAAACGTAGGTTTAGTCTTTTCACATAGTC                   CCATATTTCTTAGAGG               chr14   51352342   51352418   CCTTAATGCATTCATATTTCATATTTTAAATA   2   509                   AAACCATGGTTTCCCACAGAGTGACTTCTAC                   TCTAAGAAATGGGG               chr14   51352342   51352417   CCTTAATGCATTCATATTTCATATTTTAAATA   4   510                   AAACCATGGTTTCCCACAGAGTGACTTCTAC                   TCTAAGAAATGGG               chr14   60835842   60835919   CCGTTCTTTCCGTCACTTTCAGGTACACCAGT   1   511                   CAAACGTAGGTTTGGTCTTTTCACATAGTCC                   CATATTTCTTGGAGG               chr14   66529072   66529148   CCATTCTCCCCATCACTTTCATGTACACCAAT   3   512                   CAAACGTAGGTTTGGTCTTTGTTAACATAGT                   CCCATATTTCTTGG               chr14   79210873   79210949   CCCTATAAAGCTTAGAGAAACACAGGGCTCT   3   513                   TTAAACGATCCTTTTTCTCTTTTCTGTTTTAA                   ATTTCATCACTTGG               chr14   79210874   79210949   CCTATAAAGCTTAGAGAAACACAGGGCTCTT   4   514                   TAAACGATCCTTTTTCTCTTTTCTGTTTTAAA                   TTTCATCACTTGG               chr14   85371541   85371618   CCATTCTCCCCATCACTTTCAGGTACACTAA   1   515                   TCAAAGGTAGGTTTGGTCTTTTCACATGGTC                   CTATATTTCTTGGAGG               chr14   92918713   92918790   CCCCATAGCACGATCACATGGGACATTCAGG   1   516                   GGAAAGCAACCTTTTCCAGGAAGGAAAACC                   CAATGCTGGGACCCAGG               chr14   92918714   92918790   CCCATAGCACGATCACATGGGACATTCAGG   2   517                   GGAAAGCAACCTTTTCCAGGAAGGAAAACC                   CAATGCTGGGACCCAGG               chr14   103386821   103386897   CCCTTTCAGCGCTCACAGGCTATGGTTTTAT   2   518                   AAAAGGAACCTTTGATTTTGTTCATGTGAAA                   CTACAAAATGCCAGG               chr14_KI270847v1_alt   33275   33352   CCCCATAGCACGATCACATGGGACATTCAGG   1   519                   GGAAAGCAACCTTTTCCAGGAAGGAAAACC                   CAATGCTGGGACCCAGG               chr14_KI270847v1_alt   33276   33352   CCCATAGCACGATCACATGGGACATTCAGG   2   520                   GGAAAGCAACCTTTTCCAGGAAGGAAAACC                   CAATGCTGGGACCCAGG               chr15   20630566   20630643   CCTCCAAGAAATATTGGAGTATGTGATAAGA   1   521                   CCAAACCTTCGTTTGACTGGTGTACCTGAAA                   GTGATGGGGAGAATGG               chr15   21675103   21675180   CCATTCTCCCCGTCACTTTCAGGTACACCAA   1   522                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr15   22117571   22117648   CCATTCTCCCCGTCACTTTCAGGTACACCAA   1   523                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr15   22369744   22369821   CCATTCTCCCCATCACTTTCAGGTACACCAG   1   524                   TCAAACGAAGGTTTGGTCTTATCACATACTC                   CAATATTTCTTGGAGG               chr15   42302832   42302909   CCTCCAAGATATATGGGACTATGTGAAAAG   1   525                   GCCAAACCTACCTTTGATTGATACACCTGAA                   AATGACAGGGAGAATGG               chr15   49967601   49967678   CCTCCAAGAAATATGCGACTATGTGAAAAG   1   526                   ACCAAACCTACGTTTCATTGGTGTACCTGAA                   AGTGATGGGGAGAATGG               chr15   83964501   83964577   CCTCCAAGAAATATGGGACTATGTGGAAAG   3   527                   ACCAAACCTACGTTTGTTTGGTGTACCTGAA                   AGTGAGGGGAGAATGG               chr15   87261388   87261465   CCATTCTCCTCATCACTTTCAAGTACACCAA   1   528                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   TTATATTTCTTGGAGG               chr15_KI270727v1_random   409348   409425   CCATTCTCCCCGTCACTTTCAGGTACACCAA   1   529                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr15_KI270851v1_alt   14235   14312   CCATTCTCCCCATCACTTTCAGGTACACCAG   1   530                   TCAAACGAAGGTTTGGTCTTATCACATACTC                   CAATATTTCTTGGAGG               chr15_KI270852v1_alt   440099   440176   CCATTCTCCCCGTCACTTTCAGGTACACCAA   1   531                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr16   22123671   22123748   CCAGCAGAAGAATCTGGGGCACAGTCTGTG   1   532                   AAAAAAGGTACCTTTCTTAAGCAGGGTTCTT                   ATCCTTCATGGGTCTGG               chr16   25557623   25557700   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   533                   ACCAAACCTACGTTTGATTGTTGTACCTGAA                   AGTGAGGGGGAGAATGG               chr16   36427179   36427255   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   534                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   36476450   36476526   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   535                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   36512469   36512545   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   536                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   36520964   36521040   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   537                   TAAACGATCCTTTACACACAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   36524704   36524780   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   538                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   36566812   36566888   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   539                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   36573603   36573679   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   540                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   36667694   36667770   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   541                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   36677320   36677396   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   542                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   36683096   36683172   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   543                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   36691251   36691327   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   544                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   36710951   36711027   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   545                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   36750364   36750440   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   546                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   36791455   36791531   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   547                   TAAACGATCCTTTACACACAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   36856683   36856759   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   548                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   36926655   36926731   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   549                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   36931752   36931828   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   550                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   36948058   36948134   CCTTGTGTTGTGTGTATTCAACTCACCGAGTT   2   551                   AAACGATCCTTTACACAGAGCAGATTTGAAA                   CACTGTTTTTCTGG               chr16   36974541   36974617   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   552                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   36981331   36981407   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   553                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   36990839   36990915   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   554                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37021075   37021151   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   555                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37042812   37042888   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   556                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37085971   37086047   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   557                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37129462   37129538   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   558                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37146110   37146186   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   559                   TAAACGATCCTTTACACACAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37157309   37157385   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   560                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37183118   37183194   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   561                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37190924   37191000   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   562                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37221808   37221884   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   563                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37259501   37259577   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   564                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37272409   37272485   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   565                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37281923   37281999   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   566                   TAAACGATCCTTTACACAGAGCAGATTTGTA                   ACACTGTTTTTCTGG               chr16   37346472   37346548   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   567                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37357000   37357076   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   568                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37373301   37373377   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   569                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37419498   37419574   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   570                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37430714   37430790   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   571                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37455845   37455921   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   572                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37458558   37458634   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   573                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37486127   37486203   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   574                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37525183   37525259   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   575                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTGTGG               chr16   37536735   37536811   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   576                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37554730   37554806   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   577                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37575784   37575860   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   578                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37577483   37577559   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   579                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37583598   37583674   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   580                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37696368   37696444   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   581                   TAAACGATCCTTTCCACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37704524   37704600   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   582                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37706223   37706299   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   583                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37708941   37709017   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   584                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37763622   37763698   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   585                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37772115   37772191   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   586                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37791815   37791891   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   587                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37796229   37796305   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   588                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37797928   37798004   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   589                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37843453   37843529   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   590                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37848548   37848624   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   591                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37864846   37864922   CCTTGTGTTGTGTGTATTCAACTCACCGAGTT   2   592                   AAACGATCCTTTACACAGAGCAGATTTGAAA                   CACTGTTTTTCTGG               chr16   37902550   37902626   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   593                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37907307   37907383   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   594                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37928033   37928109   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   595                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37959262   37959338   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   596                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37964355   37964431   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   597                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37974881   37974957   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   598                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   AAACTGTTTTTCTGG               chr16   37987789   37987865   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   599                   AAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   37994586   37994662   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   600                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTGTGG               chr16   38006479   38006555   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   601                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   38011567   38011643   CCTTGTGTTGTGTGTATTTAACTCACAGAGTT   2   602                   AAACGATCCTTTACACAGAGCAGATTTGAAA                   CACTGTTTTTCTGG               chr16   38040096   38040172   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   603                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   38041456   38041532   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   604                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   38062179   38062255   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   605                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   38102937   38103013   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   606                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   38128412   38128488   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   607                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   38131809   38131885   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   608                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   38144723   38144799   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   609                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   38168845   38168921   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   610                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   38209287   38209363   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   611                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   38210986   38211062   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   612                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   38229667   38229743   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   613                   TAAACGATCCTTTACACAGAGCAGATTTGAA                   ACACTGTTTTTCTGG               chr16   47424037   47424114   CCATTCTCCCTATCACTTTCAGGTACACCAA   1   614                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr16   60730549   60730625   CCTCGTCACTGCCAGATTTTGTGGCTACCAG   2   615                   CAAAGGATCGTTTTAAGCTGCAACTCAGGAA                   ATTGAGAAAATATGG               chr16   72545014   72545091   CCTCCAAGAAATATGGGACTATGTGAAAAA   1   616                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGACAGGGAGAATGG               chr16   81945503   81945579   CCCTGTGTTCTTTTATACTAAAACAAGCCAG   2   617                   CAAACCAACCTTTGAGATGTGTTGCCTTAAA                   CATTACTGAATGGGG               chr16   81945503   81945578   CCCTGTGTTCTTTTATACTAAAACAAGCCAG   4   618                   CAAACCAACCTTTGAGATGTGTTGCCTTAAA                   CATTACTGAATGGG               chr17   16474024   16474100   CCGAGAAACGGCTTTAGCAACAAATAAATA   3   619                   TCAAAAGGATGCTTTCTCTTCAGAATAATCT                   AAAGTAAGTTGGGAGG               chr17   34438512   34438589   CCATGTTACTCCGGATAAGGACAGCAAAGG   1   620                   AGGAAAGGAACCTTTTCTGGGCCACCAGAA                   GGATGAGCTTGGGCTTGG               chr17   43690782   43690859   CCCAGGGATATGCTGGCCACGGGGAGGAGC   1   621                   CGGAAACCAACCTTTGTGTCACTGTGTAGTG                   ACAAGTGCCTTTGGAGG               chr17   43690783   43690859   CCAGGGATATGCTGGCCACGGGGAGGAGCC   2   622                   GGAAACCAACCTTTGTGTCACTGTGTAGTGA                   CAAGTGCCTTTGGAGG               chr17   69156298   69156375   CCTTAGGGACCCATAATGGCCACAACCAGG   1   623                   AGAAAAGCAAGCTTTGATGCTTAAACACTAC                   TTACAGACATGTACAGG               chr17   74595228   74595305   CCTGCCTCTGTTCCTCCTTCCTGATGGTGGCG   1   624                   GAAAGGATGCTTTTGCCAGATCAACAGTCAC                   ACACAACACACCAGG               chr17   83191644   83191721   CCTGACTCCAGCCCTCCTTGACAAGGTCTCC   1   625                   GTAAAGCATGCTTTCTCTTAGGGACCCTCAG                   AGGGAGGCTTGGTGGG               chr17   83191644   83191720   CCTGACTCCAGCCCTCCTTGACAAGGTCTCC   3   626                   GTAAAGCATGCTTTCTCTTAGGGACCCTCAG                   AGGGAGGCTTGGTGG               chr18   35135224   35135300   CCTTATTTGGAATGTGACAAGACCCATTTGT   3   627                   TTAAACCTTGGTTTTTATGCAGAAAGAAAAG                   GAAGGCTGCAGTGGG               chr18   38918861   38918938   CCATTCTCCCTGTCACTTTCAGGTACACTAAT   1   628                   CAAACGTAGGTTTGCTGTTTTTACATAGGCT                   CATATTTCTTGGAGG               chr18   45476589   45476666   CCATTCTCCCCATCACTTTCAGGTACACCAG   1   629                   TCAAACGTAGGTTTGGTCTTTTCACATAGTC                   CCATATTTCTTGGAGG               chr18   48640821   48640896   CCTGTTTGTTATTTTAGCTAATGTCAAAAAG   4   630                   AAAACCTTGCTTTTTCTGAACCCTTTCAGAG                   GCAGAAAGTGGGGG               chr18   71096732   71096808   CCATTTTCCCCACCACTTTCACGTACAGCAA   3   631                   TCAAACGTAGGTTTGGTCTTTTCACTAGTCC                   CATATTTCTTGGAGG               chr19   24957844   24957920   CCTTGTAGTGTGTGTATTCAACTCACAGAGT   2   632                   TAAACGATCCTTTACACAGAGCAGACTTGAA                   ACACTCTTGTTGTGG               chr19   25015316   25015392   CCTTGTAGTGTGTGTATTCAACTCACAGAGT   2   633                   TAAACGATCCTTTACACAGAGCATACTTGAA                   ACACTCTTTTTGTGG               chr19   25074119   25074195   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   634                   TAAACGATCCTTTACACAGAGCAGACTTGAA                   ACACTCTTTTTGTGG               chr19   25827861   25827937   CCTTGTGTTGTGTTTATTCAACTCACAGAGTT   2   635                   AAACGATCCTTTACACAGAGCAGACTTGAA                   ATACTCTTTTTGTGG               chr19   26054056   26054132   CCTTGTAGTGTGTGTATTCAACTCACAGAGT   2   636                   TAAACGATCCTTTACACAGAGCATACTTGAA                   ACACTCTTTTTGTGG               chr19   26211777   26211853   CCTTGTATTGTGAGTATTCAACTCACAGAGT   2   637                   TAAACGATCCTTTACACAGAGCAGACTTGAA                   ACACTCTTTTTGTGG               chr19   26483670   26483746   CCTTGTGTTGTGTGTCTTCAACTCACAGAGTT   2   638                   AAACGATGCTTTACACAGAGTAGACTTGAA                   ACACTCTTTTTCTGG               chr19   26636516   26636592   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   639                   TAAACGATCCTTTACACAGAGCAGACTTGTA                   ACACTCTTTTTGTGG               chr19   26637877   26637953   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   640                   TAAACGATCCTTTACACAGAGCAGACGTGA                   AACACTCTTTTTGTGG               chr19   26750223   26750299   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   641                   TAAACGATCCTTTACACAGAGCAGACTTGAA                   ACACTCTTTTTGTGG               chr19   26841158   26841234   CCTTGTGTTGTGTGTATTCAACTCACAGAGT   2   642                   TAAACGATCCTTTACACAGAGGAGACTTGTA                   ACACTCTTTTTGTGG               chr19   28517220   28517297   CCAGGAAAAAATTTAAACTTTCTTAACTTGA   1   643                   TAAAAGGTAGCTTTCAAAACCTACAATAAAT                   AACATACTTAGAGTGG               chr19   34566821   34566898   CCATTCTCCTCGTCACTTTCAGGTACACCAA   1   644                   ACAAACGTAGGTTTGGTCTTTTTACGTAGTC                   CCATATTTCTTGGAGG               chr19   52261770   52261847   CCCTCTTGAAGTTAGGGAAGTAGCATTTAAG   1   645                   GGAAACGTAGCTTTACTATTAAGAATTTCAA                   ACAGCACTTGTCAGGG               chr19   52261770   52261846   CCCTCTTGAAGTTAGGGAAGTAGCATTTAAG   3   646                   GGAAACGTAGCTTTACTATTAAGAATTTCAA                   ACAGCACTTGTCAGG               chr19   52261771   52261847   CCTCTTGAAGTTAGGGAAGTAGCATTTAAGG   2   647                   GAAACGTAGCTTTACTATTAAGAATTTCAAA                   CAGCACTTGTCAGGG               chr19   52261771   52261846   CCTCTTGAAGTTAGGGAAGTAGCATTTAAGG   4   648                   GAAACGTAGCTTTACTATTAAGAATTTCAAA                   CAGCACTTGTCAGG               chr20   11151392   11151469   CCATTCTCCCCGTCACTTTCAGGTACACCAA   1   649                   TCAAACGTAGGTTTGGTCTTTTCACATATTCC                   CATATTTCTTGGAGG               chr20   14027067   14027143   CCATTCTCCCTTCACTTTCAGGTACACCAATC   2   650                   AAACGTAGGTTTGGTCTTTTCACATAGTCCC                   ATATTTTTTGGAGG               chr20   50615399   50615476   CCTATAGTCTCAGTTACTTGGGAGGCTGAGG   1   651                   TAAAAGGATCGTTTGAGCCCAGGAGGTGGA                   GGTTGCAGTGAGCCGGG               chr20   50615399   50615475   CCTATAGTCTCAGTTACTTGGGAGGCTGAGG   3   652                   TAAAAGGATCGTTTGAGCCCAGGAGGTGGA                   GGTTGCAGTGAGCCGG               chr20   60909414   60909490   CCTTTCCCAACTCTGCTATTGCCCCCACATCC   3   653                   TAAAGGAACCTTTCTTTTTTTATATATTTTAT                   TTTAAGTTCCAGG               chr21   16226086   16226163   CCTCCAAGAAATATGGAACTATGTGAAAAG   1   654                   ACCAAACCTACGTTTGATTGACGTACCTGAA                   AGTGACAGGGAGAATGG               chr21   17835234   17835309   CCTCTTCTGAAAGCATTGATAATCAACATTT   4   655                   TAAACGTAGCTTTTCCCCATATTGCTAGGAA                   GGCTCATTCCCGGG               chr21   19425636   19425713   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   656                   GCCAAACCTACGTTTGATTGCTGTACCCGAG                   AGTGACGGGGAGAATGG               chr21   32220958   32221035   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   657                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGATGGGGAGAATGG               chr21   34335877   34335953   CCCGGGGCCTGGGTGCCCAGTGCCAGTGGTC   3   658                   AGAAAGGTTGCTTTGGTGTTTTTCATTGTTA                   GTGAGACAGAGATGG               chr21   34335878   34335953   CCGGGGCCTGGGTGCCCAGTGCCAGTGGTCA   4   659                   GAAAGGTTGCTTTGGTGTTTTTCATTGTTAGT                   GAGACAGAGATGG               chr21   36315276   36315353   CCATTCTCCCCATCATTTTCAGGTACACCAA   1   660                   TCAAACGTAGGTTTGATCTTTTCACATAGCC                   CCATATTTCTTGGAGG               chr21   41547952   41548028   CCACCAGCACTTCTGTTAGAAGTTGCAGCAG   3   661                   AGAAAGGATCCTTTAGGCACATCTCCCAGAT                   CCTTGCGAAGAGGGG               chr22   18973194   18973271   CCTGTGCCAGGGTCCTTCCACTGGGACTGGC   1   662                   AGAAACGTAGGTTTGCATGGAGTGAGAAGC                   AGGGGAGAGGTTGAGGG               chr22   18973194   18973270   CCTGTGCCAGGGTCCTTCCACTGGGACTGGC   3   663                   AGAAACGTAGGTTTGCATGGAGTGAGAAGC                   AGGGGAGAGGTTGAGG               chr22   20265462   20265539   CCCTCAGCCTCTCCCCTGCTTCTCACTCCATG   1   664                   CAAACCTACGTTTCTGCCAGTCCCAGCAGAA                   GGACCCTGGCACGGG               chr22   20265462   20265538   CCCTCAGCCTCTCCCCTGCTTCTCACTCCATG   3   665                   CAAACCTACGTTTCTGCCAGTCCCAGCAGAA                   GGACCCTGGCACGG               chr22   20265463   20265539   CCTCAGCCTCTCCCCTGCTTCTCACTCCATGC   2   666                   AAACCTACGTTTCTGCCAGTCCCAGCAGAAG                   GACCCTGGCACGGG               chr22   20265463   20265538   CCTCAGCCTCTCCCCTGCTTCTCACTCCATGC   4   667                   AAACCTACGTTTCTGCCAGTCCCAGCAGAAG                   GACCCTGGCACGG               chrX   27300998   27301075   CCTCCAAGAAATATGGGGCTATGTGAAAAG   1   668                   ACCAAACCTACCTTTGATTGGTGTATCTGAA                   AGTGACGGGGAGAATGG               chrX   28456666   28456743   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   669                   ACCAAACCTACGTTTGATTTGTGTACCTGAA                   AGTGATGGGGAGAATGG               chrX   35634985   35635062   CCATTCTCCCCGTCACTTTCAGGTACACCAA   1   670                   TCAAACGTAGGTTTGGTCTTTTCTCATTGTCC                   CATATTTCTTGGAGG               chrX   39460148   39460223   CCCATCAAGAGCGGTTGTGCATGGCAACAGT   4   671                   AAAAGGATGGTTTGTTACACTAGTACAAAA                   AGAGGTGGCCAGAGG               chrX   43926403   43926480   CCATTCTCTCTGTCACTTTCAGGTACACCAAT   1   672                   CAAACGTAGGTTTGGTCTTTTCACATAGTCC                   CATATTTCTTGGAGG               chrX   44254600   44254677   CCTCCAAGAAATACGGGACTATGTGAAAAG   1   673                   ACCAAACGTACGTTTGATTGGTGTACCTGAA                   AGTGATAGGGAGAATGG               chrX   46088602   46088679   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   674                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGACTGGGAGAATGG               chrX   50222874   50222951   CCATTCTCCCTGTCACTTTCAGGTACACGAA   1   675                   TCAAACGTAGGTTTCATCTTTTCACATAGTC                   CCATATTTCTTAGAGG               chrX   57416835   57416911   CCATTCTCTCTGTCACTTTCTGGTACACCAAT   3   676                   CAAACGTAGGTTTGGTCTTTTCACATAGTTT                   CACATATTTCTTGG               chrX   57856466   57856543   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   677                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGACAAGGAAAATGG               chrX   62702479   62702556   CCTGAAAAACATTGTTTCCAACCTGGTAAAT   1   678                   CAAAAGGAAGGTTTAACTTTGTTAGATAAGT                   CCACATATCACCAAGG               chrX   63067129   63067206   CCTCCAAGAAATGTGGGACTATGGGAAAAG   1   679                   ACCAAACCTACCTTTGTTTGGTGTACCTGAA                   AGTGACGGGGAGAAAGG               chrX   64936250   64936327   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   680                   ACCAAACCTACGTTTCATTGGTGTACCTGAA                   AGTGATGGGTAGAATGG               chrX   66720099   66720176   CCTACAAGAAATATGGGACTATGGGAAAAG   1   681                   ACCAAACCTACGTTTGATTGGTACACTGGAA                   AGTGACAGGGATAATGG               chrX   68529086   68529163   CCATTCTCCCTGTCACTTTCTGGTACACCAAT   1   682                   CAAAGGTAGGTTTGGTCTTTTCACATAGTCC                   CATATTTCTTGGAGG               chrX   73893994   73894071   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   683                   ACCAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGATGGGGAGAATGG               chrX   75723201   75723278   CCATTCTCTTTGTCACTTTCAGGTATACCAAT   1   684                   CAAACGTTGGTTTGGTCTTTTTGCATAGTCCC                   ATATTTTGTGGAGG               chrX   75815659   75815736   CCTCCAAGAAATATGAGACTATGTGAAAAG   1   685                   ACCAAACCTACGTTTGATTAGTGTACCTGAA                   AATGATGGGGAGAATGG               chrX   80967103   80967180   CCATTCTTTCTGTCACTTTCAGGTACACCAAT   1   686                   CAAACGTAGGTTTGGTCTTTTCACATAGTCC                   CATATTTCTTGGAGG               chrX   89936425   89936502   CCATTCTCCCTGTCACTTTCAGGTACACCAA   1   687                   TCAAACGTAGGTTTGTTCTTTTCACATAGTCC                   CATATTTCTTGGAGG               chrX   91038768   91038845   CCATTATCCCCATCACTTTCAGGTACACCAA   1   688                   TCAAACGTAGGTTTGGTTTTTTCACATAGTTC                   AATATTTCTTTGAGG               chrX   91471271   91471348   CCTCCAAGAAATATGGGACTATCTGAAAAG   1   689                   ATCAAACCTACGTTTGATTGGTGTACCTGAA                   AGTGACAGGGAGAATGG               chrX   96428180   96428257   CCTTTCTCCCCATCACTTTCAGGTACACCAAT   1   690                   CAAACGTAGGTTTGGTCTTTTCATATAGTCC                   CATATTTCTTGGAGG               chrX   100268291   100268368   CCTCCAAGAAATATGGGACTATGTGCAAAG   1   691                   ATCAAACCTACGTTTGATTGCTGTACCTGAA                   AGTGATGGGGAGAATGG               chrX   105811046   105811123   CCATTCTCCCCATCACTTTCAGGTACACCAG   1   692                   TCAAACGTAGGTTTGGTCTTTTCACATAATC                   CCATATTTCTTGGAGG               chrX   115673065   115673141   CCTCCAAGAAGTATGGGACCATGGAAAAGA   2   693                   TCAAACCTACGTTTGACTGGTGTACCTGAAA                   GTGACTGGGAGAATGG               chrX   117269846   117269923   CCTCCAAGAAATATGGGACTATGTGAAAAG   1   694                   ACCAAACCTACGTTTGATTGGAGTACTTGAA                   AATGACAGGGATAATGG               chrX   139191369   139191445   CCTTTAAAGACATGCTCTTTGTGCCAGAAAT   3   695                   TCAAAGGTTGCTTTTATGTCCAGTGGGGTGG                   AGGGAGGAAGCTCGG               chrX   147988614   147988691   CCATTCTCCCCGTCACTTTCAGGGACCTCAA   1   696                   TCAAACGTAGGTTTTGTCTTTTCACATAGTCC                   CATATTTCTTGGAGG               chrX   155321041   155321118   CCTCCAAGAAATATAGGACTATGTGAAAAG   1   697                   ACCAAACCTACGTTTGACTGGTGTACCTGAA                   AGTGACAGGGAGAATGG               chrY   15109391   15109468   CCATTCTCCCCATCACTTTCAGGTACACCAA   1   698                   TCAAAGGTAGGTTTGGTCTTTTCACATAGTC                   CGATATTTCCTGCAGG               Chromosomal sites were identified by searching for CCX (30-31) -AAASSWWSSTTT-X (30-31) -GG (SEQ ID NO: 699) where W is T or A and S is G or C. Pattern 1 is CCX (31) -AAASSWWSSTTT-X (31) -GG (SEQ ID NO: 699), 2 is CCX (30) -AAASSWWSSTTT-X (31) -GG (SEQ ID NO: 699), 3 is CCX (31) -AAASSWWSSTTT-X (30) -GG (SEQ ID NO: 699), and 4 is CCX (30) -AAASSWWSSTTT-X (30) -GG (SEQ ID NO: 699). Only the + strand is shown and the start and end corresponds to the first and last base pair in the chromosome (GRCh38) or alternate assembly when applicable.            
DNA Sequencing
 
     Transfections of 293T cells were performed as above in sextuplet and incubated for 72 hours. Cells were harvested and replicates were combined. Episomal DNA was extracted using a modified HIRT extraction involving alkaline lysis and spin column purification essentially as described (Quan et al., Circular polymerase extension cloning of complex gene libraries and pathways. PloS one 4, e6441 (2009); and Hillson (2010), vol. 2015, pp. CPEC protocol; the entire contents of each of which are hereby incorporated by reference). Briefly, after harvesting, HEK293T cells were washed in 500 μL of ice cold PBS, resuspended in 250 μL GTE Buffer (50 mM glucose, 25 mM Tris-HCl, 10 mM EDTA and pH 8.0), incubated at room temperature for 5 minutes, and lysed on ice for 5 minutes with 200 μL lysis buffer (200 mM NaOH, 1% sodium dodecyl sulfate). Lysis was neutralized with 150 μL of a potassium acetate solution (5 M acetate, 3 M potassium, pH 6.7). Cell debris were pelleted by centrifugation at 21,130 g for 15 minutes and lysate was applied to Econospin Spin columns (Epoch Life Science, Missouri City, Tex.). Columns were washed twice with 750 μL wash buffer (Omega Bio-tek, Norcross, Ga.) and eluted in 45 μL TE buffer, pH 8.0. 
     Isolated episomal DNA was digested for 2 hours at 37° C. with RecBCD (10 U) following the manufacturer&#39;s instructions and purified into 10 μL EB with a MinElute Reaction Cleanup Kit (Qiagen, Valencia, Calif.). Mach1-T1 chemically competent cells were transformed with 5 μL of episomal extractions and plated on agarose plates selecting for carbenicillin resistance (containing 50 μg/mL carbenicillin). Individual colonies were sequenced with primer pCALNL-for-1 to determine the rate of recombination. Sequencing reads revealed either the ‘left’ intact non-recombined recCas9 site, the expected recombined product, rare instances of ‘left’ non-recombined site with small indels, or one instance of a large deletion product. 
     Analysis of recCas9 Catalyzed Genomic Deletions 
     HEK293T cells were seeded at a density of 6×10 5  cells per well in 24 well collagen-treated plates and grown overnight (Corning, Corning, N.Y.). Transfections reactions were brought to a final volume of 100 μL in Opti-MEM (ThermoFisher Scientific, Waltham, Mass.). For each transfection, 90 ng of each guide RNA expression vector, 20 ng of pmaxGFP (Lonza, Allendale, N.J.) and 320 ng of recCas9 expression vector were combined with 2 μL Lipofectamine 2000 in Opti-MEM (ThermoFisher Scientific, Waltham, Mass.) and added to individual wells. After 48 hours, cells were harvested and sorted for the GFP transfection control on a BD FACS AriaIIIu cell sorter. Cells were sorted on purity mode using a 100 μm nozzle and background fluorescence was determined by comparison with untransfected cells. Sorted cells were collected on ice in PBS, pelleted and washed twice with cold PBS. Genomic DNA was harvested using the E. Z. N. A. Tissue DNA Kit (Omega Bio-Tek, Norcross, Ga.) and eluted in 100 μL EB. Genomic DNA was quantified using the Quant-iT PicoGreen dsDNA kit (ThermoFisher Scientific, Waltham, Mass.) measured on a Tecan Infinite M1000 Pro fluorescence plate reader. 
     Nested PCR was carried out using Q5 Hot-Start Polymerase 2× Master Mix supplemented with 3% DMSO and diluted with HyClone water, molecular biology grade (GE Life Sciences, Logan, Utah). Primary PCRs were carried out at 25 uL scale with 20 ng of genomic DNA as template using the primer pair FAM19A2-F1 and FAM19A2-R1 (Table 5). The primary PCR conditions were as follows: 98° C. for 1 minute, 35 cycles of (98° C. for 10 seconds, 59° C. for 30 seconds, 72° C. for 30 seconds), 72° C. for 1 minute. A 1:50 dilution of the primary PCR served as template for the secondary PCR, using primers FAM19A2-F2 and FAM19A2-R2. The secondary PCR conditions were as follows: 98° C. for 1 minute, 30 cycles of (98° C. for 10 seconds, 59° C. for 20 seconds, 72° C. for 20 seconds), 72° C. for 1 minute. DNA was analyzed by electrophoresis on a 1% agarose gel in TAE alongside a 1 Kb Plus DNA ladder (ThermoFisher Scientific, Waltham, Mass.). Material to be Sanger sequenced was purified on a Qiagen Minelute column (Valencia, Calif.) using the manufacturer&#39;s protocol. Template DNA from 3 biological replicates was used for three independent genomic nested PCRs. 
     The limit of detection was calculated given that one complete set of human chromosomes weighs approximately 
             3.6   ⁢           ⁢   pg   ⁢           ⁢       (       3.3   ·     10   9       ⁢           ⁢   bp   ×     1   ·     10     -   21         ⁢     g   bp       )     .           
Therefore, a PCR reaction seeded with 20 ng of genomic DNA template contains approximately 5500 sets of chromosomes.
 
     For quantification of genomic deletion, nested PCR was carried out using the above conditions in triplicate for each of the 3 biological replicates. A two-fold dilution series of genomic DNA was used as template, beginning with the undiluted stock (for sample 1, 47.17 ng/uL; for sample 2, 75.96 ng/uL; and for sample 3, 22.83 ng/uL) to reduce potential sources of pipetting error. The lowest DNA concentration for which a deletion PCR product could be observed was assumed to contain a single deletion product per total genomic DNA. 
     The number of genomes present in a given amount of template DNA can be inferred, and thus an estimate a minimum deletion efficiency for recCas9 at the FAM19A2 locus can be determined. For example, take the case of a two-fold dilution series, beginning with 20 ng genomic DNA template. After nested PCR, only the well seeded with 20 ng yielded the correct PCR product. At 3.6 pg per genome, that PCR contained approximately 5500 genomes, and since at least one recombined genome must have been present, the minimum deletion efficiency is 1 in 5500 or 0.018%. 
     The levels of genomic DNA were quantified using a limiting dilution of genomic template because using quantitative PCR (qPCR) to determine the absolute level of genome editing would require a set of PCR conditions that unambiguously and specifically amplify only from post-recombined genomic DNA. As shown in  FIG.  5 B , primary PCR using genomic DNA as a template results in a roughly 2.5 kb off-target band as the dominant species; a second round of PCR using nested primers is required to reveal guide RNA- and recCas9-dependent genome editing. 
     Results 
     Fusing Gin Recombinase to dCas9 
     It has been recently demonstrated that the N-terminus of dCas9 may be fused to the FokI nuclease catalytic domain, resulting in a dimeric dCas9-FokI fusion that cleaved DNA sites flanked by two guide RNA-specified sequences (see, e.g., Guilinger et al., Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nature biotechnology, (2014); Tsai et al., Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nature biotechnology, (2014); the entire contents of each of which are hereby incorporated by reference). The same fusion orientation was used to connect dCas9 to Ginβ, a highly active catalytic domain of dimeric Gin invertase previously evolved by Barbas and co-workers (Gaj et al., A comprehensive approach to zinc-finger recombinase customization enables genomic targeting in human cells. Nucleic acids research 41, 3937-3946 (2013), the entire contents of which is hereby incorporated by reference). Ginβ promiscuously recombines several 20-bp core “gix” sequences related to the native core sequence CTGTAAACCGAGGTTTTGGA (SEQ ID NO: 700) (Gaj et al., A comprehensive approach to zinc-finger recombinase customization enables genomic targeting in human cells. Nucleic acids research 41, 3937-3946 (2013); Klippel et al., The DNA Invertase Gin of Phage Mu—Formation of a Covalent Complex with DNA Via a Phosphoserine at Amino-Acid Position-9. Embo Journal 7, 1229-1237 (1988); Mertens et al., Site-specific recombination in bacteriophage Mu: characterization of binding sites for the DNA invertase Gin. The EMBO journal 7, 1219-1227 (1988); Plasterk et al., DNA inversions in the chromosome of  Escherichia coli  and in bacteriophage Mu: relationship to other site-specific recombination systems. Proceedings of the National Academy of Sciences of the United States of America 80, 5355-5358 (1983); the entire contents of each of which are hereby incorporated by reference). The guide RNAs localize a recCas9 dimer to a gix site flanked by two guide-RNA specified sequences, enabling the Ginβ domain to catalyze DNA recombination in a guide RNA-programmed manner ( FIG.  1 D ). 
     To assay the resulting dCas9-Ginβ (recCas9) fusions, a reporter plasmid containing two recCas9 target sites flanking a poly-A terminator that blocks EGFP transcription was constructed ( FIGS.  1 A- 1 C ). Each recCas9 target site consisted of a gix core pseudo-site flanked by sites matching a guide RNA protospacer sequence. Recombinase-mediated deletion removed the terminator, restoring transcription of EGFP. HEK293T cells were cotransfected with this reporter plasmid, a plasmid transcribing a guide RNA(s), and a plasmid producing candidate dCas9-Ginβ fusion proteins, and the fraction of cells exhibiting EGFP fluorescence was used to assess the relative activity of each fusion construct. 
     Parameters influencing the architecture of the recCas9 components, including the spacing between the core gix site and the guide RNA-binding site (from 0 to 7 bp), as well as linker length between the dCas9 and Ginβ moieties ((GGS) 2  (SEQ ID NO: 182), (GGS) 5  (SEQ ID NO: 701), or (GGS) 8  (SEQ ID NO: 183)) were varied ( FIGS.  2 A- 2 F ). Most fusion architectures resulted in no observable guide RNA-dependent EGFP expression ( FIGS.  1 C- 1 D ). However, one fusion construct containing a linker of eight GGS repeats and 3- to 6-base pair spacers resulted in approximately 1% recombination when a matched, but not mismatched, guide RNA was present ( FIGS.  2 E- 2 F ). Recombination activity was consistently higher when 5-6 base pairs separated the dCas9 binding sites from the core ( FIG.  2 F ). These results collectively reveal that specific fusion architectures between dCas9 and Ginβ can result in guide RNA-dependent recombination activity at spacer-flanked gix-related core sites in human cells. The 8×GGS linker fusion construct is referred to as “recCas9”. 
     Targeting DNA Sequences Found in the Human Genome with recCas9 
     Low levels of observed activity may be caused by a suboptimal guide RNA sequence or core gix sequence, consistent with previous reports showing that the efficiency of guide RNA:Cas9 binding is sequence-dependent (see, e.g., Xu et al., Sequence determinants of improved CRISPR sgRNA design. Genome research 25, 1147-1157 (2015), the entire contents of which is hereby incorporated by reference). Moreover, although the present optimization was conducted with the native gix core sequence (see, e.g., Klippel et al., The DNA Invertase Gin of Phage Mu—Formation of a Covalent Complex with DNA Via a Phosphoserine at Amino-Acid Position-9. Embo Journal 7, 1229-1237 (1988); Mertens et al., Site-specific recombination in bacteriophage Mu: characterization of binding sites for the DNA invertase Gin. The EMBO journal 7, 1219-1227 (1988); Plasterk et al., DNA inversions in the chromosome of  Escherichia coli  and in bacteriophage Mu: relationship to other site-specific recombination systems. Proceedings of the National Academy of Sciences of the United States of America 80, 5355-5358 (1983); the entire contents of each of which are hereby incorporated by reference), several studies have shown that zinc finger-Gin or TALE-Gin fusions are active, and in some cases more active, on slightly altered core sites. See, e.g., Gordley et al., 3rd, Synthesis of programmable integrases. Proceedings of the National Academy of Sciences of the United States of America 106, 5053-5058 (2009); Gersbach et al., Targeted plasmid integration into the human genome by an engineered zinc-finger recombinase. Nucleic acids research 39, 7868-7878 (2011); Mercer et al., Chimeric TALE recombinases with programmable DNA sequence specificity. Nucleic acids research 40, 11163-11172 (2012); Gaj et al., A comprehensive approach to zinc-finger recombinase customization enables genomic targeting in human cells. Nucleic acids research 41, 3937-3946 (2013); Gordley et al., 3rd, Evolution of programmable zinc finger-recombinases with activity in human cells. J Mol Biol 367, 802-813 (2007); Gersbach et al., 3rd, Directed evolution of recombinase specificity by split gene reassembly. Nucleic acids research 38, 4198-4206 (2010); and Gaj et al., Structure-guided reprogramming of serine recombinase DNA sequence specificity. Proceedings of the National Academy of Sciences of the United States of America 108, 498-503 (2011); the entire contents of each of which are hereby incorporated by reference). Thus, sequences found within the human genome were targeted in order to test if unmodified human genomic sequences were capable of being targeted by recCas9 and to test if varying the guide RNA and core sequences would increase recCas9 activity. 
     To identify potential target sites, previous findings that characterized evolved Gin variants (see, e.g., Gaj et al., A comprehensive approach to zinc-finger recombinase customization enables genomic targeting in human cells. Nucleic acids research 41, 3937-3946 (2013), the entire contents of which is hereby incorporated by reference) as well as the observations above were used. Using this information, the human genome was searched for sites that contained CCN (30-31) -AAASSWWSSTTT-N (30-31) -GG (SEQ ID NO: 699), where W is A or T, S is G or C, and N is any nucleotide. The N (30-31)  includes the N of the NGG protospacer adjacent motif (PAM), the 20-base pair Cas9 binding site, a 5- to 6-base pair spacing between the Cas9 and gix sites, and the four outermost base pairs of the gix core site. The internal 12 base pairs of the gix core site (AAASSWWSSTTT, SEQ ID NO: 699) were previously determined to be important for Ginβ activity (see, e.g., Gaj et al., Nucleic acids research 41, 3937-3946 (2013). 
     The search revealed approximately 450 such loci in the human genome (Table 9). A reporter construct was created, containing the sequence identical to one of these genomic loci, found in PCDH15, and then guide RNA expression vectors were constructed to direct recCas9 to this sequence ( FIG.  3 A ). These vectors encoded two pairs of guide RNAs, each of which contain spacer sequences that match the 5′ and 3′ regions flanking the PCDH15 psuedo gix sites. Co-transfection of the reporter plasmid, combinations of these flanking guide RNA expression vectors, and the recCas9 expression vector resulted in EGFP expression in 11%-13% of transfected cells ( FIG.  3 B ), representing a &gt;10-fold improvement in activity over the results shown in  FIG.  2   . These findings demonstrate that a more judicious choice of recCas9 target sequences can result in substantially improved recombination efficiency at DNA sequences matching those found in the human genome. 
     Next, whether both guide RNA sequences were required to cause recCas9-mediated deletion was determined. HEK293T cells were co-transfected with just one of the guide RNA vectors targeting the 5′ or 3′ flanking sequences of the PCDH15 psuedo-gix core site, the PCDH15 reporter plasmid, and a recCas9 expression vector. These co-transfections resulted in 2.5-3% EGFP expression ( FIG.  3 B ). The low levels of activity observed upon expression of just one of the targeting guide RNAs and recCas9 may be caused by the propensity of hyperactivated gix monomers to form dimers (see, e.g., Gaj et al., Enhancing the Specificity of Recombinase-Mediated Genome Engineering through Dimer Interface Redesign. J Am Chem Soc 136, 5047-5056 (2014), the entire contents of which is hereby incorporated by reference); transient dimerization may occasionally allow a single protospacer sequence to localize the dimer to a target site. No activity was detected above background when using off-target guide RNA vectors or when the recCas9 vector was replaced by pUC ( FIG.  3 B ). 
     These findings demonstrate that recCas9 activity can be increased substantially over the modest activity observed in the initial experiments by choosing different target sites and matching guide RNA sequences. A greater than 10-fold increase in activity on the PCDH15 site compared to the original target sequences was observed (compare  FIG.  3 B  with  FIG.  2 F ). Further, maximal recombination activity is dependent on the presence of both guide RNAs and recCas9. 
     Orthogonality of recCas9 
     Next, whether recCas9 could target multiple, separate loci matching sequences found in the human genome in an orthogonal manner was tested. A subset of the recCas9 target sites in the human genome based on their potential use as a safe-harbor loci for genomic integration, or in one case, based on their location within a gene implicated in genetic disease, were selected. 
     To identify these sites, ENSEMBL (release 81) was searched to identify which predicted recCas9 target sites fall within annotated genes (see, e.g., Cunningham et al., Ensembl 2015. Nucleic acids research 43, D662-669 (2015), the entire contents of which is hereby incorporated by reference). One such site fell within an intronic region of FGF14. Mutations within FGF14 are believed to cause spinocerebellar ataxia 27 (SCA 27) (see, e.g., van Swieten et al., A mutation in the fibroblast growth factor 14 gene is associated with autosomal dominant cerebellar ataxia [corrected]. Am J Hum Genet 72, 191-199 (2003); Brusse et al., Spinocerebellar ataxia associated with a mutation in the fibroblast growth factor 14 gene (SCA27): A new phenotype. Mov Disord 21, 396-401 (2006); Choquet et al., A novel frameshift mutation in FGF14 causes an autosomal dominant episodic ataxia. Neurogenetics 16, 233-236 (2015); Coebergh et al., A new variable phenotype in spinocerebellar ataxia 27 (SCA 27) caused by a deletion in the FGF14 gene. Eur J Paediatr Neurol 18, 413-415 (2014); Shimojima et al., Spinocerebellar ataxias type 27 derived from a disruption of the fibroblast growth factor 14 gene with mimicking phenotype of paroxysmal non-kinesigenic dyskinesia. Brain Dev 34, 230-233 (2012); the entire contents of each of which are incorporated herein by reference). Finally, a fraction of the predicted recCas9 target sites that did not fall within genes were manually interrogated to determine if some sequences fell within safe harbor loci. Using annotations in ENSEMBL genomic targets that matched most of the five criteria for safe harbor loci described by Bushman and coworkers were identified (Cunningham et al., Ensembl 2015. Nucleic acids research 43, D662-669 (2015); and Sadelain et al., Safe harbours for the integration of new DNA in the human genome. Nat Rev Cancer 12, 51-58 (2012); the entire contents of each of which are incorporated herein by reference). Five reporters and corresponding guide RNA vector pairs containing sequences identical to those in the genome were constructed. To evaluate the orthogonality of recCas9 when programmed with different guide RNAs, all combinations of five guide RNA pairs with five reporters were tested. 
     Cotransfection of reporter, guide RNA plasmids, and recCas9 expression vectors revealed that three of the five reporters tested resulted in substantial levels of EGFP-positive cells consistent with recCas9-mediated recombination. This EGFP expression was strictly dependent upon cotransfection with a recCas9 expression vector and guide RNA plasmids matching the target site sequences on the reporter construct ( FIG.  4 A ). The same guide RNA pairs that caused recombination when cotransfected with cognate reporter plasmids and a recCas9 vector were unable to mediate recombination when cotransfected with non-cognate reporter plasmids ( FIG.  4 A ). These results demonstrate that recCas9 activity is orthogonal and will only catalyze recombination at a gix related core sites when programmed with a pair of guide RNAs matching the flanking sequences. No recombinase activity above the background level of the assay was observed when reporter plasmids were transfected without vectors expressing recCas9 and guide RNAs. 
     Characterization of recCas9 Products 
     The products of recCas9-mediated recombination of the reporter plasmids were characterized to confirm that EGFP expression was a result of recCas9-mediated removal of the poly-A terminator sequence. Reporter plasmids were sequenced for chromosome 5-site 1, chromosome 12, and chromosome 13 (FGF14 locus) after cotransfection with recCas9 expression vectors and with plasmids producing cognate or non-cognate guide RNA pairs. After incubation for 72 hours, episomal DNA was extracted (as described above) and transformed into  E. coli  to isolate reporter plasmids. Single colonies containing reporter plasmids were sequenced ( FIG.  4 B ). 
     Individual colonies were expected to contain either an unmodified or a recombined reporter plasmid ( FIG.  4 C ). For each biological replicate, an average of 97 colonies transformed with reporter plasmid isolated from each transfection condition were sequenced. Recombined plasmids were only observed if reporter plasmids were previously cotransfected with cognate guide RNA plasmids and recCas9 expression vectors ( FIG.  4 D ). In two separate experiments, the percent of recombined plasmid ranged from 12% for site 1 in chromosome 5 to an average of 32% for the FGF14 locus in chromosome 13. The sequencing data therefore were consistent with the earlier flow cytometry analysis in  FIG.  4 A . The absolute levels of recombined plasmid were somewhat higher than the percent of EGFP-positive cells ( FIG.  4   ). This difference likely arises because the flow cytometry assay does not report on multiple recombination events that can occur when multiple copies of the reporter plasmid are present in a single cell; even a single recombination event may result in EGFP fluorescence. As a result, the percentage of EGFP-positive cells may correspond to a lower limit on the actual percentage of recombined reporter plasmids. Alternatively, the difference may reflect the negative correlation between plasmid size and transformation efficiency (see, e.g., Hanahan, Studies on transformation of  Escherichia coli  with plasmids. J Mol Biol 166, 557-580 (1983), the entire contents of which is hereby incorporated by reference); the recombined plasmid is approximately 5,700 base pairs and may transform slightly better than the intact plasmid, which is approximately 6,900 base pairs. 
     Since zinc finger-recombinases have been reported to cause mutations at recombinase core-site junctions (see, e.g., Gaj et al., A comprehensive approach to zinc-finger recombinase customization enables genomic targeting in human cells. Nucleic acids research 41, 3937-3946 (2013), the entire contents of which is hereby incorporated by reference), whether such mutagenesis occurs from recCas9 treatment was tested. In the reporter construct, recCas9 should delete kanR and the poly-A terminator by first cleaving the central dinucleotide of both gix core sites and then religating the two cores to each other ( FIG.  4 C ). Thus, the recombination product should be a single recombination site consisting of the first half of the ‘left’ target site and the second half of the ‘right’ target site. Erroneous or incomplete reactions could result in other products. Strikingly, all of the 134 recombined sequences examined contained the expected recombination products. Further, a total of 2,317 sequencing reads from two separate sets of transfection experiments revealed only three sequencing reads containing potential deletion products at otherwise non-recombined plasmids. 
     One of these deletion-containing reads was observed in a chromosome 12 reporter plasmid that was transfected with the pUC control and lacked both recCas9 target sites as well as the polyA terminator. This product was attributed to DNA damage that occurred during the transfection, isolation, or subsequent manipulation. Because recCas9 may only localize to sequences when cotransfected with reporter and cognate guide RNA expression vectors, a more relevant metric may be to measure the total number of deletion products observed when reporter plasmids are cotransfected with cognate guide RNA vectors and recCas9 expression vectors. A single indel was observed out of a total of 185 plasmids sequenced from cotransfections with the chromosome 5-site 1 reporter and cognate guide RNA. Similarly, one indel was observed out of 204 plasmids from the chromosome 12 reporter following transfection with cognate guide RNA and recCas9 expression vectors. Notably, out of 202 sequencing reads, no indels were observed from the chromosome 13 reporter following cognate guide RNA and recCas9 cotransfection, despite resulting in the highest observed levels of recombination. These observations collectively suggest that recCas9 mediates predominantly error-free recombination. 
     Taken together, these results establish that recCas9 can target multiple sites found within the human genome with minimal cross-reactivity or byproduct formation. Substrates undergo efficient recombination only in the presence of cognate guide RNA sequences and recCas9, give clean recombination products in human cells, and generally do not result in mutations at the core-site junctions or products such as indels that arise from cellular DNA repair. 
     RecCas9-Mediated Genomic Deletion 
     Finally, whether recCas9 is capable of operating directly on the genomic DNA of cultured human cells was investigated. Using the list of potential recCas9 recognition sites in the human genome (Table 9), pairs of sites that, if targeted by recCas9, would yield chromosomal deletion events detectable by PCR, were sought. Guide RNA expression vectors were designed to direct recCas9 to those recCas9 sites closest to the chromosome 5-site 1 or chromosome 13 (FGF14 locus), sites which were both shown to be recombined in transient transfection assays ( FIG.  4   ). The new target sites ranged from approximately 3 to 23 Mbp upstream and 7 to 10 Mbp downstream of chromosome 5-site 1, and 12 to 44 Mbp upstream of the chromosome 13-FGF14 site. The recCas9 expression vector was cotransfected with each of these new guide RNA pairs and the validated guide RNA pairs used for chromosome 5-site 1 or chromosome 13-FGF14, but evidence of chromosomal deletions by genomic PCR was not observed. 
     It was thought that genomic deletion might be more efficient if the recCas9 target sites were closer to each other on the genome. Two recCas9 sites separated by 14.2 kb within an intronic region of FAM19A2 were identified; these sites also contained identical dinucleotide cores which should facilitate deletion. FAM19A2 is one of five closely related TAFA-family genes encoding small, secreted proteins that are thought to have a regulatory role in immune and nerve cells (see, e.g., Parker et al., Admixture mapping identifies a quantitative trait locus associated with FEV1/FVC in the COPDGene Study. Genet Epidemiol 38, 652-659 (2014), the entire contents of which is hereby incorporated by reference). Small nucleotide polymorphisms located in intronic sequences of FAM19A2 have been associated with elevated risk for systemic lupus erythematosus (SLE) and chronic obstructive pulmonary disease (COPD) in genome-wide association studies (see, e.g., Parker et al., Admixture mapping identifies a quantitative trait locus associated with FEV1/FVC in the COPDGene Study. Genet Epidemiol 38, 652-659 (2014), the entire contents of which is hereby incorporated by reference); deletion of the intronic regions of this gene might therefore provide insights into the causes of these diseases. Four guide RNA sequences were cloned in expression vectors designed to mediate recCas9 deletion between these two FAM19A2 sites. Vectors expressing these guide RNAs were cotransfected with the recCas9 expression vector ( FIG.  5 A ). RecCas9-mediated recombination between the two sites should result in deletion of the 14.2 kb intervening region. Indeed, this deletion event was detected by nested PCR using gene-specific primers that flank the two FAM19A2 recCas9 targets. The expected PCR product that is consistent with recCas9-mediated deletion was observed only in genomic DNA isolated from cells cotransfected with the recCas9 and all four guide RNA expression vectors ( FIG.  5 B ). The deletion PCR product was not detected in the genomic DNA of cells transfected without either the upstream or downstream pair of guide RNA expression vectors alone, without the recCas9 expression plasmid, or for the genomic DNA of untransfected control cells ( FIG.  5 B ). The estimated limit of detection for these nested PCR products was approximately 1 deletion event per 5,500 chromosomal copies. The 415-bp PCR product corresponding to the predicted genomic deletion was isolated and sequenced. Sequencing confirmed that the PCR product matched the predicted junction expected from the recombinase-mediated genomic deletion and did not contain any insertions or deletions suggestive of NHEJ ( FIG.  5 C ). 
     A lower limit on the minimum genomic deletion efficiency was estimated using nested PCR on the serial dilutions of genomic template (see above or, e.g., Sykes et al., Quantitation of targets for PCR by use of limiting dilution. Biotechniques 13, 444-449 (1992), the entire contents of which is hereby incorporated by reference, for greater detail). A given amount of genomic DNA that yields the recCas9-specific nested PCR product must contain at least one edited chromosome. To establish a lower limit on this recCas9-mediated genomic deletion event, nested PCR was performed on serial dilutions of genomic DNA (isolated from cells transfected with recCas9 and the four FAM19A2 guide RNA expression vectors) to determine the lowest concentration of genomic template DNA that results in a detectable deletion product. These experiments revealed a lower limit of deletion efficiency of 0.023±0.017% (average of three biological replicates) ( FIG.  5 D ), suggesting that recCas9-mediated genomic deletion proceeds with at least this efficiency. Nested PCR of the genomic DNA of untransfected cells resulted in no product, with an estimated limit of detection of &lt;0.0072% recombination. 
     Use of Other Alternative Recombinases 
     A Cre recombinase evolved to target a site in the Rosa locus of the human genome called “36C6” was fused to to dCas9. This fusion was then used to recombine a plasmid-based reporter containing the Rosa target site in a guide-RNA dependent fashion.  FIG.  7 A  demonstrates the results of linker optimization using wild-type Cre and 36C6. The 1×2×, 5×, and 8× linkers shown are the number of GGS repeats in the linker. Reversion analysis demonstrated that making mutations to 36C6 fused to dCas9 could impact the relative guide dependence of the chimeric fusion ( FIG.  7 B ). Reversions are labeled with their non-mutated amino acids. For example, position 306, which had been mutated to an M, was reverted to an I before the assay was performed. A GinB construct, targeting its cognate reporter, was used as a control for the experimental data shown in  FIGS.  7 A and  7 B . The on-target guides were the chr13-102010574 guides (plasmids BC165 and 166). Abbreviations shown are GGS-36C6: dCas9-GGS-36C6; 2GGS-36C6 (using linker SEQ ID NO: 182): sdCas9-GGSGGS-36C6 (using linker SEQ ID NO: 182). 
     The target sequence used for 36C6 and all variant transfections is shown below: (guides—italics; Rosa site—bold): 
     
       
         
           
               
            
               
                 (SEQ ID NO: 760) 
               
            
           
           
               
            
               
                 CCT AGGGAAGTGATCATAGCTGAG TTTCT ATCTCATGGTTTATGCTAAA   
               
               
                   
               
               
                   CTATATGTTGACAT GTTGA GGAGACTTAAGTCCAAAACC TGG 
               
            
           
         
       
     
     In  FIGS.  7 A,  7 B,  8 ,  9 A, and  9 B , the on-target guides for GinB were the chr13-102010574 guides (plasmids BC165 and 166). All off-target guides in  FIGS.  7 A,  7 B,  8 ,  9 A, and  9 B  were composed of the chr12-62418577 guides (BC163 and BC164). 
     PAMs were identified flanking the Rosa26 site in the human genome that could support dCas9 binding ( FIG.  8   , top). Guide RNAs and a plasmid reporter were then designed to test whether the endogenous protospacers could support dCas9-36C6 activity. A GinB construct, targeting its cognate reporter, was used as a control. See  FIG.  8   . Mix: equal parts mixture of all 5 linker variants between Cas9 and 36C6. For hRosa, the target sequence, including guide RNA tagets, are below: (guides—italics; Rosa site—bold) 
     
       
         
           
               
            
               
                 (SEQ ID NO: 767) 
               
            
           
           
               
            
               
                 CCT GAAATAATGCAAGTGTAGAA TAACTTTTTAAA ATCTCATGGTTTAT   
               
               
                   
               
               
                   GCTAAACTATATGTTGACAT AAG AGTGGTGATAAGGCAACAGT AGG 
               
            
           
         
       
     
     The on target guide plasmids for hRosa are identical to the other gRNA expression plasmids, except the protospacers are replaced with those shown above ( FIG.  8   ). 
     Several tested Cre truncations of dCas9-Cre recombinase fusions are shown in  FIG.  9 A . Truncated variants of Cre recombinase fused to dCas9 showed both appreciable recombinase activity as well as a strict reliance on the presence of guide RNA in a Lox plasmid reporter system ( FIG.  9 B ). Truncated variants are labeled with the residue at which the truncated Cre begins. The linker for all fusion proteins shown in  FIGS.  9 A and  9 B  is 8×GGS. Wild type Cre fused to dCas9 was used as a positive control. The target sequence used for 36C6 and all variant transfections is shown below: (guides—italics; Rosa site—bold): 
     
       
         
           
               
            
               
                 (SEQ ID NO: 768) 
               
            
           
           
               
            
               
                 CCT AGGGAAGTGATCATAGCTGA GTTTCT ATCTCATGGTTTATGCTAAA   
               
               
                   
               
               
                   CTATATGTTGACAT GTTGA GGAGACTTAAGTCCAAAACC TGG 
               
            
           
         
       
     
     The on-target guides used were the chr13-102010574 guides (plasmids BC165 and 166) and the off-target guides were the chr12-62418577 guide (BC163 and BC164). 
     REFERENCES 
     
         
         1. J. A. Doudna, E. Charpentier, Genome editing. The new frontier of genome engineering with CRISPR-Cas9 . Science  346, 1258096 (2014). 
         2. M. R. Capecchi, Altering the genome by homologous recombination.  Science  244, 1288-1292 (1989). 
         3. K. R. Thomas, K. R. Folger, M. R. Capecchi, High frequency targeting of genes to specific sites in the mammalian genome. Cell 44, 419-428 (1986). 
         4. A. Choulika, A. Perrin, B. Dujon, J. F. Nicolas, Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of  Saccharomyces cerevisiae. Mol Cell Biol  15, 1968-1973 (1995). 
         5. D. Carroll, Progress and prospects: zinc-finger nucleases as gene therapy agents.  Gene Ther  15, 1463-1468 (2008). 
         6. J. C. Miller et al., A TALE nuclease architecture for efficient genome editing.  Nature biotechnology  29, 143-U149 (2011). 
         7. J. K. Joung, J. D. Sander, TALENs: a widely applicable technology for targeted genome editing.  Nat Rev Mol Cell Biol  14, 49-55 (2013). 
         8. P. Mali et al., RNA-guided human genome engineering via Cas9 . Science  339, 823-826 (2013). 
         9. L. Cong et al., Multiplex genome engineering using CRISPR/Cas systems.  Science  339, 819-823 (2013). 
         10. J. P. Guilinger, D. B. Thompson, D. R. Liu, Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification.  Nature biotechnology , (2014). 
         11. S. Q. Tsai et al., Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing.  Nature biotechnology , (2014). 
         12. H. Fung, D. M. Weinstock, Repair at single targeted DNA double-strand breaks in pluripotent and differentiated human cells.  PloS one  6, e20514 (2011). 
         13. W. D. Heyer, K. T. Ehmsen, J. Liu, Regulation of homologous recombination in eukaryotes.  Annu Rev Genet  44, 113-139 (2010). 
         14. D. Branzei, M. Foiani, Regulation of DNA repair throughout the cell cycle.  Nat Rev    
       
    
       Mol Cell  Bio 9, 297-308 (2008).
     15. V. T. Chu et al., Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells.  Nature biotechnology , (2015).   16. T. Maruyama et al., Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining.  Nature biotechnology,  (2015).   17. S. Lin, B. T. Staahl, R. K. Alla, J. A. Doudna, Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery.  eLife  3, e04766 (2014).   18. S. Turan, C. Zehe, J. Kuehle, J. H. Qiao, J. Bode, Recombinase-mediated cassette exchange (RMCE)—A rapidly-expanding toolbox for targeted genomic modifications.  Gene  515, 1-27 (2013).   19. T. Gaj, S. J. Sirk, C. F. Barbas, Expanding the Scope of Site-Specific Recombinases for Genetic and Metabolic Engineering.  Biotechnology and bioengineering  111, 1-15 (2014).   20. N. D. F. Grindley, K. L. Whiteson, P. A. Rice, Mechanisms of site-specific recombination.  Annu Rev Biochem  75, 567-605 (2006).   21. C. R. Sclimenti, B. Thyagarajan, M. P. Calos, Directed evolution of a recombinase for improved genomic integration at a native human sequence.  Nucleic acids research  29, 5044-5051 (2001).   22. R. Shah, F. Li, E. Voziyanova, Y. Voziyanov, Target-specific variants of Flp recombinase mediate genome engineering reactions in mammalian cells.  The FEBS journal  282, 3323-3333 (2015).   23. J. Karpinski et al., Directed evolution of a recombinase that excises the provirus of most HIV-1 primary isolates with high specificity.  Nature biotechnology , (2016).   24. F. Buchholz, A. F. Stewart, Alteration of Cre recombinase site specificity by substrate-linked protein evolution.  Nature biotechnology  19, 1047-1052 (2001).   25. B. Thyagarajan, E. C. Olivares, R. P. Hollis, D. S. Ginsburg, M. P. Calos, Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase.  Mol Cell Biol  21, 3926-3934 (2001).   26. B. Thyagarajan, M. J. Guimaraes, A. C. Groth, M. P. Calos, Mammalian genomes contain active recombinase recognition sites.  Gene  244, 47-54 (2000).   27. A. Akopian, J. He, M. R. Boocock, W. M. Stark, Chimeric recombinases with designed DNA sequence recognition.  Proceedings of the National Academy of Sciences of the United States of America  100, 8688-8691 (2003).   28. R. M. Gordley, C. A. Gersbach, C. F. Barbas, 3rd, Synthesis of programmable integrases.  Proceedings of the National Academy of Sciences of the United States of America  106, 5053-5058 (2009).   29. M. M. Prorocic et al., Zinc-finger recombinase activities in vitro.  Nucleic acids research  39, 9316-9328 (2011).   30. C. A. Gersbach, T. Gaj, R. M. Gordley, A. C. Mercer, C. F. Barbas, Targeted plasmid integration into the human genome by an engineered zinc-finger recombinase.  Nucleic acids research  39, 7868-7878 (2011).   31. A. C. Mercer, T. Gaj, R. P. Fuller, C. F. Barbas, Chimeric TALE recombinases with programmable DNA sequence specificity.  Nucleic acids research  40, 11163-11172 (2012).   32. T. Matsuda, C. L. Cepko, Controlled expression of transgenes introduced by in vivo electroporation.  Proceedings of the National Academy of Sciences of the United States of America  104, 1027-1032 (2007).   33. N. E. Sanjana et al., A transcription activator-like effector toolbox for genome engineering.  Nature protocols  7, 171-192 (2012).   34. T. Gaj, A. C. Mercer, S. J. Sirk, H. L. Smith, C. F. Barbas, A comprehensive approach to zinc-finger recombinase customization enables genomic targeting in human cells.  Nucleic acids research  41, 3937-3946 (2013).   35. Y. Fu et al., High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.  Nature biotechnology  31, 822-826 (2013).   36. J. Quan, J. Tian, Circular polymerase extension cloning of complex gene libraries and pathways.  PloS one  4, e6441 (2009).   37. N. Hillson. (2010), vol. 2015, pp. CPEC protocol.   38. R. C. Gentleman et al., Bioconductor: open software development for computational biology and bioinformatics.  Genome Biol  5, R80 (2004).   39. K. Motmans, S. Thirion, J. Raus, C. Vandevyver, Isolation and quantification of episomal expression vectors in human T cells.  Biotechniques  23, 1044-1046 (1997).   40. B. Hirt, Selective extraction of polyoma DNA from infected mouse cell cultures.  J Mol Biol  26, 365-369 (1967).   41. A. Klippel, G. Mertens, T. Patschinsky, R. Kahmann, The DNA Invertase Gin of Phage Mu—Formation of a Covalent Complex with DNA Via a Phosphoserine at Amino-Acid Position-9 . Embo Journal  7, 1229-1237 (1988).   42. G. Mertens et al., Site-specific recombination in bacteriophage Mu: characterization of binding sites for the DNA invertase Gin.  The EMBO journal  7, 1219-1227 (1988).   43. R. H. Plasterk, A. Brinkman, P. van de Putte, DNA inversions in the chromosome of  Escherichia coli  and in bacteriophage Mu: relationship to other site-specific recombination systems.  Proceedings of the National Academy of Sciences of the United States of America  80, 5355-5358 (1983).   44. H. Xu et al., Sequence determinants of improved CRISPR sgRNA design.  Genome research  25, 1147-1157 (2015).   45. R. M. Gordley, J. D. Smith, T. Graslund, C. F. Barbas, 3rd, Evolution of programmable zinc finger-recombinases with activity in human cells.  J Mol Biol  367, 802-813 (2007).   46. C. A. Gersbach, T. Gaj, R. M. Gordley, C. F. Barbas, 3rd, Directed evolution of recombinase specificity by split gene reassembly.  Nucleic acids research  38, 4198-4206 (2010).   47. T. Gaj, A. C. Mercer, C. A. Gersbach, R. M. Gordley, C. F. Barbas, Structure-guided reprogramming of serine recombinase DNA sequence specificity.  Proceedings of the National Academy of Sciences of the United States of America  108, 498-503 (2011).   48. T. Gaj et al.,  Enhancing the Specificity of Recombinase - Mediated Genome  Engineering through Dimer Interface Redesign.  J Am Chem Soc  136, 5047-5056 (2014).   49. F. Cunningham et al., Ensembl 2015 . Nucleic acids research  43, D662-669 (2015).   50. J. C. van Swieten et al., A mutation in the fibroblast growth factor 14 gene is associated with autosomal dominant cerebellar ataxia [corrected].  Am J Hum Genet  72, 191-199 (2003).   51. E. Brusse et al., Spinocerebellar ataxia associated with a mutation in the fibroblast growth factor 14 gene (SCA27): A new phenotype.  Mov Disord  21, 396-401 (2006).   52. K. Choquet, R. La Piana, B. Brais, A novel frameshift mutation in FGF14 causes an autosomal dominant episodic ataxia.  Neurogenetics  16, 233-236 (2015).   53. J. A. Coebergh et al., A new variable phenotype in spinocerebellar ataxia 27 (SCA 27) caused by a deletion in the FGF14 gene.  Eur J Paediatr Neurol  18, 413-415 (2014).   54. K. Shimojima et al., Spinocerebellar ataxias type 27 derived from a disruption of the fibroblast growth factor 14 gene with mimicking phenotype of paroxysmal non-kinesigenic dyskinesia.  Brain Dev  34, 230-233 (2012).   55. M. Sadelain, E. P. Papapetrou, F. D. Bushman, Safe harbours for the integration of new DNA in the human genome.  Nat Rev Cancer  12, 51-58 (2012).   56. D. Hanahan, Studies on transformation of  Escherichia coli  with plasmids.  J Mol Biol  166, 557-580 (1983).   57. M. M. Parker et al., Admixture mapping identifies a quantitative trait locus associated with FEV1/FVC in the COPDGene Study.  Genet Epidemiol  38, 652-659 (2014).   58. P. J. Sykes et al., Quantitation of targets for PCR by use of limiting dilution.  Biotechniques  13, 444-449 (1992).   59. A. Rath, R. Hromas, A. De Benedetti, Fidelity of end joining in mammalian episomes and the impact of Metnase on joint processing.  BMC Mol Biol  15, 6 (2014).   60. P. Rebuzzini et al., New mammalian cellular systems to study mutations introduced at the break site by non-homologous end-joining.  DNA Repair  ( Amst ) 4, 546-555 (2005).   61. J. Smith, C. Baldeyron, I. De Oliveira, M. Sala-Trepat, D. Papadopoulo, The influence of DNA double-strand break structure on end-joining in human cells.  Nucleic acids research  29, 4783-4792 (2001).   62. S. Turan et al., Recombinase-mediated cassette exchange (RMCE): traditional concepts and current challenges.  J Mol Biol  407, 193-221 (2011).   63. S. J. Sirk, T. Gaj, A. Jonsson, A. C. Mercer, C. F. Barbas, Expanding the zinc-finger recombinase repertoire: directed evolution and mutational analysis of serine recombinase specificity determinants.  Nucleic acids research  42, 4755-4766 (2014).   64. B. P. Kleinstiver et al., Broadening the targeting range of  Staphylococcus aureus  CRISPR-Cas9 by modifying PAM recognition.  Nature biotechnology  33, 1293-1298 (2015).   65. B. P. Kleinstiver et al., Engineered CRISPR-Cas9 nucleases with altered PAM specificities.  Nature  523, 481-U249 (2015).   66. K. M. Esvelt et al., Orthogonal Cas9 proteins for RNA-guided gene regulation and editing.  Nature methods  10, 1116-1121 (2013).   67. B. Zetsche et al., Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System.  Cell  163, 759-771 (2015).   68. K. Dormiani et al., Long-term and efficient expression of human beta-globin gene in a hematopoietic cell line using a new site-specific integrating non-viral system.  Gene Ther  22, 663-674 (2015).   69. E. Wijnker, H. de Jong, Managing meiotic recombination in plant breeding.  Trends in plant science  13, 640-646 (2008).   70. J. F. Petolino, V. Srivastava, H. Daniell, Editing Plant Genomes: a new era of crop improvement.  Plant Biotechnol J  14, 435-436 (2016).   
     EQUIVALENTS AND SCOPE 
     Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above description, but rather is as set forth in the appended claims. 
     In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. 
     Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. 
     Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, steps, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, steps, etc. For purposes of simplicity those embodiments have not been specifically set forth in haec verba herein. Thus for each embodiment of the invention that comprises one or more elements, features, steps, etc., the invention also provides embodiments that consist or consist essentially of those elements, features, steps, etc. 
     Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range. 
     In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein. 
     All publications, patents and sequence database entries mentioned herein, including those items listed above, are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.