Targeted gene editing constructs and methods of using them

Abstract

This disclosure provides nucleic acid constructs for use in enhancing the site-specific insertion of an exogenous nucleic acid into a genome. In some embodiments, the nucleic acid construct comprises a first polynucleotide sequence encoding a DNA-binding protein engineered to bind to a specific genomic DNA sequence, a second polynucleotide comprising a modified integrase or a modified transposase that enables insertion of the exogenous nucleic acid into the genome, and a nucleic acid sequence encoding a linker between the two nucleotides. In some embodiments, the nucleic acid construct encodes a fusion protein, for example, a fusion protein for delivery to a cell via a lentiviral particle.

Description

TARGETED GENE EDITING CONSTRUCTIONS AND METHODS OF USE THEREOF Reference to sequence listing presented electronically The content of the electronically submitted sequence listing in the ASCII text file (Name: 4349.001 PC01_Seqlisting_ST25; Size: 389,120 bytes; and Creation Date: June 11, 2020) that was submitted with the application is incorporated into the This document is for reference in its entirety. Background of the Invention Many diseases, such as cancer, developmental disorders, and some infections, have genetic and epigenetic aberrations in common. Gene therapy is designed to introduce genetic material into cells in order to directly target and edit the genome in order to correct genetically dysfunctional cells and therefore cure associated diseases. Zinc finger nucleases (ZFNs), Talen and Crispr-cas9 gene editing technologies represent some of the recently developed tools for editing DNA. Methods such as electroporation, cationic lipids, microinjections or viruses have been used to deliver genetic material to a genome. Current gene delivery strategies are typically based on adenoviruses, retroviruses, or naked DNA plasmids. Lentiviruses, which include HIV, are a powerful tool when used as a vector for the delivery of nucleic acids. Lentiviruses are capable of stably infecting dividing and non-dividing cells. Lentiviral vectors are prone to random integration into the host genome, and can often integrate at the site of highly transcribed genes, increasing the risk of insertional mutagenesis. HIV-1 integrase catalyzes the insertion of viral DNA into the host genome. In general, HIV-1 integrase consists of an N-terminus domain (NTD), a catalytic core domain (CCD), and a C-terminus domain (CTD). for its acronym in English). The NTD is used to bind and coordinate a Zn2+ cation as an important cofactor, while the CTD is used for binding to DNA. The CCD forms the catalytic nucleus in which the integration process is catalyzed. Challenges with the insertion mechanisms that are used by viral vectors include low efficiency and a lack of specificity, which can result in unintentional insertion mutagenesis and genotoxicity. Brief Description of the Invention Some aspects of this disclosure provide constructs, plasmids, vectors, particles, fusion proteins, compositions, methods and kits that are useful for targeted editing of nucleic acids, including editing of a single site or region within the genome. of a subject, for example, the human genome. The processing examples present herein provide detailed experimental data that plausibly demonstrate the successful generation of programmable transposase and integrase fusion protein constructs with the Cas9 / finger proteins. 7CICI Π / I 7Π7 / Ε / Υ zinc. Furthermore, such constructs were able to cause site-specific integration of an exogenous nucleic acid sequence into the genome of transfected cells. Without being bound by theory, the present inventors believe that this is the first time that fusion proteins of this type have been generated, with the capacity for site-specific integration of an exogenous nucleic acid into a genome and that they are suitable for gene therapy, especially that involving large genes. The inventors have also identified modified hyperactive PiggyBac transposases that carry out specific targeted transpositions. Accordingly, one aspect of this disclosure relates to a nucleic acid construct comprising: a) a first polynucleotide sequence comprising a nucleic acid encoding a first DNA binding protein designed to bind to a specific genomic DNA sequence in a genome; wherein the first DNA binding protein is a zinc finger protein or a Cas9 protein; b) a second polynucleotide sequence comprising a nucleic acid encoding a second DNA-binding protein that allows insertion of an exogenous nucleic acid into a genome, wherein the second DNA-binding protein is: Yo. a hyperactive PiggyBac transposase, or a modified hyperactive PiggyBac with improved specificity of insertion of the exogenous nucleic acid into the genome compared to the hyperactive PiggyBac, or i. a human immunodeficiency virus (HIV) integrase or a modified HIV integrase with improved specificity of exogenous nucleic acid insertion into the genome compared to the HIV integrase; and c) an optional polynucleotide sequence comprising a nucleic acid encoding a linker; wherein the nucleic acid construct encodes a fusion protein comprising the first DNA binding protein, the second DNA binding protein and the optional linker between the first DNA binding protein and the second DNA binding protein; and wherein the fusion protein allows the insertion of the exogenous nucleic acid to be made at a specific site in the genome. Also provided is a composition comprising a nucleic acid construct, a vector or a fusion protein as described herein, and a polynucleotide sequence encoding an exogenous nucleic acid for insertion into a genome, wherein the composition is contained in, or linked to, a packing vector. The present disclosure also provides a method for controlled, site-specific integration of a single copy or multiple copies of an exogenous nucleic acid sequence into a cell, wherein the method comprises: (a) delivering the nucleic acid construct, the vector or fusion protein described herein to the cell, and (b) delivering the exogenous nucleic acid to the cell; wherein the binding of the fusion protein to the specific genomic DNA sequence in the 7CICI Π / I 7Π7 / Ε / Υ genome of the cell, results in the cleavage of the genome and the integration of one or more copies of the exogenous nucleic acid into the genome of the cell. Another aspect relates to the provision of the modified hyperactive PiggyBac transposases comprising the amino acid sequence of SEQ ID NO: 9, wherein: the amino acid at position 245 is A, the amino acid at position 275 is R or A, the amino acid at position 277 is R or A, the amino acid at position 325 is A or G, the amino acid at position 347 is N or A, the amino acid at position 351 is E, P or A, the amino acid at position 372 is R, the amino acid at position 375 is A, the amino acid at position 450 is D or N, the amino acid at position 465 is W or A, the amino acid at position 560 is T or A, the amino acid at position 564 is P or S, the amino acid at position 573 is S or A, the amino acid at position 592 is G or S, and the amino acid at position 594 is L or F. In some embodiments, fusion proteins of (i) an integrase, a modified integrase, a transposase or a modified transposase linked to (ii) a Cas9 or a zinc finger protein are provided; and the nucleic acid constructs that encode the same. Certain aspects of the application are directed to a nucleic acid construct comprising: (a) a first polynucleotide sequence encoding a first DNA binding protein that is designed in such a way as to bind to a DNA sequence specific genomic in a genome; (b) a second polynucleotide sequence encoding a second DNA binding protein that allows insertion of an exogenous nucleic acid into the genome, wherein the second DNA binding protein is (i) an integrase or a modified integrase that is modified relative to a wild-type integrase, or (i) a transposase or a modified transposase that is modified relative to a wild-type transposase; and (c) a third polynucleotide sequence comprising a nucleic acid encoding a linker; wherein the nucleic acid construct encodes a fusion protein comprising the first DNA binding protein, the second DNA binding protein and the linker between the first DNA binding protein and the second DNA binding protein. In some embodiments, the nucleic acid construct comprises: (a) a first polynucleotide sequence encoding a Cas9 protein; and (b) a second polynucleotide sequence encoding a transposase or a modified hyperactive PiggyBac of the disclosure or a functional fragment thereof. In some embodiments, the nucleic acid construct comprises: (a) a first polynucleotide sequence encoding a zinc finger protein; and (b) a second polynucleotide sequence encoding an integrase or a modified integrase of the disclosure or a functional fragment thereof. In some embodiments, the application is directed to a plasmid, vector or host cell comprising a nucleic acid construct of the disclosure. Some aspects of the application are directed to a fusion protein comprising: a first DNA binding protein that is designed in such a way as to bind to a specific genomic DNA sequence in a genome; a second DNA-binding protein that allows it to be made 7CICI n / l 7Π7 / Ε / Υ the insertion of an exogenous nucleic acid into the genome, in which the second DNA-binding protein is an integrase, a transposase, or a modified integrase or transposase; and a linker that connects the first protein and the second protein. In some embodiments, the fusion protein comprises: (a) a Cas9 protein; and (b) a hyperactive PiggyBac or a modified hyperactive PiggyBac of the disclosure or a functional fragment thereof. In some embodiments, the fusion protein comprises: (a) a zinc finger protein; and (b) an integrase or a modified integrase of the disclosure or a functional fragment thereof. Some aspects of the application are directed to a lentiviral particle comprising a fusion protein of the disclosure. Some aspects of the application are directed to a method for inserting an exogenous nucleic acid sequence into the genomic DNA of an organism, which comprises: administration of a lentiviral particle comprising a nucleic acid construct or a fusion protein of disclosure to the organism, in a manner such that the first and second DNA binding proteins bind to a specific genomic DNA sequence and insert the exogenous nucleic acid into the genomic DNA; where the exogenous nucleic acid becomes integrated into the specific genomic DNA sequence. Some aspects of the disclosure are directed to a method for the controlled and site-specific integration of a single copy or multiple copies of an exogenous nucleic acid sequence into a cell, wherein the method comprises: (a) delivering the protein of fusion disclosure to the cell, and (b) delivering the exogenous nucleic acid to the cell; wherein binding of the fusion protein to the specific genomic DNA sequence in the genome of the cell results in cleavage of the genome and integration of one or more copies of the exogenous nucleic acid into the genome of the cell; and wherein the fusion protein is delivered to the cell by a lentiviral particle. Throughout the description and claims, the word comprise and its variations are not intended to exclude other technical characteristics, additives, components or steps. Additional objects, advantages and features of the invention will become apparent to persons skilled in the art after examining the description or may be learned by practice of the invention. Furthermore, the present invention encompasses all possible combinations of the particular and preferred embodiments described herein. The following examples and drawings are provided herein for illustrative purposes, and are not intended to limit the present invention. Brief Description of the Drawings Figures 1A and 1B show the percentage of cells that have the exogenous nucleic acid sequence integrated into their genome after transfection with (Figure 1A) the Cas9-PiggyBac fusion proteins (human Cas9 (hCas9), Cas9 nicase (nCas9 ), or dead Cas9 (dCas9) and overactive PiggyBac (PB) transposase) and (Figure 1B) Cas9-SB100 fusion proteins (human Cas9 (hCas9), nicase Cas9 (nCas9), or dead Cas9 (dCas9) and the transposase hyperactive Sleeping Beauty (SB100)). Vectors were created in which the 3' end of Cas9 was connected to the 5' end of each of the transposases. 7CICI n / l 7Π7 / Ε / Υ via a GGS linker (SEQ ID NO: 48, 49) (hCas9PB, nCas9PB, dCas9PB, hCas9SB, nCas9SB and dCas9SB). Other vectors were created in which the 3' end of each transposase was connected to the 5' end of Cas9 via a GGS linker (SEQ ID NO: 48, 49) (PBhCas9, PBnCas9, PBdCas9, SBhCas9, SbnCas9 and SBdCas9). PiggyBac (Figure 1 A) and SB100 (Figure 1 B) were used as the positive controls, and the transposon alone encoding an RFP (denoted as the episomal RFP in Figure 1 A) and GFP (denoted as the episomal GFP) were used. in Figure 1B) as the negative controls. Figure 1C is a different representation of Figure 1A showing the transposition activity with PB and Cas9 in different configurations. Figure 2A shows a plasmid construct encoding a Cas9 / PB fusion protein. Figure 2B shows the percentage of cells that have the exogenous nucleic acid sequence integrated into their genome by fusion constructs formed by a human Cas9-PiggyBac (targeted HCas9) or a Cas9-PiggyBac nicase (targeted NCas9). The 3' end of Cas9 was connected to the 5' end of the transposase via a linker. Non-targeting is the control for general insertion (PiggyBac alone) and Episomal is the negative control for non-integration (transposon alone). Figure 3 shows an example ZFP-integrase fusion protein. ZFP and integrase are linked via a GGS sequence. NLS refers to nuclear localization sequence. Figure 4 shows the lentivirus titer of wild-type integrase lentivirus (LV), empty viral particles (LVO), non-integrating lentivirus (NILV), non-integrating lentivirus integrative with wild-type integrase (NILV + IN), non-integrative lentivirus with ZFP-integrase fusion protein (NILV + ZP-IN (AAVS1)), non-integrative lentivirus with Cas9-integrase fusion protein (NILV + Cas-IN), and wild-type integrase lentivirus with wild-type integrase (LV + IN). (') denotes a technical replica. Figure 5 shows the percentage of cells that integrated (overall integration) the exogenous nucleic acid sequence into their genome after infection with wild-type integrase lentivirus (LV), empty viral particles (LVO). non-integrative lentivirus (NILV), non-integrative lentivirus with wild-type integrase (NILV + IN), non-integrative lentivirus with ZFP-integrase fusion protein (NILV + ZP -IN (AAVS1)), the non-integrative lentivirus with the Cas9-integrase fusion protein (NILV + Cas-IN), and the wild-type integrase lentivirus with the wild-type integrase (LV + IN). For each condition, from left to right, the first column refers to day 3, the second column to day 5, the third column to day 7, the fourth column to day 10, and the fifth column to day 12. Figure 6 shows an image of the chromosomes with representative sites of AAVS1 integration and non-integration. A star symbol represents the site for AAVS1 on chromosome 19, a triangle symbol means non-targeted integration sites; and a diamond symbol means the specific integration. Figure 7A shows the virus titer generated by wild-type integrase lentivirus (LV), empty viral particles (LVO), non-integrating lentivirus (NILV). JCICI n / l 7Π7 / Ε / Υ in English), non-integrative lentivirus with wild-type integrase (NILV + IN), non-integrative lentivirus with AAVS1 site-directed ZFP-IN fusion protein (NILV + ZP -IN(AAVSI)), and the non-integrative lentivirus with the ZFP-IN fusion protein directed at the CCR5 site (NILV + ZP-IN (CCR5)). Figure 7B shows the percentage of cells that integrated (overall integration) the exogenous nucleic acid sequence into their genome after infection with the wild-type integrase lentivirus (LV), non-integrating lentivirus (NILV). English), non-integrative lentivirus with wild-type integrase (NILV + IN), non-integrative lentivirus with AAVS1 site-directed ZFP-IN fusion protein (NILV + ZP-IN (AAVS1)), and non-integrative lentivirus integrative with the CCR5 site-directed ZFP-IN fusion protein (NILV + ZP-IN (CCR5)). Figure 7C shows the percentage of cells that integrated the exogenous nucleic acid sequence into their genome after infection with wild-type integrase lentivirus (LV), empty viral particles (LVO), non-integrative lentivirus (NILV), non-integrative lentivirus with wild-type integrase (NILV + IN), non-integrative lentivirus with AAVS1 site-directed ZFP-IN fusion protein (NILV + ZP -IN (AAVS1)) and the non-integrative lentivirus with the CCR5 site-directed ZFP-IN fusion protein (NILV + ZP-IN (CCR5)). Figure 7D shows the percentage of cells that integrated the exogenous nucleic acid sequence into their genome after infection with the wild-type integrase lentivirus (LV), non-integrating lentivirus (NILV), non-integrative lentivirus with the wild-type integrase (NILV + IN), the non-integrative lentivirus with the AAVS1 site-directed ZFP-IN fusion protein (NILV + ZP- IN (AAVS1)), and the non-integrative lentivirus with the AAVS1 site-directed fusion protein of ZFP-IN fusion directed to the CCR5 site (NILV + ZP-IN (CCR5)). Figures 8A to 8C show the lentivirus titer (Figure 8A) and the percentage of CAR-expressing cells on day 3 and day 14 (Figure 8B), and the percentage of CD3-expressing cells is shown in Figure 8A. figure 8C. Jurkat cells were infected with several lentivirus conditions: wild-type integrase lentivirus (LV), empty viral particles (LVO), non-integrating lentivirus (NILV). , non-integrative lentivirus with wild-type integrase (NILV + IN), non-integrative lentivirus with ZFP-integrase fusion protein (NILV + ZFP-IN (TRCa-1), non-integrative lentivirus with Cas9-integrase fusion (NILV + Cas-IN). NILV showed a drastic decrease in titer; and transcomplementation with expression of WT IN or ZNF-IN fusion in virus-producing cells had no effect. rescue effect on the titer, nor on the integration capacity. Furthermore, the cells did not lose CD3 expression when integration was directed towards the TCR locus (CD3 protein expression). This denotes the need to use factors additional for transcomplementation, such as the VPR protein; especially in the context of this cell line. Figures 9A and 9B show the titer for the WT lentivirus and for two different integrase-deficient virus systems (NILV and TAA, the latter indicating that a stop codon has been introduced at the beginning of the IN coding region in the plasmid of lentiviral packaging) alone or transcomplemented with the IN or VPR_IN fusion. Titers were detected by fluorescent cytometry analysis in the 7CICI n / l 7Π7 / Ε / Υ day 3 after infection (Figure 9A). Figure 9B shows the relative integration efficiencies of the transcomplemented integration machines showing the advantage of VPR protein fusion with IN for transcomplementation. WT: the lentivirus produced with WT IN; NILV: the lentivirus produced with non-integrating IN, which harbors two mutations in its catalytic center; TAA: the lentivirus produced with a defective IN, where the protein is not expressed; + IN: the lentivirus transcomplemented with IN; + VPR-IN: the lentivirus transcomplemented with the IN fused to VPR at the end of the C terminus. Figure 10A shows a schematic of the nucleic acid construct formed by an insertion domain with a DNA binding domain and a programmable DNA recognition domain fused via a linker. Figure 10B is a schematic showing the fusion of Cas9 and a linker-linked transposase in different configurations. Figure 11 shows the results of Cas9 activity on hyPB-linked Cas9 through the use of different linker sizes and compositions. Cas9 activity was measured by gRNA target site sequencing and using CRISPR-GA to analyze indel frequency. 2 different gRNAs targeting the AAVS1 site were used. The linkers that were used are SEQ ID NO: 50 to 63. Figure 12 shows the results of the programmable transposition efficiency of the genetrap transposase. RFP fluorescence was measured by flow cytometry 10 days after transfection. Different linkers were used to determine the length of the linkers and the importance of composition in targeted insertion. Average of 2 independent experiments. The linkers that were used are SEQ ID NO: 50 to 63. Figure 13 shows the results of transposition directed to the linkers of hcas9_PB. Efficiencies of targeted transposition of different cas9-PB linker constructs through the use of split GFP cell line by using 2 different gRNAs. GFP expression was measured by flow cytometry at 72 h posttransfection. Figure 14 shows a schematic of the split GFP reporter cell line generated for high-throughput screening of the library of the different hyPB mutations as well as validation of the individual mutants. A splice acceptor (SA) followed by half of the coding sequence of GFP (Ct-GFP) was introduced downstream of a target region site in the genome of Hek293T cells. through the use of the Sleeping Beauty 10Ox system. The PiggyBac transposon flanked by the inverted terminal repeats (ITR) for this selection was a complete RPF expression cassette followed by a promoter and the other half of GFP (Nt-GFP) and a donor of splicing (SD); or, only the middle fragment of the GFP; as shown in the figure. Figure 15 shows the results of targeted transposition of the hcas9_PB-selected mutants. Efficiencies of directed transposition of hcas9_PB D450N and hcas9_PB R372A K375A D450. GFP expression was measured by flow cytometry at 72 h posttransfection. Average of 4 independent experiments. 7CICI n / l 7Π7 / Ε / Υ Figure 16 shows the results of random and directed transposition of the mutants selected by hcas9_PB. Specific and random transposition efficiencies of hcas9_PB D450N and hcas9_PB R372A K375A D450. GFP expression was measured by flow cytometry at 72 h posttransfection, and RFP expression was measured by flow cytometry at 15 days posttransfection and normalized by RFP fluorescence at 48 h after transfection assumed as transfection efficiency. Figure 17 is a scheme showing the fusion of ZFP and a linker-linked transposase in different configurations. Figure 18 shows the results of targeted rearrangement of ZFP-PB fusion proteins. Specific transposition efficiencies of ZFP_hyPB or ZFP_hyPBD450N in the N- and C-terminus conformations. GFP expression was measured by flow cytometry at 5 days after transfection. More than 1 independent repetition. ZFP_PB: Fusion of ZFP and hyPB in the C terminus configuration through the use of the XTEN linker; PB_ZFP: Fusion of ZFP and hyPB in the N-term configuration through the use of the XTEN linker, ZFP 450: Fusion of ZFP and hyPB (D450N) in the C-term configuration through the use of the XTEN linker; 450_ZFP: Fusion of ZFP and hyPB (D450N) in the N-terminus configuration through the use of the XTEN linker; hyPB: hyPB without modifications; 1 / 2 GFP: Control transposon alone. Figure 19 shows a schematic of the analysis method used in screening a library of PiggyBac mutations. In Figure 20, the 1116 bp PiggyBac region with all library variants was sequenced using lllumina NGS technology. The I7 index primer was replaced with a custom-made primer to allow complete sequencing of the different variants, with the exception of the 450 and 465 variants. Figures 21A and 21B show the results of diversity generation of the hyPB library. Figure 21A is an example of the classification diagram. Positive hits from targeted integration (GFP fluorescence) were selected at gate P4, while negative hits from targeted integration (no GFP fluorescence) were selected at gate P5. Nonviable cells and debris were selectively negative in anterior gates with DAPI staining. Figure 21B shows the results of the double plasmid transfection efficiency. Transfection efficiency was measured by transfection of a GFP and an equimolar RFP plasmid with 1 / 2 GFP and transfection of the gRNA on the same day and under the same conditions. Gate P8 selects for double plasmid transfection. Nonviable cells and debris were selectively negative in anterior gates with DAPI staining. Figures 22A to 22K show the results of the library screening analysis by comparing positive hits with negative hits. Figures 22A and 22B: Bulk library sequencing shown as quality control; where the vast majority of variants were shown only once. The logo of the representative bulk PiggyBac library is shown, where the positions correspond to the amino acid positions: 1- R245; 2- R275; 3-R277; 7CICI n / l 7Π7 / Ε / Υ 4-G325; 5-Ν347; 6- S351; 7- R372; 8-Κ375; 9- R388; 10-Τ560; 11- S564; 12- S573; 13- Μ589; 14- S592; 15-F594. Additionally, the logo for negative selected cells is displayed in a pattern similar to that of the bulk library. Figures 22C to 22K correspond to 3 independent repetitions of positive hits; variant calling positive logos (below), as well as the Top 1 variant after selection (above). Also shown are the logos for the top 5 and top 10 variants. Panels to the left of B and C show the relative enrichment of PiggyBac variants in populations classified as positive versus negative on the Iog2 scale. Figure 23A shows the Top 1 and Top 3 positive variants of independent repeat 3. There is a difference of only 1 amino acid at position 254. Figure 23B shows the 3 top 1 variants identified in 3 independent repeats. WT hyPB is also shown for reference. Figure 24A shows the most overrepresented variants in GFP-positive cells versus RFP-positive cells. GPF clustering, directed insertion; FPR, random insertion, and negative population are shown. Figures 24B and 24C show the variants found between the positive hit in more than 1 independent repetition. Rep: Independent Experimental Repetition; Pos: Positive cells with directed integration; Neg: Negative cells where directed integration did not occur. Figure 25 shows a variant covariation histogram. This shows the percentage of one vanant seen together with another in the positive sample divided by the negative sample. In addition to the variants included in the library design, the variants that were randomly introduced by the lentiviral reverse transcriptase during the generation of the viral library were analyzed. Some of these new variants are associated in the positive hits and carry out directed integration in combination. Example of D450N and W465A. Figure 26 shows that the modified hyPB demonstrated a greater increase in targeted integration compared to WT hyPB when fused to Cas9. Cas9 was fused with hyPB or with different combinations of hyPB mutants (Unilarge-A: D450N; Unilarge-B: R245A / D450N; UnilargeC: R245A / G325A / D450N / S573P; Unilarge-D: R245A / G325A / S573P) through the use of a 4GGS linker and reporter cell line system. Figure 27 shows the results of integrase-deficient transcomplementation. The viral production efficiency measured on day 2 and the integration capacity measured on day 7 were evaluated for different systems in Hek293T cells. Western blots showed the presence of IN in trans in the viral particles. The efficiency of viral production and its integration capacity were evaluated through the infection of the different conditions of the integration-deficient virus and the virus transcomplemented in Hek293T. Cells were passaged for 7 days until no more episomal signal was detected, and the GFP signal was analyzed by flow cytometry on days 2, 5 and 7. Different production efficiencies could be detected for the different systems, where NILV was the closest to the WT in production. In all cases, a clear rescue of integration activity was seen when transcomplementation with WT-HIVJN was carried out. Proof that IN was loaded into the transcomplementation system was obtained by Western blot. WT: the lentivirus 7CICI Π / I 7Π7 / Ε / Υ produced with WT IN; NILV: the lentivirus produced with non-integrating IN, which harbors two mutations in its catalytic center; TAA: the lentivirus produced with an IN-defective IN, where the protein is not expressed due to the presence of a stop codon at the beginning of the IN coding sequence, TAAx3: the lentivirus produced with an IN-defective IN , where the protein is not expressed due to the presence of 3 consecutive stop codons at the beginning of the IN coding sequence; Delta-IN: the lentivirus produced with a defective IN in IN, where the coding sequence of the IN has been removed; Delta-IN_cPPT: the lentivirus produced with an IN-defective IN, where the IN coding sequence has been replaced by the core polypyrimidine trac (cPPT) sequence; + VPR-IN: the trans lentivirus complemented with the IN fused to the VPR at the end of the C terminus. Detailed description of the invention I. Definitions As used herein, the singular forms an, an, and the include reference in the singular and plural, unless the context otherwise clearly indicates. Therefore, for example, a reference to an agent includes a single agent and a plurality of such agents. The terms nucleic acid, polynucleotide and oligonucleotide are used interchangeably and refer to a deoxyribonucleotide or ribonucleotide polymer, in a linear or circular conformation, and in a single-stranded or double-stranded form. For the purposes of the present disclosure, these terms should not be construed as limiting the length of a polymer. The terms may encompass known analogues of natural nucleotides, as well as nucleotides that are modified into base, sugar and / or phosphate moieties (e.g., phosphorothioate base structures). In general, an analog of a particular nucleotide has the same base pairing specificity; that is, an analogue of A will make a base pair with T. The terms polypeptide, peptide, and protein are used interchangeably to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of the corresponding naturally occurring amino acids. The term binding protein, as used herein, refers to a protein that is capable of binding in a non-covalent manner to another molecule. A binding protein can bind, for example, to a DNA molecule (a DNA binding protein), to an RNA molecule (an RNA binding protein) and / or to a protein molecule (a binding protein). to proteins). In the case of a protein-binding protein, it can bind to itself (to form homodimers, homotrimers, etc.) and / or can bind to one or more molecules of a different protein or proteins. A binding protein may have more than one type of binding activity. For example, zinc finger proteins have DNA-binding, RNA-binding, and protein-binding activity. The term zinc finger protein, as used herein, is a protein, or a domain within a larger protein, that binds to DNA in a sequence-specific manner through one or more zinc fingers. zinc, which are amino acid sequence regions within 7CICI n / l 7Π7 / Ε / Υ a zinc finger protein binding domain whose structure is stabilized through the coordination of a zinc ion. The term zinc finger protein is often abbreviated as ZFP. The term zinc finger nucleases refers to artificial restriction enzymes generated by the fusion of a zinc finger DNA binding domain to a DNA cleavage domain. Zinc finger domains can be designed in such a way as to target specific desired DNA sequences and this allows zinc finger nucleases to target unique sequences within complex genomes. Zinc finger nuclease is often abbreviated as ZFN or ZNP. The terms nucleic acid sequence or polynucleotide sequence or gene sequence, as used herein, refer to a nucleotide sequence of any length, which may be DNA or RNA; This can be linear, circular or branched and can be single stranded or double stranded. The terms amino acid sequence or polypeptide or protein, as used herein, refer to a polymer of amino acid residues. Unless specified, a polymer of amino acid residues can be of any length. The term exogenous, as used herein, refers to a molecule that is not normally present in a cell, but that can be introduced into a cell by one or more genetic, biochemical or other methods. The normal presence in the cell is determined with respect to the particular stage of development and the environmental conditions of the cell. Therefore, for example, a molecule that is present only during embryonic muscle development is an exogenous molecule with respect to an adult muscle cell. In a similar way, a heat shock-induced molecule is an exogenous molecule with respect to a cell without heat shock. An exogenous molecule may comprise, for example, a functional version of a malfunctioning endogenous molecule or a malfunctioning version of a normally functioning endogenous molecule. In contrast, an endogenous molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions. For example, an endogenous nucleic acid may comprise a chromosome, the genome of a mitochondria, chloroplast or other organelle, or a naturally occurring episomal nucleic acid. Additional endogenous molecules may include proteins, for example, transcription factors and enzymes. A target site or target sequence is a sequence that defines a portion of a nucleic acid or polypeptide to which a binding molecule will bind, provided that sufficient conditions exist for binding. For example, the 5'-GAATTC-3' sequence is a target site for the EcoRI restriction endonuclease. The term fusion, as used herein, refers to a molecule in which two or more subunit molecules are linked, preferably in a covalent manner. Subunit molecules can be the same chemical type of molecule, or they can be different chemical types of molecules. The term fusion protein, as used herein, refers to a hybrid polypeptide 7CICI n / l 7Π7 / Ε / Υ comprising protein domains from at least two different proteins. A protein can be located in the amino terminus portion (N terminus) of the fusion protein or in the carboxyl terminus protein (C terminus), thereby forming an amino terminus fusion protein or a protein. fusion of the carboxyl term, respectively. The terms gene or genome, as used herein, include a region of DNA that encodes a gene product, as well as all regions of DNA that regulate the production of the gene product, whether or not such regulatory sequences are adjacent. to the coding and / or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translation regulatory sequences, such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators. , boundary elements, origins of replication, matrix binding sites and locus control regions. The term eukaryotic cells includes, but is not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells and human cells (e.g., T cells). The term linked, as used herein, refers to the juxtaposition of two or more components (such as sequence elements), in which the components are arranged in such a way that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted on at least one of the other components. A functional fragment of a protein, polypeptide or nucleic acid is a protein, polypeptide or nucleic acid, respectively, whose sequence is not identical to that of the full-length protein, polypeptide or nucleic acid, and yet retains the same function as the protein, polypeptide or full-length nucleic acid. A functional fragment may possess more, fewer, or the same number of residues as the corresponding native molecule, and / or may contain one or more amino acid or nucleotide substitutions. The term transfect, as used herein, refers to the introduction of nucleic acids (either DNA or RNA) into eukaryotic or prokaryotic cells or organisms. The term cleavage, as used herein, refers to the breakage of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-strand cleavage and double-strand cleavage are possible, and double-strand cleavage can occur as a result of two distinct single-strand cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends. In certain embodiments, fusion polypeptides are used for targeted double-stranded DNA cleavage. The term integrase, as used herein, refers to an enzyme that is produced by a virus that allows genetic material to be integrated into DNA, for example, genomic DNA, 7CICI Π / I 7Π7 / Ε / Υ an infected cell. The term specificity, as used herein, refers to the ability to selectively bind a sequence that shares a degree of sequence identity with a selected sequence. The terms insertion and integration, as used herein, refer to the addition of one nucleic acid sequence into a second nucleic acid sequence or genome. The terms specific, site-specific, directed and on-target in relation to insertion or integration, are used interchangeably herein to refer to the insertion of a nucleic acid at a specific site of a second nucleic acid or genome. The terms random, untargeted, and off-target refer to nonspecific, unintentional genetic insertion. The terms total or global refer to the total number of insertions. The term mutation, as used herein, refers to a substitution of a residue within a sequence, for example, 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 identification of 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, for example, by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Coid Spring Harbor Laboratory Press, Coid Spring Harbor, N.Y. (2012). The term transposase, as used herein, refers to an enzyme that binds to the end of a transposon and catalyzes its movement to another part of the genome by means of a cut-and-paste mechanism or a replicative transposition mechanism. . The term modified, as used herein, refers to a protein or nucleic acid sequence that is different from a corresponding unmodified protein or nucleic acid sequence. The term linker, as used herein, refers to a chemical group or molecule that links two adjacent molecules or parts. The terms vector and plasmid, as used herein, refer to any polynucleotide that can carry, for example, a second polynucleotide of interest and, for example, that can transfer gene sequences to target cells. Therefore, the term includes cloning and expression vehicles as well as integration vectors. In particular, the term expression vector, as used herein, refers to any polynucleotide capable of directing the expression of a nucleic acid. In some aspects, the terms vector and plasmid are used interchangeably with the term nucleic acid construct. The term percent identity, as used herein, refers to the percent identity of two sequences, whether nucleic acid or amino acid sequences, and is the number of exact matches between two aligned sequences divided by the length of the shortest sequences and multiplied by 100. 7CICI n / l 7Π7 / Ε / Υ The terms recombinant or engineered, as used herein, refer to a protein or nucleic acid sequence that has been created in an artificial way. The term subject, as used herein, refers to an individual organism, for example, an individual mammal. In some embodiments, the subject is a human being. 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, goat, beef, cat, or dog. In some embodiments, the subject is a vertebrate, amphibian, reptile, fish, insect, fly, or nematode. In some embodiments, the subject is a research animal. The terms treatment, treating and treating refer to a clinical intervention that aims to reverse, alleviate, delay the onset or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described in this document. As used herein, the terms treatment, treating, and treating refer to a clinical intervention that aims to reverse, alleviate, delay the onset, or inhibit the progression of a disease or disorder, or one or more symptoms. of the same, as described in this document. In some embodiments, the treatment may be administered after one or more symptoms have developed and / or after a disease has been diagnosed. In other embodiments, the treatment may be administered in the absence of symptoms, for example, to prevent, reduce the likelihood of developing or delay the onset of a symptom, or inhibit the 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. II. Construction of nucleic acids Targeted editing of nucleic acid sequences, for example, the introduction of a specific modification (e.g., insertion of an exogenous nucleic acid) into genomic DNA, is a promising approach for the treatment of human genetic diseases. To this end, the inventors aim to provide improved nucleic acid constructs for use in genome editing that are highly efficient in installing a desired modification; minimal off-target activity; and the ability to be programmed in such a way as to precisely edit a site within the human genome. Certain aspects of the present application are directed to a nucleic acid construct for use in improving the site-specific insertion of an exogenous nucleic acid, for example, a gene of interest (GOI), into a genome. In some embodiments, the GOI is a therapeutic gene, for example, a gene that encodes a therapeutic protein. Examples of therapeutic genes of interest include the CFTR (cystic fibrosis transmembrane conductance regulator) gene for treating cystic fibrosis disease; the SMN1 (Survival Motor Neuron 1) gene to treat spinal muscular atrophy (SMA); the G171V variant 7CICI n / l 7Π7 / Ε / Υ of the LRP5 (LDL receptor-related protein 5) gene to prevent osteoporosis and bone fractures; and the A673T variant of the APP gene (amyloid beta precursor protein) to reduce the predisposition to Alzheimer's disease. In some embodiments, the exogenous nucleic acid for insertion (e.g., the GOI) can be up to about 10 kb, up to about 15 kb, up to about 20 kb in length, up to about 25 kb in length, up to approximately 30 kb in length, up to approximately 35 kb in length or up to approximately 40 kb in length. In some embodiments, the polynucleotide sequence encoding a DNA-binding protein that allows insertion of an exogenous nucleic acid into the genome comprises an integrase or an integrase that is modified relative to a wild-type integrase, and The exogenous nucleic acid for insertion may be up to 10 kb, up to 15 kb or up to 20 kb in length, for example, about 1 kb to about 20 kb, about 1 kb to about 19 kb, about 1 to about 18 kb, from about 1 kb to about 17 kb, from about 1 kb to about 16 kb, or from about 1 kb to about 15 kb. In some embodiments, the polynucleotide sequence encoding a second DNA-binding protein that allows insertion of an exogenous nucleic acid into the genome comprises a transposase or a transposase that is modified relative to a wild-type transposase, and the exogenous nucleic acid for insertion may be up to 10 kb, up to 15 kb, up to 20 kb in length, up to 25 kb in length, up to 30 kb in length, up to 35 kb in length or up to 40 kb in length, for example, about 1 kb to about 40 kb, about 1 kb to about 39 kb, about 1 to about 38 kb, about 1 kb to about 37 kb, about 1 kb to about 36 kb, or from about 1 kb to about 35 kb. In some embodiments, the nucleic acid construct comprises a polynucleotide sequence encoding a first DNA-binding protein, e.g., a gene editing polypeptide, and a polynucleotide sequence encoding a second DNA-binding protein, e.g. For example, an integrase or a transposase, wherein the nucleic acid construct encodes the first and second binding proteins as a fusion protein. In some embodiments, the nucleic acid construct further comprises a nucleic acid sequence that encodes a linker between the first and second binding proteins. In some embodiments, the nucleic acid construct encodes a fusion protein that allows and / or promotes site-specific insertion of exogenous nucleic acid into a genome. In some embodiments, the first or second binding protein is an integrase that is modified relative to the wild type. In some embodiments, the first or second binding protein is a transposase that is modified relative to the wild type. In some embodiments these are directed to a vector or plasmid comprising a nucleic acid construct of the disclosure. In certain aspects, the nucleic acid construct of the disclosure encodes a fusion protein that improves the specificity of the insertion of a nucleic acid, for example, a GOI, 7CICI n / l 7Π7 / Ε / Υ in the genome. In some embodiments, the fusion protein and exogenous nucleic acid are delivered to a cell through the use of a lentivirus particle. In some embodiments, the first and second binding proteins are in separate nucleic acid constructs, for example, the transposase or integrase (e.g., a transposase and / or integrase modified with respect to the wild type) is in a construct. of nucleic acid separated from Cas9 or ZFP. Certain aspects are directed to a plasmid or vector comprising a nucleic acid construct disclosed herein. In some embodiments, the plasmid comprising the nucleic acid construct is a packaging plasmid. In some embodiments, the plasmid comprising the nucleic acid construct further comprises a polynucleotide encoding capsid proteins, for example, gag and pol. In some embodiments, (i) the plasmid comprising the nucleic acid construct is combined with: (i) a plasmid comprising a polynucleotide that encodes proteins for a viral envelope (envelope plasmid); and (iii) a plasmid comprising an exogenous nucleic acid sequence (e.g., a GOI), wherein when the combination is introduced into a production cell line (e.g., eukaryotic cells, prokaryotic cells and / or cell lines) , a virus particle is produced comprising the exogenous nucleic acid, for example, GOI, and the fusion protein comprising the first and second binding proteins. In some embodiments, (i) the plasmid comprising the nucleic acid construct is combined with: (ii) a plasmid comprising the nucleic acid construct further comprising a polynucleotide encoding capsid proteins, e.g., gag and pol (a packaging plasmid, wherein the packaging plasmid lacks a functional integrase); (iii) a plasmid comprising a polynucleotide that encodes proteins for a viral envelope (envelope plasmid), and (iv) a plasmid comprising an exogenous nucleic acid sequence (for example, a GOI), wherein when the combination A virus particle comprising the exogenous nucleic acid, for example, GOI, is introduced into a production cell line (e.g., eukaryotic and prokaryotic cells and / or cell lines), and the fusion protein comprising the first is produced. and the second binding proteins. The nucleic acid construct comprises a first polynucleotide sequence encoding a first DNA-binding protein that is designed in such a way as to bind to a specific DNA sequence, a second polynucleotide sequence encoding a second DNA-binding protein. DNA binding that allows insertion of the exogenous nucleic acid into the genome wherein the second DNA binding protein is an integrase or a transposase (for example, a transposase and / or integrase that is modified relative to the type wild), and the third polynucleotide sequence comprising a nucleic acid sequence that encodes a linker between the first and second polynucleotides. In some embodiments, the first DNA binding protein is a zinc finger protein or a Cas9 protein. In some embodiments, the nucleic acid construct comprises a linker that is selected from the group consisting of a (GGS)n, a (GGGGS)n (SEQ ID NO: 133), a (G)n, a (EAAAK )n (SEQ ID NO: 134), an XTEN-based linker, or an (XP)n motif, or a combination of 7CICI n / l 7Π7 / Ε / Υ any of these, where n is independently an integer between 1 and 50. In some embodiments, the nucleic acid encodes a linker comprising an XTEN sequence or a GGS. In some embodiments, the linker nucleic acid sequence is between 3 and 150 nucleotides in length. In some embodiments, the linker is 12 to 24 amino acids, or 36 to 72 nucleic acids in length. In some embodiments, the nucleic acid construct comprises a linker nucleic acid sequence that is 6 to 120, 6 to 90, 6 to 78, 6 to 72, 9 to 120, 9 to 90, 9 to 78, from 9 to 72, from 12 to 120, from 12 to 90, from 12 to 78, from 12 to 72, from 15 to 120, from 15 to 90, from 15 to 78, from 15 to 72, 18 to 120, from 18 to 90, from 18 to 78, from 18 to 72, from 21 to 120, from 21 to 90, from 21 to 78, from 21 to 72, from 24 to 120, from 24 to 90, 24 to 78, from 24 to 72, from 27 to 120, from 27 to 90, from 27 to 78, from 27 to 72, from 30 to 120, from 30 to 90, from 30 to 78, from 30 to 72, 33 to 120, 33 to 90, 33 to 78, 33 to 72, 36 to 120, 36 to 90, 36 to 78 or 36 to 72 nucleotides in length. In some embodiments, the nucleic acid encoding the linker is between 9 and 150 nucleic acids in length. In some embodiments, a zinc finger protein is linked to a modified integrase of the disclosure with a linker comprising a GGS sequence. In some embodiments, the linker is between 1 and 50 amino acids in length. In some embodiments, the linker is 3 to 40, 3 to 30, 3 to 29, 3 to 24, 4 to 40, 4 to 30, 4 to 29, 4 to 24, 5 to 40, from 5 to 30, from 5 to 29, from 5 to 24, from 6 to 40, from 30, from 6 to 29, from 6 to 24, from 7 to 40, from 7 to 30, from 7 to 29 , from 7 to 24, from 8 to 40, from 8 to 30, from 8 to 29, from 24, from 9 to 40, from 9 to 30, from 9 to 29, from 9 to 24, from 10 to 40, from 10 to 30, from 10 to 29, from 10 to 24, from 11a, from 11 to 30, from 11 to 29, from 11 to 24, from 12 to 40, from 12 to 30, from 12 to 29, or 12 to 24 amino acids long. In some embodiments, the 3' end of the first polynucleotide sequence is connected to the 5' end of the second polynucleotide sequence via nucleic acid encoding a linker. In some embodiments, the 5' end of the first polynucleotide sequence is connected to the 3' end of the second polynucleotide sequence via nucleic acid encoding a linker. In some embodiments, the 3' end of the Cas9 protein is connected to the 5' end of the transposase via a linker. In some embodiments, the 5' end of the Cas9 protein is connected to the 3' end of the transposase via a linker. In some embodiments, the 3' zinc finger protein is connected to the 5' end of the integrase via a linker. In some embodiments, the 5' zinc finger protein is connected to the 3' end of the integrase via a linker. In some embodiments, a linker is not needed because the modified integrase or modified transposase is expressed from a plasmid separate from Cas9 or ZFP. Certain aspects of the disclosure are directed to a vector or a plasmid (e.g., an expression vector or a packaging vector) comprising a nucleic acid construct of the disclosure suitable for expression in a host cell, e.g., cells. of mammals, yeast cells, insect cells, plant cells, fungal cells or algal cells. In some embodiments, the nucleic acid construct comprises: (a) a first polynucleotide sequence comprising a nucleic acid encoding a first DNA-binding protein 7CICI Π / I 7Π7 / Ε / Υ that is designed in such a way as to bind to a specific genomic DNA sequence in a genome; wherein the first DNA binding protein is a zinc finger protein or a Cas9 protein; (b) a second polynucleotide sequence comprising a nucleic acid encoding a second DNA-binding protein that allows insertion of an exogenous nucleic acid into a genome, wherein the second DNA-binding protein is: ( i) a hyperactive PiggyBac transposase, or a modified hyperactive PiggyBac with improved specificity of exogenous nucleic acid insertion into the genome compared to the hyperactive PiggyBac, or (i) a human immunodeficiency virus (HIV) integrase, or a modified HIV integrase with improved specificity of exogenous nucleic acid insertion into the genome compared to HIV integrase; and (c) an optional polynucleotide sequence comprising a nucleic acid encoding a linker; wherein the nucleic acid construct encodes a fusion protein comprising the first DNA binding protein, the second DNA binding protein and the optional linker between the first DNA binding protein and the second DNA binding protein; and wherein the fusion protein allows the insertion of the exogenous nucleic acid to be made at a specific site in the genome. In one embodiment, (a) the first DNA binding protein is a Cas9 protein or a zinc finger protein; and (b) the second DNA binding protein is a hyperactive PiggyBac transposase, or a modified hyperactive PiggyBac transposase with improved specificity of insertion of the exogenous nucleic acid into the genome compared to the hyperactive PiggyBac transposase. In another embodiment, (a) the first DNA binding protein is a Cas9 protein or a zinc finger protein; and (b) the second DNA binding protein is an HIV integrase, or a modified HIV integrase with improved specificity of insertion of the exogenous nucleic acid into the genome compared to the HIV integrase. In some embodiments, the Cas9 protein is one described in this disclosure and is particularly selected from the group consisting of a human Cas9, a Cas9 nicase and a dead Cas9, and more particularly is a Cas9 human or a single Cas9 house. In one embodiment, when dCas9 is used, the second DNA binding protein is not a Gin, Hin or Tn3 recombinase catalytic domain or a Fokl DNA cleavage domain. Such recombinases and Fokl need a known site (an acceptable sequence in the genome) to be able to integrate; Therefore, the possibilities of targeting sites are much more limited; and they also need the formation of dimers, for example, of Gin, to be functional. In another embodiment, the zinc finger protein is one described in this disclosure and in particular is a C2H2 zinc finger protein comprising 6 binding domains. In another embodiment, the linker is one described in this disclosure and in particular the linker comprises an XTEN sequence (for example, SEQ ID NO: 61, encoded by SEQ ID NO: 60) or a GGS sequence, more particularly a GGSx3 (SEQ ID NO: 49, encoded by SEQ ID NO: 48), GGSx4 (SEQ ID NO: 51, encoded by SEQ ID NO: 50), GGSx5 (SEQ ID NO: 53, encoded by SEQ ID NO: 52), GGSx6 (SEQ ID NO: 55, encoded by SEQ ID NO: 54), GGSx7 (SEQ ID NO: 57, encoded by SEQ ID NO: 56) or GGSx8 (SEQ ID NO: 59, encoded by SEQ ID 7CICI Π / I 7Π7 / Β / Υ NO: 58). In another embodiment, the 3' end of the first polynucleotide sequence is connected to the 5' end of the second polynucleotide. In some embodiments, the modified hyperactive PiggyBac transposase is one described in this disclosure. In other embodiments, the modified HIV integrase is one described in the disclosure. In other embodiments, a linker is not used. Instead, for example, the first and / or second polynucleotide sequences comprise nucleic acids that encode a first and a second DNA-binding protein and further comprise additional nucleic acids at at least one of their ends that carry out the linker function. In one embodiment, (a) the first DNA binding protein is a Cas9 protein or a zinc finger protein, and (b) the second DNA binding protein is a hyperactive PiggyBac transposase, or a modified hyperactive PiggyBac. an improved specificity of the insertion of the exogenous nucleic acid into the genome compared to the hyperactive PiggyBac, wherein the nucleic acid construct comprises (c) the polynucleotide sequence comprising a nucleic acid encoding a linker comprising an XTEN sequence or a GGS sequence, and wherein the 3' end of the first polynucleotide sequence is connected to the 5' end of the second polynucleotide. In one embodiment, (a) the first DNA binding protein is a Cas9 protein, and (b) the second DNA binding protein is a hyperactive PiggyBac transposase, or a modified hyperactive PiggyBac with the proviso that when Cas9 is an inactive Cas9 (dCas9) the linker is not KLAGGAPAVGGGPK (SEQ ID NO: 130). In one embodiment, (a) the first DNA binding protein is a zinc finger protein, and (b) the second DNA binding protein is a hyperactive PiggyBac transposase, or a modified hyperactive PiggyBac, wherein the Zinc finger is capable of recognizing multiple recognition sites, because, as explained in this disclosure, the binding domain of the zinc finger protein can be designed in such a way as to bind to a sequence of choice. In one embodiment, (a) the first DNA binding protein is a zinc finger protein, and (b) the second DNA binding protein is a hyperactive PiggyBac transposase, or a modified hyperactive PiggyBac, and the linker is XTEN. . In one embodiment, (a) the first DNA binding protein is a zinc finger protein, and (b) the second DNA binding protein is a hyperactive PiggyBac transposase, or a modified hyperactive PiggyBac, in which the protein zinc-binding domain does not have a DNA-binding domain of Gal4. Gal4 binds to CGG-Nn-CCG, where N can be any base. This protein is a positive regulator for the genetic expression of galactose-induced genes, such as GAL1, GAL2, GAL7, GAL10 and MEL1 that encode enzymes used to convert galactose to glucose. It recognizes a 17 base pair sequence in (5'-CGGRNNRCYNYNCNCCG-3j (SEQ ID NO: 135) the upstream activation sequence (UAS-G) of these genes. Therefore, Gal4 recognizes a short and very frequent in the genome, so it is not site specific. In a particular embodiment, the protein 7CICI n / l 7Π7 / Ε / ΥΙΛΙ zinc binding has a Gal4 DNA binding domain designed to be site specific. In one embodiment, (a) the first DNA binding protein is a zinc finger protein, and (b) the second DNA binding protein is a hyperactive PiggyBac transposase, or a modified hyperactive PiggyBac transposase, with the condition of that the linker is not from EFGGGGSGGGGGGGGGGSQF (SEQ ID NO: 131). In another embodiment, (a) the first DNA binding protein is a Cas9 protein or a zinc finger protein, and (b) the second DNA binding protein is an HIV integrase, or a modified HIV integrase. with improved specificity of insertion of the exogenous nucleic acid into the genome compared to HIV integrase, wherein the nucleic acid construct comprises (c) the polynucleotide sequence comprising a nucleic acid encoding a linker comprising a XTEN sequence or a GGS sequence, and wherein the 3' end of the first polynucleotide sequence connects to the 5' end of the second polynucleotide. In some embodiments, the nucleic acid construct is in the form of DNA or RNA. Also provided herein are vectors comprising any of the nucleic acid constructs provided in this disclosure. In particular, the vectors are suitable for expression in mammalian cells, yeast cells, insect cells, plant cells, fungal cells or algal cells. Also provided herein are host cells comprising any of the nucleic acid constructs or vectors provided in this disclosure. III. Integrase and modified integrase Integrase is a key enzyme for stable integration of the viral genome into a host cell, but integrase is also associated with insertional mutagenesis, because the site of integration by wild-type integrase is unpredictable. Integration has been shown to be preferred for highly transcribed genes, increasing the risk of mutation of important genes and regulators. In general, HIV-1 integrase consists of an N-terminus domain (NTD), a catalytic core domain (CCD), and a C-terminus domain (CTD). for its acronym in English). The NTD is used to bind and coordinate a Zn2+ cation as an important cofactor, while the CTD is used for binding to DNA. The CCD domain forms the catalytic core where the integration process is catalyzed. After entering the host cell and reverse transcribing the viral RNA genome, four integrase molecules form a tetramer and bind to the ends of the viral DNA, which is then referred to as the intasome. The preintegration complex (PIC) digests the 3ΌΗ end of the DNA forming a 5ΌΗ overhang, which is then necessary for a nucleophilic attack on the host DNA. During the formation of this PIC, the complex is transported to the nucleus. After transport to the nucleus, PIC forms a complex with host DNA, which is termed a strand transfer complex (STC). In this case, the two 3ΌΗ flaps of the viral DNA attack both sites of the host DNA backbone with a gap of approximately 5 nucleotides. This leads to a target duplication of the 5 nucleotides. After the nucleophilic attack, 7CICI n / l 7Π7 / Β / Υ is integrated into the viral DNA, and the single-stranded DNA parts are repaired by the host cell's DNA repair machinery. The present disclosure provides nucleic acid constructs comprising polynucleotides encoding integrases and modified integrases for insertion of exogenous nucleic acid into a specific site of a genome. In some embodiments, the exogenous nucleic acid for insertion may be up to 10 kb, up to 15 kb, or up to 20 kb in length, e.g., about 1 kb to about 20 kb, about 1 kb to about 19 kb. , from about 1 to about 18 kb, from about 1 kb to about 17 kb, from about 1 kb to about 16 kb, or from about 1 kb to about 15 kb. In some embodiments, the polynucleotide sequence encoding a DNA-binding protein that allows insertion of an exogenous nucleic acid into the genome comprises an integrase that can be modified relative to a wild-type integrase, and the acid Exogenous nucleic acid for insertion can be up to 10 kb or up to 15 kb in length. Some aspects of this disclosure provide for integrase fusion proteins that are designed through the use of the methods and strategies described herein. Some embodiments of this disclosure provide nucleic acids encoding integrases or modified integrases and / or fusion proteins comprising same. Some embodiments of this disclosure provide plasmids or expression vectors comprising such nucleic acid constructs encoding integrases or modified integrases and / or fusion proteins comprising the same. The integrase or modified integrase of the invention can be any integrase that can insert an exogenous nucleic acid into a specific site of a genome. Non-limiting examples of integrases include HIV integrase, lentiviral integrase, adenoviral integrase, retroviral integrase, and mouse mammary tumor virus integrase. In some embodiments, the integrase (e.g., a modified integrase comprising one or more modifications relative to the wild type) is an HIV integrase, in particular the HIV integrase sequence corresponding to NC_001802.1 (SEQ ID NO : 1 and 2, amino acid and nucleic acid sequences, respectively). In some embodiments, the modified integrase comprises one or more modifications relative to the wild-type HIV integrase (SEQ ID NO: 1 and 2). In some embodiments, the integrase is a modified HIV integrase. The modified HIV integrase may comprise a mutation of one or more amino acids selected from amino acids: 10, 13, 64, 94, 116, 117, 119, 120, 122, 124, 128, 152, 168, 170 , 185, 231,264, 266 or 273 corresponding to the amino acid numbering of SEQ ID NO: 1. The modified HIV integrase mutation may comprise one or more of the amino acid modifications listed in Table 8. The mutation of The modified HIV integrase may comprise one or more of the amino acid modifications selected from D10K, Ε13K, D64A, D64E, G94D, G94E, G94R, G94K, D116A, D116E, N117D, N117E, N117R, N117K, S119A , S119P, S119T, S119G, S119D, S119E, S119R, S119K, N120D, N120E, N120R, N120K, T122K, T122I, T122V, T122A, T122R, A124D, A124E, A124R, A124K, 7CICI Π / I 7Π7 / Β / Υ Α128Τ, Ε152Α, E152D, Q168L, Q168A, E170G, F185K, R231G, R231K, R231D, R231E, R231S, K264R, K266R or K273R corresponding to the amino acid numbering of SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the modified integrase may comprise one or more mutations relative to the wild type that impair DNA binding, for example, at amino acids 94, 117, 119, 120, 124 and / or 231 (e.g., G94D , G94E, G94R, G94K, N117D, N117E, N117R, N117K, S119A, S119P, S119T, S119G, S119D, S119E, S119R, S119K, N120D, N120E, N120R, N120K, A124D, A12 4E, A124R, A124K, A124K, R231G , R231K, R231D, R231 E and / or R231K) that correspond to the amino acid numbering of SEQ ID NO: 1 or SEQ ID NO: 4. In some embodiments, the modified integrase may comprise one or more mutations relative to the wild type that improve DNA binding, for example, at amino acid 94, 117, 119, 120, 122, 124 and / or 231 (e.g. , G94D, G94E, G94R, G94K, N117D, N117E, N117R, N117K, S119A, S119P, S119T, S119G, S119D, S119E, S119R, S119K, N120D, N120E, N120R, N120K, T122 K, T122I, T122V, T122A, T122R , A124D, A124E, A124R, A124K, R231G, R231K, R231D, R231E and / or R231S) that correspond to the amino acid numbering of SEQ ID NO: 1 or SEQ ID NO: 5. In some embodiments, the modified integrase may comprise one or more mutations relative to the wild type that are involved in acetylation of the integrase by p300, for example, at amino acids 264, 266 and / or 273 (e.g., K264R, K266R and / or K273R) that correspond to the amino acid numbering of SEQ ID NO: 1 or SEQ ID NO: 6. In some embodiments, the modified integrase may comprise one or more mutations in highly conserved amino acids that are critical for retroviral integrative recombination, for example, in amino acids 10, 13, 64,116, 128, 152, 168 and / or 170 (e.g. example, D10K, E13K, D64A, D64E, D116A, D116E, A128T, E152A, E152D, Q168L, Q168A and / or E170G) corresponding to the amino acid numbering of SEQ ID NO: 1 or SEQ ID NO: 7 . In some embodiments, the modified integrase may comprise one or more mutations that interfere with the interaction with LEDGF / p75 and impair chromosome docking and HIV-1 replication, for example, amino acid 168 (e.g., Q168L or Q168A) which corresponds to the amino acid numbering of SEQ ID NO: 1 or SEQ ID NO: 8. In some embodiments, the modified HIV integrase comprises an amino acid sequence of at least 80 percent, at least 85 percent, at least 90 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, or at least 99 percent identical to the sequence set forth in SEQ ID NO: 1. In some embodiments, the integrase of Modified HIV comprises an amino acid sequence that has one or more of the modifications disclosed herein in relation to SEQ ID NO: 1, 3, 4, 5, 6, 7 or 8, and retains at least 80 percent, at least 85 percent, at least 90 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, or at least 99 percent identity with the sequence set forth in SEQ ID NO: 1,3, 4, 5, 6, 7 or 8, respectively. In some embodiments, the integrase of 7CICI n / l 7Π7 / Β / Υ Modified HIV is selected for its high specificity of integrating DNA into a genome compared to wild-type HIV integrase. Certain aspects of the disclosure are directed to a vector or a plasmid (e.g., an expression vector or a packaging vector) comprising a nucleic acid construct comprising an integrase or a modified integrase of the disclosure suitable for expression. in a host cell, for example, mammalian cells, yeast cells, insect cells, plant cells, fungal cells or algal cells. In some embodiments, the integrase or modified integrase is expressed as a fusion protein with a Cas9 protein or as a zinc finger protein. In some embodiments, the integrase or modified integrase is coexpressed with a Cas9 protein or a zinc finger protein from separate vectors, but is delivered to the same cell. In some embodiments, the integrase or the modified integrase or the fusion protein comprising the same is packaged into a lentivirus particle for delivery to a cell. IV. Transposase and modified transposase Transposons are chromosomal segments that can undergo transposition, for example, DNA that can translocalize as a whole in the absence of a complementary sequence in the host DNA. Transposons can be used to carry out long-range DNA engineering in human cells. Common transposon systems used in mammalian cells include Sleeping Beauty (SB), which was reconstructed from inactive transposons, and PiggyBac (PB), which was isolated from the Trichoplusia moth. PiggyBac has a higher transposition activity than SB and can be removed without scarring. Native DNA transposons usually contain a single gene encoding the transposase protein, which is flanked by inverted terminal repeats (ITRs) that carry transposase binding sites. During their transposition, the transposase protein recognizes these ITRs to catalyze the cleavage and subsequent reintegration of the element elsewhere in a random manner. Furthermore, some of these transposons can be adapted for use in gene therapy protocols, where they are used as two-component systems, in which a plasmid contains an expression cassette into which a DNA sequence can be introduced, placed between the ITRs of the transposon, in a host genome directed by the cotransfected plasmid containing the sequence encoding the transposase enzyme or its mRNA synthesized in vitro. In certain aspects of the invention, a transposon base is used to efficiently mediate stable integration and persistent expression of transgenes, such as therapeutic genes. The present disclosure provides nucleic acid constructs comprising polynucleotides encoding transposases or modified transposases for insertion of exogenous nucleic acid into a specific site of a genome. In some embodiments, the exogenous nucleic acid for insertion may be up to 20 kb in length, up to 25 kb in length, up to 30 kb in length, or up to 40 kb in length, for example, from about 1 kb to about 40 kb, from about 1 kb to about 39 kb, from about 1 to about 38 kb, 7CICI n / l 7Π7 / Β / Υ from approximately 1 kb to approximately 37 kb, from approximately 1 kb to approximately 36 kb, from approximately 1 kb to approximately 35 kb, from approximately 1 kb to approximately 30 kb, from approximately 1 kb to about 30 kb, or from about 1 kb to about 25 kb. In some embodiments, the polynucleotide sequence encoding a DNA-binding protein that allows insertion of an exogenous nucleic acid into the genome comprises a transposase or a transposase that is modified relative to a wild-type transposase, and The exogenous nucleic acid for insertion can be up to 35 kb or up to 40 kb in length. A transposase or a modified transposase of the invention can be any transposase that can insert an exogenous nucleic acid into a specific site of a genome. Some aspects of this disclosure provide transposase fusion proteins that are designed through the use of the methods and strategies described herein. Some embodiments of this disclosure provide nucleic acids encoding such transposases or modified transposases and / or fusion proteins comprising the same. Some embodiments of this disclosure provide plasmids or expression vectors comprising such nucleic acid constructs encoding transposases or modified transposases and / or fusion proteins comprising the same. Non-limiting examples of transposases include Frog Prince, Sleeping Beauty, Overactive Sleeping Beauty, PiggyBac, and Overactive PiggyBac. In some embodiments, the transposase is the hyperactive PiggyBac transposase corresponding to SEQ ID NO: 9 and 67 (also referred to in this disclosure as hyPB or simply as PB). In some embodiments, the modified transposase comprises one or more modifications relative to the overactive PiggyBac transposase (SEQ ID NO: 9). In some embodiments, the transposase is a modified hyperactive PiggyBac transposase. The modified hyperactive PiggyBac transposase may comprise a mutation of one or more of the amino acids selected from amino acids: 245, 268, 275, 277, 287, 290, 315, 325, 341, 346, 347, 350, 351 ,356,357,372,375,388,409,412,432,447,450,460,461,465,517,560,564,571,573,576,586,587,589,592,and 594 which correspond to the amino acid numbering of SEQ ID NO: 9. The modified hyperactive PiggyBac mutation may comprise one or more of the amino acid modifications listed in Table 3. The modified hyperactive PiggyBac transposase mutation may comprise one or more of amino acid modifications selected from: R245A, D268N, R275A / R277A, K287A, K290A, K287A / K290A, R315A, G325A, R341A, D346N, N347A, N347S, T350A, S351E, S351P, S351A, K3 56E, N357A , R372A, K375A, R372A / K375A, R388A, K409A, K412A, K409A / K412A, K432A, D447A, D447N, D450N, R460A, K461A, R460A / K461A, W465A, S517A, T560A, S564P, S571N, S573A, K576A, H586A , I587A, M589V, S592G or F594L that correspond to the amino acid numbering of SEQ ID NO: 9 or SEQ ID NO: 10. In some embodiments, the modified transposase may comprise one or more mutations relative to hyPB that are involved in the conserved catalytic triad, for example, at amino acids 268 and / or 346 (e.g., D268N and / or D346N) that correspond to the amino acid numbering of SEQ ID NO: 9 or SEQ ID NO: 11. 7CICI Π / I 7Π7 / Β / Υ In some embodiments, the modified transposase may comprise one or more mutations relative to hyPB that are critical for cleavage, for example, at amino acids 287, 287 / 290 and / or 460 / 461 (e.g., K287A, K287A / K290A and / or R460A / K461 A) that correspond to the amino acid numbering of SEQ ID NO: 9 or SEQ ID NO: 12. In some embodiments, the modified transposase may comprise one or more mutations relative to hyPB that are involved in binding to the target, for example, at amino acids 351,356 and / or 379 (for example, S351 E, S351 P, S351A and / or or K356E) that correspond to the amino acid numbering of SEQ ID NO: 9 or SEQ ID NO: 13. In some embodiments, the modified transposase may comprise one or more mutations relative to hyPB that are critical for integration, for example, at amino acids 560, 564, 571,573, 589, 592 and / or 594 (e.g., T560A, S564P , S571N, S573A, M589V, S592G and / or F594L) that correspond to the amino acid numbering of SEQ ID NO: 9 or SEQ ID NO: 14. In some embodiments, the modified transposase may comprise one or more mutations relative to hyPB that are involved in the alignment, for example, at amino acids 325, 347, 350, 357 and / or 465 (e.g., G325A, N347A, N347S , T350A and / or W465A) that correspond to the amino acid numbering of SEQ ID NO: 9 or SEQ ID NO: 15. In some embodiments, the modified transposase may comprise one or more mutations relative to hyPB that are well conserved, for example, at amino acids 576 and / or 587 (e.g., K576A and / or I587A) that correspond to the amino acid numbering of SEQ ID NO: 9 or SEQ ID NO: 16. In some embodiments, the modified transposase may comprise one or more mutations relative to hyPB that are involved in binding to Zn2+· e.g., 586 (e.g., H586A) which corresponds to the amino acid numbering of SEQ ID NO: 9 or SEQ ID NO: 17. In some embodiments, the programmed transposase may comprise one or more mutations relative to hyPB that are involved in integration, for example, 315, 341, 372 and / or 375 (for example, R315A, R341A, R372A and / or K375A) that correspond to the amino acid numbering of SEQ ID NO: 9 or SEQ ID NO: 18. In some embodiments, the modified hyperactive PiggyBac comprises an amino acid sequence of at least 85 percent, at least 90 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, or at least 99 percent identical to the sequence set forth in SEQ ID NO: 9. In some embodiments, the modified hyperactive PiggyBac is selected for its high integration specificity of DNA in a genome compared to the overactive PiggyBac. In some embodiments, the modified hyperactive PiggyBac comprises an amino acid sequence having one or more of the modifications disclosed herein in connection with SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18, and that retains at least 80 percent, at least 85 percent, at least 90 percent, at least 95 percent, at least 96 percent , at least 97 percent, at least 98 percent, or at least 99 percent identity with the sequence set forth in 7CICI Π / I 7Π7 / Ε / Υ SEQ ID NO: 9,10, 11, 12, 13, 14, 15, 16, 17 or 18, respectively. In some embodiments, the hyperactive PiggyBac transposase is encoded by a nucleic acid sequence having at least 85 percent, 90 percent, 95 percent, 96 percent, 97 percent, 98 percent , 99 percent or 100 percent sequence identity with SEQ ID NO: 67. In some embodiments, the SB100 transposase is encoded by a nucleic acid sequence that has at least 85 percent, 90 percent percent, 95 percent, 96 percent, 97 percent, 98 percent, 99 percent or 100 percent sequence identity with SEQ ID NO: 68. In some embodiments, the PB transposase comprises an amino acid sequence that has at least 85 percent, 90 percent, 95 percent, 96 percent, 97 percent, 98 percent, 99 percent percent or 100 percent sequence identity with SEQ ID NO: 72. In some embodiments, the SB100 transposase comprises an amino acid sequence that has at least 85 percent, 90 percent, 95 percent, 96 percent, 97 percent, 98 percent, 99 percent or 100 percent sequence identity with SEQ ID NO: 73. In some embodiments, the modified transposase is a modified Sleeping Beauty transposase comprising one or more mutations. In some embodiments, the one or more mutations in the hyperactive Sleeping Beauty transposase or SB100 correspond to: L25F, R36A, I42K, G59D, 1212K, N245S, K252A and Q271L of SEQ ID NO: 9 or SEQ ID NO: 73 . In certain embodiments, the modified transposase is not a Himar1C9 mutant. Certain aspects of the invention are directed to a vector or a plasmid (for example, an expression vector or a packaging vector) comprising a nucleic acid construct comprising a transposase or a modified transposase of the disclosure suitable for expression in a host cell, for example, mammalian cells, yeast cells, insect cells, plant cells, fungal cells or algal cells. In some embodiments, the transposase or modified transposase is expressed as a fusion protein with a Cas9. In some embodiments, the transposase or modified transposase is coexpressed with a separate Cas9 vector, but is delivered to the same cell. In some embodiments, the transposase or the modified transposase or the fusion protein comprising the same is packaged into a lentivirus particle for delivery to a cell. As shown in Example 20, a newly developed hyperactive PiggyBac transposase mutation library can be used for the identification of the modified hyperactive PiggyBac that carries out the specific targeted transpositions. The targeted rearrangement-positive hyperactive PiggyBac was identified through the use of such a library. In some embodiments, the modified hyperactive PiggyBac transposase may comprise a mutation of one or more amino acids that are selected from amino acids: 245, 275, 277, 325, 347, 351,372, 375, 388, 450, 465, 560, 564 , 573, 589, 592, 594 which correspond to the amino acid numbering of SEQ ID NO: 9. In some embodiments, the modified hyperactive PiggyBac mutation may comprise one or 7CICI n / l 7Π7 / Β / Υ more than the amino acid modifications listed in Table 11. In some embodiments, the modified hyperactive PiggyBac transposase mutation may comprise one or more of the amino acid modifications selected from: R245A, R275A, R277A, R275A / R277A, G325A, N347A, N347S, S351 E, S351 P , S351A, R372A, K375A, R388A, D450N, W465A, T560A, S564P, S573A, M589V, S592G or F594L corresponding to the amino acid numbering of SEQ ID NO: 9 or SEQ ID NO: 119. In one embodiment, the modified hyperactive PiggyBac transposase comprises the amino acid modification D450 corresponding to the amino acid numbering of SEQ ID NO: 9 or SEQ ID NO: 119. In one embodiment, the modified hyperactive PiggyBac transposase comprises the amino acid modifications R372A, K375A and D450, which correspond to the amino acid numbering of SEQ ID NO: 9 or SEQ ID NO: 119. In one embodiment, the modified hyperactive PiggyBac transposase comprises the amino acid modifications R245A and D450, which correspond to the amino acid numbering of SEQ ID NO: 9 or SEQ ID NO: 119. In one embodiment, the modified hyperactive PiggyBac transposase comprises the amino acid modifications R245A, G325A and S573P, which correspond to the amino acid numbering of SEQ ID NO: 9 or SEQ ID NO: 119. In one embodiment, the modified hyperactive PiggyBac transposase comprises the amino acid modifications R245A, G325A, D450 and S573P, which correspond to the amino acid numbering of SEQ ID NO: 9 or SEQ ID NO: 119. As stated in the previous paragraphs, modified hyperactive PiggyBac transposases are provided herein that can be fused with the elements disclosed herein, but can also be used alone or in combination with different elements. Said transposases have been generated by the inventors. Therefore, modified hyperactive PiggyBac transposases are provided comprising the amino acid sequence of SEQ ID NO: 9, wherein: Yo. the amino acid at position 245 is A, i. the amino acid at position 275 is R oA, i. the amino acid at position 277 is R oA, iv. the amino acid at position 325 is A or G, v. the amino acid at position 347 is N oA, vi. the amino acid at position 351 is E, P or A, vii. the amino acid at position 372 isR, viii. the amino acid at position 375 isA, ix. the amino acid at position 450 is D or N, x. the amino acid at position 465 is W or A, xi. the amino acid at position 560 is T or A, 7CICI n / l 7Π7 / Β / Υ xii. the amino acid at position 564 is P oS, xiii. the amino acid at position 573 is S oA, xiv. the amino acid at position 592 is G oS, and xv. the amino acid at position 594 is L oF. In some embodiments, the modified hyperactive PiggyBac comprises an amino acid sequence that is selected from the group consisting of SEQ ID NOs: 120, 121, 122, 123, 124, 125, 126, 127, 128 and 129. In some embodiments, the modified hyperactive PiggyBac comprises an amino acid sequence having one or more of the modifications disclosed herein in connection with SEQ ID NOs: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128 or 129, and that retains at least 80 percent, at least 85 percent, at least 90 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, or at least 99 percent identity with the sequence set forth in SEQ ID NO: 119, 120, 121, 122, 123 , 124, 125, 126, 127, 128 or 129, respectively. In some embodiments, the modified hyperactive PiggyBac is selected for its high specificity of DNA integration into a genome compared to the hyperactive PiggyBac. The present disclosure also relates to the modified hyperactive PiggyBac transposases provided herein for use as drugs, in particular in gene therapy, ex vivo or in vivo. V. Cas9 and zinc finger gene editing Current genome engineering tools, including engineered zinc finger proteins (ZFPs), transcription activator-like effector nucleases (TALENs), and, more recently, the RNA-guided DNA endonuclease Cas9 effect cleavage. sequence-specific DNA in a genome. This programmatic cleavage can result in mutation of the DNA at the cleavage site through non-homologous end joining (NHEJ) or replacement of the DNA surrounding the cleavage site through homology-directed repair (HDR). Certain aspects of the disclosure are directed to nucleic acid constructs comprising polynucleotides that encode a DNA-binding protein that is designed in such a way as to bind to a specific genomic DNA sequence, for example, Cas9 and ZFP. . In some embodiments, such DNA binding proteins are fused to the modified integrase or modified transposase disclosed herein for gene editing. Yo. Cas9 The CRISPR-Cas9 system is a highly effective tool for gene inactivation or modification through sequence-specific double-strand breaks (DSBs). These DSBs are recognized by the cellular DNA damage response machinery and can be repaired by endogenous DSB repair pathways. The predominant repair pathway is non-homologous end joining (NHEJ), which often results in JCICI n / l 7Π7 / Β / Υ small insertions and / or deletions that can create frameshift mutations and disrupt gene function. This pathway can be exploited to generate genetic deletion mutations. Alternatively, in the presence of repair templates, the damage can be seamlessly repaired by homology-directed repair (HDR). However, despite remarkable progress, HDR-mediated genome editing to introduce precise genetic modifications is much less efficient than NHEJ-mediated genetic disruption. Furthermore, large multi-kb replacements via HDR pathways are challenging and require selection and / or sorting of large cells in the population. Consistent with the above, the main applications for HDR pathways are the local replacement of key regions within genes. The terms Cas9 and Cas9 nuclease refer 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 or the gRNA-binding domain of Cas9). A Cas9 nuclease is also sometimes known as a casnl nuclease or as 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, complementary sequences for the background mobile elements, and invading target nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, correct processing of the pre-crRNA requires a small transcoded RNA (tracrRNA), an endogenous ribonuclease 3 (rnc), and a Cas9 protein. TracrRNA serves as a guide for ribonuclease 3-assisted processing of pre-crRNA. Subsequently, Cas9 / crRNA / tracrRNA endonucleolytically cleaves the linear or circular dsDNA target complementary to the spacer. The non-complementary target strand for the cRNA is first cleaved in an endonucleolytic manner, and then trimmed 3'-5' in an exonucleolytic manner. In nature, DNA binding and cleavage usually requires proteins and both RNAs. However, single guide RNAs (sgRNAs or simply gRNAs) can be designed in such a way as to incorporate aspects of both crRNA and tracrRNA into a single RNA species. Cas9 recognizes a short motif in CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self from non-self. The sequences and structures of the Cas9 nuclease are well known to those skilled in the art. Cas9 orthologs have been described in several species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those skilled in the art based on this disclosure, and such Cas9 nucleases and sequences include the Cas9 sequences from the organisms and loci disclosed in Chylinski, et al., The tracrRNA and Cas9 families of type II CRISPR-Cas immunity Systems (2013) RNA Biology 10:5, 726737; the full contents of which are incorporated herein by reference. In some embodiments, a Cas9 nuclease has an inactive DNA cleavage domain (e.g. 7CICI n / l 7Π7 / Ε / Υ example, inactivated). A nuclease-inactivated Cas9 protein can be interchangeably referred to as a dCas9 protein (for nuclease-dead Cas9). Methods for generating a Cas9 protein (or a fragment thereof) having an inactive DNA cleavage domain are known (see, for example, Jinek et al., Science. 337: 816-821 (2012). ; Q¡ et al., Repurposing CRISPR as an RNAGuided Platform for Sequence-Specific Control of Gene Expression (2013) Cell. 28; 152 (5): 1173-83, the entire content of each of which is incorporated herein document for 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 complementary strand for the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the D10A and H841A mutations completely inactivate the nuclease activity of S. pyogenes Cas9. The Cas9 nicase is a variant of the Cas9 nuclease that is differentiated by a point mutation (D10A) in the RuvC nuclease domain, allowing it to cut, but not cleave, DNA. The term Cas9 also includes variants and functional fragments of it. In some embodiments, proteins comprising Cas9 fragments are provided. For example, in some embodiments, a protein comprises one of the two domains of Cas9: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, the protein comprising Cas9 or fragments thereof is known as a Cas9 variant. A variant of Cas9 shares homology with Cas9, or with a fragment of it. For example, a Cas9 variant may be at least about 70 percent identical, at least about 80 percent identical, at least about 90 percent identical, at least about 95 percent identical, for at least about 96 percent identical, at least about 97 percent identical, at least about 98 percent identical, at least about 99 percent identical, at least about 99.5 percent identical, or at least approximately 99.9 percent identical 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 percent identical, at least about 80 percent identical, at least about 90 percent identical, at least about 95 percent identical, at least about 96 percent identical, at least about 97 percent identical, at least about 98 percent identical, at least about 99 percent identical, at least about 99.5 percent identical, or at least about 99.9 percent to the corresponding fragment of wild-type Cas9. In some embodiments, Cas9 refers to Cas9 of: Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1) (SEQ ID NO: 19); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1) (SEQ ID NO: 20); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1) (SEQ ID NO: 21); Prevotella intermedia (NCBI Ref: NC_017861.1) (SEQ ID NO: 22); Spiroplasma taiwanense (NCBI Ref: NC_021846.1) (SEQ ID NO: 23); Streptococcus iniae (NCBI Ref: NC_021314.1) (SEQ ID NO: 24); Belliella 7CICI Π / I 7Π7 / Β / Υ Baltic (NCBI Ref: NC_018010.1) (SEQ ID NO: 25); Psychroflexus torquisi (NCBI Ref: NC_018721.1) (SEQ ID NO: 26); Streptococcus thermophilus (NCBI Ref: YP_820832.1) (SEQ ID NO: 27); Listeria innocua (NCBI Ref: NP_472073.1) (SEQ ID NO: 28); Campylobacter jejuni (NCBI Ref: YP_002344900.1) (SEQ ID NO: 29); or Neisseria. meningitidis (NCBI Ref: YP_002342100.1) (SEQ ID NO: 30). In some embodiments, wild type Cas9 corresponds to Streptococcus pyogenes Cas9 (NCBI Reference Sequence: NC_017053.1) (SEQ ID NO: 31). Among the known Cas9 proteins, Cas9 from S. pyogenes has been widely used as a tool for genome design. This Cas9 protein is a large, multi-domain protein that contains two distinct nuclease domains. Point mutations can be introduced into Cas9 to abolish nuclease activity, resulting in a dead Cas9 (dCas9) that still retains its ability to bind to DNA in a manner programmed by sgRNA. In principle, when fused to another protein or domain, dCas9 can target that protein to virtually any DNA sequence simply by co-expression with an appropriate sgRNA. The present disclosure provides nucleic acid constructs comprising polynucleotides encoding Cas9 proteins for insertion of exogenous nucleic acid into a specific site of a genome. Some aspects of this disclosure provide fusion proteins comprising a Cas9 protein and a modified integrase or a modified transposase of the invention. Some embodiments of this disclosure provide nucleic acids encoding such proteins or Cas9 fusion proteins. Some embodiments provide a plasmid or expression vector comprising said nucleic acids. The Cas9 encoded by the nucleic acid construct disclosed herein may be any Cas9 that can bind to a specific genomic DNA sequence in a genome. Non-limiting examples of Cas9 proteins include human Cas9 (hCas9), nicase Cas9 (nCas9), dead Cas9 (dCas9), Streptococcus pyogenes Cas9, Staphylococcus aureus Cas9, Casi 2a, Casi 2b and dead Cas9 (dCas9), and variants and functional fragments thereof. In some embodiments, Cas9 is a human Cas9 or a variant or functional fragment thereof. In some embodiments, hCas9 is encoded by a nucleic acid sequence that is at least about 70 percent, at least about 75 percent, at least about 80 percent, at least about 85 percent percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent, or about 100 percent sequence identity with SEQ ID NO: 64. In some embodiments, nCas9 is encoded by a nucleic acid sequence that has at least about 70 percent, for at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least 7CICI n / l 7Π7 / Β / Υ about 97 percent, at least about 98 percent, at least about 99 percent, or about 100 percent sequence identity with SEQ ID NO: 65 In some embodiments, dCas9 is encoded by a nucleic acid sequence that is at least about 70 percent, at least about 75 percent, at least about 80 percent, at least about 85 percent. percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, minus about 99 percent, or about 100 percent sequence identity with SEQ ID NO: 66. In some embodiments, hCas9 comprises an amino acid sequence that is at least about 70 percent, at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent, or about 100 percent sequence identity with SEQ ID NO: 69. In some embodiments, nCas9 comprises an amino acid sequence that has at least about 70 percent, at least about 75 percent percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent, or about 100 percent sequence identity with SEQ ID NO: 70. In some embodiments, dCas9 comprises a amino acid sequence having at least about 70 percent, at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent, or about 100 percent percent sequence identity with SEQ ID NO: 71. Certain aspects of the disclosure are directed to a vector or a plasmid (e.g., an expression vector or a packaging vector) comprising a nucleic acid construct comprising a Cas9 suitable for expression in a host cell, e.g. , mammalian cells, yeast cells, insect cells, plant cells, fungal cells or algal cells. In some embodiments, the nucleic acid construct comprises a polynucleotide sequence encoding a Cas9 that is expressed as a fusion protein with a modified transposase of the disclosure. ii. Zinc finger proteins The present disclosure also provides nucleic acid constructs that 7CICI n / l 7Π7 / Β / Υ comprise polynucleotides that encode a zinc finger protein (ZFP) for the insertion of exogenous nucleic acid at a specific site in a genome. Some aspects of this disclosure provide fusion proteins comprising a ZFP and a modified integrase or a modified transposase of the invention. Some embodiments of this disclosure provide nucleic acids encoding such ZFPs or fusion proteins. Some embodiments of this disclosure provide plasmids or expression vectors comprising said coding nucleic acids. The zinc finger proteins used herein are proteins that can bind to DNA in a sequence-specific manner. ZFPs are distributed unevenly in eukaryotes. ZFPs have been identified that are involved in DNA recognition, RNA binding, and protein binding. Certain classifications for zinc finger proteins are based on fold groups in view of the general shape of the basic structure of the proteins in the folded domain. The most common zinc finger fold groups are the C2H2 or CyszHisz-like (the classic zinc finger), the treble clef, and the zinc ribbon. The representative motif that characterizes one class of these proteins (C2H2 class) is, -Cys- (X) 2-4 -Cys- (X) 12 -His- (X) 3-5 His (where in X is a is any amino acid). The ZFP of the invention may be any ZFP, variant or functional fragment thereof, that can be linked to a specific genomic DNA sequence in a genome. Non-limiting examples of the ZFP include ZFPs comprising a group of folds or a zinc finger motif selected from C2H2, gag knuckle, treble clef, zinc ribbon, Znz / Cyse-like or similar. to a TAZ2 domain, or any combination thereof. In some embodiments, ZFP is a C2H2 zinc finger protein. In some embodiments, the ZFP is a designed ZFP. The designed zinc finger arrays can be fused to a DNA cleavage domain (usually the Fokl cleavage domain) for the generation of zinc finger nucleases. Such zinc finger and Fokl fusions have become useful reagents for manipulating genomes. The ZFP of the disclosure may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more zinc finger domains. The ZFP may comprise 2 to 12, 2 to 10, 2 to 8, 3 to 8, 4 to 8, or 5 to 8 zinc finger domains. In some embodiments, the ZFP comprises 6 zinc finger domains. A common modular assembly process involves combining separate zinc fingers where each can recognize a 3-base DNA sequence for the generation of 3-finger, 4-, 5-, or 6-finger arrays that recognize target sites that They range from 9 base pairs to 18 base pairs in length. Another method uses 2-finger modules for the generation of zinc finger arrays with up to six individual zinc fingers. In some embodiments, the binding domain of the ZFP can be designed in such a way as to bind to a sequence of choice. An engineered zinc finger binding domain may have improved binding specificity, compared to a naturally occurring ZFP. In some embodiments, the nucleic acid sequence encoding the ZFP corresponds to SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36 or SEQ ID NO: 38. In some embodiments, the amino acid sequence of the 7CICI n / l 7Π7 / Β / Υ ZFP corresponds to SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37 or SEQ ID NO: 39. In some embodiments, the ZFP comprises an amino acid sequence that has at least about 70 percent, at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent, or about 100 percent sequence identity to any of SEQ ID NO: 33 , 35, 37 or 39. Certain aspects of the disclosure are directed to a vector or a plasmid (e.g., an expression vector or a packaging vector) comprising a nucleic acid construct comprising a ZFP suitable for expression in a host cell, e.g. , mammalian cells, yeast cells, insect cells, plant cells, fungal cells or algal cells. In some embodiments, the nucleic acid construct comprises a polynucleotide sequence encoding a ZFP that is expressed as a fusion protein with a modified integrase or a modified transposase of the disclosure. Vile. fusion protein The present disclosure provides fusion proteins for site-specific insertion of exogenous nucleic acids into a genome. In certain embodiments, the fusion protein comprises a first DNA binding protein that is designed in such a way as to bind to a specific genomic DNA sequence, a second DNA binding protein that allows the insertion to be made of an exogenous nucleic acid in the genome wherein the second DNA binding protein is an integrase or a transposase of this disclosure, and a linker that connects the first and second proteins. In some embodiments, the first DNA binding protein is a Cas9 protein or a zinc finger protein. In some embodiments, the first DNA binding protein is a Cas9 and the second binding protein is a modified transposase disclosed herein, wherein the first and second binding proteins can be oriented in the construction in any order. In some embodiments, the first DNA binding protein is a zinc finger protein and the second binding protein is a modified integrase, wherein the first and second binding proteins can be oriented in the construct in any order. In some embodiments, the fusion protein comprises a linker between the first binding protein and the second binding protein, wherein the linker comprises a (GGS)n motif, a (GGGGS)n motif (SEQ ID NO: 133) , a (G)n motif, an (EAAAK)n motif (SEQ ID NO: 134), an XTEN-based motif or an (XP)n motif, or a combination of any of the same, where n is independently an integer between 1 and 50. In some embodiments, the linker is 12 to 24 amino acids, or is encoded by a nucleic acid sequence having a length of 36 to 72 nucleic acids. In some embodiments, the linker comprises an XTEN sequence or a GGS sequence. In some embodiments, the fusion protein comprises a zinc finger protein linked to a modified integrase of the disclosure, wherein the linker comprises a GGS sequence or 7CICI n / l 7Π7 / Β / Υ an XTEN sequence, and in which the modified integrase can be 5' or 3' to the linker. In some embodiments, the fusion protein comprises a Cas9 protein linked to a modified transposase of the invention, wherein the linker comprises a GGS sequence or an XTEN sequence, and wherein the modified transposase may be 5' or 3' for the linker. In some embodiments, the linker is a linker shown in Table 1. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 49. In some embodiments, the linker comprises an amino acid sequence that is selected from the group consisting of SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, and SEQ ID NO: 63, or any combination thereof. In some embodiments, the linker is encoded by a nucleic acid sequence comprising SEQ ID NO: 48. In some embodiments, the linker is encoded by a nucleic acid sequence comprising a sequence that is selected from the group consisting in SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, or any combination thereof. 7CICI n / l 7Π7 / Ε / Υ Table 1: Linkers Linker Nucleic acid sequence (SEQ ID NO) Amino acid sequence (SEQ ID NO) GGSx3 ggtggatctggcggtggatctggtggcggt (SEQ ID NO: 48) GGSGGGSGGG (SEQ ID NO: 49) GGS4x ggagggagtggtgggtccggtggtagtggcggatcc (SEQ ID NO: 50) GGSGGSGGSGGS ( SEQ ID NO: 51) GGS5x ggaggctccggtgggtctggtgggagcggtggtagtggcggatcc (SEQ ID NO: 52) GGSGGSGGSGGSGGS (SEQ ID NO: 53) GGS6x ggaggcagtggtgggagcggtggttccgggggtagtggtggttcc gggggatcc (SEQ ID NO: 54) GGSGGSGGSGGSGG SGGS (SEQ ID NO: 55) GGS7x ggaggttctggaggctccggtgggtccgggggaagtggggggtc aggcggatcaggaggatcc (SEQ ID NO: 56) GGSGGSGGSGGSGGSGGSGG S ( SEQ ID NO: 57) GGS8x ggaggtagcggaggttccggagggagcggcgggagtggggga agcgggggaagtggaggatccgggggaggatcc (SEQ ID NO: 58) GGSGGSGGSGGSGGSGGSGG S (SEQ ID NO: 59) Linker Nucleic acid sequence (SEQ ID NO) Amino acid sequence (SEQ ID NO) : 62) GSAGSAAGSGEF (SEQ ID NO: 63) 7CICI n / l 7Π7 / Β / Υ In some embodiments, the 3' end of the first DNA binding protein is connected to the 5' end of the second DNA binding protein via a linker. In some embodiments, the 3' end of the second DNA binding protein is connected to the 5' end of the first DNA binding protein via a linker. In some embodiments, the 3' end of the Cas9 protein is connected to the 5' end of the transposase via a linker. In some embodiments, the 5' end of the Cas9 protein is connected to the 3' end of the transposase via a linker. In some embodiments, the 3' zinc finger protein is connected to the 5' end of the integrase via a linker. In some embodiments, the 5' zinc finger protein is connected to the 3' end of the integrase via a linker. Also provided herein are fusion proteins that are obtained from the expression of any of the nucleic acid constructs provided in this disclosure. VIII. Cells / host organism In some embodiments, the nucleic acid construct of the disclosure is expressed in a host cell. Suitable host cells include, but are not limited to, eukaryotic and prokaryotic cells and / or cell lines. Non-limiting examples of such host cells or cell lines generated from such cells include COS, CHO (e.g., CHO-S, CHOK1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NSO, SP2 / 0-Ag14, HeLa, HEK293 (e.g. HEK293-F, HEK293-H, HEK293-T) and perC6, as well as insect cells such as Spodoptera fugiperda (Sf), or fungal cells such as Saccharomyces, Pichia and Schizosaccharomyces. In some embodiments, the host cell is from a microorganism. Microorganisms that are useful for certain methods disclosed herein include, for example, bacteria (e.g., E coli), yeasts (e.g., Saccharomyces cerevisiae), and plants. The host cell can be prokaryotic or eukaryotic. In some embodiments, the host cell is eukaryotic. Suitable eukaryotic host cells include, but are not limited to, yeast cells, insect cells, plant cells, fungal cells and algal cells. In some embodiments, the host cell is a competent host cell. In some embodiments, the host cell is naturally competent. In some embodiments, host cells are made competent, for example, through a process using calcium chloride and heat shock. The cells used may be competent for any cell, in particular eukaryotic cells, in particular those of mammals, for example, human or animal. They can be somatic or embryonic or differentiated stem cells. In some aspects, the cells include 293T cells, fibroblast cells, hepatocytes, muscle cells (skeletal, cardiac, smooth, blood vessel, etc.), nerve cells (neurons, glial cells, astrocytes), epithelial cells, kidney cells, ocular cells, etc It may also include insect cells, plant cells, yeast or prokaryotic cells. Additionally, primary cells can be isolated and used ex vivo for reintroduction into the subject to be treated after treatment with nucleases (e.g., ZFN or TALEN) or nuclease systems (e.g., CRISPR / Cas). . Suitable primary cells include peripheral blood mononuclear cells (PBMCs) and other blood cell subsets such as, but not limited to, T lymphocytes such as CD4+ T cells or CD8+ T cells. Suitable cells also include stem cells such as, but are not limited to, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells (CD34+), neural stem cells, and mesenchymal stem cells. In some embodiments, the host cell is transfected with a plasmid comprising a nucleic acid construct disclosed herein. In some embodiments, the plasmid comprising the nucleic acid construct is a packaging plasmid. In some embodiments, the plasmid comprising the nucleic acid construct further comprises a polynucleotide encoding capsid proteins, for example, gag and pol. In some embodiments, the host cell is transfected with (i) the plasmid comprising the nucleic acid construct that is combined in the host cell with (i) a plasmid comprising a polynucleotide that encodes proteins for a viral envelope (plasmid wrapper); and (iii) a plasmid comprising an exogenous nucleic acid sequence (e.g., a GOI), wherein a virus particle is produced comprising the exogenous nucleic acid, e.g., the GOI, and the fusion protein comprising the first and second binding proteins. In some embodiments, the host cell is transfected with (i) the plasmid comprising the nucleic acid construct is combined with (i) a plasmid comprising the nucleic acid construct further comprising a polynucleotide encoding capsid proteins. , for example, gag and pol (a packaging plasmid, where the packaging plasmid lacks a functional integrase); (iii) a plasmid comprising a polynucleotide that encodes proteins for a viral envelope (envelope plasmid), and (iv) a plasmid comprising an exogenous nucleic acid sequence (for example, a GOI), wherein a virus particle comprising the exogenous nucleic acid, for example, GOI, and the fusion protein comprising the first and second binding proteins. In other embodiments, a vector, for example, a lentiviral vector according to the disclosure, can be used to deliver a fusion protein encoded by a nucleic acid construct of the disclosure and an exogenous nucleic acid to an organism, for example, to a mammal, and in a way 7CICI n / l 7Π7 / Β / Υ more particular to a mammalian target cell of interest. Lentiviral vectors comprising the fusion proteins of the disclosure are capable of transducing various types of cells, such as, for example, liver cells (e.g., hepatocytes), muscle cells, brain cells, kidney cells, retinal cells and hematopoietic cells. In some embodiments, the target cells of the present disclosure are non-dividing cells. These cells include cells such as neuronal cells that do not normally divide. However, the present disclosure is not intended to be limited to non-dividing cells (including, but not limited to, muscle cells, white blood cells, spleen cells, liver cells, eyes, epithelial cells, etc.). In certain embodiments, a packaged fusion protein of the disclosure is administered to an organism, for example, for gene editing of the organism's DNA. In some embodiments, the organism is a human being. In some embodiments, the organism is a non-human mammal. In some embodiments, the organism is a non-human primate. In some embodiments, the organism is a rodent. In some embodiments, the organism is a sheep, goat, cattle, cat, or dog. In some embodiments, the organism is a vertebrate, amphibian, reptile, fish, insect, fly, or nematode. In some embodiments, the organism is a research animal. In some embodiments, the organism is genetically modified, for example, a genetically modified non-human subject. The organism can be of either sex and can be at any stage of development. IX. Method for insertion into the genome Methods for inserting exogenous nucleic acids into a genome have been described. See, for example, Yusa et al. PNAS 4 (108): 1531-1536 (2011); Feng et al. Nuc. Acid Res. 4 (38): 1204-1216 (2009); Kettlun et al. Amer. Soc. Gene and Cell Ther. 9 (19): 1636-1644 (2011); Skipper et al. 20 (92): 1-23 (2013); Lí etal. PNAS 25: E2279-E2287 (2013); Mátés et al. Nature Genetics 41 (6): 753-761 (2009); Mali etal. Nat Methods 10 (10): 957-963; Vargas et al. J. Trans. Med. 14 (288): 1-15 (2016); Gersbach et al. Acc. Chem. Res. 47: 2309-2318 (2014); Chandrasegaran et al. Ther cellular gene. Ins. 3 (1): 33-41 (2017); Wilson et al. 649: 353-363 (2010); Zhao Zhang, et al. Mol Ther Nucleic Acids. 9: 230-241 (2017); Naldini L. EMBO Mol Med. 11 (3) (2019); and Naldini L, et al. Hum Gene Ther. 27 (10): 727-728 (2016), each of which is incorporated herein by reference. The present disclosure provides a nucleic acid construct that encodes a fusion protein for insertion of exogenous nucleic acid into a specific site of a genome. The present invention also provides fusion proteins for insertion of exogenous nucleic acid at a specific site in the genome. In some embodiments, the exogenous nucleic acid for insertion may be up to 5 kb in length, up to 10 kb in length, up to 15 kb in length, up to 20 kb in length, up to 25 kb in length, up to 30 kb in length, up to 35 kb in length, or up to 40 kb in length. In another embodiment, methods for inserting the site-specific nucleic acid into the genome are provided. In some embodiments, the methods comprise contacting a target DNA with any of the fusion proteins that make up a Cas9 and a transposase described in the 7CICI n / l 7Π7 / Β / Υ present document. For example, in some embodiments, the method consists of contacting a DNA with a fusion protein comprising two linked polypeptides: (i) a Cas9; and (i) a transposase, where active Cas9 binds to a gRNA that hybridizes to a region of DNA, for example, to genomic DNA. In some embodiments, the methods comprise contacting a target DNA with any of the fusion proteins that make up a Cas9 and an integrase described herein. For example, in some embodiments, the method consists of contacting a DNA with a fusion protein comprising two linked polypeptides: (i) a Cas9; and (i) an integrase, where active Cas9 binds to a gRNA that hybridizes to a region of DNA, for example, to genomic DNA. In some embodiments, the methods comprise contacting a target DNA with any of the fusion proteins that make up a ZFP and an integrase described herein. For example, in some embodiments, the method consists of contacting a DNA with a fusion protein comprising two linked polypeptides: (i) ZFP; and (i) an integrase, where the active ZFP hybridizes to a region of DNA, for example, to genomic DNA. In some embodiments, the fusion protein is delivered to an organism and / or a cell comprising target DNA, e.g., genomic DNA, through the use of a viral vector, e.g., a lentiviral particle. X. Lentiviral packaging Methods for lentiviral packaging have been described. See, Grandchamp et al. 9 (6): 1-13 (2014); Voelkel et al. 107 (17): 7805-7810 (2010); Tan et al. 80 (4) 1939-1948; Lí et al. 9 (8): 1-9 (2014); Mátés et al. Nature Genetics 41 (6): 753-761 (2009); and Robert H Kutnerl, et al. NATURE PROTOCOLS 4 (4): 495 (2009), each of which is incorporated herein by reference. Typically, lentiviral delivery systems use a split system with different lentiviral genes on separate plasmids that are used to produce a complete virus that does not contain the genetic components necessary to cause viral disease. For example, a plasmid (an envelope plasmid) may encode proteins for the viral envelope (env); another plasmid (a packaging plasmid) may encode capsid proteins (for example, gag and pol) and enzymes such as reverse transcriptase and / or integrase; and an additional plasmid comprising the gene of interest (GOI) flanked by the long terminal repeats (for genome integration) and a psi sequence (showing a signal to package the gene into the virus) ( a transfer plasmid). If these plasmids are introduced simultaneously into a cell, viruses containing GOI will be produced without the viral genes needed to cause the disease. In certain aspects of the disclosure, the lentiviral vector (or particle) of the invention can be obtained by means of a split system, for example, a transcomplementation system (vector or packaging system), by means of in vitro transfection. of a permissive cell (such as 293T cells) with a plasmid containing certain components of the lentiviral vector genome, and at least one other plasmid providing, in trans, the gag, pol and env sequences encoding the polypeptides 7CICI n / l 7Π7 / Β / Υ of GAG, ROL and envelope proteins, or for a portion of these polypeptides sufficient to allow the formation of retroviral particles. As an example, host cells are transfected with: a) a packaging plasmid, comprising a gag and pol lentiviral sequence, b) a second plasmid (an envelope expression plasmid or a pseudotyping env plasmid) comprising a gene encoding an envelope protein (such as VSV-G), c) a plasmid vector comprising, between the 5' and 3' LTR sequences, a psi packaging sequence and a transgene, and d) a plasmid comprising a nucleic acid construct encoding a modified fusion protein disclosed herein. In some embodiments, the nucleic acid construct encoding the modified fusion protein disclosed herein is on the packaging plasmid rather than on a separate plasmid. The nucleic acids encoding the gag, pol and env cDNA can be conveniently prepared according to conventional techniques, from the viral gene sequences available in the state of the art and from databases. In some embodiments, a lentiviral vector comprises a nucleic acid construct as described herein. In some embodiments, a lentiviral vector comprises a fusion protein as described herein. The promoters used in the plasmids can be identical or different. In some embodiments, in the plasmid transcomplementation system, the envelope plasmid and the plasmid vector, respectively, to promote the expression of gag and pol of the coat protein, the mRNA of the vector genome and the transgene are promoters that can be identical or different. Such promoters can be conveniently chosen from ubiquitous or specific promoters, for example, from the CMV viral promoters, TK, the RSV LTR promoter and the RNA polymerase III promoter such as U6 or H1, or the promoters of helper viruses encoding env, gag, and pol (i.e., adenoviral, baculoviral, or herpes viruses). For production of the lentiviral vector of the invention, the plasmids described herein can be introduced into host cells, and viruses are produced and harvested. Suitable cells include, but are not limited to, eukaryotic and prokaryotic cells and / or cell lines. Non-limiting examples of such cells or cell lines that are generated from such cells include, for example, COS, CHO cells (e.g., CHO-S, CHO-K1, CHO-DG44, CHODUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NSO, SP2 / 0Ag14, HeLa, HEK293 (e.g. HEK293-F, HEK293-H, HEK293-T), and perC6, as well as insect cells such as Spodoptera fugiperda (Sf), or fungal cells such as Saccharomyces, Pichia and Schizosaccharomyces. Once the host cells are transfected with the plasmids and a lentiviral vector (or particle) of the invention is produced, the lentiviral vectors (or particles) of the invention can be purified from the supernatant of the cells. Purification of the lentiviral vector to improve concentration can be achieved by any suitable method, such as by means of a density gradient purification (for example, cesium chloride (CsCl)), by means of the techniques of 7CICI n / l 7Π7 / Β / Υ chromatography (e.g. column or batch chromatography), or by ultracentrifugation. For example, the vector of the invention can be subjected to two or three CsCl density gradient purification steps. The vector is desirably purified from infected cells through the use of a method comprising Usado cells, application of Usado to a chromatography resin, elution of the virus from the chromatography resin, and collection of a fraction containing the lentiviral vector of the disclosure. XI. Delivery method Lentiviral vector delivery methods have been described. See, for example, Vargas et al. J. Trans. Med. 14 (288): 1-15 (2016); Mali etal. Nat Methods 10(10):957-963; Mátés et al. Nature Genetics 41 (6): 753-761 (2009); Skipper et al. 20 (92): 1-23 (2013). Lentiviral vectors comprising a fusion protein encoded by a nucleic acid construct of the invention can be delivered to a subject by any route. In some embodiments, a lentiviral vector of the invention can be delivered to the cells of a subject either in vivo or ex vivo. In some embodiments, the lentiviral vector of the invention can be administered in vivo. In some embodiments, a lentiviral vector comprising a fusion protein encoded by a nucleic acid construct of the invention can be used to deliver a GOI and / or to target a genetic defect in the DNA of a subject. In some embodiments, the lentiviral vector is administered to the subject parenterally, preferably intravascularly (including intravenously). When administered parenterally, it is preferred that the vectors be administered in a pharmaceutical vehicle suitable for injection, such as a sterile aqueous solution or dispersion. In some embodiments, the lentiviral vector of the invention can be used ex vivo. In some embodiments, a lentiviral vector comprising a fusion protein encoded by a nucleic acid construct of the invention can be used to deliver a GOI and / or to target a genetic defect in the DNA of a subject. In some embodiments, cells are removed from a subject and the lentiviral vector comprising a fusion protein encoded by a nucleic acid construct of the disclosure is administered to the cells ex vivo to modify the DNA of the cells. The cells carrying the modified DNA are expanded and infused back into the subject. In certain embodiments, a lentiviral vector comprising a fusion protein encoded by a nucleic acid construct of the invention can be used for chimeric antigen receptor (CAR) T cell therapy in order to genetically modify T cells. autologous from a patient to express a CAR specific for a tumor antigen. In a further embodiment, the modified CAR-T cells are expanded ex vivo and re-perfused into the patient. In some embodiments, the altered T cells are more specifically targeted to cancer cells. Unlike antibody therapies, CAR-T cells are capable of replicating in vivo, resulting in long-term persistence. Following administration of a lentiviral vector of the invention or of the cells modified ex vivo through the use of a lentiviral vector of the disclosure, the subject may be monitored for 7CICI n / l 7Π7 / Β / Υ detect transgene expression. The dose and duration of treatment are determined on an individual basis depending on the condition or disease being treated. A variety of conditions or diseases can be treated based on the genetic expression produced by the administration of the gene of interest in the vector of the present invention. The dose of the vector that is delivered through the use of the method of the invention will vary depending on the desired response on the part of the host and the vector used. In some gene therapy applications, it is desirable that the gene therapy vector be delivered with a high degree of specificity to a particular tissue type. Accordingly, a viral vector can be modified to have specificity for a given cell type that expresses a ligand such as a fusion protein with a viral coat protein on the outer surface of the virus. The ligand is chosen in such a way as to have affinity for a receptor known to be present on the cell type of interest. Certain aspects of the disclosure are directed to a method for inserting an exogenous nucleic acid sequence into the genomic DNA of an organism, which comprises: identifying the specific genomic DNA sequence in the genome of the organism; administering a lentiviral particle comprising the nucleic acid construct of the disclosure to the organism to bind to the specific genomic DNA sequence, and inserting the exogenous nucleic acid into the genomic DNA; where the exogenous nucleic acid becomes integrated into the specific genomic DNA sequence. Certain aspects of the disclosure are directed to a method for the controlled and site-specific integration of a single copy or multiple copies of an exogenous nucleic acid sequence into a cell, wherein the method comprises: a) providing the construct of nucleic acid, vector or fusion protein disclosure to the cell, and (b) delivery of the exogenous nucleic acid to the cell; wherein binding of the fusion protein to the specific genomic DNA sequence in the genome of the cell results in cleavage of the genome and integration of one or more copies of the exogenous nucleic acid into the genome of the cell. In some aspects, delivery to the cell is by means of a lentiviral particle. XII. Method of use and applications Several strategies can be used to test integration sites and to search for the best machinery for targeted integration. For the analysis of the modified integrase and transposons disclosed herein, a reporter cell line with a promoter, half of the GFP coding sequence, and a splice site donor can be used. downstream of the targeted insertion site in the genome. For example, the lentiviral payload may have a fusion integrase variant followed by the inverted splice site acceptor and the other half of the GFP. GFP expression will occur when direct insertion and splicing of GFP containing mRNA generated from the insertion site occurs and the integrated payload gives rise to the complete GFP CDS. VPR transcomplementation systems can also be used to detect and compare 7CICI Π / I 7Π7 / Β / Υ the integration mulants. The transcomplementation system can be used for targeted insertion of the lentiviral payload containing a fusion integrase variant that, when expressed and loaded into the particle promotes its own integration, will be loaded into the viral particle through use of a VPR merger. This will complement in trans the defective integration encoded in the IN in the packaging vector used for particle production. Other methods that can be used for integration mapping include IC or FISH probes. Targeted insertion can also be detected via targeted disruption of TCRa or RFP, or activation of GFP via targeted splice site integration. For the FISH approach to common staining of the insertion and target region in chromatin, fluorescence in situ hybridization can be performed to locate the GOI transposon in the Hek293T genome. Hek293T can be transfected with 1) the GOI transposon, 2) the programmed transposase, and 3) the gRNA for PPP1R12. The probes are designed to target the PPP1R12 gene, the CD46 gene (as the negative control), and GOI, and can be synthesized with Nick's translation mix (Sigma) from DNA amplified by means of a PCR. In some embodiments, a fusion protein comprising a modified transposase or a modified integrase as disclosed herein improves the specificity of insertion of the exogenous nucleic acid into the genome compared to a fusion protein containing the protein corresponding wild type, for example, as determined by means of a Genetrap assay. In some embodiments, HEK293T cells, or any other permissible cells, are transfected or transduced with the lentiviral particles with the following plasmids or payloads: (i) a plasmid comprising a gRNA that targets a specific region of DNA, (i) a plasmid comprising the nucleic acid construct of the disclosure encoding a modified transposase fusion protein or a modified integrase fusion protein, and (iii) a genetrap plasmid comprising a nucleic acid sequence encoding a reporter protein, for example, GFP, which lacks a promoter. In some embodiments, the genetrap plasmid further comprises a transposon with inverted repeats. In some embodiments, the percentage of cells containing the GFP insert can be determined by flow cytometry. In some embodiments, the programmed transposase fusion protein increases the percentage of cells containing the GFP insert by at least 5 percent, by at least 10 percent, by at least 15 percent, by at least 20 percent, by at least 25 percent or by at least 30 percent compared to the corresponding wild-type protein. In some embodiments, the programmed transposase fusion protein increases the percentage of cells containing the GFP insert by about 15 to 30 percent. In some embodiments, the percentage of insertions at the targeted site and the percentage of coderture at the blank site (number of reads per insertion site) can be determined by extraction of genomic DNA and targeted sequencing with oligonucleotides specific for the targets. Viral LTRs. In some embodiments, the modified transposase fusion protein increases the percentage 7CICI Π / I 7Π7 / Β / Υ of targeted site insertions at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold or at least 100-fold compared to the corresponding wild-type protein. In some embodiments, the percentage of insertions at the targeted site increases by about 10 to 100 times. In some embodiments, the modified transposase fusion protein increases the percentage coverage at the target site (number of reads per insertion site) by at least 10-fold, by at least 20-fold, by at least 30-fold, in at least 40 times, in at least 50 times, in at least 60 times, in at least 70 times, in at least 80 times, in at least 90 times, in at least 100 times, in at least 110 times, at least 120 times, at least 130 times, at least 140 times, at least 150 times, at least 160 times, at least 170 times, at least at least 180-fold, at least 190-fold or at least 200-fold compared to the corresponding wild-type protein. In some embodiments, the percentage coverage at the target site (number of reads per insertion site) is at least 100-fold. In some embodiments, the modified integrase fusion protein improves the specificity of insertion of the exogenous nucleic acid into the genome compared to the corresponding wild-type protein as quantified by GFP integration. In some embodiments, the lentivirus containing the modified integrase fusion protein was generated by transfection of HEK293T cells, or any other permissible cell, with (i) a plasmid containing a nucleic acid sequence encoding the GFP, (i) a plasmid containing the packaging proteins, (iii) a plasmid containing an envelope protein, and (iv) a plasmid containing the nucleic acid construct encoding the modified integrase fusion protein . The supernatant containing the lentivirus was collected at 48 hours after transfection. For targeted insertion, HEK293T cells were infected with lentivirus containing the modified integrase fusion protein. In some embodiments, the percentage of GFP-positive cells was quantified by flow cytometry at 3, 5, 7, 10, and 12 days postinfection. In some embodiments, the modified integrase fusion protein increases the percentage of cells containing the GFP insert by at least 5 percent, by at least 10 percent, by at least 15 percent, by at least 20 percent, by at least 25 percent, or by at least 30 percent compared to the corresponding wild-type protein. In some embodiments, the percentage of insertions at the targeted site and the percentage of coverage at the target site (number of reads per insertion site) can be determined by extraction of genomic DNA and targeted sequencing with specific oligonucleotides. the viral inserted LTR. In some embodiments, the modified integrase fusion protein increases the percentage of insertions at the targeted site by at least 10-fold, by at least 20-fold, by at least 30-fold, by at least 40-fold, by at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold or at least 100-fold compared to the corresponding wild-type protein. In some embodiments, the protein 7CICI n / l 7Π7 / Β / Υ modified integrase fusion increases percentage coverage at the target site (number of reads per insertion site) by at least 10-fold, by at least 20-fold, by at least 30-fold times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times , at least 110 times, at least 120 times, at least 130 times, at least 140 times, at least 150 times, at least 160 times, at least 170 times, by at least 180-fold, by at least 190-fold or by at least 200-fold compared to the corresponding wild-type protein. Possible applications of lentiviral vectors comprising the fusion proteins of the disclosure include gene therapy, that is, gene transfer into any mammalian cell, in particular human cells. These can be dividing cells or inactive cells, and cells belonging to central organs or peripheral organs such as the liver, pancreas, muscle, heart, etc. Gene therapy can allow the expression of proteins, for example, neurotrophic factors, enzymes, transcription factors, receptors, etc. Lentiviral vectors according to the invention may also be particularly suitable for research purposes. In some embodiments, a nucleic acid construct, a fusion protein and / or a lentiviral vector of the disclosure is administered to a subject for the treatment of a disease. In some embodiments, the disease is a genetic disorder that may benefit from gene therapy. In some embodiments, lentiviral vectors comprising the fusion proteins according to the disclosure can be used as a medicament. The lentiviral vector according to the disclosure may be in particular suitable for the treatment of a genetic disease in a subject. XIII. Compositions and kits The present disclosure also provides compositions for practicing the methods disclosed as described herein. In some embodiments, a composition comprises a nucleic acid construct or a vector as defined in this disclosure, and a polynucleotide sequence encoding an exogenous nucleic acid for insertion into a genome, contained in, or linked to, a vector. packing. In some embodiments, the nucleic acid construct is in a form of RNA, DNA or protein, and the polynucleotide sequence encoding the exogenous nucleic acid is in a form of RNA or DNA, depending on the method of administration. In particular, the polynucleotide sequence encoding the exogenous nucleic acid is in an RNA form. In some embodiments, the composition is virus-free, and the packaging vector is a nanoparticle, for example, a polymeric or lipid nanoparticle. The packaging vector can also be a carrier that is linked to the elements of the composition. In some embodiments, the composition is contained in a viral vector, in particular a lentiviral particle. In some embodiments, the composition comprises (a) the nucleic acid construct described herein (e.g., comprising Cas9 and a transposase) in the form of RNA, (b) a guide RNA if necessary (for example, such as a linear RNA molecule from a JCICI n / l 7Π7 / Β / Υ single strand separated), and (c) a polynucleotide comprising the exogenous gene for insertion into the form of DNA (e.g., into a vector), contained in, or linked to, a packaging vector. In some embodiments, the composition comprises (a) the fusion protein described herein (for example, comprising Cas9 and a transposase) in the form of a protein, (b) a guide RNA if necessary (e.g., such as a separate single-stranded linear RNA molecule), wherein the fusion protein and the guide RNA form a ribonucleic protein (RNP) complex, and (c) a polynucleotide comprising the exogenous gene for insertion into the form of DNA (for example, in a vector) contained in or linked to a packaging vector. In some embodiments, the composition comprises (a) the nucleic acid construct described herein (e.g., comprising Cas9 and a transposase) in the form of DNA, (b) a guide RNA if necessary (e.g., such as a separate linear RNA molecule or as DNA in a vector), and (c) a polynucleotide comprising the exogenous gene for insertion into the form of DNA (e.g., in a vector) contained in a packaging vector or linked to it. In some embodiments, the composition comprises (a) the fusion protein described herein (e.g., comprising Cas9 and an integrase) in a protein form, (b) a guide RNA if necessary ( for example, such as a separate RNA molecule that forms a complex with the fusion protein), and (c) a polynucleotide comprising the exogenous gene for insertion, contained in, or linked to, a packaging vector. In a particular embodiment, the packaging vector is a lentiviral particle. In some embodiments, (a) the fusion protein is linked to the lentiviral capsid via gag-pol or VPR (viral protein R). In some embodiments, (c) the polynucleotide is in an RNA form as a payload of the integrase. In a particular embodiment, when the ZFP is used, (b) the guide RNA may not be needed. Also provided in the present disclosure are kits for practicing the methods disclosed, as described herein. The kit may contain the nucleic acid constructs or fusion proteins as described herein. In some aspects, the kit may contain lentiviral particles containing nucleic acid constructs or fusion proteins as described herein. This kit may also include instructions for using the components of the kit to practice the present methods. Instructions for practicing these methods are usually recorded on a suitable recording medium. For example, instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kit as a package insert, in the labeling of the container of the kit or components of the kit (i.e., associated with the packaging or subpackage), etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, for example, CD-ROM, floppy disk, etc. In other embodiments, the actual instructions are not present in the kit, but means are provided to obtain the instructions from a remote source, for example, over the Internet. An example of this modality is a kit that includes a 7CICI n / l 7Π7 / Β / Υ web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means of obtaining the instructions is recorded on a suitable substrate. XIV. Modalities E1. A nucleic acid construct comprising: a) a first polynucleotide sequence encoding a first DNA binding protein that is designed in such a way as to bind to a specific genomic DNA sequence in a genome; b) a second polynucleotide sequence that encodes a second DNA binding protein that allows the insertion of an exogenous nucleic acid into the genome, wherein the second DNA binding protein is: (i) an integrase that is modifies relative to a wild-type integrase, or (i) a transposase that is modified relative to a wild-type transposase; and c) a third polynucleotide sequence comprising a nucleic acid encoding a linker; wherein the nucleic acid construct encodes a fusion protein comprising the first DNA binding protein, the second DNA binding protein, and the linker between the first DNA binding protein and the second DNA binding protein. E2. The nucleic acid construction according to embodiment E1, wherein the second DNA binding protein is modified to improve the specificity of the insertion of the exogenous nucleic acid into the genome compared to the corresponding wild type protein. E3. The nucleic acid construction according to the Ε1 or E2 embodiment, wherein the exogenous nucleic acid for insertion can be up to about 20 kb in length. E4. The nucleic acid construction according to either embodiment Ε1 or E3, wherein the first polynucleotide sequence encodes a protein that is selected from the group consisting of a zinc finger protein, a Cas9 protein, and any variant or functional fragment thereof. E5. The nucleic acid construction according to the E4 embodiment, wherein the Cas9 protein is selected from the group consisting of a human Cas9, a Cas9 nicase, a Streptococcus pyogenes Cas9, a Staphylococcus aureus Cas9, a Casi2a, a Casi2b, and a dead Cas9. E6. The nucleic acid construction according to embodiment E4, wherein the zinc finger protein is a C2H2 zinc finger protein. E7. The nucleic acid construction according to any of embodiments E1 to E6, wherein the modified integrase is a modified human immunodeficiency virus (HIV) integrase or a functional fragment thereof. E8. The nucleic acid construction according to embodiment E7, wherein the modified HIV integrase comprises a mutation of one or more of the amino acids 10, 13, 64, 94, 116, 117, 119, 120, 122, 124, 128 , 152, 168, 170, 185, 231,264, 266 or 273 that correspond to the number of amino acids 7CICI Π / I 7Π7 / Β / Υ from the wild-type HIV integrase sequence (SEQ ID NO: 1). E9. The nucleic acid construction according to embodiment E8, wherein the modified HIV integrase mutation comprises one or more of D10K, Ε13K, D64A, D64E, G94D, G94E, G94R, G94K, D116A, D116E, N117D, N117E , N117R, N117K, S119A, S119P, S119T, S119G, S119D, S119E, S119R, S119K, N120D, N120E, N120R, N120K, T122K, T122I, T122V, T122A, T122R, A124D, A124E, A124R, A124K, A128T, E152A , E152D, Q168L, Q168A, E170G, F185K, R231G, R231K, R231 D, R231E, R231S, K264R, K266R or K273R, which correspond to the number of amino acids of the wild-type HIV integrase sequence (SEQ ID NO: 1 ). E10. The nucleic acid construction according to any of embodiments E7 to E9, wherein the modified HIV integrase comprises an amino acid sequence of at least 85 percent, at least 90 percent, or at least 95 percent identical to the sequence set forth in SEQ ID NO: 3. E11. The nucleic acid construction according to any of embodiments E1 to E6, wherein the modified transposase is selected from the group consisting of a modified Frog Prince, a modified Sleeping Beauty, a modified hyperactive Sleeping Beauty (SB100X), a modified PiggyBac, a modified hyperactive PiggyBac and any functional fragment thereof. E12. The nucleic acid construction according to embodiment E11, wherein the modified transposase is a modified hyperactive PiggyBac or a functional fragment thereof. E13. The nucleic acid construction according to embodiment E12, wherein the modified hyperactive PiggyBac comprises a mutation of one or more of the amino acids 245, 268, 275, 277, 287, 290, 315, 325, 341,346, 347, 350, 351,356, 357, 372, 375, 388, 409, 412, 432, 447, 450, 460, 461, 465, 517, 560, 564, 571,573, 576, 586, 587, 589, 592 and 594 which correspond to the number of amino acids of the hyperactive PiggyBac sequence (SEQ ID NO: 9). E14. The nucleic acid construct according to embodiment E13, wherein the modified hyperactive PiggyBac mutation comprises one or more of R245A, D268N, R275A / R277A, K287A, K290A, K287A / K290A, R315A, G325A, R341A, D346N, N347A , N347S, T350A, S351E, S351P, S351A, K356E, N357A, R372A, K375A, R372A / K375A, R388A, K409A, K412A, K409A / K412A, K432A, D447A, D447N, D450N, R460A, K461A, R460A / K461A, W465A , S517A, T560A, S564P, S571N, S573A, K576A, H586A, I587A, M589V, S592G or F594L that correspond to the number of amino acids of the hyperactive PiggyBac sequence (SEQ ID NO: 9). E15. The nucleic acid construction according to any of embodiments E12 to E14, wherein the modified hyperactive PiggyBac comprises an amino acid sequence of at least 85 percent, at least 90 percent, or at least 95 percent percent identical to the sequence set forth in SEQ ID NO: 10. E16. The nucleic acid construction according to any of embodiments E1 to E15, wherein the linker comprises an XTEN sequence or a GGS sequence. E17. The nucleic acid construction according to any of embodiments E1 to E16, wherein the sequence encoding the linker is between about 9 and about 150 7CICI n / l 7Π7 / Β / Υ nucleic acids in length. E18. The nucleic acid construct according to any of embodiments E1 to E17, wherein the 3' end of the first polynucleotide sequence is connected to the 5' end of the second polynucleotide via the nucleic acid linker. E19. The nucleic acid construct according to any of embodiments E1 to E17, wherein the 3' end of the second polynucleotide sequence is connected to the 5' end of the first polynucleotide sequence via the nucleic acid linker. E20. A vector comprising the nucleic acid construct according to any of embodiments E1 to E19, wherein the expression vector is suitable for expression in mammalian cells, in yeast cells, in insect cells, in plant cells, fungal cells or algae cells. E21. The construction of nucleic acid according to the E1 modality, wherein: a) the first polynucleotide sequence encodes a Cas9 protein; and b) the second polynucleotide sequence encodes a modified transposase that is a modified hyperactive PiggyBac or a functional fragment thereof. E22. The nucleic acid construction according to embodiment E21, wherein the Cas9 protein is selected from the group consisting of a human Cas9, a Cas9 nicase, a Streptococcus pyogenes Cas9, a Staphylococcus aureus Cas9, a Casi2a, a Casi 2b, and a dead Cas9. E23. The nucleic acid construction according to any of embodiments E21 or E22, wherein the modified hyperactive PiggyBac comprises a mutation of one or more of amino acids 245, 268, 275, 277, 287, 290, 315, 325, 341,346, 347, 350, 351,356, 357, 372, 375, 388, 409, 412, 432, 447, 450, 460, 461, 465, 517, 560, 564, 571, 573, 576, 586, 587, 58 9, 592 and 594 which correspond to the number of amino acids of the hyperactive PiggyBac sequence (SEQ ID NO: 9). E24. The nucleic acid construct according to embodiment E23, wherein the modified hyperactive PiggyBac mutation comprises one or more of R245A, D268N, R275A / R277A, K287A, K290A, K287A / K290A, R315A, G325A, R341A, D346N, N347A , N347S, T350A, S351E, S351P, S351A, K356E, N357A, R372A, K375A, R372A / K375A, R388A, K409A, K412A, K409A / K412A, K432A, D447A, D447N, D450N, R460A, K461A, R460A / K461A, W465A , S517A, T560A, S564P, S571N, S573A, K576A, H586A, I587A, M589V, S592G or F594L that correspond to the number of amino acids of the hyperactive PiggyBac sequence (SEQ ID NO: 9). E25. The nucleic acid construct according to any of embodiments E21 or E22, wherein the modified hyperactive PiggyBac comprises an amino acid sequence of at least 85 percent, at least 90 percent, or at least 95 percent. percent identical to the sequence set forth in SEQ ID NO: 10. E26. The nucleic acid construction according to any of embodiments E21 to E25, wherein the nucleic acid encoding the linker comprises an XTEN sequence or a GGS sequence. 7CICI n / l 7Π7 / Β / Υ Ε27. The nucleic acid construction according to any of embodiments E21 to E26, wherein the sequence encoding the linker is between 9 and 150 nucleic acids in length. E28. The nucleic acid construct according to any of embodiments E22 to E27, wherein the 3' end of the second polynucleotide sequence is connected to the 5' end of the first polynucleotide sequence via the linker. E29. The construction of nucleic acid according to the E1 modality, wherein: a) the first polynucleotide sequence encodes a zinc finger protein; and b) the second polynucleotide sequence encodes a modified integrase or a functional fragment thereof. E30. The nucleic acid construction according to embodiment E29, wherein the zinc finger protein is a C2H2 zinc finger protein. E31. The nucleic acid construction according to any of embodiments E29 or E30, wherein the modified integrase is a modified human immunodeficiency virus (HIV) integrase or a functional fragment thereof. E32. The nucleic acid construction according to embodiment E31, wherein the modified HIV integrase comprises a mutation of one or more of amino acids 10, 13, 64, 94, 116, 117, 119, 120, 122, 124, 128 , 152, 168, 170, 185, 231, 264, 266 or 273 corresponding to the number of amino acids of the wild-type HIV integrase sequence (SEQ ID NO: 1). E33. The nucleic acid construction according to embodiment E32, wherein the modified HIV integrase mutation comprises one or more of D10K, Ε13K, D64A, D64E, G94D, G94E, G94R, G94K, D116A, D116E, N117D, N117E , N117R, N117K, S119A, S119P, S119T, S119G, S119D, S119E, S119R, S119K, N120D, N120E, N120R, N120K, T122K, T122I, T122V, T122A, T122R, A124D, A124E, A124R, A124K, A128T, E152A , E152D, Q168L, Q168A, E170G, F185K, R231G, R231K, R231 D, R231E, R231S, K264R, K266R or K273R corresponding to the number of amino acids of the wild-type HIV integrase sequence (SEQ ID NO: 1) . E34. The nucleic acid construction according to any of embodiments E31 to E33, wherein the modified HIV integrase comprises an amino acid sequence of at least 85 percent, at least 90 percent, or at least 95 percent identical to the sequence set forth in SEQ ID NO: 3. E35. The nucleic acid construction according to any of embodiments E29 to E34, wherein the linker comprises an XTEN sequence or a GGS sequence. E36. The nucleic acid construction according to any of embodiments E29 to E35, wherein the sequence encoding the linker is 9 to 150 nucleic acids in length. E37. The nucleic acid construct according to any of embodiments E29 to E37, wherein the 3' end of the second polynucleotide sequence is connected to the 5' end of the first polynucleotide sequence via the linker. E38. A vector comprising the nucleic acid construct according to any of embodiments E21 to E37, wherein the expression vector is suitable for expression in cells of 7CICI n / l 7Π7 / Β / Υ mammals, in yeast cells, in insect cells, in plant cells, in fungal cells or in algal cells. E39. A host cell comprising the nucleic acid construct or the vector according to any of embodiments E1 to E38. E40. A fusion protein comprising: a first DNA binding protein that is designed in such a way as to bind to a specific genomic DNA sequence in a genome; a second DNA binding protein that allows insertion of an exogenous nucleic acid into the genome, wherein the second DNA binding protein is an integrase or a transposase that is modified relative to the wild type; and a linker that connects the first protein and the second protein. E41. The fusion protein according to embodiment E40, wherein the second DNA binding protein is modified to improve the specificity of the insertion of the exogenous nucleic acid into the genome compared to the corresponding wild type protein. E42. The fusion protein according to any of embodiments E40 or E41, wherein the exogenous nucleic acid may be up to approximately 20 kb in length. E43. The fusion protein according to any of embodiments E40 to E42, wherein the first DNA binding protein is selected from the group consisting of a zinc finger protein, a Cas9 protein and any variant or portion of functional fragment of them. E44. The fusion protein according to embodiment E43, wherein the Cas9 protein is selected from the group consisting of a human Cas9, a Cas9 nicase, a Streptococcus pyogenes Cas9, a Staphylococcus aureus Cas9, a Casi2a, a Casi2b, and a dead Cas9. E45. The fusion protein according to embodiment E43, wherein the zinc finger protein is a C2H2 zinc finger protein. E46. The fusion protein according to any of embodiments E40 to E45, wherein the modified integrase is a modified human immunodeficiency virus (HIV) integrase or a functional fragment thereof. E47. The fusion protein according to embodiment E46, wherein the modified HIV integrase comprises a mutation of one or more of amino acids 10, 13, 64, 94, 116, 117, 119, 120, 122, 124, 128, 152, 168, 170, 185, 231,264, 266 or 273 which correspond to the number of amino acids of the wild-type HIV integrase sequence (SEQ ID NO: 1). E48. The fusion protein according to embodiment E47, wherein the modified HIV integrase mutation comprises one or more of D10K, E13K, D64A, D64E, G94D, G94E, G94R, G94K, D116A, D116E, N117D, N117E, N117R, N117K, S119A, S119P, S119T, S119G, S119D, S119E, S119R, S119K, N120D, N120E, N120R, N120K, T122K, T122I, T122V, T122A, T122R, A124D, A1 24E, A124R, A124K, A128T, E152A, E152D, Q168L, Q168A, E170G, F185K, R231G, R231K, R231D, R231E, R231S, K264R, K266R or K273R that correspond to the number of amino acids in the integrase sequence of 7CICI n / l 7Π7 / Β / Υ Wild type HIV (SEQ ID NO: 1). E49. The fusion protein according to any of embodiments E46 to E48, wherein the modified HIV integrase comprises an amino acid sequence of at least 85 percent, at least 90 percent, or at least 95 percent percent identical to the sequence set forth in SEQ ID NO: 3. E50. The fusion protein according to any of embodiments E40 to E45, wherein the modified transposase is selected from the group consisting of a modified Frog Prince, a modified Sleeping Beauty, a modified hyperactive Sleeping Beauty (SB100X), a PiggyBac modified, a modified hyperactive PiggyBac, and any functional fragment thereof. E51. The fusion protein according to embodiment E50, wherein the modified transposase is a modified hyperactive PiggyBac or a functional fragment thereof. E52. The fusion protein according to embodiment E51, wherein the modified hyperactive PiggyBac comprises a mutation of one or more of the amino acids 245, 268, 275, 277, 290, 315, 325, 341,346, 347, 350, 351,356, 357 , 372, 375, 388, 409, 412, 432, 447, 450, 460, 461,465, 517, 560, 564, 571,573, 576, 586, 587, 589, 592, and 594 that correspond to the number of amino acids in the sequence of hyperactive PiggyBac (SEQ ID NO: 9). E53. The fusion protein according to embodiment E52, wherein the modified hyperactive PiggyBac mutation comprises one or more of R245A, D268N, R275A / R277A, K287A, K290A, K287A / K290A, R315A, G325A, R341A, D346N, N347A, N347S, T350A, S351E, S351P, S351A, K356E, N357A, R372A, K375A, R372A / K375A, R388A, K409A, K412A, K409A / K412A, K432A, D447A, D447N, D450N, R4 60A, K461A, R460A / K461A, W465A, S517A, T560A, S564P, S571N, S573A, K576A, H586A, I587A, M589V, S592G or F594L corresponding to the number of amino acids of the hyperactive PiggyBac sequence (SEQ ID NO: 9). E54. The fusion protein according to any of embodiments E50 to E53, wherein the modified hyperactive PiggyBac comprises an amino acid sequence of at least 85 percent, at least 90 percent, or at least 95 percent identical to the sequence set forth in SEQ ID NO: 10. E55. The fusion protein according to any of embodiments E40 to E54, wherein the linker comprises an XTEN sequence or a GGS sequence. E56. The fusion protein according to any of embodiments E40 to E55, wherein the linker is between 3 and 50 amino acids in length. E57. The fusion protein according to embodiment E40, wherein: a) the first DNA binding protein is a Cas9 protein; and b) the second DNA binding protein is a modified hyperactive PiggyBac or a functional fragment thereof. E58. The fusion protein according to embodiment E57, wherein the Cas9 protein is selected from the group consisting of a human Cas9, a Cas9 nicase, a Streptococcus pyogenes Cas9, a Staphylococcus aureus Cas9, a Casi2a, a Almost 2b, and a Cas9 7CICI n / l 7Π7 / Β / Υ dead. E59. The fusion protein according to any of embodiments E57 or E58, wherein the modified hyperactive PiggyBac comprises a mutation of one or more of amino acids 245, 268, 275, 277, 287, 290, 315, 325, 341,346, 347 , 350, 351,356, 357, 372, 375, 388, 409, 412, 432, 447, 450, 460, 461, 465, 517, 560, 564, 571, 573, 576, 586, 587, 589, 592 and 594 which correspond to the number of amino acids of the hyperactive PiggyBac sequence (SEQ ID NO: 9). E60. The fusion protein according to embodiment E59, wherein the modified hyperactive PiggyBac mutation comprises one or more of R245A, D268N, R275A / R277A, K287A, K290A, K287A / K290A, R315A, G325A, R341A, D346N, N347A, N347S, T350A, S351E, S351P, S351A, K356E, N357A, R372A, K375A, R372A / K375A, R388A, K409A, K412A, K409A / K412A, K432A, D447A, D447N, D450N, R4 60A, K461A, R460A / K461A, W465A, S517A, T560A, S564P, S571N, S573A, K576A, H586A, I587A, M589V, S592G or F594L corresponding to the number of amino acids of the hyperactive PiggyBac sequence (SEQ ID NO: 9). E61. The fusion protein according to any of embodiments E57 to E60, wherein the modified hyperactive PiggyBac comprises an amino acid sequence of at least 85 percent, at least 90 percent, or at least 95 percent identical to the sequence set forth in SEQ ID NO: 10. E62. The fusion protein according to embodiment E40, wherein: a) the first DNA-binding protein is a zinc finger protein; and b) the second DNA binding protein is a modified integrase or a functional fragment thereof. E63. The fusion protein according to embodiment E62, wherein the zinc finger protein is a C2H2 zinc finger protein. E64. The fusion protein according to any of embodiments E62 or E63, wherein the modified integrase is a modified human immunodeficiency virus (HIV) integrase or a functional fragment thereof. E65. The fusion protein according to embodiment E64, wherein the modified HIV integrase comprises a mutation of one or more of amino acids 10, 13, 64, 94, 116, 117, 119, 120, 122, 124, 128, 152, 168, 170, 185, 231,264, 266 or 273 which correspond to the number of amino acids of the wild-type HIV integrase sequence (SEQ ID NO: 1). E66. The fusion protein according to embodiment E65, wherein the modified HIV integrase mutation comprises one or more of D10K, E13K, D64A, D64E, G94D, G94E, G94R, G94K, D116A, D116E, N117D, N117E, N117R, N117K, S119A, S119P, S119T, S119G, S119D, S119E, S119R, S119K, N120D, N120E, N120R, N120K, T122K, T122I, T122V, T122A, T122R, A124D, A1 24E, A124R, A124K, A128T, E152A, E152D, Q168L, Q168A, E170G, F185K, R231G, R231K, R231D, R231E, R231S, K264R, K266R or K273R corresponding to the number of amino acids of the wild-type HIV integrase sequence (SEQ ID NO: 1). E67. The fusion protein according to the E62 modality, wherein the HIV integrase 7CICI n / l 7Π7 / Β / ΥΙΛΙ modified comprises an amino acid sequence that is at least 85 percent, at least 90 percent, or at least 95 percent identical to the sequence set forth in SEQ ID NO: 3. E68. The fusion protein according to any of embodiments E57 to E67, wherein the linker comprises an XTEN sequence or a GGS sequence. E69. The fusion protein according to any of embodiments E57 to E68, wherein the linker is 3 to 50 amino acids in length. E70. The fusion protein according to any of embodiments E40 to E69, wherein the 3' end of the second DNA binding protein is connected to the 5' end of the first DNA binding protein via the linker. E71. A lentiviral particle comprising the fusion protein according to any of embodiments E40 to E69. E72. A method for producing a lentiviral particle for gene editing, which comprises the expression in a host cell of: a) a polynucleotide comprising the nucleic acid construct according to any of embodiments E1 to E38; and b) a polynucleotide that encodes proteins for a lentiviral envelope. E73. The method according to embodiment E72, which further comprises the expression of c) a polynucleotide sequence comprising the exogenous nucleic acid. E74. The method according to any of embodiments E72 or E73, wherein the polynucleotide comprising the nucleic acid construct further comprises a nucleic acid sequence encoding lentiviral capsid proteins. E75. The method according to any of embodiments E72 to E74, which further comprises the recovery of the lentiviral particle from the host cell. E76. The method according to any of embodiments E72 to E75, which further comprises the purification of the lentiviral particle. E77. A method for inserting an exogenous nucleic acid sequence into the genomic DNA of an organism, which comprises: administration of a lentiviral particle comprising the nucleic acid construct according to any of embodiments E1 to E38 or a protein fusion according to any of embodiments E40 to E71 to the organism, in a manner such that the first and second DNA binding proteins bind to a specific genomic DNA sequence and insert the exogenous nucleic acid into the genomic DNA; where the exogenous nucleic acid becomes integrated into the specific genomic DNA sequence. E78. A method for controlled, site-specific integration of a single copy or multiple copies of an exogenous nucleic acid sequence into a cell, wherein the method comprises: a) delivering the fusion protein according to any of embodiments E40 to E71 to the cell, and b) the delivery of the exogenous nucleic acid to the cell; wherein the binding of the fusion protein to the specific genomic DNA sequence in the 7CICI n / l 7Π7 / Β / Υ genome of the cell, results in the cleavage of the genome and the integration of one or more copies of the exogenous nucleic acid into the genome of the cell; and wherein the fusion protein is delivered to the cell by a lentiviral particle. E79. A nucleic acid construct comprising: a) a first polynucleotide sequence comprising a nucleic acid encoding a first DNA binding protein that is designed in such a way as to bind to a specific genomic DNA sequence in a genome; wherein the first DNA binding protein is a zinc finger protein or a Cas9 protein; b) a second polynucleotide sequence comprising a nucleic acid encoding a second DNA-binding protein that allows insertion of an exogenous nucleic acid into a genome, wherein the second DNA-binding protein is: (i) a hyperactive PiggyBac transposase, or a modified hyperactive PiggyBac with improved specificity of exogenous nucleic acid insertion into the genome compared to the hyperactive PiggyBac, or (i) a human immunodeficiency virus (HIV) integrase or a modified HIV integrase with improved specificity of exogenous nucleic acid insertion into the genome compared to HIV integrase; and c) an optional polynucleotide sequence comprising a nucleic acid encoding a linker; wherein the nucleic acid construct encodes a fusion protein comprising the first DNA binding protein, the second DNA binding protein and the optional linker between the first DNA binding protein and the second DNA binding protein; and wherein the fusion protein allows the insertion of the exogenous nucleic acid to be made at a specific site in the genome. E80. The nucleic acid construction according to embodiment E79, wherein the Cas9 protein is selected from the group consisting of a human Cas9, a Cas9 nicase and a dead Cas9. E81. The nucleic acid construction according to embodiment E79, wherein the zinc finger protein is a C2H2 zinc finger protein comprising 6 domains. E82. The nucleic acid construct according to any of embodiments E79 to E81, wherein the linker comprises an XTEN sequence or a GGS sequence. E83. The nucleic acid construction according to any of embodiments E79 to E82, wherein the 3' end of the first polynucleotide sequence is connected to the 5' end of the second polynucleotide. E84. The nucleic acid construction according to any of embodiments E79 to E83, wherein: (a) the first DNA binding protein is a Cas9 protein or a zinc finger protein, and (b) the second DNA binding protein is binding to DNA is a hyperactive PiggyBac transposase, or a modified hyperactive PiggyBac with improved specificity of exogenous nucleic acid insertion into the 7CICI Π / I 7Π7 / Β / Υ genome compared to the hyperactive PiggyBac, wherein the nucleic acid construct comprises (c) the polynucleotide sequence comprising a nucleic acid encoding a linker comprising an XTEN sequence or a sequence of GGS, and wherein the 3' end of the first polynucleotide sequence is connected to the 5' end of the second polynucleotide. E85. The nucleic acid construction according to any of embodiments E79 to E83, wherein: (a) the first DNA binding protein is a Cas9 protein or a zinc finger protein, and (b) the second DNA binding protein is DNA binding is an HIV integrase, or a modified HIV integrase with improved specificity of insertion of the exogenous nucleic acid into the genome compared to the HIV integrase, wherein the nucleic acid construct comprises (c) the sequence of polynucleotides comprising a nucleic acid encoding a linker comprising an XTEN sequence or a GGS sequence, and wherein the 3' end of the first polynucleotide sequence is connected to the 5' end of the second polynucleotide. E86. The nucleic acid construction according to any of embodiments E79 to E84, wherein the modified hyperactive PiggyBac transposase comprises a mutation of one or more of amino acids 245, 268, 275, 277, 287, 290, 315, 325, 341,346 , 347, 350, 351,356, 357, 372, 375, 388, 409, 412, 432, 447, 450, 460, 461, 465, 517, 560, 564, 571, 573, 576, 586, 587, 589, 592 and 594 that correspond to the amino acid sequence of SEQ ID NO: 9 of the hyperactive PiggyBac. E87. The nucleic acid construction according to embodiment E86, wherein the modified hyperactive PiggyBac transposase mutation comprises one or more of the amino acid modifications selected from: R245A, D268N, R275A / R277A, K287A, K290A, K287A / K290A, R315A, G325A, R341A, D346N, N347A, N347S, T350A, S351 E, S351 P, S351A, K356E, N357A, R372A, K375A, R372A / K375A, R388A, K409A, K4 12A, K409A / K412A, K432A, D447A, D447N, D450N, R460A, K461A, R460A / K461A, W465A, S517A, T560A, S564P, S571 N, S573A, K576A, H586A, I587A, M589V, S592G, or F594L that correspond to the amino acid sequence of the SEQ ID NO: 9 of the overactive PiggyBac. E88. The nucleic acid construction according to any of embodiments E79 to E84, wherein the modified hyperactive PiggyBac transposase comprises a mutation of one or more of amino acids 245, 275, 277, 325, 347, 351, 372, 375, 388 , 450, 465, 560, 564, 573, 589, 592, 594 that correspond to the amino acid sequence of SEQ ID NO: 9 of the hyperactive PiggyBac. E89. The nucleic acid construction according to embodiment E88, wherein the modified hyperactive PiggyBac transposase mutation comprises one or more of the amino acid modifications selected from: R245A, R275A, R277A, R275A / R277A, G325A, N347A, N347S, S351E, S351P, S351A, R372A, K375A, R388A, D450N, W465A, T560A, S564P, S573A, M589V, S592G or F594L that correspond to the amino acid sequence of SEQ ID NO: 9 of the hyperactive PiggyBac. E90. The nucleic acid construction according to embodiment E88, wherein the modified hyperactive PiggyBac transposase comprises the amino acid sequence of SEQ ID NO: 9, wherein: the amino acid at position 245 is A, the amino acid at position 275 is R or A, the amino acid in 7CICI n / l 7Π7 / Β / Υ position 277 is R or A, amino acid at position 325 is A or G, amino acid at position 347 is N or A, amino acid at position 351 is E, P or A , the amino acid at position 372 is R, the amino acid at position 375 is A, the amino acid at position 450 is D or N, the amino acid at position 465 is W or A, the amino acid at position 560 is T or A, the amino acid at position 564 is P or S, the amino acid at position 573 is S or A, the amino acid at position 592 is G or S, and the amino acid at position 594 is Lo F. E91. The nucleic acid construction according to embodiment E88, wherein the modified hyperactive PiggyBac transposase comprises an amino acid sequence that is selected from the group consisting of SEQ ID NO: 120, 121, 122, 123, 124,125, 126 , 127, 128 and 129. E92. The nucleic acid construction according to embodiment E88, wherein the modified hyperactive PiggyBac transposase comprises an amino acid sequence that is at least 80 percent identical to a sequence that is selected from the group consisting of SEQ ID NO: 119, 120, 121,122, 123, 124, 125, 126, 127, 128 and 129, where the modified hyperactive PiggyBac shows improved specificity of DNA integration into a genome compared to the hyperactive PiggyBac. E93. The nucleic acid construction according to any of embodiments E79 to E83 or E85, wherein the modified HIV integrase comprises a mutation of one or more of amino acids 10,13, 64, 94,116,117,119,120,122, 124,128, 152, 168,170,185, 231,264 , 266 or 273 that correspond to the amino acid sequence of SEQ ID NO: 1 of wild-type HIV integrase. E94. The nucleic acid construction according to embodiment E93, wherein the modified HIV integrase mutation comprises one or more of D10K, Ε13K, D64A, D64E, G94D, G94E, G94R, G94K, D116A, D116E, N117D, N117E , N117R, N117K, S119A, S119P, S119T, S119G, S119D, S119E, S119R, S119K, N120D, N120E, N120R, N120K, T122K, T122I, T122V, T122A, T122R, A124D, A124E, A124R, A124K, A128T, E152A , E152D, Q168L, Q168A, E170G, F185K, R231G, R231K, R231 D, R231E, R231S, K264R, K266R or K273R, corresponding to the amino acid sequence of SEQ ID NO: 1 of wild-type HIV integrase . E95. A vector comprising the nucleic acid construct according to any of embodiments E79 to E95, wherein the vector is suitable for expression in mammalian cells, in yeast cells, in insect cells, in plants, in fungal cells or in algae cells. E96. A host cell comprising the nucleic acid construct or the vector according to any of embodiments E79 to E95. E97. A fusion protein obtained from the expression of the nucleic acid construct according to any of embodiments E79 to E94. E98. A composition comprising a nucleic acid construct, a vector or a fusion protein according to any of embodiments E79 to E95 or E97, and a polynucleotide sequence encoding an exogenous nucleic acid for insertion into a genome, wherein The composition is contained in, or linked to, a packing vector. 7CICI n / l 7Π7 / Β / Υ Ε99. The composition according to embodiment E98, wherein the nucleic acid construct is in a form of RNA, DNA or protein, and the polynucleotide sequence encoding the exogenous nucleic acid is in the form of DNA or RNA. E100. The composition according to any of embodiments E98 and E99, wherein the packaging vector is a nanoparticle or a lentiviral particle. E101. A method for the controlled and site-specific integration of a single copy or multiple copies of an exogenous nucleic acid sequence into a cell, wherein the method comprises: (a) delivery of the nucleic acid construct, the vector or the fusion protein according to any of embodiments E79 to E95 or E97 to the cell, and (b) delivering the exogenous nucleic acid to the cell; wherein binding of the fusion protein to the specific genomic DNA sequence in the genome of the cell results in cleavage of the genome and integration of one or more copies of the exogenous nucleic acid into the genome of the cell. E102. A modified hyperactive PiggyBac transposase comprising the amino acid sequence of SEQ ID NO: 9, wherein: the amino acid at position 245 is A, the amino acid at position 275 is R or A, the amino acid at position 277 is R or A, the amino acid at position 325 is A or G, the amino acid at position 347 is N or A, the amino acid at position 351 is E, P or A, the amino acid at position 372 is R, the amino acid at position 375 is A, amino acid at position 450 is D or N, amino acid at position 465 is W or A, amino acid at position 560 is T or A, amino acid at position 564 is P or S, the amino acid at position 573 is S or A, the amino acid at position 592 is G or S, and the amino acid at position 594 is L or F. E103. The modified hyperactive PiggyBac transposase according to embodiment E102, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 120, 121, 122, 123, 124, 125, 126, 127, 128y 129. E104. The modified hyperactive PiggyBac transposase of claim E012, comprising an amino acid sequence that is at least 80 percent identical to a sequence selected from the group consisting of SEQ ID NOs: 119, 120, 121,122, 123, 124, 125, 126, 127, 128 and 129, where the modified hyperactive PiggyBac shows improved specificity of DNA integration into a genome compared to the hyperactive PiggyBac. The content of all references that are cited (including bibliographic references, patents, patent applications and websites) or that may be cited throughout this application are expressly incorporated by reference in their entirety for any purpose, as well as the references cited therein. The following examples are provided by way of illustration and not by way of limitation. Examples PB and hyPB are used interchangeably to refer to the overactive PiggyBac transposase. Examples 1 to 3 below relate to the generation and performance in terms of targeted integration of Cas9 and programmable transposase fusion protein constructs. In example 1, different DNA constructions of the 7CICI n / l 7Π7 / Β / Υ hyperactive PiggyBac and Sleeping Beauty transposases fused to different versions of Cas9, resulting in the integration of the transposon into the genome of the transfected cells. Surprisingly, the PiggyBac and Cas9 constructs were able to promote targeted integration at the site of interest in the genome (Example 2). Example 3 provides modified transposases that are generated to increase the specificity of insertion of exogenous nucleic acid sequences into the genome. Example 1: DNA Vectors for Expression of Programmable Transposase Fusion Proteins This experiment aims to test different configurations of the fusion of the hyperactive transposases PiggyBac (referred to herein as hyPB or PB) and Sleeping Beauty (referred to herein as SB100x) to nuclease (h). , to nicase (n) and to dead Cas9 (d) for the performance of transposon integration. Programmable transposase fusion proteins were created by incorporating into a pcDNA3.3-TOPO expression vector (the backbone of the Invitrogen plasmid, Addgene plasmid #41815), the DNA sequences encoding human Cas9. wild-type (hCas9), nicase Cas9 (nCas9) or dead Cas9 (dCas9) (SEQ ID NO: 64 to 66, respectively) and the overactive PiggyBac transposase (PB) or the overactive Sleeping Beauty transposase (SB100) (SEQ ID NO: 67 and 68, respectively). Vectors were created in which the 3' end of Cas9 was connected to the 5' end of each of the transposases via a nucleic acid linker sequence (SEQ ID NO: 48) encoding a GGS linker (hCas9PB , nCas9PB, dCas9PB, hCas9SB, nCas9SB and dCas9SB). Other vectors were created in which the 3' end of each of the transposases was connected to the 5' end of Cas9 via a nucleic acid linker sequence (SEQ ID NO: 48) encoding a GGS linker (PBhCas9 , PBnCas9, PBdCas9, SBhCas9, SBnCas9 and SBdCas9). A compendium of fusion constructs is provided in Table 2. 7CICI n / l 7Π7 / Β / Υ Table 2 List of programmable transposase proteins that are generated in example 1 Programmable Transposase Fusion Proteins Cas9 Transposase Targeting Linker Human Cas9 Hyperactive PiggyBac hCas9-PB GGS Linker Nicasa Cas9 Hyperactive PiggyBac nCas9-PB GGS Linker Dead Cas9 Hyperactive PiggyBac dCas9-PB GGS Linker Human Cas9 Hyperactive PiggyBac PB-hCas9 GGS Linker Nicasa Cas9 PiggyBac Overactive PB-nCas9 GGS Linker Programmed Transposase Fusion Proteins Cas9 Transposase Targeting Linker Dead Cas9 Overactive PiggyBac PB-dCas9 GGS Linker Human Cas9 Sleeping Beauty Overactive hCas9-SB GGS Linker Nicasa Cas9 Sleeping Beauty Overactive nCas9-SB GGS Linker Dead Cas9 Sleeping Beauty Overactive dCas9-SB GGS Linker Human Cas9 Sleeping Beauty Hyperactive SB-hCas9 GGS Linker Nicasa Cas9 Sleeping Beauty Hyperactive SB-nCas9 GGS Linker Dead Cas9 Sleeping Beauty Hyperactive SB-dCas9 GGS Linker 7CICI Π / I 7Π7 / Β / Υ Before transfection, frozen HEK293T cells were quickly thawed at 37°C, then resuspended in 5 mL of prewarmed medium and pelleted by centrifugation at 1,000 rpm for 4 min. The pellet was resuspended in fresh medium and ~1.6 x 106 cells were seeded into a new T75 flask. When the cells reached 95 percent confluency they were passed through using trypsin and plated at 40 percent confluency. Cells were passaged twice before using them for experiments. For transfection experiments, 5 cent, 2 mM glutamine and penicillin in 100 U per 0.1 mg per mL of streptomycin). Before transfection, the medium was replaced with 2.7 mL of fresh complete DMEM medium. OptiMEM I reduced serum medium was mixed with each plasmid combination, as well as with the linear polyethyleneimine (PEI 25K) solution at 1 mg per mL. A 3:1 ratio of PEI 25K (pg): total DNA (pg) was used. The two solutions were mixed and incubated at room temperature for 15 min. After incubation, 300 pL of the mixture was applied dropwise to the cells. 24 hours after transfection, the medium was replaced with fresh complete medium. Cells were harvested after transfection for flow cytometry or for cell sorting and DNA extraction. HEK293T cells were cotransfected with a plasmid encoding a programmatic transposase fusion protein from Table 2, a plasmid encoding the nucleic acid to be integrated, which was an RFP (red fluorescent protein) or GFP transposon ( green fluorescent protein), and a guide RNA directed to the AAVS1 site (adeno-associated virus integration site 1) in the human genome. Overactive PiggyBac and SB100 were used as a positive control, and transposon alone as a negative control for the detection of episomal expression (i.e., expression from the non-inserted plasmid). Fluorescence was analyzed by flow cytometry until day 14, after which episomal fluorescence could not be detected. Cells were then selected for GFP expression and, two days after selection, target DNA integration was quantified by counting the percentage of fluorescent cells. Results and Conclusions: The results for the Cas9-PB fusions are shown in Figure 1A and Figure 1C; and the results for Cas9-SB100 fusions are shown in Figure 1B. Human Cas9 fused to hyperactive PiggyBac (hCas9PB) and Cas9 nicase fused to hyperactive PiggyBac (nCas9PB) increased the percentage of fluorescent cells by approximately 8 percent compared to the episomal RFP negative control after 14 days ( Figures 1A and 1C). Therefore, such fusion proteins were able to successfully integrate exogenous DNA into the cellular genome. The tested Cas9-Sleeping Beauty fusion proteins were unable to produce more fluorescent cells than the negative control for episomal GFP after 14 days (Figure 1B). Example 2: Efficiency of targeted transposition of programmable transposase fusion proteins Following the previous example, we studied whether there was any targeted (versus non-targeted) insertion with the configurations that had the best overall insertion in Example 1. To this end, HEK293T were cotransfected through the use of Lipofectamine 3000 with a plasmid (pSico) encoding hCas9PB or nCas9PB, a genetrap plasmid encoding a transposon with inverted repeats, and a promoterless GFP, and a guide RNA (gRNA) directed to the AAVS1 site or to a site within the CD46 gene after the promoter in the human genome. The 3' end of Cas9 was connected to the 5' end of the transposase via a linker (SEQ ID NO: 48). An example of the structure of the Cas9PB expression vector is shown in Figure 2A. The transposase contained a splice acceptor and a promoterless GFP between the 3' and 5' repeats. The gRNA and Cas9 direct the transposase to integrate the transposon into a promoter region. Through use of this approach, cells only become fluorescent if the transposon is inserted into the target site. Results and Conclusions: Quantification of the percentage of cells expressing GFP showed that Cas9-PiggyBac programmable transposase (HCas9 targeted) and Cas9-PiggyBac nicase (NCas9 targeted) fusion proteins had higher targeted delivery of target DNA. compared to the Non-targeted (control for general insertion (PiggyBac alone)) and Episomal (negative control for non-integration (transposon alone)) controls (Figure 2B). In this case the 3-fold and 4-fold increase in signal over background was significant; especially considering that not all cells were efficiently transformed with all vectors necessary for transposon insertion; and the efficiency of random insertion for hyPB under non-optimized conditions like those used here is 10 to 15 percent. Example 3: Generation of modified hyperactive PiggyBac transposases Modified hyperactive PiggyBac transposases were generated to increase specificity 7CICI n / l 7Π7 / Β / Υ of the insertion of exogenous nucleic acid sequences into the genome. A list of transposase amino acid mutations is provided in Table 3. Table 3 Mutation sites for hyperactive PiggyBac vs hyperactive PiggyBac of SEQ ID NO: 9 7CICI n / l 7Π7 / Β / Υ Position Wild Type Amino Acid Mutation Classifications 245 R A Alanine Detection 268 D N Conserved Catalytic Triad 275 R A Alanine Detection 277 R A Alanine Detection 275 / 277 R / R A / A Alanine Detection 287 K A Alanine Detection: Decreased Cleavage 290 K A Alanine sensing 287 / 290 K / K A / A Alanine sensing: decreased cleavage 315 R A Alanine sensing: integration competent 325 G A Alignment integrase 341 R A Alanine sensing: integration competent 346 D N Conserved catalytic triad 347 N A, S Alignment integrase 350 T A Alignment integrase 351 s E, P, A Mutant comparable to integrase mutations that impair target binding -> k351 Position Wild-type Amino Acid Mutation Classifications is integration competent 357 N A Alignment integrase 356 K E Mutant comparable to integrase mutations that impair target binding -> k356 is integration competent 372 R A Alanine detection: integration competent 375 K A Detection of Alanine: Integration Proficient 372 / 375 R / K A / A Alanine Detection 388 R A Alanine Detection 409 K A Alanine Detection 412 K A Alanine Detection 409 / 412 K / K A / A Alanine Detection 432 K A Alanine Detection 447 D A, N Conserved catalytic triad 460 R A Alanine detection: Decreased cleavage 461 K A Alanine detection: Decreased cleavage 460 / 461 R / K A / A Alanine detection: Decreased cleavage 7CICI Π / I 7Π7 / Β / Υ Position Wild-type Amino Acid Mutation Classifications 465 W A Alignment Integrase 517 S N Int- / Exc+ 560 T A Int- / Exc+ 564 s P Int- / Exc+ 571 N S Int- / Exc+ 573 S A Int- / Exc+ 576 K A Well-conserved residues, other Important functions are not bound to DNA because it is a flexible tail. 586 H 589 M V Int- / Exc+ 592 S G Int- / Exc+ 594 F L Int- / Exc+ JCICI n / l 7Π7 / Β / Υ In example 4 below, several constructs were generated with the goal that the zinc finger protein (ZFP) was capable of binding to a chromosomal target site for the insertion of the gene of interest. ZFP constitutes an alternative to Cas9 as the DNA binding protein. Examples 5 to 13 generally relate to the generation and performance in terms of targeted integration of HIV-1 integrase and Cas9 / ZFP fusion protein constructs. In particular, in example 5 the fusion proteins of ZFP and integrase were generated. Examples 6 to 10 provide different integrase-defective packaging systems (i.e., non-integrating vectors) created to serve as a basis for in vitro studies to demonstrate recovery of integration function with integrase fusion proteins. integrase created in example 11. In example 12 it is observed that the targeted integrase fusion proteins increased the percentage of targeted insertion. Example 4: Generation of a targeted zinc finger protein (ZFP) The objective was to generate several ZFPs that bind to a chromosomal target site for the insertion of the gene of interest. A 6-domain zinc finger protein was generated to target the AAVS1 site (SEQ ID NO: 40) in the human genome. The target DNA sequences and corresponding ZFP helices are shown in Table 4. A construct encoding the target sites and ZFP (AAVS1-6dZFP) was prepared. The nucleic acid and amino acid sequences encoding ZFP are SEQ ID NOs: 32 and 33, respectively. 7CICI n / l 7Π7 / Β / Υ Table 4 List of AAVS1 target sites and corresponding ZFP helices Finger Tripod Propeller SEQ ID NO: 1 AGC ERSHLRE 41 2 CAG RADNLTE 42 3 CGT SRRTCRA 43 4 CCG RNDTLTE 44 5 CGG RSDKLTE 45 6 AGA QLAHLRA 46 Example 5: Generation of a ZFP-integrase fusion protein Integrase fusion proteins were generated with the ZFPs having 6 domains (effectively sequence specific). For the generation of a site-specific integrase, the ZFP generated in Example 4 (AAVS1-6d-ZFP) was cloned into a pcDNA3.1 expression vector together with the HIV-1 integrase (SEQ ID NO: 1) ( pZFP-AAVS1-6d-IN). The sequence encoding the fusion protein contained a nuclear localization signal at the N terminus (SEQ ID NO: 47) and a GGS linker sequence (SEQ ID NO: 48) between the ZFP and the integrase (Figure 3). Additional integrase fusion vectors were generated such as pZFP-TRCa-IN (which includes SEQ ID NO: 38, targeting the TRCa locus) and pZFP-AAVs1-TEX-IN (which includes a TEX linker (SEQ ID NO: 61 )), which were prepared through the use of similar methods. Example 6: Generation of DNA vectors with defective integrase Integrase-defective packaging systems were created to serve as a basis for in vitro studies through the use of an engineered integrase. Defective integrase constructs were created from the non-integrative packaging (NILV) plasmid psPAX2. psPAX2 plasmids have a single N64D mutation and two N64D / N116D mutations. An integrase-deleted plasmid (ΔΙΝ) was created that lacked the entire integrase coding region. A non-coding plasmid was created containing a stop codon before the integrase coding sequence (Example 8 below). Plasmids containing the truncated integrases were created, including a construct containing the C-terminus domain and the DNA binding domain without the cPPT / CTS (Example 10 below). General cloning protocols were followed, as briefly described below. ΚΑΡΑ HiFi HotStart Protocol For PCR experiments employing ΚΑΡΑ HiFi HotStart, the PCR reaction mixture was prepared according to the manufacturer's protocol of the ΚΑΡΑ HiFi PCR kit. ΚΑΡΑ HiFi PCR reactions were carried out using the Mastercycler Pro. Plasmid DNA extraction Plasmid DNA was extracted through the use of the QIAprep Spin Miniprep kit according to the manufacturer's protocol. Bacterial cultures were harvested by centrifugation at 5,000 rpm for 3 min. The cell pellet was resuspended in 250 pL of buffer P1 and mixed by inverting the tube 4 to 6 times with 250 pL of buffer P2. 350 pL of buffer N3 was added and mixed by inverting the tube. The Eppendorf tube was centrifuged for 10 minutes at 12,000 rpm to extract cellular debris and chromosomal DNA. The supernatant was transferred to the supplied QIAprep spin column and centrifuged for 1 min (12,000 rpm). The sample was washed twice with 0.5 mL of PB buffer and 0.75 mL of PE buffer, and each time centrifuged for 1 min at 12,000 rpm. Additional centrifugation for 1 min at 12,000 rpm removed the residual wash solution buffer. The QIAprep spin column was transferred to a new 1.5 mL microcentrifuge tube and 50 pL of water was added to elute the plasmid by letting the tube sit for 1 min and centrifugation followed for 1 min at 12,000 rpm. The concentration was measured with a NanoDropOne. Isolation and purification of plasmid DNA Bacterial strains (DH5a or DH10B) containing the desired plasmid were grown overnight in LB medium containing 100 pg per mL carbenicillin. Plasmids were isolated through the use of NZYTech plasmid mini or maxi kits, according to the manufacturer's protocol. Plasmids were eluted in 30 pL (miniprep) or 500 pL (maxiprep) of hot water at 65 °C. Plasmids were stored at −20°C. For PCR purification, the reaction mixture was processed through the use of the PCR purification kit. DNA was eluted in 30 pL of hot water at 65 °C. DNA gel electrophoresis The agarose was dissolved in 100 mL of TAE buffer by boiling. The liquid gels were supplemented with 4 pL of greensafe per 100 mL of the agarose solution and poured into a tray. To visualize DNA preparations, DNA was mixed with a 6x loading dye and ycici η / ι ζηζ / Ε / γ was loaded onto a 1 percent agarose gel. Additionally, one chamber was loaded with 1 pL of a genetic ladder per 1 mm lane of gel. Gels were run for 1.5 hours at 100 V and visualized through the use of a transilluminator. Transformation For DH5a transformation experiments, plasmids were transformed into 50 pL of DH5a cells according to the manufacturer's protocol. After recovery in s.o.c. medium, bacteria were pelleted at 15,000 g for 30 seconds and resuspended in 50 pL of LB medium. Cells were spread on an LB-agar plate containing 100 pg per mL carbenicillin and incubated at 37°C overnight. Cultures were harvested and inoculated overnight into LB medium containing 100 pg per mL carbenicillin. The liquid culture was used either for isolation of the plasmids once again or for a glycerol supply. For glycerol supply, 500 pL of the liquid culture was mixed with 500 pL of 50 percent glycerol and stored at −80°C. For transformation experiments with ultracompetent XL-10 Gold cells, cells were first thawed on ice and 45 pL of the cells were added to a pre-chilled 14 mL Falcon polypropylene round bottom tube. 2 pL of the βME mixture provided with the kit was added to the cells. The contents of the tube were gently rotated and the cells were incubated on ice for 10 minutes (rotating every 2 minutes). 1.5 pL of the Dpnl-treated DNA was added to an aliquot of the cells, mixed, and incubated on ice for 30 minutes. The mixture of cells and DNA was thermally pulsed into the tube at 42 °C for 30 seconds. The tubes were then incubated on ice for 2 minutes. Then, 0.5 mL of prewarmed (42 °C) NZY+ broth was added to each tube and then incubated at 37 °C for 1 hour with shaking at 225–250 rpm. The mixture was then plated on agar plates containing the appropriate antibiotic for the plasmid vector. Five colonies were selected for DNA extraction and the sequences were verified. Colony 1 was selected and maintained. Example 7: Generation of non-integrating vectors containing PPT or a ZFP-modified integrase fusion protein In order to create an integrase (IN)-defective but otherwise fully functional psPAX2 plasmid, the polypyrimidine tract (PPT) domain (SEQ ID NO: 74, which is crucial for the subsequent formation of double-stranded cDNA from all retroviral RNA genomes, such as lentivirus) was cloned into a psPAX2 vector that did not contain an integrase (psPAX2-AIN). The AAVS1-targeting synthetic zinc finger construct generated in Example 4 (AAVS1-6d-ZFP-IN) was cloned into psPAX2-ÁIN. Two different forward primers and the same reverse primer (SEQ ID NO: 75 to 77) were designed for the PPT with and without a stop codon (IN + PPT and IN + PPT (STOP)). Two different forward primers (SEQ ID NO: 78 to 80) and the same reverse primer were designed for AVS1-6d-ZFP-IN with and without a nuclear localization signal (AAVS1-6d-ZFPIN and AAVS1 -6d- ZFP-IN (-NLS)). Inserts were amplified by PCR using standard Kappa conditions, an annealing temperature of 62°C, and extension times of 40 seconds for PPT and 90 seconds for AAVS1 -6d-ZFP. -IN. The PCR products were separated 7CICI n / l 7Π7 / Β / Υ by gel electrophoresis. The amplified products were purified and an assembly protocol was carried out with a 1:2.5 ratio of base structure to insert and 5 cycles. 50 pL of the competent cells were transformed with 4 pL of the ligation product, and 60 percent of the competent cells were plated on carbenicillin plates. Initial colony verification was determined by restriction digestion and DNA gel electrophoresis. The following colonies were collected: colonies 1 and 2 (IN + PPT F1 + R, AAVS1 -6d-ZFP-IN F1 + R, AAVS1-6d-ZFP-IN (-NLS) F2 + R) and colonies 7 and 8 ( IN + PPT (STOP) F2 + R). In order to further verify that colonies contained the correct insert, colony PCR was performed with 4 mM Mg, 62-STS, and NEB standard taq. Example 8: Generation of non-integrating vectors through the insertion of a stop codon A non-integrating vector was generated by inserting a stop encoding prior to the integrase open reading frame (psPAX2-TAA-IN). psPAX2-TAA-IN was generated by site-directed mutagenesis by adding two stop codons after the protease cleavage site at the start of the integrase. PCR conditions were used for site-directed mutagenesis to create psPAX2-TAA-IN. After PCR, the reaction tubes were placed on ice for 2 min to cool. 1 pL of Dpnl was then added directly to each amplification reaction and incubated at 37°C for 5 minutes to digest the parent (unmutated) double-stranded DNA. Plasmid DNA was digested to confirm that site-directed mutagenesis did not produce any unwanted modifications. Digestion of psPAX2 and psPAX2-TAA-IN with Sacl and Agel should give rise to three bands of 7,500, 1,900 and 1,300 bp. Digestion of psPax2-AIN with Sacl and Agel should give rise to three bands of 7,500, 1,300 and 800 bp. The digestion reaction was carried out, and the digestion resulted in the correct banding pattern. Example 9: Reconstitution of wild-type integrase in an integrase-defective vector The objective was to develop the methodology to see if a non-integrating vector could recover insertion activity with the expression of different forms of the integrase fusion proteins. In order to confirm that psPAX2-ÁIN was fully functional, an integrase was added to the vector through the use of Gibson Assembly. Furthermore, in order to test whether the assembly sites are good for cloning the IN fusion, a wt-IN was cloned with the additional N terminus of IN that is in the base structure before the site (with the Leu that is not I should be there). This was also done with an additional protease target sequence to avoid this domain at the false N terminus. A PCR reaction was carried out to amplify the fragments of IN-1, IN-2 and IN-3. The products amplified by PCR were separated by DNA gel electrophoresis. Amplified bands were purified and assembly was carried out with a 1:2.5 ratio of base structure to insert and 5 cycles at 37 °C. 50 pL of the competent cells were transformed with 4 pL of the ligation product and plated on carbenicillin plates. For the generation of the construct containing IN-3, Gibson assembly was carried out following the standard protocol for the Gibson Assembly HIFi 1 step kit (via the use of the CRG 7CICI n / l 7Π7 / Β / Υ MM) (SGI-DNA, Inc., www.sgidna.com / products / gibson-assembly-reagents / ). Reaction mixtures were created and assembled for 1 h at 50 °C. Competent cells were transformed with 2 pL of the reaction mixture. pL of competent cells were transformed with 2 pL of the ligation product and plated on carbenicillin plates. Example 10: Generation of non-integrating vectors containing a truncated integrase in the C-terminus domain The C-terminus domain (CTD) integrase fragments (nucleic acids 83 to 118 of SEQ ID NO: 74) and CppT + CTD (SEQ ID NO: 74) were cloned into the vector. psPAX2. The products amplified by PCR were separated by DNA gel electrophoresis. Ligation of CppT + CTD was carried out through the use of the conditions that were used in example 9. Ligation was carried out for 5 cycles at 65 °C and the ligation product was transformed. No colonies grew. Ligation and transformation were carried out again, and three colonies were verified by sequencing with an IN-fw primer (SEQ ID NO: 81). Example 11: Generation of integrase fusion proteins Targeted integrase fusion proteins were created by incorporating HIV-1 integrase and either targeted ZFP or human Cas9 into a pcDNA3.3 expression vector. A vector was created in which the 3' end of ZFP or Cas9 was connected to the 5' end of integrase via a nucleic acid linker. A second vector was created in which the 3' end of integrase was connected to the 5' end of ZFP or Cas9 via a nucleic acid linker. The linkers used were XTEN or GGS in the range of 13,16,19, 22, 25 or 28 amino acids in length. The ZFP-integrase fusion protein was designed in such a way as to target the AAVS1 site or the T cell receptor alpha (TCRa) locus in the human genome. The Cas9-integrase fusion protein was used in combination with guide RNAs targeting the AAVS1 site or the TCRa locus in the human genome. A list of the modified integrase fusion proteins is shown in Table 5. 7CICI n / l 7Π7 / Β / Υ Table 5 List of the modified integrase fusion proteins that were generated in Example 11 Integrase DNA binding protein Target site Linker Targeting HIV-1 integrase Zinc finger protein AAVS1 XTEN or GGS 12, 16, 19,22,25,0 28 amino acids long ZFP-integrase Integrase DNA binding protein Target site Linker Targeting HIV-1 integrase Zinc finger protein AAVS1 GGS Integrase-ZFP HIV-1 integrase Zinc finger protein TCRa XTEN or GGS 12, 16, 19,22,25, 0 28 amino acids long ZFP-Integrase HIV-1 Integrase TCRa Zinc Finger Protein GGS Integrase-ZFP HIV-1 Integrase CCR5 Zinc Finger Protein GGS ZFP-Integrase HIV-1 Cas9 AAVS1 XTEN Cas9-Integrase Integrase HIV-1 Cas9 AAVS1 GGS Integrase-Cas9 HIV-1 Integrase Cas9 TCRa Example 12: Cis and trans complementation of integrase-defective lentivirus with targeted integrase fusion proteins The targeted integrase fusion proteins of Example 11 were used to complement the lack of integration capacity of the non-integrative lentivirus, with the expression of an IN with two mutations in the catalytic domain (D64V / D116N). For this experiment, targeting integrase fusion proteins were cloned into a pcDNA3.1 vector. Lentivirus was produced by cotransfection of cells with pSICO (GFP expression payload), pmd2.g (VSVG for envelope expression), pax2 (containing packaging proteins and integrase), or NILV-pax2 (containing the packaging proteins), and the pcDNA3.1 vector containing either the wild-type integrase or the targeted integrases (Table 6). 7CICI n / l 7Π7 / Β / Υ Table 6 Conditions for complementation of integrase-defective lentivirus with targeted integrase fusion proteins 7CICI n / l 7Π7 / Β / Υ Packaging / Plasmids LV LVO NILV NILV + IN NILV+ZPIN (AAVS1) NILV+Cas9_ IN (AAVS1) pSICO + 4- 4- 4- 4- psPAX2 + + psPAX2-NILV 4- 4- 4- 4- pMD2.G + 4- 4- 4- 4- 4- pHIV1 -IN 4- pZFP-AAVS1-R 4- pCas9_IN(AAVS1) 4- 6 x 105 HEK293T cells (passage 8) were seeded per well in a 6-well plate and incubated overnight. 5 hours before starting virus production, the medium was changed to 1.7 mL of medium containing 1:1000 chloroquine diphosphate (CD; supply = 25 mM). Plasmids were infected at a molar ratio of 1.6:1.32:0.72:3.32 (pSICO:pax2:VSVG:wtIN-rescue). PEI (polyethyleneimine; supply = 1 mg per mL) was used as the transfection reagent, while 3 pL of PEI was used for 1 pg of the total DNA used for transfection. DNA was diluted in 83 pL of Opti-MEM and 83 pL of PEI, mixed, and incubated for 15 to 20 minutes at room temperature. Each transfection mixture was added dropwise to the cells with the CD medium. The cells were incubated overnight and the medium was replaced the next day with 2.5 mL of fresh medium. The next day, the cell supernatant was centrifuged for 5 minutes at 1,000 rpm and passed through a 45 pm filter. The supernatant containing the virus was stored at −80°C. The first step was confirmation that the different lentivirus packages maintained the ability to infect cells in a way independent of their content. In order to determine the virus titer, 75,000 HEK293T cells were seeded per well in a 6-well plate. The cells were infected with a mixture of 1 mL of the medium containing 1:100 polybrene and 500 pL of the previously produced virus supernatant (1:3). The medium was changed the next day. The next day, the medium was aspirated and the cells were detached through the use of 200 pL of trypsin. The reaction was stopped by adding 800 pL of normal medium and analyzed by flow cytometry. Virus titer was quantified for wild-type integrase lentivirus (LV), empty viral particles (LVO), non-integrative lentivirus (NILV), non-integrative lentivirus with wild-type integrase (NILV + IN), non-integrative lentivirus with ZFP-integrase fusion protein (NILV + ZP-IN (AAVS1)), non-integrative lentivirus with Cas9integrase fusion protein (NILV + Cas-IN) and wild-type integrase lentivirus with wild-type integrase (LV + IN). LV and LVO were used as the positive and negative controls, respectively. HEK293T cells were infected and the virus titer was quantified by counting the number of GFP-positive cells (Figure 4). Results: The virus titer was within the same order of magnitude for all conditions. The overall integrative capacity of the targeted integrase fusion proteins was then determined by flow cytometry and next-generation sequencing of the target insert. HEK293T cells were infected at the same multiplicity of infection for all conditions, and GFP fluorescence was monitored at 3, 5, 7, 10, and 12 days postinfection. Seven days postinfection, cells were sorted for GFP expression. Results: On day 12, cells infected with non-complemented NILV had a lower percentage of cells expressing GFP (Figure 5) indicating a reduction in the capacity for viral production. In order to evaluate the ability for targeted integration of the tested integrase fusion proteins, genomic DNA was extracted according to the DNeasy Blood and Tissue Kit (Qiagen) protocol at day 12. Cell cultures were harvested by of centrifugation at 190 rpm for 5 min (maximum 5 x 105). The pellet was dissolved in 200 μL of PBS (phosphate buffered saline). 20 pL of proteinase K was added along with 200 pL of AL regulator. After vortexing, samples were incubated at 56 °C for 10 min. After the addition of 200 pL of ethanol (96 to 100 percent) and brief vortexing, the mixture was transferred to a DNeasy Mini spin column, placed in a 3 mL collection tube, and centrifuged at 8,000. rpm for 1 minute. The spin column was moved to a new 2 mL collection tube and 500 pL of buffer AW1 was added. The tubes were centrifuged at 8,000 rpm for 1 min. This washing step was repeated for buffer AW2 (3 min centrifugation). The centrifuge was then transferred to a new 1.5 mL microcentrifuge tube and 200 pL of buffer AE was added to the center of the spin column membrane to elute the DNA by allowing the tube to stand for 1 minute and this was followed by a 1-minute centrifugation at 8,000 rpm. The concentration of genomic DNA was quantified with a NanoDrop One. Reverse cloning was performed with oligos specific for the viral inserted LTR. Targeted next-generation sequencing was analyzed by the following parameters: read filtering, such as R1 and R2 contain the corresponding sequencing primer, restriction of checking with the leftmost bases (as many bp as the primer), allowing 2 mismatches, trimming the primer sequences (SEQ ID NO: 82 to 89), filtering the reads, such as R1 and R2, contain the corresponding LTR bases, restricting the checking with the 5 bases of the leftmost read, using the first 5 bases of LTR (following the sequencing primer) with K = 3 (means that for the ACTGA sequence 7CICI n / l 7Π7 / Β / Υ will check the read for the presence of one of the following k-mers: ACT, CTG, TGA), allow 2 bad pairings, trimming of the corresponding LTR base pairs, mapping the reads with the reference genome, the recovery of the coverage (number of reads per insertion site), the division by 2 regions where there are R1 and R2 overlapping, the addition of only one of the insertion sites if there is no overlap of R1 and R2, applying a coverage threshold, calculating coverage per 10 mb of the reference genome, and carrying out coverage plots, calculating percentage coverage for each insertion site. Results: Targeted integrase fusion proteins increased AAVS1 site coverage and the percentage of targeted insertion (Table 7 and Figure 6). As seen in Table 7, there are more numbers of reads in the target site when the insertion is performed by integrase fusion proteins; compared to IN WT, which is indicative of directed insertion. Figure 6 is a representation of the most common targeting sites in the genome for IN and ZFPJN (AAVS1); which denotes the presence of the directed insertion only in the fusion condition. ycici η / ι ζηζ / Ε / γ Table 7 Number of AAVS1 reads and percentage of insertion targeted by targeted integrase fusion proteins Sample Number of reads in AAVS1 % targeted insertion Native (LV) 6 0 Non-integrative + Native (NILV+IN) 3 0 Non-integrative (NILV) + ZFP-IN (AAVS1) 216 30 Non-integrative (NILV) + Cas9-IN (AAVS1) 71 10 A second ZFP was also generated to target a nucleic acid segment within the CCR5 gene. This zinc finger protein was fused to the HIV-1 integrase to create a CCR5-targeting integrase. Lentivirus containing this ZFP-IN was produced as described in the previous paragraphs and transduced into HEK293T (NILV + ZP-IN (CCR5)) cells (Table 6). Results: The virus titer of NILV + ZP-IN (CCR5) was similar to that of LV and NILV + IN (Figure 7A). This construct was able to produce the viral particles with the same efficiency as the other ZFPJN fusion tested (Figures 7B and C). Its ability to integrate DNA in a site-specific manner for CCR5 was not tested. In another experiment, newly cloned expression vectors for the fusion of ZFP-IN with 6d targeted to the TCRa locus and gRNA targeted to the same site (see Example 11). The assay tested whether wild-type integrase and the ZFP-integrase fusion can complement the capacity of NILV and promote selective integration of a CAR-T cassette. Jurkat cells were infected at the same multiplicity of infection for all insert particles targeting TCRa. In this experiment, virus particles were loaded with a CD19 CAR-T cassette that would result in loss of CD3 protein expression (encoded by the TCRa gene) after targeted insertion. The percentage of CD19-positive and CD3-negative cells was tracked over time. The lentivirus titer is shown in Figure 8A and the percentage of cells expressing CAR on day 3 and day 14 is shown in Figure 8B. The percentage of CD3 expressing cells is shown in Figure 8C. This indicates that transcomplementation did not work in the context of this cell line, in the absence of the VPR, an important factor for efficient IN transcomplementation. Example 13: Generation of a modified integrase by means of site-directed mutagenesis and saturation mutagenesis Modified HIV-1 integrases were generated by site-directed mutagenesis and saturation mutagenesis. For site-directed mutagenesis, a modified HIV-1 integrase will be created by mutating amino acids through site-directed mutagenesis. The QuikChange Lightning multi-site directed mutagenesis kit will be used, and the primers were designed according to the manufacturer's recommendations (SEQ ID NO: 90 to 97). The plasmid to be mutated is approximately 7,000 bp. Approximately 5 colonies per approach will be examined by sequencing. Glycerol stocks will be prepared from the colonies containing the desired plasmids. Saturation mutagenesis of HIV-1 integrase will be performed for the generation of a large pooling library of different HIV-1 integrase molecules. The protocol was adopted from Cornell et al., (Biochemistry, 57 (5) 604-613, 2018). Several forward primers containing a degenerate NNS sequence at the mutational site and a reverse primer will be used in a PCR reaction (SEQ ID NO: 90 to 97). The entire plasmid will be amplified for the generation of the mutated integrase molecules. The primers will be optimized at a melting temperature of 68°C. During the cycles, the annealing temperature will increase by 0.3 °C per cycle. A list of the amino acid mutation is provided in Table 8. Table 8 Sites of mutation of the aa sequence of HIV-1 integrase vs HIV-1 type integrase 7CICI n / l 7Π7 / Β / Υ wild NC_001802.1 - NP 705928 (SEQ ID NO: 1) Amino acid position Wild type amino acid Amino acid mutation Classifications 10 D K Residue critical for retroviral integrative recombination in a region that is highly conserved 13 E K Residue critical for retroviral integrative recombination in a region that is highly conserved Amino acid position Wild type amino acid Amino acid mutation Classifications 64 D A, E Critical residue for retroviral integrative recombination in a region that is highly conserved 94 G D, E Negative amino acids that could affect DNA binding (tested for 231E) 94 G R , K Positive amino acids that could improve DNA binding 116 D A, E Critical residue for retroviral integrative recombination in a region that is highly conserved 117 N D, E Negative amino acids that could affect DNA binding (tested for 231E) 117 N R , K Positive amino acids that could improve DNA binding 119 S A, P, T, G Positions found in other integrase vanants (taken from an alignment by Gijbers et al 2014) 119 S D, E Negative amino acids that could affect DNA binding (tested for 231E) 119 S R, K Positive amino acids that could improve DNA binding 120 N D, E Negative amino acids that could affect DNA binding (tested for 231E) 120 N R, K Positive amino acids that could improve DNA binding 7CICI Π / I 7Π7 / Β / Υ Amino acid position Wild type amino acid Amino acid mutation Classifications 122 T K, I, V, A Positions found in other integrase variants (taken from an alignment by Gijbers et al 2014) 122 T R Positive amino acids that could improve binding to DNA 124 A D, E Negative amino acids that could affect DNA binding (tested for 231E) 124 A R, K Positive amino acids that could improve DNA binding 128 A T Residue critical for retroviral integrative recombination in a region that is highly conserved 152 E A, D Residues critical for retroviral integrative recombination in a region that is highly conserved 168 Q L, A Residues critical for retroviral integrative recombination in a region that is highly conserved and integrase mutants defective for interaction with LEDGF / p75 are affected in chromosome docking and HIV-1 replication 170 E G Critical residue for retroviral integrative recombination in a region that is highly conserved 185 F K 7CICI Π / I 7Π7 / Β / Υ Amino acid position Wild type amino acid Amino acid mutation Classifications 231 R G, K Positions found in other integrase vanants (taken from an alignment by Gijbers et al 2014) 231 R D, E Positive amino acids that could improve DNA binding 231 R K Negative amino acids that could affect DNA binding (tested for 231E) 231 R S Negative amino acids that could affect DNA binding (tested for 231E) 264 K R Acetylation of IN Acetylation of HIV-1 integrase by p300 regulations of viral integration 266 K R Acetylation of IN Acetylation of HIV-1 integrase by p300 regulations of viral integration 273 K R Acetylation of IN Acetylation of HIV-1 integrase by p300 regulations of viral integration JCICI n / l 7Π7 / Β / Υ Example 14: Generation of pRRLVPR integrase constructs and testing of transcomplementation efficiency in HEK293T cells The pRRLIN, pRRLVPRIN and pRRLINGFP vectors will be generated to be used in VPR transcomplementation (Table 9). Table 9 pRRL constructs GFP(-) GFP(+) VPR(-) pRRL_IN pRRL_IN_GFP VPR(+) PrrIVIN pRRLVINGFP The constructs were tested through the use of a GFP expression assay. HEK293T cells were transfected with pSICO MAXI, pSICO MINI, and pRRL INGFP to test episomal expression of pRRLINGFP. Expression of the VPRINGFP construct in lentivirus-producing cells was detected as positive. The transcomplementation efficiency was then tested in HEK293T cells. The LV medium was ultracentrifuged, allowed to settle for resuspension, and cells were seeded. Infection was carried out in a volume of 0.6 mL (1.5 * 0.4). Polybrene was added. The titer was determined by cytometry. The title (1:100) is shown in figure 9. The VPR transcomplementation system will be used to compare integrase sequences modified for integration. In Examples 15 to 19 later in this document, different fusion protein constructs were generated with the modified hyperactive PiggyBac transposase. The total and targeted transposition activity of the constructs was determined, leading to relevant results especially for the hCas9-mutated PB constructs. Tests for the generation and determination of targeted transposition activity of mutated PB and ZFP fusion protein constructs are also provided. Different linkers are tested, which show that XTEN performed better than the rest of the linkers tested. 5GGS and 7GGS also performed correctly, indicating that the length of the linker and its flexibility play an important role in its performance. Example 15: Methods for the generation of fusion proteins with the modified hyperactive PiggyBac transposases and determination of the efficiency of directed transposition Transfections: Hek293T cells were seeded the day before to reach 70 to 80 percent confluence on the day of transfection (typically 290,000 cells in the p12 well plate). Transfections were carried out through the use of Lipofectamine 3000 reagent following the manufacturer's instructions or PEI in a 1:3 DNA-PEI ratio in the OptiMem. Programmable transposase (PT), gRNA, and transposon plasmids were transfected together in a ratio of 1 PT:2.5 gRNA:2.5 transposon. Cells were passaged and maintained to the desired end point depending on the experiment. Generation of PB mutants: Different mutations were introduced into the hyPB sequence fused to Cas9 (hCas9_PB plasmid) by site-directed mutagenesis following the instructions of the Agilent Quickchange Lightning Mutagenesis Kit. The primers were designed with the QuikChange primer design to achieve the following mutations: PB R245A, PB R275-277A, PB R388A, PB S351 A, PB W465A, PB R372A-K375A, PB D450N (SEQ ID NO: 100 to 106) . Cas9 activity: The transposase plasmid programmed with Cas9 nuclease and the gRNA plasmid were transfected together at a ratio of 1:2.5. Cells were harvested after 48 h and genomic DNA was extracted. PCR was carried out with primers targeting 150-200 bp around the gRNA target site (NGS-aavs fw and NGS-aavs rv, SEQ ID NO: 98 and 99). IIlumina adapters and barcodes were introduced into a second PCR and miseq sequencing was typically carried out in one cell 7CICI n / l 7Π7 / Β / Υ 2 x 250 Nano flow. Results were analyzed with the CRISPR-GA web tool. Genetrap Test: A promoterless RFP transposon preceded by a splicing acceptor was produced and gRNAs targeting PPR1 alpha and CD46 intron 1 were designed and cloned under the regulation of the U6 promoter. RFP fluorescence would only be detected if the transposon were inserted into the targeting regions or into other regions of the promoter by chance. For the genetrap assay, Hek293T cells were transfected with genetrap transposon, programmable transposase, and gRNA, and the RFP signal was analyzed by flow cytometry. Split GFP Reporter Cell Line: The 293T reporter cell lines were produced for targeted transposition evidence experiments. Briefly, the cell line has a target region (with different gRNA and ZFP target sequences) and a splice acceptor sequence followed by half of a GFP coding sequence. This cell line was generated by random insertion of the reporter cassette through the use of the hyperactive version of the Sleeping Beauty transposase, SB100X. Targeted introduction of a transposon with the first half of the GFP sequence with a promoter and a splice donor results in a GFP signal detectable by flow cytometry. A second transposon containing half of the GFP sequence and a complete RFP sequence preceded by the constitutive EF1 alpha promoter was generated in order to evaluate targeted versus random insertion. Around 15 days after transfection there was good decay of the episomal signal allowing analysis of total insertion (RFP signal) versus targeted insertion (GFP signal). Example 16: Generation of plasmid constructs of fusion proteins with modified hyperactive PiggyBac transposases Different plasmid constructs were cloned to achieve a fusion between a DNA-targeting programmable element (Cas9, ZNF) and a mammalian transposase (PiggyBac, SB100). The linker between the two modules was variable in the different constructions, chosen from a library of linkers with SEQ ID NO: 50 to 63. The constructions are shown in Table 10. 7CICI n / l 7Π7 / Β / Υ Table 10 List of fusion proteins generated Cas9 and hyPB fusions Cas9 and SB100 fusions ZFN and hyPB fusions Fusions with hyPB mutations - hcas9_hyPB - hcas9_SB100 - ZFN_hyPB - hcas9_ hyPB_D450N 4GGS linker, - ncas9_hyPB - ncas9_SB100 -hyPB_ZFN ncas9_hyPB_D4 50N 4 ggs linker, - dcas9_hyPB - dcas9_SB100 dcas9_hyPB, D450N 4 GGS linker - hyPB_hcas9 - SB100_hcas9 - hcas9_hyPB_D450N-R372-375A 4 GGS - hyPB_ncas9 - SB100_ncas9 linker, ncas9_ hyPB_D450N-R372-375A 4 Cas9 and hyPB fusions Cas9 and SB100 fusions ZFN and hyPB fusions Fusions with hyPB mutations - hyPB_dcas9 - SB100_dcas9 GGS linker, dcas9_ hyPB_D450N-R372375A 4 GGS linker - hcas9_hyPB with the following mutations: R245A, R275-277A, R388 A, S351A , W465A - ZFP_ hyPB D450N - hyPB D450N ZFP - ZFP hyPB D450N-R372-375A - hyPB D450N-R372-375A_ZFP 7CICI Π / I 7Π7 / Β / Υ hcas9: human cas9 nuclease codon optimized; ncas9: human nicase cas9 codon optimized; dcas9: human dead cas9 codon optimized. Example 17: Transposition efficiency of different linkers Hek 293T cells were transfected with hcas9_PB constructs with different linkers in length and structure (linker library) and with 2 different gRNAs (AAVS1 1 and AAVS1 2). Genomic DNA was extracted 48 hours after transfection, and the target region was amplified by PCR and sequenced with miseq lllumina sequencing. Results: Constructs with different lengths and linker structure do not obstruct cas9 nuclease activity. The 4GGS linker provides higher cas9 activity at both gRNA target sites compared to hcas9 activity (Figure 11). Example 18: Targeted rearrangement of fusion proteins with modified hyperactive PiggyBac transposases 18.1. GeneTrap: The targeted transposition activity of the hcas9_PB construct (hcas9 linked to hyPB through the use of different linkers described in the previous paragraphs) was evaluated by means of a transposon genetrap. The genetrap transposon contains a promoterless RFP sequence preceded by a splice acceptor sequence that can only be expressed if inserted into a promoter region after a splice donor. The genetrap transposon was cotransfected with PPR 1 intron 1 gRNA and the transposase programmed with different linker constructs. The results were analyzed 10 days after transfection by RFP fluorescence through the use of flow cytometry. Results: Targeting activity was increased with programmed transposase compared to the random insertion of hyPB which has more fluorescence in the condition transfected with programmed transposase than in the condition transfected with wild-type hyPB. 8ggs, XTEN linkers increased genetrap targeting activity compared to the other linkers (Figure 12). Split GFP Reporter Cell Line: 18.2 Directed transposition of hcas9_PB with different linkers The targeted transposition activity of the hcas9_PB construct was evaluated through the use of a reporter cell line. The hcas9_PB construct with different linkers was transfected with AAVS1 3 or TCR1 alpha gRNA and GFP half transposon. Results: No major differences were observed with respect to the transposition of the constructions of the different linkers (Figure 13). 18.3. Directed transposition of the selected mulants: PB 450 and PB 372-375-450 were selected for further directed transposition experiments due to their good directed transposition efficiencies. The experiments were carried out as mentioned in the previous paragraphs through the use of aavsl 3 and tcr 1 gRNA. Results: The targeted rearrangement of hcas9_PB 450 and hcas9_PB 372-374-450 was 6 to 10 times higher compared to hcas9_PB with the WT hyPB sequence. The hcas9 + hyPB transfected on separate plasmids showed some targeting activity, while hyPB without hCas9 showed 0 activity, indicating that the split GFP reporter cell line is a robust method for targeted insertion for selection of variants carrying perform this function on the noise of Ther methods that are not specific enough (figure 15). 18.4. PB mulants selected by directed and random transposition: Targeted and random transposition were evaluated through the use of a dual RFP-GFP transposon mentioned in the previous paragraphs for the selected mulants of example 19.4. Red fluorescence indicates full insertion (where RFP is constitutively expressed) around 15 days after transfection (to ensure a non-episomic signal), and GFP fluorescence indicates targeted transposition. Results: Figure 16 illustrates that greater directed transposition compared to random transposition was demonstrated in both the hcas9_PB D450N selected mulants and the hcas9_PB R372A K375A D450 selected mulants compared to hcas9:PB with the wt hyPB sequence. The total transposition efficiency is lower in both muienles and the directed results are consistent with Figure 15. 18.5. Directed transposition ZFP-PB constructs: Constructs for zinc finger fusion proteins with hyperactive PiggyBac were cloned through the use of ZFP targeting the tcr4 sequence present in the split GFP reporter cell line and hyPB or hyPB with the D450N mutations. Cells were transfected with the combinations of ZFP-PB and GFP transposon medium following the protocol of Example 15. The GFP signal was analyzed 5 days after transfection. Results: Directed transposition above background (random insertion of hyPB) was observed in all constructs. Results: Directed transposition is greater in ZFP in the N-terminus position for both hyPB and hyPB D450N (Figure 18). The ZFP sequence for these experiments corresponds to a 6-domain finger protein with the nucleic acid and amino acid sequences of SEQ ID NO: 117 and 118, respectively. 7CICI Π / I 7Π7 / Β / Υ In Example 20 below in this document, a library of PB mutations was designed and subjected to a screening method to identify PB modified for positive targeted transposition. Some hits were identified and validated for the modified PB with positive directed transposition. Example 20: Generation of a hyperactive PiggyBac mutation library and detection of targeted transposition Methods: A hyPB mutation library was designed and purchased from Twist Biosciences. 7CICI n / l 7Π7 / Β / Υ Table 11 Mutation sites for hyPiggyBac Position Wild-type amino acid Mutation 245 R A 275 R A 277 R A 325 G A 347 N A, S 351 S E, P, A 372 R A 375 K A 388 R A 450 D N 465 W A 560 T A 564 s P 573 s A Position Wild-type amino acid Mutation 589 M V 592 S G 594 F L 7CICI n / l 7Π7 / Β / Υ Detection method: A screening method was designed to identify PiggyBac variants from the library of engineered mutants that bound to a targetable DNA binding protein such as Cas9 and specific targeted rearrangements were carried out. A schematic of the detection method is shown in Figure 19. The PB library was cloned by Golden Gate assembly through the use of the Esp3l enzyme into a SIN lentiviral transfer plasmid containing hCas9 and an XTEN linker followed by the Esp3l cloning sites before an NLS to achieve hCas9_XTEN_PB_NLS fusion protein under the regulation of CMV promoter. About 6,000,000 colonies were harvested after ElectroMAXMRStbl4MRa competent cells from Invitrogen electroporation, and the plasmid was extracted with Maxiprep through the use of HiPure Maxiprep kit, LifeTechnologies. Lentiviruses were produced (through the use of the helper plasmids pMD2.G and psPAX2 purchased from Addgene) through the use of the Addgene lentivirus production protocol. The lentiviruses were ultracentrifuged and titrated by copy number analysis qPCR (with the oligonucleotides of SEQ ID NO: 107 to 110). Briefly, 80,000 Hek293T cells were seeded the day before in 12-well plates. Cells were infected with the library lentiviruses and the standard GFP lentivirus at dilutions of 1 / 2, 1 / 10 for the library lentiviruses and 1 / 50, 1 / 100, 1 / 1000 for the GFP lentiviruses. The GFP signal was analyzed 3 days postinfection by flow cytometry. The cells were harvested and gDNA was extracted. The qPCR assay was designed in such a way as to assess the copy number of the WPRE gene and was normalized by the copy number of the RNase gene. Hek293T reporter cells were infected at an MOI of 0.8, in 500 cm2 square dishes through the use of polybrene at 1:1000, and 10M cells were plated the day before. At 3 to 4 days postinfection, cells were transfected with 8.1 pmol of AAVS1 gRNA plasmid and 1 / 2 GFP transposon through the use of PEI at 1:3. cm. From 3 to 4 days after transfection the cells were selected through the use of the FACSAria cytometer with a 0.70 pm nozzle. A control transfection was carried out in the 10 cm dish using RFP and GFP plasmids with the same molarity, and analyzed on a Fortessa cytometer to determine GFP-RFP positive cells. After selection, gDNA was extracted directly. Different sequencing methods were used to analyze PB mutants with a positive targeted rearrangement: PiggyBac Library Region Targeted Sequencing: The 1116 bp PiggyBac region with all library variants was amplified by PCR with the NGS cluster 1 forward and NGS cluster 2 reverse primer cluster through the use of ΚΑΡΑ HiFI Hotstart ReadyMix. The lllumina adapters and barcodes were added in a second PCR, and the NEBNext 9 primer and custom lllumina barcodes (SEQ ID NO: 111 to 114) were used. Targeted sequencing was performed on lllumina v2 or v3 miseq flow cells. The I7 index primer was replaced with a custom-made primer to allow complete sequencing of the different variants. Generation and sequencing of the genomic library of PiggyBac and Cas9 sequences: A 6000 bp PCR was carried out from the genomic DNA of the cells classified as positive for GFP with the primers of CMV-F and SV40 pA in reverse (SEQ ID NO: 115 and 132), and with the amplification of Cas9 and PB sequences with ΚΑΡΑ HiFi HotStart ReadyMix. DNA was then purified using the Qiagen Gel Extraction Kit and fragmented to 500 bp using Covaris S220 and Crimp-Cap Microtube AFA Fiber. The genomic library was prepared using the ΚΑΡΑ Hyperprep kit according to the manufacturer's instructions. RESULTS: 20.1. HyPB Library Diversity Generation: 1 / 2 GFP reporter cell line was infected at an MOI of 0.8 with lentiviruses containing hcas9_PB with mutations from the PB library. At 3 days postinfection, cells were transfected with the AAVS1 gRNA transposon 3 and 1 / 2 GFP with a transfection efficiency of 75 to 90 percent. In a first experiment, a total of 254M cells were classified and 185,757 positive cells were obtained that showed 0.073 percent of positive variants for directed transposition. In a second experiment, 120M cells were sorted and 70,974 positive cells were obtained that showed 0.059 percent positive variants for targeted transposition (Figures 21A and 21B). Genomic DNA was extracted directly from cells classified as positive and negative. 2 / 3 of the DNA obtained was processed for targeted sequencing analysis and 1 / 3 was processed as a genomic library sequencing as specified in the previous paragraphs in the Methods section of this example. 20.2. Screening analysis of the hyPB library by targeted variable region sequencing: Analysis of Cas9-PB variants positive and negative cells was analyzed as follows. Reads from targeted sequencing were mapped against the reference sequence. All library variation positions were recovered through the use of two different approaches: by position, through the use of aligned reads, and by sequence, through the use of a pattern match to the surrounding sequence. . The log fold change of all variant counts was calculated between positive (GFP-positive cells with targeted integration) and negative (non-targeted integration samples, regardless of whether 7CICI n / l 7Π7 / Β / Υ integration occurred or not), and the superior variants were recovered. Additionally, the negative selection of those samples with random integration was carried out with a positive selection for RFP; where the transposon was inserted randomly into the genome. The results are shown in Figures 22A to 22K. Therefore, through the use of an unsupervised high-throughput screening approach of a pooling library of variants, we identified a collection of PiggyBac mutants capable of performing site-directed insertion with high efficiency. , as indicated by the comparison of the presence of positive versus negative cells in the population. Targeted and random transposition of the top positive hit in repeat 1 was then evaluated through the use of a dual RFP-GFP transposon mentioned in the previous paragraphs. Red fluorescence indicates full insertion (where RFP is constitutively expressed) around 15 days after transfection, and GFP fluorescence indicates targeted transposition. Results: Higher directed transposition compared to random transposition was shown in Top 1 of repeat variant 1 compared to hcas9_PB and wt hyPB (Figures 23A and 23B). Independent validation of the insertion into the target was carried out through the use of our reporter cell line, and significant activity was observed in the target compared to the WT version and the D450N mutant. 20.3. Identification of overrepresented positive hits: The selection identified several positive hits that are overrepresented in the GFP population against the variants that were selected as negative. Some of them were also not found in the RFP population representing general insertion, indicating an increase in the capacity for integration. Additionally, the RFP includes random and targeted integration. Therefore, a collection of combination mutants for PiggyBac capable of carrying out site-directed insertion with high efficiency was identified (Figures 24A to 24C). 20.4. Screening analysis of the hyPB library by means of library sequencing: For library sequencing, reads were mapped against the reference sequence, variant calling was performed with recovery of all variations from the reference, and Euclidean distance and correlation between allele counts were calculated. positive and negative. The most different positions were recovered as variants; and the association between these variables was calculated. Results: In addition to the variants included in the library design, the variants that were introduced randomly by lentiviral reverse transcriptase during the generation of the viral library were analyzed. Some of these new variants were associated with the positive hits and probably carry out targeted integration on the pool, and perhaps need to be present in the mutant form in the variant version of hyPB to carry out targeted integration. The example of D450N and W465A is shown in figure 25. ycici η / ι ζηζ / Ε / γ The mutated PB sequences identified in Example 20 are listed in Table 12 (SEQ ID NO: 120 to 129). 20.5. Validation of hyPB library selection: Directed and random transposition of several combinations of unique mutations observed in Top 1 -1 identified in the positive hits of the selection (Unilarge-A, -B, -C and UnilargeD) were evaluated through the use of a dual RFP transposon -GFP mentioned in the previous paragraphs. Red fluorescence indicates full insertion (where RFP is constitutively expressed) around 15 days after transfection, and GFP fluorescence indicates targeted transposition. Results: In all cases an increase in targeted insertion relative to overall integration was observed for Cas9 fused with different combinations of hyPB mutants with the 4GGS linker (Unilarge-A: D450N; Unilarge-B: R245A / D450N; Unilarge-C: R245A / G325A / D450N / S573P; Unilarge-D: R245A / G325A / S573P) compared to the fusion of Cas9 to the WT version of hyPB. Some of the mutant combinations tested (R245A / G325A / D450N / S573P) had a large increase in targeted insertion that was up to 30 percent of total integrative events instead of 3 percent in the hyPB fusion (Unilarge C) (figure 26). Example 21 below provides an overview of the development status of the different integration-deficient viral vectors, as well as the best transcomplementation system; and data on transcomplementation with IN fusion proteins. Example 21: Transcomplementation of different integrase-deficient systems For the generation of an efficient transcomplementation system to test IN fusion proteins, the efficiency of viral production and its capacity for integration were evaluated through the infection of the different conditions of the integration-deficient virus and the transcomplemented virus. in Hek293T and Jurkat cells. Cells were passaged for 7 days until no episomal signal was detected, and GFP signal was analyzed by flow cytometry on days 2, 5, and 7. Results: Different production efficiencies could be detected for different systems, where the NILV was the closest to the WT after production. In all cases, a clear rescue of the integration activity could be seen when transcomplementation with WTHIVJN was carried out. (figure 27). Proof that IN was loaded into the transcomplementation system was obtained by Western blot. 7CICI n / l 7Π7 / Β / Υ Table 12. Sequences, na sequence denotes nucleic acid sequence and aa sequence denotes amino acid sequence. SEQ ID NO: SEQUENCE NAME SEQUENCE 1 Wild-type HIV-1 integrase, aa sequence NC_001802.1 - NP 705928 FLDGIDKAQDEHEKYHSNWRAMASDFNLPPVVAKEIVA SCDKCQLKGEAMHGQVDCSPGIWQLDCTHLEGKVILVA VHVASGYIEAEVIPAETGQETAYFLLKLAGRWPVKTIHTD NGSNFTGATVRAACWWAGIKQEFGIPYNPQSQGVVES SEQ ID NO: SEQUENCE NAME SEQUENCE MNKELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIG GYSAGERIVDIIATDIQTKELQKQITKIQNFRVYYRDSRNP LWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYG KQMAG D DCVASRQDE D 2 Wild-type HIV-1 integrase, na sequence NC_00 1802.1 tttttag atg g aatag ataaggcccaag atg aacatg ag aaatatcacag taat tggagagcaatggctagtgattttaacctgccacctgtagtagcaaaagaaat agtagccagctgtgataaatgtcagctaaaaggagaagccatgcatggaca agtagactgtagtccaggaatatggcaactagattgtacacatttagaagga a aagttatcctggtagcagttcatgtagccagtggatatatagaagcagaagttat tccagcagaaacagggcaggaaacagcatattttcttttaaaattagcaggaa gatggccagtaaaaacaatacatactgacaatggcagcaatttcaccggtgct acggttagggccgcctgttggtgggcgggaatcaagcaggaatttggaattcc ctaca atccccaaagtcaaggagtagtagaatctatgaataaagaattaaag aaaattataggacaggtaagagatcaggctgaacatcttaagacagcagtac aaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtac agtgcaggggaaagaatagtagacataatagcaacagacatacaaactaa agaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacaggga cagcagaaatccactttggaaaggaccagcaaagctcctctggaaaggtga aggggcagtagtaata caagataatagtgacataaaagtagtgccaagaag aaaagcaaagatcattagggattatggaaaacagatggcaggtgatgattgt gtggcaagtagacaggatgaggattag 3 Modified HIV-1 integrase, aa sequence SEQ ID NO: 1 With D10K, E13K, D64A, D64E, G94D, G94E, G94R, G94K, D116A, D116E, N117D, N117E, N117R, N117K, S119A, S119P, S119T, S119G, S119D, S119E, S119R, S119K, N120D, N120E, N120R, N120K, T122K, T122I, T122V, T122A, T122R, A124D, A124E, A1 24R, A124K, A128T, E152A, E152D, Q168L, Q168A, E170G, F185K, R231G, R231K, R231 D, R231E, R231S, K264R, K266R, K273R, or any combination thereof 4 Modified integrase, aa sequence with impaired DNA binding SEQ ID NO: 1 With G94D , G94E, G94R, G94K, N117D, N117E, N117R, N117K, S119A, S119P, S119T, S119G, S119D, S119E, 7CICI Π / l 7Π7 / Β / Υ SEQ ID NO: SEQUENCE NAME SEQUENCE S119R, S119K, N120D, N120E, N120R, N120K, A124D, A124E, A124R, A124K, R231G, R231K, R231D, R231E, R231K, or any combination thereof 5 Modified integrase, sequence aa with enhanced DNA binding SEQ ID NO: 1 With G94D, G94E, G94R, G94K, N117D, N117E, N117R, N117K, S119A, S119P, S119T, S119G, S119D, S119E, S119R, S119K, N120D, N120E, N12 0R, N120K, T122K, T122I, T122V, T122A, T122R, A124D, A124E, A124R, A124K, R231G, R231K, R231D, R231E, R231S, or any combination thereof 6 Modified integrase, aa sequence with acetylation mutations SEQ ID NO : 1 With K264R, K266R, K273R, or any combination thereof 7 Modified integrase, aa sequence with mutations in retroviral integrative recombination SEQ ID NO: 1 With D10K, E13K, D64A, D64E, D116A, D116E, A128T, E152A , E152D, Q168L, Q168A, E170G, or any combination thereof 8 Modified integrase with mutations in HIV-1 replication, aa sequence SEQ ID NO: 1 With Q168L and / or Q168A 9 Hyperactive PiggyBac, aa sequence MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDD VQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSL ASNRILTLPQRTIRGKNKHCWSTSKPTRRSRVSALNIVR SQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISL KRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMST DDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTL RENDVFTPV RKIWDLFIHQCIQNYTPGAHLTIDEQLLGFR GRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLG RGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSI PLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVG TSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINEST 7CICI Π / I 7Π7 / Β / Υ SEQ ID NO: SEQUENCE NAME SEQUENCE GKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWP MALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNL YMGLTSSFMRKRLEAPTLKRYLRDNISNILPKEVPGTSD DSTEEPVMKKRTYCPSKIRRKASASCKKCKKVICRE HNIDMCQSCF 10 PiggyB modified hyperactive ac, aa sequence SEQ ID NO: 9 With R245A, D268N, R275A / R277A, K287A, K290A, K287A / K290A, R315A, G325A, R341A, D346N , N347A, N347S, T350A, S351E, S351P, S351A, K356E, N357A, R372A, K375A, R372A / K375A, R388A, K409A, K412A, K409A / K412A, K432A, D447A, D447N, D450N, R460A, K461A, R460A / K461A , W465A, S517A, T560A, S564P, S571N, S573A, K576A, H586A, I587A, M589V, S592G, F594L, or any combination thereof With D268N and / or D346N 12 Modified hyperactive PiggyBac, aa sequence with mutations in the amino acids that are critical for cleavage SEQ ID NO: 9 With K287A, K287A / K290A, R460A / K461A, or any combination thereof 13 Hyperactive PiggyBac modified, aa sequence with mutations that are involved in target binding SEQ ID NO: 9 With S351E, S351P, S351A, K356E, or any combination thereof 14 Modified hyperactive PiggyBac, aa sequence with mutations that are SEQ ID NO : 9 With T560A, S564P, S571N, S573A, M589V, S592G, F594L, or any combination thereof 7CICI Π / I 7Π7 / Β / Υ SEQ ID NO: SEQUENCE NAME SEQUENCE critical for integration 15 Modified hyperactive PiggyBac, aa sequence with mutations that are involved in the alignment SEQ ID NO: 9 With G325A, N347A, N347S, T350A, W465A, or any combination thereof 16 Modified hyperactive PiggyBac, aa sequence with mutations in well-conserved amino acids SEQ ID NO: 9 With K576A and / or I587A 17 Modified hyperactive PiggyBac, aa sequence with mutations involved in binding to Zn2+ SEQ ID NO: 9 With H586A 18 Modified hyperactive PiggyBac, aa sequence with mutations that are involved in integration SEQ ID NO: 9 With R315A, R341A, R372A, K375A, or any combination thereof YFQAQRRNKKEEWITLRDAIQKAGFPLSNGTSALFEETK EKRRHENITLSILGWEITKFLQVKDVWPQLAIIGHHGNFS APGFLSDEDDLEDIEDIFDDNGWSPTHELLVSSLLQAVG LEKQPEIKHISPASAILISGLVVLADRIASQSEMASDGLQA LQKEELFFHQPEKWIANRKAFCREIIENTVGTYHPWESE AAGIRAVLGDYEPRFTQKAALNAGDGLFNVMETTGAGK TEAALLRHVKRKERLLFFLPTQATTNAIMDRIGKIFDGTP NVASLAHGLAVTEDFYAHPILPVQGSSDDANYKDNGGL YPTEFVRSAGTPRLLAPVCVGTIDQALMGALPSKFNHLR 7CICI Π / I 7Π7 / Β / Υ SEQ ID NO: SEQUENCE NAME SEQUENCE LLALANAHVVVDEVHTMDQYQSELMSGLLEWWSATDT PVTLLTATMPAWQREKFHLSYTGKDPHFKGVFPSLEDW STPSKNTETSQENIPTEAFTIPINIDKIAHNEIVDSHVQWV IEQRKLFPQARIGIICNTVGRAQSIAEALAHESPIVLHSRM TAGHRKEAATKLEQAIGKK GTANATLVIGTQAIEASLDID LDLLRTELCPAPSLIQRAGRLWRRLDPQREVRVPGMVG KKLTIAVVDSPSTGQTLPYLRSQLYRVESWLKQRDRIEF PADIQDFIDATTPGLQELFQKVSLPEDCGSAEEREALAD DYLNEVASWVTKQRQAGTSRIDFAKHGKPRQVLASDC VVEDFLQITSANNLEESATRLIDYPSISAILCDPTGTIP GA WTDSVEKLIAISAKDSESLRRALRASISIPHSKKFLPITSR EIPLSEAKTLLSGYSAVHIQPDEYDLQSGLKGPQK 20 Cas9 from Corynebacterium diphtheria, aa sequence MNPHEELWAKQKGLAKPYPLLAHLLDSAAVAGALWDH WLRQDLRQMFIEELGSNAREIIQFVVGSHDIGKATPLFQ YQKAQKG EVWDSIRYAIDR TG RYQKPLPSSYLVKKTSG GPNRHEQWSSFASKNEYLKPSAAAKENWIGLAIGGHH GRFEPVGYGRHQRKAAEDLAKSGWSAAQQDLLRALEK ASGITRASLPSELSPELTLVLSGLTILADRISSTESFVITGA RMIDDGTLHLATPIDWLKTRKLDSEKHVAKTVGIYHGWN NHESAIHSILKGYDPRPLQTIALQNQVGLLNLMAPTGNG KTEAAILRHSLKENDRLIFLLPTQATSNAIMRRVQGIYSD TPNAAALAHSLASVEDFYQTPLSVFDDHYDPSKEQFES SMSGGLYPSSFVCSGAARLLAPICIGTVDQALATALPGK WIHLRILALANAHIVIDEVHTLDHYQTALLENILPILAKLKT KITFLTATMPSWQRTKLLTAYGGEDLQIPPTVFPAAETVL PGQFNRTLIDSDST TIDFTMEETSYDHLVESHVKWHQTT RLNAPHARIGLICNTVKRAQEIAAALEKTNDRIVLLHSRM TTEHRRRSAELLESLLGPNGNRKTITVVGTQAIEASLDID LDILRTELCPAPSLVQRAGRVWRRNDPYRSSRITADHK PISVVFIAEAKDWQVLPYLRAETSRTQRWLEKHNQMFL PQMAQEFIDAATVDLDTATSEMDLDA LALMGIHLMKAD GAKARIQDVLNSDSKVSDFALLTSKNEIDEAQTRLIEEGT HLRIILGDENESIPGGWKHGLSSLLKLKASDRESLRTALL ASIPLLVSEKQKQLLYQHNLVPLSSSKTVLAGFYFLPKA QNFYSKNLGFIWPEEKD 7CICI Π / I 7Π7 / Β / Υ SEQ ID NO: SEQUENCE NAME SEQUENCE 21 Cas9 from Spiroplasma syrphidicola, aa sequence MNYKKLILGLDLGIASCGWAVTGQMEDGNWVLDDFGV RLFQTPENSKDGTTNAAARRLKRGARRLIKRRKNRIKDL KNLFEKINFINKASLDKYINEHSATNLVEDFNRHELYNPY FLRSIGITEKLTREELVWSLIHIANRRGYKNKFAFDIEGD GKKRETKLDEAISNALISSNLTISQEIVRNKKFRDAKNKK ALLVRNKGGKEGENNFQFLFARDDYKKEVDLLLAKQAK FYPELTEEIRAKAADIIFRQRDFEDGPGPKKQELREIYKK ENKQFSKNFTQLEGRCTFLRELSVGYKSSILFDLFHIISE VSKISKYIEENDQLAQDIISSFLYNEAGKKGKTLLKEILKK HHINDDIFDTN AYKNIDFKTNYLNLLKEVFGNDVLKNLSL NRLEDNIYHQLGFIIHTNITPERKEKAINQWLLENNIILAKE KLNILLKPNSSISTTVKTSFKWMSIAISNFLKGIPYGKFQA QFIKEDNFKLPESYAKQYQKYLTGEKTFEMFAPIIDPDL WRNPIVFRAINQARKVIKKLFEKYTFIDQINIELTREMGLS FSDRKKVKER QDDSLKENAKAKEFLMANGIIVNDTNVLK YKLWIQQNKKSLYSGKEITIADLGASNVLQIDHIIPYSKLA DDSFNNKVLVFSKENQEKGNQFADQYVKSLGTENYNN YKKRVNYLLFQNQINQKKAEYLLCSNQNEEILNDFVSRN LNDTRYITRYVTNWLKAEFELQSRFGLAKPKIMTLNGAIT SRFRRTWLRNSPWGLEKKS 22 Cas9 from Prevotella intermedia, aa sequence MKRILGLDLGTTSIGWALVNEAENNNEASSIVRLGVRVN PLTVDEKSNFEKGKAITTNADRQLRHGARINLQRYKLRR QNLHDCLQKQGWLGTEAMYEEGKASTFETYKLRAKAA EEEISLHEFARVLFMLNKKRGYKSNRKANNKEDGQLFD GMTIAKKLYEEHLTPAEYSLQLLNKGKKFTQGYYRSDLN AELERIWDEQKKYYPEILTDEFKQQLEGKTKTNTSKIFLA KYGIYSADLKGLDRKFQPLKWRVEALQQQVDKEVLAFVI SDLKGQIANTSGLLGAISDRSKELYFNKQTVGQYLWASL EENPHISIKNKPFYRQDYLDEFEKIWETQAAFHK QLTPE LKQEIRDIIIFYQRPLKSKKSLISVCELEQRKVKATIDGKE KEITIGPKVAPKSSPVFQEFRIWQNLNNVLLIDNDTNEKR PLDEVERNLLYKELSIKAKLSKTEALKILNKKGKQWDLNY RELEGNRTQAILFDCYNRIITLTGHEECDFKKIKASEIRHY VSTIFKNLGFSTEILDFDPSLKKHELEKQPMYQLWHL LY SYESDNSRTGNESLLRKLETTFGFPEEYATVLCDVVFEE 7CICI Π / l 7Π7 / Β / Υ SEQ ID NO: SEQUENCE NAME SEQUENCE D YG N LS V KAM R EIL P YLQ AG N D YSQ AC A YAG YN H S R H SLTKEELDQKVYKERLELLPKNSLRNPVVEKILNQMINVI NAIIDEYGKPDEIRIEMARELKSSAADRKKTTHAISQGNA ENQRIREILEKEFSLSYISRNDIIKYKLYEELEPNYYKTLY SD TYITKDKLFSKDFDIEHIIPKARLFDDSFSNKTLEARNI NLEKSNKTAFDFIKEKYGEDGAEAYKKKLDMLLENDAIS RPKYNNLLRAEADIPSDFINRDLRNTQYIAKKACEILGEL VKTVTPTTGKITNRLREDWQLVDVMKELNFEKYEKLGLT EIVEDRDGRKIKRIKDWTKRNDHRHHAMDALAIAFTKPS FIQYLNNLNARSNKG DSIYAIENKELHYEEGKLRFNAPIP VNEFRAEAKRHLSAILVSIKAKNKVMTQNVNKIKTKHGIIK KIQLTPRGPLHNETIYGTKMRPIIKMVKVGAALDEATINK VSSPAIREALLKRLNEYSGNAKKAFTGKNTLEKNPIYLNA GRTKTVPSLVKTVEWESFHPTRKLIDKDLNVDKVVDKGI REILKARLEEFNGDAKK AFSNLEENPIYLDEAKKIALKRV SIEGVLSAIPLHTLKNQAGKPITGKDGKPVLGNYVQTSN NHHIAFYYDEDGNLQDNAVSFFEAAERKSQGIPVIDKDY NRDKGWRFLFTMKQNEYFVFPNEATGFIPSEVDLTDEA NYGIISPNLYRVQKVSRIDKGTSASRDYWFRHHLETILN DDAKLKNLAFKRIRGLLELKDIIKVR INSTGKIVAVGEYD 23 Cas9 from Spiroplasma taiwanense , aa sequence MWSRKILKAGSRLFDEANLSDKIASKRREQRGRRRNLR RKITWKQDLINLFVKYNFLQKENDFYELDFNFDLLELRKK AINSKIELEQLLIILFNYIKHRGSFNYREDLSELKNISQEEL ETSSEFKLPVDIQFELKEENNKFREINNEKSLINHEWYVK EINLILDAQIENKLINLDFKKDYLKLFNRKREY YDGPGPK DKNLLNPSKYGWKNQEEFFDRFAGKDTYDSKEQRAPK HSLTSYLFNILNDLNNLSINGDRNQLTYENKKDLINLTLIN QKEKAENITLKKIAKYLKINEKNITGYRLKPNSNESIFTVF ESANKMRSILVKNNKSIDFICLENIDKIDKIVDILTKYQSIE DKSLKLEELNFDFFDKETCEKLAVISLTGTH ALSKKTMSK LIEEMFHDNLNHMEALAKLKIKPDYKLKVDLTNFKTIPILR EKINEMYISPVVKRALIESLKIIKELERHFKDFEIKDIVIEMA KKNSAEKKQFISKIQRQNVDLVKKLSNDYSLDENKLNFK MKEKFLLLSEQ 7CICI Π / l 7Π7 / Β / Υ SEQ ID NO: SEQUENCE NAME SEQUENCE 24 Cas9 of Streptococcus iniae, aa sequence MRKPYSIGLDIGTNSVGWAVITDDYKVPSKKMRIQGTTD RTSIKKNLIGALLFDNGETAEATRLKRTTRRRYTRRKYRI KELQKIFSSEMNELDIAFFPRLSESFLVSDDKEFENHPIF GNLKDEITYHNDYPTIYHLRQTLADSDQK ADLRLIYLALA HIIKFRGHFLIEGNLDSENTDVHVLFLNLVNIYNNLFEEDI VETASIDAEKILTSKTSKSRRLENLIAEIPNQKRNMLFGNL VSLALGLTPNFKTNFELLEDAKLQISKDSYEEDLDNLLAQ IGDQYADLFIAAKKLSDAILLSDIITVKGASTKAPLSASMV QRYEEHQQDLALLKNLVKKQIPEKYKEIFDNKE KNGYAG YIDGKTSQEEFYKYIKPILLKLDGTEKLISKLEREDFLRKQ RTFDNGSIPHQIHLNELKAIIRRQEKFYPFLKENQKKIEKL FTFKIPYYVGPLANGQSSFAWLKRQSNESITPWNFEEV VDQEASARAFIERMTNFDTYLPEEKVLPKHSPLYEMFM VYNELTKVKYQTEGMKRPVFLSSEDKEEIVNL LFKKERK VTVKQLKEEYFSKMKCFHTVTILGVEDRFNASLGTYHDL LKIFKDKAFLDDEANQDILEEIVWTLTLFEDQAMIERRLV KYADVFEKSVLKKLKKRHYTGWGRLSQKLINGIKDKQT GKTILGFLKDDGVANRNFMQLINDSSLDFAKIIKNEQEKT IKNESLEETIANLAGSPAIKKGILQSIKIV DEIVKIMGQNPD NIVIEMARENQSTMQGIKNSRQRLRKLEEVHKNTGSKIL KEYNVSNTQLQSDRLYLYLLQDGKDMYTGKELDYDNLS QYDIDHIIPQSFIKDNSIDNTVLTTQASNRGKSDNVPNIET VNKMKSFWYKQLKSGAISQRKFDHLTKAERGALSDFDK AGFIKRQLVETRQITKHVAQILDSRFNS NLTEDSKSNRN VKIITLKSKMVSDFRKDFGFYKLREVNDYHHAQDAYLNA VVGTALLKKYPKLEAEFVYGDYKHYDLAKLMIQPDSSLG KATTRMFFYSNLMNFFKKEIKLADDTIFTRPQIEVNTETG EIVWDKVKDMQTIRKVMSYPQVNIVMKTEVQTGGFSKE SIWPKGDSDKLIARKKSWDPKKYGGFDS PIIAYSVLVVA KIAKGKTQKLKTIKELVGIKIMEQDEFEKDPIAFLEKKGYQ DIQTSSIIKLPKYSLFELENGRKRLLASAKELQKGNELAL PNKYVKFLYLASHYTKFTGKEEDREKKRSYVESHLYYF DVRLSQVFRVTNVEF 25 Cas9 from Belliella baltica, aa sequence MKKILGLDLGTTSIGWAFIKEPEKDVVGSEIVDMGVRIVP LSSDEENDFAKGNTISINADRTLKRGARRNLQRFKQRR 7CICI Π / I 7Π7 / Β / Υ SEQ ID NO: SEQUENCE NAME SEQUENCE NALLEIFKEKKLISTNFKYAEDGPSSTFSTLNLRAKAAKE KIELQDLVKVLLQINKKRGYKSSRKAKSEEDDGSAIDSM GIAKELYENDLTPGQWVYEALQKGRKNVPDFYRSDLQE EFKKIVNYQSEFFPDIFNASFVEDWMGKASTPTKQYFNK KGVQLAENKGKREE RRLQEYKWRAEAVNFKIDLSEIALI LSQINSQISNSSGYLGAISDRSKELYFKNLTVGQYLYQQI KKNPHTRLKGQVFYRQDYLDEFERIWSVQSSFYPQLND ALKREVRDITIFFQRRLKSQKHLISNCEFEDHHKVVPKSH PVFQEFRIWQNLNNLLLIKKDNLNEKFDLELESKIALANE LAF KRELNVKDALKILGLKPNEWEFNFTKIEGNRTNQAF FDAFAKIIELEDGEPIDLGDLKADDILDQFSEAFLRIGIDTE LLQVNSDIEGAEYEKQSYIQFWHLLYSSEDDQKLKLNLI RKFGFKPEHAKILASISLQDDHASLSSRAIKKILPHLQSGL IYDKACTYAGYNHSSSFTKDENEKRELRAELELLKKNSL RNPV VEKILNQMINVVNAILKDPELGRPDEIRVEMARELK ANAEQRKNMTSNIASATRDHDKYREILKSEFGLKRVTKN DLLRYKLWLETDGISLYTGKPIEASKLFSKEYDIEHIIPKA RLFDDSFSNKTICERQLNIDKANVTAFSFLQNKLSADEF EQYQSRVKSLYGKLSKAKIQKLLMANDKIPEDFIARQLQ ETRYISKKAKEILFEISRR VSVTTGTITDKLREDWGLVEIM KELNWEKYDKLGLTYTIEGKHGERLNKIKDWSKRNDHR HHAMDALTVALTKPAYIQYLNNLNAKGLNNKKGTEVFAI EQKYLKRENGKLCFIPPIENIRSEAKKHLSRILVSYKAKN KVVTINKNKTKSKAGLNEQIALTPRGQLHKETVYGKSFH YSTKFEKIGASFNVQKINTVA KKEEREALLKRLAENGND PKKAFTGKNTLNKMPIYLDLGKNIKLSEKVKTVVLEQNYT IRKNIDPDLKVDKVIDVGIKRILESRLEEFGGNAKLAFSNL EENPIWLNKEKGISIKRVKISGVSNVESLHVKKDHFGEPI LDQEGNEIPVDFVSTGNNHHVAIYEDENGNLQEEVVSF FEAVVRQNQGLPIIKKNHTLGWK FLFTLKQNEYFVFPSD DFVPADVDLMDEQNYHLISPNLFRVQKIARKNYVFNNHL ETKAVDNDLLKSKKELSKITYHFYQTPEHLRGIIKIRINHL GKIIQIGEY 26 Cas9 from Psychroflexus torquisi, aa sequence MKRILGLDLGTNSIGWSLIEHDFKNKQGQIEGLGVRIIPM SQEILGKFDAGQSISQTADRTKYRGVRRLYQRDNLRRE 7CICI Π / I 7Π7 / Β / Υ SEQ ID NO: SEQUENCE NAME SEQUENCE RLHRVLKILDFLPKHYSESIDFQDKVGQFKPKQEVKLNY RKNEKNKHEFVFMNSFIEMVSEFKNAQPELFYNKGNGE ETKIPYDWTLYYLRKKALTQQITKEELAWLILNFNQKRGY YQLRGEDIDEDKNKKYMQLKVNNLIDSGAKVKGKVLY No RS HPLFQEFRIWQWLQNLKIYNKEKIENGKLEDVTTQLLPN NEAYVTLFDFLNTKKELEQKQFIEYFVKKKLIDKKEKEHF RWNFVEDKKYPFSETRAQFLSRLAKVKGIKNTEDFLNK NTQVGSKENSPFIKRIEQLWHIIYSVSDLKEYEKALEKFA EKHNLEKDSFLKNFKKFPPFVSDYASYSKKAISKLLPIMR MGKYWSESAVPTQVKERSLSIMERVKVLPLKEGYSDKD LADLLSRVSDDDIPKQLIKSFISFKDKNPLKGLNTYQANY LVYGRHSETGDIQHWKTPEDIDRYLNNFKQHSLRNPIVE QVVMETLRVVRDIWEHYGNNEKDFFKEIHVELGREMKS PAGKREKLSQRNTENENTNHRIREVLKELMNDAS VEGG VRDYSPSQQEILKLYEEGIYQNPNTNYLKVDEDEILKIRK KNNPTQKEIQRYKLWLEQGYISPYTGKIIPLTKLFTHEYQI EHIIPQSRYYDNSLGNKIICESEVNEDKDNKTAYEYLKVE KGSIVFGHKLLNLDEYEAHVNKYFKKNKTKLKNLLSEDIP EGFINRQLNDSRYISKLVKGLLSNIVRENGEQ EATSKNLI PVTGVVTSKLKQDWGLNDKWNEIIAPRFKRLNKLTNSN DFGFWDNDINAFRIQVPDSLIKGFSKKRIDHRHHALDAL VVACTSRNHTHYLSALNAENKNYSLRDKLVIKNENGDYT KTFQIPWQGFTIEAKNNLEKTVVSFKKNLRVINKTNNKF WSYKDENGNLNLGKDGKPKKKLRKQTKGYNWAIRKPL HKETVSGIYNINAPKNKIATSVRTLLTEIKNEKHLAKITDL RIRETILPNHLKHYLNNKGEANFSEAFSQGGIEDLNKKIT TLNEGKKHQPIYRVKIFEVGSKFSISEDENSAKSKKYVEA AKGTNLFFAIYLDEENKKRNYETIPLNEVITHQKQVAGFP KSERLSVQPDSQKGTFLFTLSPNDLVYVPNNEELENRD LFNLGN LNVEQISRIYKFTDSSDKTCNFIPFQVSKLIFNLK 7CICI Π / I 7Π7 / Β / Υ SEQ ID NO: SEQUENCE NAME SEQUENCE KKEQKKLDVDFIIQNEFGLGSPQSKNQKSIDDVMIKEKCI KLKIDRLGNISKA 27 Cas9 from Streptococcus thermophilus, aa sequence MTKPYSIGLDIGTNSVGWAVTTDNYKVPSKKMKVLGNT SKKYIKKNLLGVLLFDSGITAEGRRLKRTARRRYTRRRN RILYL QEIFSTEMATLDDAFFQRLDDSFLVPDDKRDSKY PIFGNLVEEKAYHDEFPTIYHLRKYLADSTKKADLRLVYL ALAHMIKYRGHFLIEGEFNSKNNDIQKNFQDFLDTYNAIF ESDLSLENSKQLEEIVKDKISKLEKKDRILKLFPGEKNSGI FSEFLKLIVGNQADFRKCFNLDEKASLHFSKESYDEDLE TLLGYIGDDYSDVFLKA KKLYDAILLSGFLTTVDNETEAP LSSAMIKRYNEHKEDLALLKEYIRNISLKTYNEVFKDDTK NGYAGYIDGKTNQEDFYVYLKKLLAEFEGADYFLEKIDR EDFLRKQRTFDNGSIPYQIHLQEMRAILDKQAKFYPFLA KNKERIEKILTFRIPYYVGPLARGNSDFAWSIRKRNEKIT PWNFEDVIDKESSAEAFINRMTS FDLYLPEEKVLPKHSL LYETFNVYNELTKVRFIAESMRDYQFLDSKQKKDIVRLY FKDKRKVTDKDIIEYLHAIYGYDGIELKGIEKQFNSSLSTY HDLLNIINDKEFLDDSSNEAIIEEIIHTLTIFEDREMIKQRLS KFENIFDKSVLKKLSRRHYTGWGKLSAKLINGIRDEKSG NTILDYLIDDGISNRNFMQLIHD DALSFKKKIQKAQIIGDE DKGNIKEVVKSLPGSPAIKKGILQSIKIVDELVKVMGGRK PESIVVEMARENQYTNQGKSNSQQRLKRLEKSLKELGS KILKENIPAKLSKIDNNALQNDRLYLYYLQNGKDMYTGD DLDIDRLSNYDIDHIIPQAFLKDNSIDNKVLVSSASNRGK SDDVPSLEVVKKRKTFWYQLLK SKLISQRKFDNLTKAER GGLSPEDKAGFIQRQLVETRQITKHVARLLDEKFNNKKD ENNRAVRTVKIITLKSTLVSQFRKDFELYKVREINDFHHA HDAYLNAVVASALLKKYPKLEPEFVYGDYPKYNSFRER KSATEKVYFYSNIMNIFKKSISLADGRVIERPLIEVNEETG ESVWNKESDLATVRRVLSYPQ VNVVKKVEEQNHGLDR GKPKGLFNANLSSKPKPNSNENLVGAKEYLDPKKYGGY AGISNSFTVLVKGTIEKGAKKKITNVLEFQGISILDRINYR KDKLNFLLEKGYKDIELIIELPKYSLFELSDGSRRMLASIL STNNKRGEIHKGNQIFLSQKFVKLLYHAKRISNTINENHR KYVENHKKEFEELFYYILEFNENYVGAKKNGKLLNSAFQ 7CICI Π / I 7Π7 / Β / Υ SEQ ID NO: SEQUENCE NAME SEQUENCE SWQNHSIDELCSSFIGPTGSERKGLFELTSRGSAADFEF LGVKIPRYRDYTPSSLLKDATLIHQSVTGLYETRIDLAKL GEG 28 Cas9 from Listeria innocua, aa sequence MKKPYTIGLDIGTNSVGWAVLTDQYDLVKRKMKIAGDSE KKQIKKNFWGVRLFDEGQTAADRRMARTARRR IERRR NRISYLQGIFAEEMSKTDANFFCRLSDSFYVDNEKRNSR HPFFATIEEEVEYHKNYPTIYHLREELVNSSEKADLRLVY LALAHIIKYRGNFLIEGALDTQNTSVDGIYKQFIQTYNQVF ASGIEDGSLKKLEDNKDVAKILVEKVTRKEKLERILKLYP GEKSAGMFAQFISLIVGSKGNFQKPFDLIEKSDIECAKDS YEED LESLLALIGDEYAELFVAAKNAYSAVVLSSIITVAET ETNAKLSASMIERFDTHEEDLGELKAFIKLHLPKHYEEIF SNTEKHGYAGYIDGKTKQADFYKYMKMTLENIEGADYFI AKIEKENFLRKQRTFDNGAIPHQLHLEELEAILHQQAKYY PFLKENYDKIKSLVTFRIPYFVGPLANGQSEFAWLTRKA DGEI RPWNIEEKVDFGKSAVDFIEKMTNKDTYLPKENVL PKHSLCYQKYLVYNELTKVRYINDQGKTSYFSGQEKEQI FNDLFKQKRKVKKKDLELFLRNMSHVESPTIEGLEDSFN SSYSTYHDLLKVGIKQEILDNPVNTEMLENIVKILTVFEDK RMIKEQLQQFSDVLDGVVLKKLERRHYTGWGRLSA KLL MGIRDKQSHLTILDYLMNDDGLNRNLMQLINDSNLSFKS IIEKEQVTTADKDIQSIVADLAGSPAIKKGILQSLKIVDELV SVMGYPPQTIVVEMARENQTTGKGKNNSRPRYKSLEK AIKEFGSQILKEHPTDNQELRNNRLYLYYLQNGKDMYTG GDDVPPLEIVRKRKVFWEKLYQGNLMSKRKFDYLTKA ERGGLTEADKARFIHRQLVETRQITKNVANILHQRFNYE KDDHGNTMKQVRIVTLKSALVSQFRKQFQLYKVRDVND YHHAHDAYLNGVVANTLLKVYPQLEPEFVYGDYHQFD WFKANKATAKKQFYTNIMLFFAQKDRIIDENGEILWDKK YLDTVKKV MSYRQMNIVKKTEIQKGEFSKATIKPKGNSS KLIPRKTNWDPMKYGGLDSPNMAYAVVIEYAKGKNKLV FEKKIIRVTIMERKAFEKDEKAFLEEQGYRQPKVLAKLPK YTLYECEEGRRRMLASANEAQKGNQQVLPNHLVTLLHH AANCEVSDGKSLDYIESNREMFAELLAHVSEFAKRYTLA JCICI η / Ι 7Π7 / Β / Υ SEQ ID NO: SEQUENCE NAME SEQUENCE EANLNKINQLFEQNKEGDIKAIAQSFVDLMAFNAMGAPA SFKFFETTIERKRYNNLKELLNSTIIYQSITGLYESRKRLD D 29 Cas9 from Campylobacter jejuni, aa sequence MARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKT GESLALPRRLARSARKRLARRKARLNHLKHLIANEFKLN YEDYQSFDESLAKAYKGSLISPYELRFRALNELLSKQDF ARVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLAN YQSVGEYLYKEYFQKFKENSKEFTNVRNKKESYERCIA QSFLKDELKLIFKKQREFGFSFSKKFEEEVLSVAFYKRAL KDFSHLVGNCSFFTDEKRAPKNSPLAFMFVALTRIINLLN NLKNTEG ILYTKDDLNALLNEVLKNGTLTYKQTKKLLGLS DDYEFKGEKGTYFIEFKKYKEFIKALGEHNLSQDDLNEIA KDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISF KALKLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLPA FNETYYKDEVTNPVVLRAIKEYRKVLNALLKKYGKVHKIN IEL AREVGKNHSQRAKIEKEQNENYKAKKDAELECEKLG LKINSKNILKLRLFKEQKEFCAYSGEKIKISDLQDEKMLEI DHIYPYSRSFDDSYMNKVLVFTKQNQEKLNQTPFEAFG NDSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQKNFK DRNLNDTRYIARLVLNYTKDYLDFLPLSDDENTKLND tq Neisseria meningitidis Cas9 , aa sequence MAAFKPNPINYILGLDIGIASVGWAMVEIDEDENPICLIDL GVRVFERAEVPKTGDSLAMARRLARSVRRLTRRRAHRL LRARRLLKREGVLQAADFDENGLIKSLPNTPWQLRAAAL DRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELG ALLKGVADNAHALQTGDFRTPAELALNKFEKESGHIRN QRGDYSHTFSRK DLQAELILLFEKQKEFGNPHVSGGLK 7CICI Π / I 7Π7 / Β / Υ 100 SEQ ID NO: SEQUENCE NAME SEQUENCE EGIETLLMTQRPALSG DAVQKMLG HCTFE PAE PKAAKN TYTAERFIWLTKLNNLRILEQGSERPLTDTERATLMDEP YRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTL MEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAF SLFKTDEDITGRLKDRIQPEILEALL KHISFDKFVQISLKAL RRIVPLMEQGKRYDEACAEIYGDHYGKKNTEEKIYLPPI PADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAR EVGKSFKDRKEIEKRQEENRKDREKAAAKFREYFPNFV GEPKSKDILKLRLYEQQHGKCLYSGKEINLGRLNEKGYV EIDHALPFSRTWDDSFNNKV LVLGSENQNKGNQTPYEY FNGKDNSREWQEFKARVETSRFPRSKKQRILLQKFDED GFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRVFA SNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTV AMQQKITRFVRYKEMNAFDGKTIDKETGEVLHQKTHFP QPWEFFAQEVMIRVFGKPDGK PEFEEADTPEKLRTLLA EKLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVK SAKRLDEGVSVLRVPLTQLKLKDLEKMVNREREPKLYE ALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVR VEQVQKTGVWVRNHNGIADNATMVRVDVFEKGDKYYL VPIYSWQVAKGILPDRAVVQGKDEEDW QLIDDSFNFKF SLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDH KIGKNGILEGIGVKTALSFQKYQIDELGKEIRPCRLKKRP PVR 31 Cas9 from Streptococcus pyogenes, aa sequence MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTD RHSIKKNLIGALLFGSGETAEATRLKRTARRRYTRRKNRI CYL QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPI FGNIVDEVAYHEKYPTIYHLRKKLADSTDKADLRLIYLAL AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEE NPINASRVDAKAILSARLSKSRRLENLIAQLPGEKRNGLF GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN LLAQIGDQYADLFLAAKNLSDAILLSDILRVNSEITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE DLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFL KD NREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITP 7CICI Π / I 7Π7 / Β / Υ 101 SEQ ID NO: SEQUENCE NAME SEQUENCE WNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSL LYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLL FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASL GAYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRGMI EERLKTYAHLFDDKVMK QLKRRRYTGWGRLSRKLINGI RDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQ KAQVSGQGHSLHEQIANLAGSPAIKKGILQTVKIVDELVK VMGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGI KELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ ELDINRLSDYDVDHIVPQS FIKDDSIDNKVLTRSDKNRGK SDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKY DENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHH AHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKY FFYSNIMNFFKTEITLANGEIRKRPLI ETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG ELQKGNELALPSKYVNFLYLASH YEKLKGSPEDNEQKQ LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS TKEVLDATLIHQSITGLYETRIDLSQLGGD 32 Zinc finger protein (ZFP), na sequence atggcccaggctgctcttgagcccggagagaaaccctacaagtgcccggag t gcggaaagtccttctctgagcggagtcacctccgagagcaccagcggactca tacgggcgaaaaaccatacaagtgcccagaatgtggtaaatctttttctcgggc tgacaacctgactgaacatcagcgcacgcacaccggtgaaaaaccttacaa gtgtccagagtgtggcaagagcttttctagtagaagga cctgtcgagcgcatca gcggactcacaccggcgaaaaaccctataagtgtccggaatgtggaaagag ctttagccgcaacgacacccttactgaacaccagcgaacacacacgggaga aaaaccatataaatgtccggaatgtggcaaaagttttagtcggagtgataaact tacggagcaccaacggacacacaccggagagaagccatataagtgtcctg aatgtggaaagtccttctcacagcttgctcatctgcgagcacatcagcgcacac a cacc 7CICI Π / I 7Π7 / Β / Υ 102 SEQ ID NO: SEQUENCE NAME SEQUENCE 33 ZFP, aa sequence MAQAALEPGEKPYKCPECGKSFSERSHLREHQRTHTG EKPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCPEC GKSFSSRRTCRAHQRTHTGEKPYKCPECGKSFSRNDT LTEHQRTHTGEKPYKCPECGKSFSRSDKLTEHQRTHTG EKPYK CPECGKSFSQLAHLRAHQRTHT 34 ZNF-E2C, seq. cagagcggcgatctgcgccgccatcagcgcacccataccggcgaaaaacc gtataaatgcccggaatgcggcaaaagctttagcgattgccgcgatctggcgc gccatcagcgcacccataccggcgaaaaaccgtataaatgcccggaatgcg gcaaaagctttagccagagcagccatctggtgcg ccatcagcgcacccatac cggcgaaaaaccgtataaatgcccggaatgcggcaaaagctttagcgattgc cgcgatctggcgcgccatcagcgcacccataccggcgaaaaaccgtataaa tgcccggaatgcggcaaaagctttagccgcagcgataaactggtgcgccatc agcgcacccataccgg caaaaaaaaccagcggccaggcgggc 35 ZNF-E2C, aa sequence MAQAALEPGEKPYKCPECGKSFSRKDSLVRHQRTHTG EKPYKCPECGKSFSQSGDLRRHQRTHTGEKPYKCPEC GKSFSDCRDLARHQRTHTGEKPYKCPECGKSFSQSSH LVRHQRTHTGEKPYKCPECGKSFSDCRD LARHQRTHT GEKPYKCPECGKSFSRSDKLVRHQRTHTGKKTSGQAG 36 ZNF-E3, na sequence atggcgcaggcggcgctggaaccgggcgaaaaaccgtataaatgcccgga atgcggcaaaagctttagcgatccgggcgcgctggtgcgccatcagcgcacc cataccggcgaaaaaccgtataaatgccc ggaatgcggcaaaagctttagc cagagcagccatctggtgcgccatcagcgcacccataccggcgaaaaacc gtataaatgcccggaatgcggcaaaagctttagcgattgccgcgatctggcgc gccatcagcgcacccataccggcgaaaaaaccgtataaatgcccggaatgcg gcaaaagctttag ccagagcagccatctggtgcgccatcagcgcacccatac cggcgaaaaaaccgtataaatgcccggaatgcggcaaaagctttagcgattgc cgcgatctggcgcgccatcagcgcacccataccggcgaaaaaccgtataaa tgcccggaatgcggcaaaagctttagccagagcagccatctggtgcg ccatc agcgcacccataccggcaaaaaaaaccagcggccaggcgggc 7CICI Π / l 7Π7 / Β / Υ 103 SEQ ID NO: SEQUENCE NAME SEQUENCE 37 ZNF-E3, aa sequence MAQAALEPGEKPYKCPECGKSFSDPGALVRHQRTHTG EKPYKCPECGKSFSQSSHLVRHQRTHTGEKPYKCPEC GKSFSDCRDLARHQRTHTGEKPYKCPECGKSFSQSSH LVRHQRTHTGEKPYKCPECGKSFSDCRDLARHQRTHT GEKPYKCPECGKSFSQSSHLVRHQRTHTGKKTSGQAG 38 ZNF-TRCa, na sequence atggcgcaggcggctcttgaacccggggagaaaccctataaatgccctgagt gtggcaagagtttttcaaccacaggaaacttgacagtccaccaacggaccca caccggcgagaaaccatacaagtgtccggagtgtggtaagtcttt ctcaagtc ctgccgaccttaccagacatcaacgcacacatacaggtgaaaaaccttacaa gtgcccagagtgcggaaaaagtttttcacaatctggcgacctccgcaggcacc agcgcactcacaccggtgaaaaaccatacaagtgtcctgagtgcgggaaga gttttagtcaacgagctcatctggagcgacaccaaagg actcatactggggag aaaccgtacaaatgtcccgaatgtgggaagagcttctctaccaagaattccctt acagagcaccagcgcacgcatacgggagagaagccgtataagtgtccgga atgtggcaagagcttttccagaagtgaccaccttacaacccaccagaggacg cacacc 39 ZNF-TRCa, aa sequence MAQAALEPGEKPYKCPEC GKSFSTTGNLTVHQRTHTG EKPYKCPECGKSFSSPADLTRHQRTHTGEKPYKCPEC GKSFSQSGDLRRHQRTHTGEKPYKCPECGKSFSQRAH LERHQRTHTGEKPYKCPECGKSFSTKNSLTEHQRTHTG EKPYKCPECGKSFSRSDHLTTHQRTHT 40 AAVS1 site agacggccgcgtcagagc 41 Zinc finger domain 1, aa sequence ERSHLRE 42 Zinc finger domain 2, aa sequence aa RADNLTE 43 Zinc finger domain 3, aa sequence SRRTCRA 44 Zinc finger domain 4 , aa sequence RNDTLTE 7CICI Π / I 7Π7 / Β / Υ 104 SEQ ID NO: SEQUENCE NAME SEQUENCE 45 Zinc finger domain 5, aa sequence RSDKLTE 46 Zinc finger domain 6, aa sequence QLAHLRA 47 Nuclear localization signal atggctccaaagaaaaagaggaaagtgggaatccacggagtccccgccgc t 48 GGSx3 linker, na sequence ggtggatctggcggtggatctgg tggcggt 49 Linker of GGSx3, aa sequence GGSGGGSGGG 50 GGS4x linker, na sequence ggagggagtggtgggtccggtggtagtggcggatcc 51 GGS4x linker, aa sequence GGSGGSGGSGGS 52 GGS5x linker, na sequence ggaggctccggtgggtctggtgggagcggtggtagtggcggatcc 53 GGS5x linker, aa sequence GGSGGSGGSGGSGGS 54 GGS6x linker, aa sequence na ggaggcagtggtgggagcggtggttccgggggtagtggtggttccgggggat cc 55 GGS6x linker, sequence of aa GGSGGSGGSGGSGGSGGS 56 GGS7x linker, sequence of na ggaggttctggaggctccggtgggtccgggggaagtggggggtcaggcgga tcaggaggatcc 57 GGS7x linker, aa sequence GGGSGGSGGSGGSGGSGGSGGS 7CICI Π / l 7Π7 / Β / Υ 105 SEQ ID NO: SEQUENCE NAME SEQUENCE 58 GGS8x linker, na sequence ggaggtagcggaggttccggagggagcggcgggagtgggggaagcgggg gaagtggaggatccgggggaggatcc 59 GGS8x linker, aa sequence GGSGGSGGSGGSGGSGGSGGS 60 XTEN linker, na tccgg sequence tagcgaaacaccggggacttcagaatcggccaccccggagtct 61 XTEN Linker, aa sequence SGSETPGTSESATPES 62 B Linker, sequence de na ggaagcgccggtagtgcggctgggtctggcgagttc 63 B linker, aa sequence GSAGSAAGSGEF 64 Human Cas9 (hCas9), na sequence atggacaagaagtactccattgggctcgatatcggcacaaacagcgtcggct gggccgtcattacggacgagtacaaggtgccgagcaaaaaattcaaagttct gggcaataccgatcgccacagcataaagaagaacctcattggcgccctcctg ttcgactccggggagacggccgaagccacgcggctcaaaagaacagcacg gcgcagatatacccgcagaaagaatcggatctgctacctgcaggagatcttta gtaatgagatggctaaggtggatgactctttcttccataggctggaggagtccttt ttgg tggagggaggataaaaagcacgagcgccacccaatctttggcaatatcgt ggacgaggtggcgtaccatgaaaagtacccaaccatatatcatctgaggaag aagcttgtagacagtactgataaggctgacttgcggttgatctatctcgcgctgg cgcatatg atcaaatttcg ggg acacttcctcatcg aggggg acctg aac cca gacaacagcgatgtcgacaaactctttatccaactggttcagacttacaatcag cttttcgaagagaacccgatcaacgcatccggagttgacgccaaagcaatcct gagcgctaggctgtccaaatcccggcggctcgaaaacctcatcgcacagctc cctggggagaagaagaacggcctgtttggtaatcttatcgccctgt cactcggg ctgacccccaactttaaatctaacttcgacctggccgaagatgccaagcttcaa ctg agcaaag acacctacgatg atg atctcg acaatctgctggcccag atcg g cgaccagtacgcagacctttttttggcggcaaagaacctgtcagacgccattct gctgagtgatattctgcgagtgaacacggagatcaccaaagctccgctgagc gctagtatg atcaagcgctatg atg agcaccaccaagacttg actttgctg aag 7CICI Π / I 7Π7 / Β / Υ 106 SEQ ID NO: SEQUENCE NAME SEQUENCE gcccttgtcagacagcaactgcctgagaagtacaaggaaattttcttcgatcag tctaaaaatggctacgccggatacattgacggcggagcaagccaggaggaa ttttacaaatttattaagcccatcttggaaaaaatggacggcaccgaggagctg ctggtaaagcttaacagagaagatctg ttgcgcaaacagcgcactttcgacaa tggaagcatcccccaccagattcacctgggcgaactgcacgctatcctcaggc ggcaagaggatttctacccctttttgaaagataacagggaaaagattgagaaa atcctcacatttcggataccctactatgtaggccccctcgcccgggggaaattcca gattcgcgtggatgactcgca aatcagaagagaccatcactccctggaacttc gaggaagtcgtggataagggggcctctgcccagtccttcatcgaaaggatga ctaactttg ataaaaatctgcctaacg aaaagg tg cttcctaaacactctctg ct gtacgagtacttcacagtttataacgagctcaccaaggtcaaatacgtcacag aagggatga gaaagccagcattcctgtctggagagcagaagaaagctatcg tggacctcctcttcaagacgaaccggaaagttaccgtgaaacagctcaaaga agactatttcaaaaagattgaatgtttcgactctgttgaaatcagcggagtggag gatcgcttcaacgcatccctgggaacgtatcacgatctcctgaaaatcattaaa ga caaggacttcctggacaatgaggagaacgaggacattcttgaggacattg tcctcacccttacgttgtttgaagatagggagatgattgaagaacgcttgaaaac ttacgctcatctctcttcgacgacaaagtcatgaaacagctcaagaggcgccgatattackggatgggggcggctgtcaagaaaactgatcaatgggatccgagaca agcagagtggaa agacaatcctggattttcttaagtccgatggatttgccaacc ggaacttcatgcagttgatccatgatgactctctcacctttaaggaggacatcca gaaagcacaagtttctggccagggggacagtcttcacgagcacatcgctaat cttgcaggtagcccagctatcaaaaaagggaatactgcagaccgttaaggtcgt ggatgaactc gtcaaagtaatgggaaggcataagcccgagaatatcgttatc gagatggcccgagagaaccaaactacccagaagggacagaagaacagta gggaaaggatgaagaggattgaagagggtataaaagaactggggtcccaa atccttaag g aacacccag ttg aaaacacccagcttcag aatg ag aag ctct acctgtactacctgcagaacggca gggacatgtacgtggatcaggaactgga catcaatcggctctccgactacgacgtggatcatatcgtgccccagtcttttctca aagatgattctattgataataaagtgttgacaagatccgataaaaatagaggg aagagtgataacgtcccctcagaagaagttgtcaagaaaatgaaaaattattg gcggcagctgctgaacgccaaactgatcacacaacggaagttcgataatctg actaaggctgaacgaggtggcctgtctgagttggataaagccggcttcatcaa aa ggcagcttgttgagacacgccagatcaccaagcacgtggcccaaattctc gattcacgcatgaacaccaagtacgatgaaaatgacaaactgattcgagagg 7CICI Π / I 7Π7 / Β / Υ 107 SEQ ID NO: SEQUENCE NAME SEQUENCE tgaaagttattactctgaagtctaagctggtctcagatttcagaaaggactttcag ttttataaggtgagagagatcaacaattaccaccatgcgcatgatgcctacctg aatgcagtggtaggcactgcacttatcaaaaatatcccaagcttgaatctgaa tttgtttacggagactataa agtgtacgatgttaggaaaatgatcgcaaagtctg agcaggaaataggcaaggccaccgctaagtacttcttttacagcaatattatga attttttcaag accg ag attacactgg ccaatgg ag agattcgg aagcg acca cttatcgaaacaaacggagaaacaggagaaatcgtgtgggacaagggtag ggatttcgcga cagtccggaaggtcctgtccatgccgcaggtgaacatcgtta aaaagaccgaagtacagaccggaggcttctccaaggaaagtatcctcccga aaaggaacagcgacaagctgatcgcacgcaaaaaagattgggaccccaa gaaatacggcggattcgattctcctacagtcgcttacagtgtactggttgtggcc aaagtggaga aagggaagtctaaaaaactcaaaagcgtcaaggaactgct gggcatcacaatcatggagcgatcaagcttcgaaaaaaaccccatcgactttc tcgaggcgaaaggatataaagaggtcaaaaaagacctcatcattaagcttcc caagtactctctctttgagcttgaaaacggccggaaacgaatgctcgctagtgc ggg cgagctgcagaaaggtaacgagctggcactgccctctaaatacgttaatt tcttgtatctggccagccactatgaaaagctcaaagggtctcccgaagataatg agcagaagcagctgttcgtggaacaacacaaacactaccttgatgagatcat cgagcaaataagcgaattctccaaaagagtgatcctcgccgacgctaacctc gataaggtgctttctgcttacaataagcacagggataagcccatcagggagca ggcagaaaacattatccacttgtttactctgaccaacttgggcgcgcctgcagc cttcaagtacttcgacaccaccatagacagaaagcggtacacctctacaaag gaggtcctggacgccacactgattcatcagtcaattacggggctctatgaaac aagaatcgacct ctctcagctcggtggagac 65 Nicasa Cas9 (nCas9), na sequence atggacaagaagtactccattgggctcgctatcggcacaaacagcgtcggct gggccgtcattacggacgagtacaaggtgccgagcaaaaaattcaaagttct gggcaataccgatcgccacagcataaagaagaacctcattggcgccctcctg ttcgactccggggagacggccgaagccacgcggctcaaaagaacagcacg gcg cagatatacccgcagaaagaatcggatctgctacctgcaggagatcttta gtaatgagatggctaaggtggatgactctttcttccataggctggaggagtccttt ttggtggaggaggataaaaagcacgagcgccacccaatctttggcaatatcgt ggacgaggtggcgtaccatgaaaagtacccaaccatatatcatctgaggaag aagctt gtagacagtactgataaggctgacttgcggttgatctatctcgcgctgg cgcatatgatcaaatttcggggacacttcctcatcgagggggacctgaaccca gacaacagcgatgtcgacaaactctttatccaactggttcagacttacaatcag 7CICI Π / I 7Π7 / Β / Υ 108 SEQ ID NO: SEQUENCE NAME SEQUENCE cttttcgaagagaacccgatcaacgcatccggagttgacgccaaagcaatcct gagcgctaggctgtccaaatcccggcggctcgaaaacctcatcgcacagctc cctggggagaagaagaacggcctgtttggtaatcttatcgccctgtcactcggg ctgacccccaactttaaat ctaacttcgacctggccgaagatgccaagcttcaa ctg agcaaag acacctacgatg atg atctcg acaatctgctggcccag atcg g cgaccagtacgcagacctttttttggcggcaaagaacctgtcagacgccattct gctgagtgatattctgcgagtgaacacggagatcaccaaagctccgctgagc gctagta tg atcaagcgctatg atg agcaccaccaagacttg actttgctg aag gcccttgtcagacagcaactgcctgagaagtacaaggaaattttcttcgatcag tctaaaaatggctacgccggatacattgacggcggagcaagccaggaggaa ttttacaaatttattaagcccatcttggaaaaaatggacggcaccgaggagctg ct ggtaaagcttaacagaagatctgttgcgcaaacagcgcactttcgacaa tggaagcatcccccaccagattcacctgggcgaactgcacgctatcctcaggc ggcaagaggatttctacccctttttgaaagataacagggaaaagattgagaaa atcctcacatttcggataccctactatgtaggccccctcgcccgggggaaattcca g attcgcgtggatgactcgcaaatcagaagagaccatcactccctggaacttc gaggaagtcgtggataagggggcctctgcccagtccttcatcgaaaggatga ctaactttgataaaaatctgcctaacgaaaaggtgcttcctaaacactctctgct gtacgagtacttcacagtttataacgagctcaccaaggtcaaatacgt cacag aagggatgagaaagccagcattcctgtctggagagcagaagaaagctatcg tggacctcctcttcaagacgaaccggaaagttaccgtgaaacagctcaaaga agactatttcaaaaagattgaatgtttcgactctgttgaaatcagcggagtggag gatcgcttcaacgcatccctgggaacgtatcacgatctcctga aaatcattaaa gacaaggacttcctggacaatgaggagaacgaggacattcttgaggacattg tcctcacccttacgttgtttgaagatagggagatgattgaagaacgcttgaaaac ttacgctcatctcttcgacgacaaagtcatgaaacagctcaagaggcgccgatattackggatgggggcggctgtcaagaaaactgatcaatgggatccgagaca a gcagagtggaaagacaatcctggattttcttaagtccgatggatttgccaaca ggaacttcatgcagttgatccatgatgactctctcacctttaaggaggacatcca gaaagcacaagtttctggccagggggacagtcttcacgagcacatcgctaat cttgcaggtagcccagctatcaaaaagggaatactgcagaccgttaaggtcgt ggatgaactcgtcaaagtaatgggaaggcataagcccgagaatatcgttatc gagatggcccgagagaaccaaactacccagaagggacagaagaacagta gggaaaggatgaagaggattgaagagggtataaaagaactggggtcccaa atccttaag g aaca cccag ttg aaaacacccagcttcag aatg ag aag ctct 7CICI Π / l 7Π7 / Β / Υ 109 SEQ ID NO: SEQUENCE NAME SEQUENCE acctgtactacctgcagaacggcagggacatgtacgtggatcaggaactgga catcaatcggctctccgactacgacgtggatcatatcgtgccccagtcttttctca aagatgattctattgataataaagtgttgacaagatccgataaaaatagaggg aagagtgataacgtcccctcagaagaagttg tcaagaaaatgaaaaattattg gcggcagctgctgaacgccaaactgatcacacaacggaagttcgataatctg actaaggctgaacgaggtggcctgtctgagttggataaagccggcttcatcaa aaggcagcttgttgagacacgccagatcaccaagcacgtggcccaaattctc gattcacgcatgaacaccaagtacgatgaaaat gacaaactgattcgagagg tgaaagttattactctgaagtctaagctggtctcagatttcagaaaggactttcag ttttataaggtgagagagatcaacaattaccaccatgcgcatgatgcctacctg aatgcagtggtaggcactgcacttatcaaaaaatatcccaagcttgaatctgaa tttgtttacggagactataaagtgtacgat gttaggaaaatgatcgcaaagtctg agcaggaaataggcaaggccaccgctaagtacttcttttacagcaatattatga attttttcaag accg ag attacactgg ccaatgg ag ag attcgg aagcg acca cttatcgaaacaaacggagaaacaggagaaatcgtgtgggacaagggtag ggatttcgcgacagtccg gaaggtcctgtccatgccgcaggtgaacatcgtta aaaagaccgaagtacagaccggaggcttctccaaggaaagtatcctcccga aaaggaacagcgacaagctgatcgcacgcaaaaaagattgggaccccaa gaaatacggcggattcgattctcctacagtcgcttacagtgtactggttgtggcc aaagtggagaaagggaag tctaaaaaactcaaaagcgtcaaggaactgct gggcatcacaatcatggagcgatcaagcttcgaaaaaaccccatcgactttc tcgaggcgaaaggatataaagaggtcaaaaaagacctcatcattaagcttcc caagtactctctctttgagcttgaaaacggccggaaacgaatgctcgctagtgc gggcgagctg cagaaaggtaacgagctggcactgccctctaaatacgttaatt tcttgtatctggccagccactatgaaaagctcaaagggtctcccgaagataatg agcagaagcagctgttcgtggaacaacacaaacactaccttgatgagatcat cgagcaaataagcgaattctccaaaagagtgatcctcgccgacgctaacctc gataaggt gctttctgcttacaataagcacagggataagcccatcagggagca ggcagaaaacattatccacttgtttactctgaccaacttgggcgcgcctgcagc cttcaagtacttcgacaccaccatagacagaaagcggtacacctctacaaag gaggtcctggacgccacactgattcatcagtcaattacggggctctatgaaac aagaatcgacctctctcagct cggtggagac 66 Dead Cas9 (dCas9), na sequence atggacaagaagtactccattgggctcgctatcggcacaaacagcgtcggct gggccgtcattacggacgagtacaaggtgccgagcaaaaaattcaaagttct gggcaataccgatcgccacagcataaagaagaacctcattggcgccctcctg 7CICI Π / I 7Π7 / Β / Υ 110 SEQ ID NO: SEQUENCE NAME SEQUENCE ttcgactccggggagacggccgaagccacgcggctcaaaagaacagcacg gcgcagatatacccgcagaaagaatcggatctgctacctgcaggagatcttta gtaatgagatggctaaggtggatgactctttcttccataggctggaggagtccttt ttggtggaggaggataaaaagcacga gcgccacccaatctttggcaatatcgt ggacgaggtggcgtaccatgaaaagtacccaaccatatatcatctgaggaag aagcttgtagacagtactgataaggctgacttgcggttgatctatctcgcgctgg cgcatatgatcaaatttcggggacacttcctcatcgagggggacctgaaccca gacaacagcgatgtcgacaaactctttat ccaactggttcagacttacaatcag cttttcgaagagaacccgatcaacgcatccggagttgacgccaaagcaatcct gagcgctaggctgtccaaatcccggcggctcgaaaacctcatcgcacagctc cctggggagaagaagaacggcctgtttggtaatcttatcgccctgtcactcggg ctgacccccaactttaaatctaact tcgacctggccgaagatgccaagcttcaa ctg agcaaag acacctacgatg atg atctcg acaatctgctggcccag atcg g cgaccagtacgcagacctttttttggcggcaaagaacctgtcagacgccattct gctgagtgatattctgcgagtgaacacggagatcaccaaagctccgctgagc gctagtatg at caagcgctatg atg agcaccaccaagacttg actttgctg aag gcccttgtcagacagcaactgcctgagaagtacaaggaaattttcttcgatcag tctaaaaatggctacgccggatacattgacggcggagcaagccaggaggaa ttttacaaatttattaagcccatcttggaaaaaatggacggcaccgaggagctg ctggtaa agcttaacagagaagatctgttgcgcaaacagcgcactttcgacaa tggaagcatcccccaccagattcacctgggcgaactgcacgctatcctcaggc ggcaag agg atttctacccctttttg aaag ataacaggg aaaag attg ag aaa atcctcacatttcggataccctactatgtaggccccctcgcccggggaaattcca gattcgcgtggatgactcgcaaatcagaagagaccatcactccctggaacttc gaggaagtcgtggataagggggcctctgcccagtccttcatcgaaaggatga ctaactttgataaaaatctgcctaacgaaaaggtgcttcctaaacact ctctgct gtacgagtacttcacagtttataacgagctcaccaaggtcaaatacgtcacag aagggatgagaaagccagcattcctgtctggagagcagaagaaagctatcg tggacctcctcttcaagacgaaccggaaagttaccgtgaaacagctcaaaga agactatttcaaaaagattgaatgtttcgactctgttgaaatcag cggagtggag gatcgcttcaacgcatccctgggaacgtatcacgatctcctgaaaatcattaaa gacaaggacttcctggacaatgaggagaacgaggacattcttgaggacattg tcctcacccttacgttgtttgaagatagggagatgattgaagaacgcttgaaaac ttacgctcatctcttcgacgacaaagtcatgaaacagctcaagaggc gccgat attacksggatgggggcggctgtcaagaaaactgatcaatgggatccgagaca 7CICI Π / l 7Π7 / Β / Υ 111 SEQ ID NO: SEQUENCE NAME SEQUENCE agcagagtggaaagacaatcctggattttcttaagtccgatggatttgccaacc ggaacttcatgcagttgatccatgatgactctctcacctttaaggaggacatcca gaaagcacaagtttctggccagggggacagtcttcacgagcacatcgctaat cttgcaggtagcccagctatcaa aaagggaatactgcagaccgttaaggtcgt ggatgaactcgtcaaagtaatgggaaggcataagcccgagaatatcgttatc gagatggcccgagagaaccaaactacccagaagggacagaagaacagta gggaaaggatgaagaggattgaagagggtataaaagaactggggtcccaa atccttaag g aacacccag ttg aaaacacccagctt cag aatg ag aag ctct acctgtactacctgcagaacggcagggacatgtacgtggatcaggaactgga catcaatcggctctccgactacgacgtggctgctatcgtgccccagtcttttctca aagatgattctattgataataaagtgttgacaagatccgataaagctagaggga agagtgataacgtcccctcagaagaagttgtcaagaa aatgaaaaattattgg cggcagctgctgaacgccaaactgatcacacaacggaagttcgataatctga ctaaggctgaacgaggtggcctgtctgagttggataaagccggcttcatcaaa aggcagcttgttgagacacgccagatcaccaagcacgtggcccaaattctcg attcacgcatgaacaccaagtacgatgaaaatgacaaact gattcgagaggt gaaagttactctgaagtctaagctggtctcagatttcagaaaggactttcagt tttataagg tg ag ag agatcaacaattaccaccatgcgcatg atgcctacctg a atgcagtggtaggcactgcacttatcaaaaaatatcccaagcttgaatctgaatt tgtttacggagactataaagtgtacgatgttag gaaaatgatcgcaaagtctga gcaggaaataggcaaggccaccgctaagtacttcttttacagcaatattatgaa ttttttcaagaccgagattacactggccaatggagagattcggaagcgaccact tatcgaaacaaacggagaaacaggagaaatcgtgtgggacaagggtaggg atttcgcgacagtccggaaggtcctgtccat gccgcaggtgaacatcgttaaa aagaccgaagtacagaccggaggcttctccaaggaaagtatcctcccgaaa aggaacagcgacaagctgatcgcacgcaaaaaagattgggaccccaaga aatacggcggattcgattctcctacagtcgcttacagtgtactggttgtggccaa agtggagaaagggaagtctaaaaactcaa aagcgtcaaggaactgctgg gcatcacaatcatggagcgatcaagcttcgaaaaaaaccccatcgactttctc gaggcgaaaggatataaagaggtcaaaaaagacctcatcattaagcttccca agtactctctctttgagcttgaaaacggccggaaacgaatgctcgctagtgcgg gcgagctgcagaaaggtaacgagctggcactgccctctaaatac gttaatttct tgtatctggccagccactatgaaaagctcaaagggtctcccgaagataatgag cag aagcagctg ttcgtgg aacaacacaaacactaccttg atg ag atcatcg agcaaataagcg aattctccaaaag ag tg atcctcgccg acg ctaacctcg at 7CICI η / Ι 7Π7 / Β / Υ 112 SEQ ID NO: SEQUENCE NAME SEQUENCE aaggtgctttctgcttacaataagcacagggataagcccatcagggagcagg cagaaaacattatccacttgtttactctgaccaacttgggcgcgcctgcagcctt caagtacttcgacaccaccatagacagaaagcggtacacctctacaaagga ggtcctggacgccacactgattcatcagtcaattacgg ggctctatgaaacaa gaatcgacctctctcagctcggtggagac 67 Overactive PiggyBac (PB) transposase, na sequence atgggcagcagcctggacgacgagcacatcctgagcgccctgctgcagagc gacgacgagctggtcggcgaggacagcgacagcgaggtgagcgaccacg tgagcgaggacgacgtgcagt ccgacaccgaggaggccttcatcgacgag gtgcacgaggtgcagcctaccagcagcggctccgagatcctggacgagcag aacgtgatcgagcagcccggcagctccctggccagcaacaggatcctgacc ctgccccagaggaccatcaggggcaagaacaagcactgctggtccacctcc aagcccaccaggcggagcagggtgtccgcc ctgaacatcgtgagaagcca gaggggccccaccaggatgtgcaggaacatctacgaccccctgctgtgcttc aagctgttcttcaccgacgagatcatcagcgagatcgtgaagtggaccaacg ccgagatcagcctgaagaggcgggagagcatgacctccgccaccttcaggg acaccaacgaggacgagatctacgccttcttc ggcatcctggtgatgaccgcc gtg agg aagg acaaccacatg ag caccg acg acctgttcg acag atccctg agcatggtgtacgtgagcgtgatgagcagggacagattcgacttcctgatcag atgcctgaggatggacgacaagagcatcaggcccaccctgcgggagaacg acgtgttcacccccgt gagaaagatctgggacctgttcatccaccagtgcatcc agaactacacccctggcgcccacctgaccatcgacgagcagctgctgggctt caggggcaggtgccccttcagggtctatatccccaacaagcccagcaagtac ggcatcaagatcctgatgatgtgcgacagcggcaccaagtacatgatcaacg gcatgccctacctgggcaggggcacccagaccaacggcgtgcccctgggcg agtactacgtgaaggagctgtccaagcccgtccacggcagctgcagaaacat cacctgcgacaactggttcaccagcatccccctggccaagaacctgctgcag gag ccctacaagctgaccatcgtgggcaccgtgagaagcaacaagagaga gatccccgaggtcctgaagaacagcaggtccaggcccgtgggcaccagcat gttctgcttcgacggccccctgaccctggtgtcctacaagcccaagcccgcca agatggtgtacctgctgtccagctgcgacgaggacgccagcatcaacgaga gcaccggcaagcccca gatggtgatgtactacaaccagaccaagggcggc gtggacaccctggaccagatgtgcagcgtgatgacctgcagcagaaagacc aacaggtggcccatggccctgctgtacggcatgatcaacatcgcctgcatcaa cagcttcatcatctacagccacaacgtgagcagcaagggcgagaaggtgca gagccggaaaaagttcatgc ggaacctgtacatgggcctgacctccagcttca 7CICI Π / I 7Π7 / Β / Υ 113 SEQ ID NO: SEQUENCE NAME SEQUENCE tgaggaagaggctggaggcccccaccctgaagagatacctgagggacaac atcagcaacatcctgcccaaagaggtgcccggcaccagcgacgacagcac cgaggagcccgtgatgaagaagaggacctactgcacctactgtcccagcaa gatcagaagaaaggccagcgccagctgcaagaagtgtaaga aggtcatctg ccgggagcacaacatcgacatgtgccagagctgtttc 68 Sleeping Beauty transposase (SB100) hyperactive, na sequence Atgggaaaatcaaaagaaatcagccaagacctcagaaaaagaattgtaga cctccacaagtctggttcatccttgggagcaatttccaaacgcctggcggtacc acgttcatctgtacaaacaa tagtacgcaagtataaacaccatgggaccacg cagccgtcataccgctcaggaaggagacgcgttctgtctcctagagatgaacg tactttggtgcgaaaagtgcaaatcaatcccagaacaacagcaaaggacctt gtgaagatgctggaggaaacaggtacaaaagtatctatatccacagtaaaac gagtcctatatcgacataac ctgaaaggccactcagcaaggaagaagccac tgctccaaaaccgacataagaaagccagactacggtttgcaactgcacatgg ggacaaagatcgtactttttggagaaatgtcctctggtctgatgaaacaaaaat agaactgtttggccataatgaccatcgttatgtttggaggaagaagggggagg cttgcaagccgaagaacaccat cccaaccgtgaagcacgggggtggcagc atcatgttgtggggggtgctttgctgcaggagggactggtgcacttcacaaaata gatggcatcatggacgccgtgcagtatgtggatatattgaagcaacatctcaag acatcagtcaggaagttaaagcttggtcgcaaatgggtcttccaacacgacaa tgaccccaagcatacttcca aagttgtggcaaaatggcttaaggacaacaaa gtcaaggtattggagtggccatcacaaagccctgacctcaatcctatagaaaa tttgtgggcagaactgaaaaagcgtgtgcgagcaaggaggcctacaaacctg actcagttacaccagctctgtcaggaggaatgggccaaaattcacccaaatta ttgtgggaagctt gtggaaggctacccgaaacgtttgacccaagttaaacaattt aaaggcaatgctaccaaatac 69 Human Cas9 (hCas9 ), aa sequence ENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGL FGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLD NLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPL SASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK 7CICI Π / I 7Π7 / Β / Υ 114 SEQ ID NO: SEQUENCE NAME SEQUENCE NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNR EDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD NREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITP WNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSL LYEYFTVYNELTKV KYVTEGMRKPAFLSGEQKKAIVDLL FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASL GTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGI RDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQ KAQVSGQGDSLHEHIAN LAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEE GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVD QELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNR GKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTK AERGGLSELDKAGFIKRQLVET RQITKHVAQILDSRMNT KYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP LIETNGETGEIVWDKGRDFATRKVLSMPQVNIVKKTEV QTGGFSKESILPKRNSDK LIARKKDWDPKKYGGFDSPT VAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNP IDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH RDKPIREQAENIIHLFTLTNLGA PAAFKYFDTTIDRKRYTS TKEVLDATLIHQSITGLYETRIDLSQLGGD 70 Nicasa Cas9 (nCas9), aa sequence MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTD RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI CYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPI FGNIVDEVAYHEKYPTI YHLRKKLVDSTDKADLRLIYLAL AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFE ENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGL FGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLD NLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPL SASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK 7CICI Π / I 7Π7 / Β / Υ 115 SEQ ID NO: SEQUENCE NAME SEQUENCE NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNR EDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD NREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITP WNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSL LYEYFTVYNELTKV KYVTEGMRKPAFLSGEQKKAIVDLL FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASL GTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGI RDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQ KAQVSGQGDSLHEHIAN LAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEE GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVD QELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNR GKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTK AERGGLSELDKAGFIKRQLVET RQITKHVAQILDSRMNT KYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP LIETNGETGEIVWDKGRDFATRKVLSMPQVNIVKKTEV QTGGFSKESILPKRNSDK LIARKKDWDPKKYGGFDSPT VAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNP IDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH RDKPIREQAENIIHLFTLTNLGA PAAFKYFDTTIDRKRYTS TKEVLDATLIHQSITGLYETRIDLSQLGGD 71 dead Cas9 (dCas9), aa sequence MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTD RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI CYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPI FGNIVDEVAYHEKYPTIYHL RKKLVDSTDKADLRLIYLAL AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFE ENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGL FGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLD NLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPL SASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK 7CICI Π / l 7Π7 / Β / Υ 116 SEQ ID NO: SEQUENCE NAME SEQUENCE NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNR EDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD NREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITP WNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSL LYEYFTVYNELTKV KYVTEGMRKPAFLSGEQKKAIVDLL FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASL GTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGI RDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQ KAQVSGQGDSLHEHIAN LAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEE GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVD QELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKAR GKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTK AERGGLSELDKAGFIKRQLVET RQITKHVAQILDSRMNT KYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRP LIETNGETGEIVWDKGRDFATRKVLSMPQVNIVKKTEV QTGGFSKESILPKRNSDK LIARKKDWDPKKYGGFDSPT VAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNP IDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH RDKPIREQAENIIHLFTLTNLGA PAAFKYFDTTIDRKRYTS TKEVLDATLIHQSITGLYETRIDLSQLGGD 72 Hyperactive PiggyBac (PB) transposase, na sequence MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDD VQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSL ASNRILTLPQRTIRGKNKHCWSTSKPTRRSRVSALNIVR SQRGPTRMCRNIY DPLLCFKLFFTDEIISEIVKWTNAEISL KRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMST DDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTL RENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFR GRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLG RGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSI 7CICI Π / I 7Π7 / Β / Υ SEQ ID NO: SEQUENCE NAME SEQUENCE PLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVG TSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINEST GKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWP MALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNL YMGLTSSFMRKRLEAPTL KRYLRDNISNILPKEVPGTSD DSTEEPVMKKRTYCTYCPSKIRRKASASCKKCKKVICRE HNIDMCQSCF 73 Sleeping Beauty transposase (SB100) hyperactive, aa sequence MGKSKEISQDLRKRIVDLHKSGSSLGAISKRLAVPRSSV QTIVRKYKHHGTTQPSYRSGRRRVLSPRDERTLVRKVQ INPRTTAKDLVKMLEET GTKVSISTVKRVLYRHNLKGHS ARKKPLLQNRHKKARLRFATAHGDKDRTFWRNVLWSD ETKIELFGHNDHRYVWRKKGEACKPKNTIPTVKHGGGSI MLWG C F AAG GTG ALH Kl DGIM D AVQY VDILKQH LKTS V RKLKLGRKWVFQHDNDPKHTSKVVAKWLKDNKVKVLE WPSQSPDLNP IENLWAELKKRVRARRPTNLTQLHQLCQ EEWAKIHPNYCGKLVEGYPKRLTQVKQFKGNATKY 74 cPPT Domain / CTS of IN, sequence of na ttttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagt agacataatagcaacagacatacaaactaaagaattacaaaaacaaattac aaaaattcaaaatttt 75 Primer GG-cPPT-Fw tcctctcgtctccattattttaaaagaaaaggggggatt 76 Primer GG-cPP T-STOP- Fw tcctctcgtctccattaatttaaaagaaaaggggggatt 77 Primer GG-cPPT-Rv tcctctcgtctccctgaaaaattttgaatttttgtaatttgtttttg 78 Primer GG-AAVS1-6d-Fw tcctctcgtctccattatatggctccaaagaaaaagagg 79 Primer dor GG -AAVS1 -6d-Rv tcctctcgtctccctgatcaatcctcatcctgtctacttgccaca 80 Primer GG-AAVS1-6d (NLS)-Fw tcctctcgtctccattatatggcccaggctgctct 81 PrimerIN-Fw ttttagatggaatagataaggccc 7CICI Π / I 7Π7 / Β / Υ 118 SEQ ID NO: SEQUENCE NAME SEQUENCE 82 Primer c 85 Primer Sacl-pSICO_IC5'Rv2 cctctctatgggcagtctagcgagctcctggtctaaccagagagaccc 86 Primer Xbal-pSICO_IC3'Fw1 ctagctctagatccctcagacccttttagtca 87 Primer Sacl-pSICO_IC3'Rv1 ctagcgagctccaacagacgggcacacacta 88 Primer CD1-A128T-F tcaccagtactacagttaagaccgcctgttggtgg 91 Primer CCD1-A128T-R ccaccaacaggcggtcttaactgtagtactggtga 92 Primer CCD2-E170G-F acaggtaagagatcaggctggccatcttaagacagcagtac 93 Primer CCD2-E170G-R gtactgctgtcttaagatggccagcctgatctcttacctgt 94 Primer NTD1 -E10 / 13K-F ggttttttagatggaatagataaggcccaaaaggaacataagaaatatcacag taattggaga 95 Primer NTD1-E10 / 13K-R tctccaattactgtgatatttctta tgttccttttgggccttatctattccatctaaaaaaa cc 7CICI Π / I 7Π7 / Β / Υ 119 SEQ ID NO: SEQUENCE NAME SEQUENCE 96 Primer Solubility- F185K-F aaatggcagtattcatccacaataagaaaagaaaaggggggattggggg 97 Primer Solubility- F185K-R cccccaatccccccttttcttttcttattgtggatgaatactgccattt 98 Primer Primer NGSaavs fw a cactctttccctacacgacgctcttccgatctaggacagcatgtttgctgcct 99 Primer NGS-aavs rv gactggagttcagacgtgtgctcttccgatctgctccaggaaatgggggtg 100 Primer PB R245A cgtgttcacccccgtggcaaag atctggg acctg 10 1 PB R275-277A Primer agctgctgggcttcgcgggcgcgtgccccttcaggg 102 PB Primer R388A gaacagcaggtccgcgcccgtgggcacc 103 PB Primer S351A gacaactggttcaccgccatccccctggccaa 104 PB Primer W465A gaaagaccaacagggcgcccatggccctgc 1 05 Primer PB R372A-K375A catcgtgggcaccgtggcaagcaacgcgagagagatccccgag 106 Primer PB D450N gcgtggacaccctgaaccagatgtgcagc 107 Primer SYBR-WPRE3_Fw acgctatgtggatacgctgct 108 Primer SYBR-WPRE- 3_Rv agcaaacacagtgcacaccac 109 Primer SYBRRNaseP_Fw ggagtgaggagggatgtgaa 110 Primer SYBRRNaseP_Rv attgagggcactggaaattg 111 Iillumina custom primer aatgatacggcgaccaccgagatctacacagctagacactctttccctacacg acgctcttccgatct 7CICI Π / l 7Π7 / Β / Υ 120 SEQ ID NO: SEQUENCE NAME SEQUENCE 112 NEBNext Index Primer 9 caagcagaagacggcatacgagatctgatcgtgactggagttcagacgtgtg ctcttccgatct 113 NGS Primer cluster 1 fw acactctttccctacacgacgctcttccgatct ctgcgggagaacgacgtgtt 114 NGS primer cluster 2 rv gactggagttcagacgtgtgctcttccgatct cctcaccttcctcttcttcttgg 115 CMV-F primer ctgcagcgcggggatctcatgctggagttcttcgcccacccc 116 Cas9 primer rv caccttcctctcttcttcttggggtca 117 ZFP_TCRa 4, sequence of na atggctcctaagaagaagcggaaagtcggcatacacggagtgcctgctgca atggcagaaaggccattccaatgcagaatatgcatgaggaacttctcagatcg cagtaacctctcaaggcatatacggacccatacgggggaaaaaccatttgcct gtgatatatgtggccgcaagttcgctcagaaagtgaccttggcagctcacacta agattcacacacatccaagagcccctatccctaagccgttccaatgtaggatat gcatgcgaaacttctctgatcggagtgcactgagtaggcacatcagaacaca cac gggagaaaagcctttcgcttgcgatatctgcgggcggaagttcgcaacat ccgggaatctcactcgccatacgaaaatacacactggcagccaaaaaccttt ccaatgccgaatatgtatgagaaattttagctacagaagttcattgaaagaaca cattagaacccataccggagaaaagccgttcgcgtgcgatatctgcggt CGG QCRICMRNFSYRS SLKEHIRTHTGEKPFACDICGRKFATSGNLTRHTKIH 119 Modified hyperactive PiggyBac, aa sequence SEQ ID NO: 9 With R245A, R275A, R277A, R275A / R277A, G325A, N347A, N347S, S351E, S351P, S351A, R372A, K375A, R388A, D450N, W465A, T560A, S564P, S573A, M589V, S592G, F594L, or any combination thereof 245, 7CICI Π / l 7Π7 / Β / Υ 121 SEQ ID NO: SEQUENCE NAME SEQUENCE sequence from a to R or A at position 275, R or A at position 277, A or G at position 325, N or A at position 347, E, P or A at position 351, R at position 372, A at position 375, D or N at position 450 W or A at position 465 T or A at position 560, P or S at position 564, S or A at position 573, G or S at position 592, L or F at position 594, or any combination thereof. 121 Modified hyperactive PiggyBac Top1.1, aa sequence MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDD VQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSL ASNRILTLPQRTIRGKNKHCWSTSKPTRRSRVSALNIVR SQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISL KRRESMTSATFRDT NEDEIYAFFGILVMTAVRKDNHMST DDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTL RENDVFTPVAKIWDLFIHQCIQNYTPGAHLTIDEQLLGFR GRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLG RGTQTNGVPLAEYYVKELSKPVHGSCRNITCDNWFTEI PLAKNLLQEPYKLTIVGTVRSNAREIPEVL KNSRSRPVG TSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINEST GKPQMVMYYNQTKGGVDTL(D / N)QMCSVMTCSRKTNR (W / A)PMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKK FMRNLYMGLTSSFMRKRLEAPTLKRYLRDNISNILPKEV PGTSDDSTEEPVMKKRTYCTYCPPKIRRKASA SCKKCK KVICREHNIDMCQGCL position 450 can be D or N position 465 can be W or A 7CICI Π / l 7Π7 / Β / Υ 122 SEQ ID NO: SEQUENCE NAME SEQUENCE 122 Modified hyperactive PiggyBac Top1.2, aa sequence MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDD VQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSL ASNRILTLPQRTIRGKNKHCWSTSKPTRRSRVSALNIVR SQRGPTRMCRNIYDPLLCFKLFFTDEIISEIV KWtnaeisl PG TSDDSTEEPVMKKRTYCAYCPSKIRRKASASCKKCK KVICREHNIDMCQSCF position 450 can be D or N position 465 can be W or A 123 Modified hyperactive PiggyBac Top1.3, aa sequence MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDD VQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSL ASNRILTLPQRTIRGKNKHCWSTSKPTRRSRVSALNIVR SQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKW TNAEISL KRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMST DDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTL RENDVFTPVAKIWDLFIHQCIQNYTPGAHLTIDEQLLGFR GRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLG RGTQTNGVPLAEYYVKELSKPVHGSCRNITCDAWFTAI PLAKNLL QEPYKLTIVGTVRSNAREIPEVLKNSRSRPVG TSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINEST GKPQMVMYYNQTKGGVDTL(D / N)QMCSVMTCSRKTNR (W / A)PMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKK FMRNLYMGLTSSFMRKRLEAPTLKRYLRDNISNILPKEV PGTSDDSTE EPVMKKRTYCAYCPSKIRRKASAACKKCK KVICREHNIDMCQSCF 7CICI Π / I 7Π7 / Β / Υ 123 SEQ ID NO: SEQUENCE NAME SEQUENCE position 450 can be D or N position 465 can be W or A 124 Regular Modified Hyperactive PiggyBac 1, aa sequence MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDD VQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSL ASNRILTLPQRTIRGKNKHCWSTSKPTRRSRVSALNI VR SQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISL KRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMST DDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTL RENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFR GRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLG RGTQTNGVPLGE YYVKELSKPVHGSCRNITCDAWFTSI PLAKNLLQEPYKLTIVGTVASNKREIPEVLKNSRSRPVGT SMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTG KPQMVMYYNQTKGGVDTL(D / N)QMCSVMTCSRKTNR ( W / A)PMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKF MRNLYMGLTSSFMRKRLEAPTLKRYLRDNISNILPKEVP GTSDDSTEEPVMKKRTYCTYCPSKIRRKASASCKKCKK VICREHNIDMCQSCF position 450 can be D or N position 465 can be W or A 125 Regular modified hyperactive PiggyBac 2, aa sequence MG SSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDD VQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSL ASNRILTLPQRTIRGKNKHCWSTSKPTRRSRVSALNIVR SQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISL KRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMST DDLFDRSLSMVYVS VMSRDRFDFLIRCLRMDDKSIRPTL RENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFR GRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLG RGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSI PLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVG TSMFCFDGPLTLVSYKPKPAKMVY LLSSCDEDASINEST GKPQMVMYYNQTKGGVDTL(D / N) QMCSVMTCSRKTNR 7CICI Π / l 7Π7 / Β / Υ 124 SEQ ID NO: SEQUENCE NAME SEQUENCE (W / A)PMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKK FMRNLYMGLTSSFMRKRLEAPTLKRYLRDNISNILPKEV PGTSDDSTEEPVMKKRTYCTYCPSKIRRKASASCKKCK KVICREHNIDMCQGCF position 450 can be D or N position 465 can be W or A 126 Piggy Regular modified hyperactive bac 3, aa sequence MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDD VQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSL ASNRILTLPQRTIRGKNKHCWSTSKPTRRSRVSALNIVR SQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISL KRRESMTSATFRDTNEDEIYAFFGILV MTAVRKDNHMST DDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTL RENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFR GRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLG RGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSI PLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVG TSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINEST GKPQMVMYYNQTKGGVDTL(D / N)QMCSVMTCSRKTNR (W / A)PMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKK FMRNLYMGLTSSFMRKRLEAPTLKRYLRDNISNILPK EV PGTSDDSTEEPVMKKRTYCTYCPSKIRRKASASCKKCK KVICREHNIDMCQSCF position 450 can be D or N position 465 can be W or A 127 PiggyBac modified hyperactive regular 4, aa sequence MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDD VQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSL ASNRILT LPQRTIRGKNKHCWSTSKPTRRSRVSALNIVR SQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISL KRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMST DDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTL RENDVFTPVAKIWDLFIHQCIQNYTPGAHLTIDEQLLGFA GACPFRVYIPNKPSKYGIKI LMMCDSGTKYMINGMPYLG 7CICI Π / I 7Π7 / Β / Υ 125 SEQ ID NO: SEQUENCE NAME SEQUENCE RGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSI PLAKNLLQEPYKLTIVGTVASNAREIPEVLKNSRSRPVGT SMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTG KPQMVMYYNQTKGGVDTL(D / N)QMCSVMTCSRKTNR( W / A)PMALLYG MINIACINSFIIYSHNVSSKGEKVQSRKKF MRNLYMGLTSSFMRKRLEAPTLKRYLRDNISNILPKEVP GTSDDSTEEPVMKKRTYCTYCPSKIRRKASASCKKCKK VICREHNIDMCQSCL position 450 can be D or N position 465 can be W or A 128 Regular Modified Hyperactive PiggyBac 5, sequence aa MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDD VQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSL ASNRILTLPQRTIRGKNKHCWSTSKPTRRSRVSALNIVR SQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISL KRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMST DDLFDRS LSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTL RENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFA GRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLG RGTQTNGVPLAEYYVKELSKPVHGSCRNITCDSWFTAI PLAKNLLQEPYKLTIVGTVASNKREIPEVLKNSRSRPVGT SMFCFDGPLTLVSYKPK PAKMVYLLSSCDEDASINESTG KPQMVMYYNQTKGGVDTL(D / N)QMCSVMTCSRKTNR( W / A)PMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKF MRNLYMGLTSSFMRKRLEAPTLKRYLRDNISNILPKEVP GTSDDSTEEPVMKKRTYCAYCPSKIRRKASASCKKCKK VICREHNIDMCQGCF With position 450 can be D or N position 465 can be W or A 129 Regular Modified Hyperactive PiggyBac 6, aa sequence MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDD VQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSL ASNRILTLPQRTIRGKNKHCWSTSKPTRRSRVSALNIVR SQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISL 7CICI Π / I 7Π7 / Β / Υ 126 SEQ ID NO: SEQUENCE NAME SEQUENCE KRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMST DDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTL RENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFA GRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLG RGTQTNGVPLGEYYVKELSKPVHG SCRNITCDNWFTAI PLAKNLLQEPYKLTIVGTVASNAREIPEVLKNSRSRPVGT SMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTG KPQMVMYYNQTKGGVDTL(D / N)QMCSVMTCSRKTNR( W / A)PMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKF MRNLYMGLTSSFMRKRLEAPTLKRY LRDNISNILPKEVP GTSDDSTEEPVMKKRTYCAYCPSKIRRKASASCKKCKK VICREHNIDMCQGCF With position 450 can be D or N position 465 can be W or A 130 Linker, sequence aa KLAGGAPAVGGGPK 131 Linker, aa sequence EFGGGGSGGGGSGGGGSQF 132 Primer SV40pA-R G aaatttg tg atgctattg c 133 Linker (GGGGS)n n is an integer between 1 and 50 134 Linker (EAAAK)n n is an integer between 1 and 50 JCICI η / Ι 7Π7 / Β / Υ

Claims

CLAIMS 1. A nucleic acid construct comprising: a) a first polynucleotide sequence comprising a nucleic acid encoding a first DNA-binding protein engineered to bind to a specific genomic DNA sequence in a genome; wherein the first DNA-binding protein is either a zinc finger protein or a Cas9 protein;(b) a second polynucleotide sequence comprising a nucleic acid encoding a second DNA-binding protein enabling the insertion of an exogenous nucleic acid into a genome, wherein the second DNA-binding protein is: (i) a hyperactive PiggyBac transposase, or a modified hyperactive PiggyBac with improved specificity for insertion of the exogenous nucleic acid into the genome compared to hyperactive PiggyBac, or (ii) a human immunodeficiency virus (HIV) integrase or an HIV integrase modified with improved specificity for insertion of the exogenous nucleic acid into the genome compared to HIV integrase; and (c) an optional polynucleotide sequence comprising a nucleic acid encoding a linker;wherein the nucleic acid construct encodes a fusion protein comprising the first DNA-binding protein, the second DNA-binding protein, and the optional linker between the first DNA-binding protein and the second DNA-binding protein; and wherein the fusion protein enables the insertion of the exogenous nucleic acid into a specific site in the genome.

2. The nucleic acid construction according to claim 1, wherein the Cas9 protein is selected from the group consisting of a human Cas9, a Cas9 nicase, and a dead Cas9.

3. The nucleic acid construction according to claim 1, wherein the zinc finger protein is a C2H2 zinc finger protein comprising 6 domains.

4. The nucleic acid construction according to what is claimed in any of claims 1 to 3, wherein the linker comprises an XTEN sequence or a GGS sequence.

5. The nucleic acid construction according to what is claimed in any of claims 1 to 4, wherein the 3' end of the first polynucleotide sequence is connected to the 5' end of the second polynucleotide.

6. The nucleic acid construct according to any one of claims 1 to 5, wherein: (a) the first DNA-binding protein is a Cas9 protein or a zinc finger protein, and (b) the second DNA-binding protein is a hyperactive PiggyBac transposase, or a modified hyperactive PiggyBac with improved specificity for insertion of the exogenous nucleic acid into the genome compared to hyperactive PiggyBac, wherein the nucleic acid construct comprises (c) the polynucleotide sequence comprising a nucleic acid encoding a linker comprising an XTEN sequence or a GGS sequence, and wherein the 3' end of the first polynucleotide sequence is connected to the 5' end of the second polynucleotide.

7. The nucleic acid construct according to any one of claims 1 to 5, wherein: (a) the first DNA-binding protein is a Cas9 protein or a zinc finger protein, and (b) the second DNA-binding protein is an HIV integrase, or an HIV integrase modified with improved specificity for insertion of the exogenous nucleic acid into the genome compared to the HIV integrase, wherein the nucleic acid construct comprises (c) the polynucleotide sequence comprising a nucleic acid encoding a linker comprising an XTEN sequence or a GGS sequence, and wherein the 3' end of the first polynucleotide sequence is connected to the 5' end of the second polynucleotide.

8. The nucleic acid construct according to any one of claims 1 to 6, wherein the modified hyperactive PiggyBac transposase comprises a mutation of one or more of the amino acids 245, 268, 275, 277, 287, 290, 315, 325, 341, 346, 347, 350, 351, 356, 357, 372, 375, 388, 409, 412, 432, 447, 450, 460, 461, 465, 517, 560, 564, 571, 573, 576, 586, 587, 589, 592, and 594 corresponding to the amino acid sequence SEQ ID NO: 9 of the hyperactive PiggyBac.

9. The nucleic acid construct according to claim 8, wherein the mutation of the modified hyperactive PiggyBac transposase comprises one or more of the amino acid modifications selected from: R245A, D268N, R275A / R277A, K287A, K290A, K287A / K290A, R315A, G325A, R341A, D346N, N347A, N347S, T350A, S351E, S351P, S351A, K356E, N357A, R372A, K375A, R372A / K375A, R388A, K409A, K412A, K409A / K412A, K432A, D447A, D447N, D450N, R460A, K461A, R460A / K461A, W465A, S517A, T560A, S564P, S571N, S573A, K576A, H586A, I587A, M589V, S592G, or F594L which correspond to the amino acid sequence of SEQ ID NO: 9 of the hyperactive PiggyBac.

10. The nucleic acid construct according to any one of claims 1 to 6, wherein the modified hyperactive PiggyBac transposase comprises a mutation in one or more of the amino acids 245, 275, 277, 325, 347, 351, 372, 375, 388, 450, 465, 560, 564, 573, 589, 592, 594 corresponding to the amino acid sequence SEQ ID NO: 9 of hyperactive PiggyBac. 7CICI n / l 7P7 / B / Y 129 11. The nucleic acid construct according to claim 10, wherein the mutation of the modified hyperactive PiggyBac transposase comprises one or more of the amino acid modifications selected from: R245A, R275A, R277A, R275A / R277A, G325A, N347A, N347S, S351E, S351P, S351A, R372A, K375A, R388A, D450N, W465A, T560A, S564P, S573A, M589V, S592G or F594L corresponding to the amino acid sequence SEQ ID NO: 9 of the hyperactive PiggyBac.

12. The nucleic acid construct according to claim 10, wherein the modified hyperactive PiggyBac transposase comprises the amino acid sequence of SEQ ID NO: 9, wherein: i. the amino acid at position 245 is A, ii. the amino acid at position 275 is R or A, iii. the amino acid at position 277 is R or A, iv. the amino acid at position 325 is A or G, v. the amino acid at position 347 is N or A, vi. the amino acid at position 351 is E, P or A, vii. the amino acid at position 372 is R, viii. the amino acid at position 375 is A, ix. the amino acid at position 450 is D or N, x. the amino acid at position 465 is W or A, xii. xii. the amino acid at position 560 is T or A, xiii. the amino acid at position 564 is P or S, xiv. the amino acid at position 573 is S or A, xiv. the amino acid at position 592 is G or S, and xv. the amino acid at position 594 is L or F.

13. The nucleic acid construct according to claim 10, wherein the modified hyperactive PiggyBac transposase comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 120, 121, 122, 123, 124, 125, 126, 127, 128 and 129.

14. The nucleic acid construct according to claim 10, wherein the modified hyperactive PiggyBac transposase comprises an amino acid sequence that is at least 80 percent identical to a sequence selected from the group consisting of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, and 129, wherein the modified hyperactive PiggyBac exhibits a higher specificity for DNA integration into a genome compared to hyperactive PiggyBac.

15. The nucleic acid construct according to what is claimed in any of claims 1 to 5 or 7, wherein the modified HIV integrase comprises a mutation of one or more of the amino acids 10, 13, 64, 94, 116, 117, 119, 120, 122, 124, 128, 152, 168, 170, 185, 231, 264, 130, 266 or 273 corresponding to the amino acid sequence of SEQ ID NO: 1 of wild-type HIV integrase.

16. The nucleic acid construct according to claim 15, wherein the modified HIV integrase mutation comprises one or more of D10K, E13K, D64A, D64E, G94D, G94E, G94R, G94K, D116A, D116E, N117D, N117E, N117R, N117K, S119A, S119P, S119T, S119G, S119D, S119E, S119R, S119K, N120D, N120E, N120R, N120K, T122K, T122I, T122V, T122A, T122R, A124D, A124E, A124R, A124K, A128T, E152A, E152D, Q168L, Q168A, E170G, F185K, R231G, R231K, R231D, R231E, R231S, K264R, K266R or K273R, which correspond to the amino acid sequence of SEQ ID NO: 1 of wild-type HIV integrase.

17. A vector comprising the nucleic acid construct according to any of claims 1 to 16, wherein the vector is suitable for expression in mammalian cells, yeast cells, insect cells, plant cells, fungal cells, or algal cells.

18. A host cell comprising the nucleic acid construct or vector according to what is claimed in any of claims 1 to 17.

19. A fusion protein obtained from the expression of the nucleic acid construct according to what is claimed in any of claims 1 to 16.

20. A composition comprising the nucleic acid construct, vector, or fusion protein according to any of claims 1 to 17 or 19, and a polynucleotide sequence encoding an exogenous nucleic acid for insertion into a genome, wherein the composition is contained in, or linked to, a packaging vector.

21. The composition according to claim 20, wherein the nucleic acid construct is in the form of RNA, DNA or protein, and the polynucleotide sequence encoding the exogenous nucleic acid is in the form of RNA or DNA.

22. The composition according to what is claimed in any of claims 20 and 21, wherein the packaging vector is a nanoparticle or a lentiviral particle.

23. A method for the site-controlled and specific integration of a single copy or multiple copies of an exogenous nucleic acid sequence into a cell, wherein the method comprises: a) supplying the nucleic acid construct, vector, or fusion protein according to any of claims 1 to 17 or 19 to the cell, and b) supplying the exogenous nucleic acid to the cell; wherein the binding of the fusion protein to the specific genomic DNA sequence in the cell's genome results in genome excision and the integration of one or more copies of the exogenous nucleic acid into the cell's genome.

24. A modified hyperactive PiggyBac transposase comprising the amino acid sequence of SEQ ID NO: 9, wherein: i. the amino acid at position 245 is A, ii. the amino acid at position 275 is R or A, iii. the amino acid at position 277 is R or A, iv. the amino acid at position 325 is A or G, v. the amino acid at position 347 is N or A, vi. the amino acid at position 351 is E, P or A, vii. the amino acid at position 372 is R, viii. the amino acid at position 375 is A, ix. the amino acid at position 450 is D or N, x. the amino acid at position 465 is W or A, xii. xii. The amino acid at position 560 is T or A, xiii. The amino acid at position 564 is P or S, xiv. The amino acid at position 573 is S or A, xiv. The amino acid at position 592 is G or S, and xv. The amino acid at position 594 is L or F. 7CICI n / l 7Π7 / Β / Y 25. The modified hyperactive PiggyBac transposase according to claim 24, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 120, 121, 122, 123, 124, 125, 126, 127, 128 and 129.

26. The modified hyperactive PiggyBac transposase according to claim 24, comprising an amino acid sequence that is at least 80 percent identical to a sequence selected from the group consisting of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, and 129, wherein the modified hyperactive PiggyBac exhibits a higher specificity for DNA integration into a genome compared to hyperactive PiggyBac.