Programmable transposases and uses thereof

Abstract

The present disclosure provides an efficient and precise programmable gene delivery technology based on a composition comprising: (i) a first protein comprising or consisting of a site-specific DNA-binding protein capable of binding and cleaving a target nucleic acid sequence; or a nucleic acid construct encoding the first protein; and (ii) a second protein comprising or consisting of a transposase; or a nucleic acid construct encoding the second protein; wherein the transposase is a modified hyperactive PiggyBac.

Description

[Technical Field]

[0001] The present invention relates to the fields of gene editing and gene therapy. [Background technology]

[0002] Many diseases, such as cancer, developmental disorders, and some infectious diseases, share common genetic and epigenetic abnormalities. Gene therapy is designed to introduce genetic material into cells to directly target and edit the genome, correcting genetically dysfunctional cells and thereby curing the associated disease.

[0003] The gene editing toolbox has expanded significantly in recent years to include promising tools for gene therapy as well as repairing defective genes to treat disorders in subjects in need.

[0004] Traditionally, gene editing is based on the design of artificial endonucleases that induce double-strand breaks (DSBs) at sequences of interest within the genome. 1 Cells repair DSBs via one of two major pathways: non-homologous end joining (NHEJ) or homology-directed repair (HDR). 2 Recently, DSB-independent editing has also been developed. Deaminases, or base editors (BEs), 3 Methods based on direct editing of DNA bases using reverse transcriptase (RT), i.e., prime editor (PE) 4 Methods based on in situ substitution of DNA bases using .

[0005] However, pathological genetic defects can range from a few bases to large deletions. Base editors or prime editors target only a few bases, and HDR-based editing does not scale well with size. 5 NHEJ-based methodologies have been developed, such as homology-independent targeted integration (HITI). 6Although this method has been demonstrated for insertions of several kilobases, it remains inefficient for very large edits. 5 Although HITI can work to deliver exons, it may not be efficient enough to reliably deliver the cDNA of genes such as dystrophin (approximately 14 kb) or laminin-α2 (approximately 9 kb).

[0006] High-precision CRISPR-programmable transposons have been reported in bacteria 7、8 However, they are not available in mammalian cells. Previous attempts to fuse zinc finger or Streptococcus pyogenes Cas9 (SpCas9) to the mammalian-compatible piggyBac (PB) or sleeping beauty transposases have delivered the systems with relatively low levels of precision. 9-11 The PB system is a system whose efficiency is properly adjusted according to size. 12 It is an attractive tool for gene therapy because it is a mutation-independent technique and works in all tissues due to its low dependency on DNA repair mechanisms.

[0007] Therefore, there remains a need to develop novel systems for target gene delivery into mammalian cells, either in vitro, ex vivo, or ex vivo. Summary of the Invention

[0008] Certain programmable transposases and their use in targeted gene editing are disclosed in WO2020250181, the contents of which are incorporated herein by reference.

[0009] The present disclosure provides a more efficient and precise programmable gene delivery technology based on a composition comprising: (i) a first protein comprising or consisting of a site-specific DNA-binding protein capable of binding and cleaving a target nucleic acid sequence; or a nucleic acid construct encoding the first protein; and (ii) a second protein comprising or consisting of a transposase; or a nucleic acid construct encoding the second protein; wherein the transposase is a modified hyperactive PiggyBac. Such a technology has the ability to deliver not only small but also large nucleic acid fragments. The inventors have tested this technology in mammalian cells and in vivo in mouse liver and surprisingly achieved high efficiency (5-10%) of site-directed integration in all cases.

[0010] In one embodiment, the composition comprises: (i) a first protein comprising or consisting of a site-specific DNA-binding protein capable of binding and cleaving a target nucleic acid sequence; or a nucleic acid construct encoding the first protein; and (ii) a second protein comprising or consisting of a transposase; or a nucleic acid construct encoding the second protein; wherein the transposase is a modified hyperactive PiggyBac comprising one or more amino acid mutations compared to the hyperactive PiggyBac of SEQ ID NO: 9.

[0011] In one embodiment, the first protein and the second protein are fused to each other, optionally via a linker, to form a fusion protein. In one embodiment, the first protein is fused to the C-terminal end of the second protein, optionally via a linker.

[0012] In one embodiment, the transposase is a modified hyperactive PiggyBac that comprises one or more amino acid mutations to increase excision activity compared to unmodified hyperactive PiggyBac and / or one or more amino acid mutations to decrease DNA binding activity compared to unmodified hyperactive PiggyBac.

[0013] In one embodiment, the one or more amino acid mutations do not consist of R372A, K375A, and D450N. In one embodiment, the one or more amino acid mutations are selected from among amino acid substitutions that increase excision activity at positions M194, D450, T560, S564, S573, S592, or F594, where the position numbers correspond to the amino acid numbering of unmodified hyperactive PiggyBac in SEQ ID NO: 9, and are preferably selected from among the amino acid substitutions M194V and / or D450N. In one embodiment, the one or more amino acid mutations are selected from among amino acid substitutions that increase excision activity at positions M194 or D450, where the position numbers correspond to the amino acid numbering of unmodified hyperactive PiggyBac in SEQ ID NO: 9, and are preferably selected from among the amino acid substitutions M194V and / or D450N. In one embodiment, the one or more amino acid mutations are selected from amino acid substitutions that reduce DNA binding activity at positions R275, R277, R347, R372, K375, R376, E377, and / or E380, where the position numbers correspond to the amino acid numbers of the unmodified, highly active PiggyBac of SEQ ID NO: 9, and are preferably selected from amino acid substitutions R275A, R277, R347S, R372A, K375A, R376A, E377A, and / or E380A. In one embodiment, the one or more amino acid mutations are selected from amino acid substitutions that reduce DNA binding activity at positions R372, K375, R376, E377, and / or E380, where the position numbers correspond to the amino acid numbers of the unmodified, highly active PiggyBac of SEQ ID NO: 9, and are preferably selected from amino acid substitutions R372A, K375A, R376A, E377A, and / or E380A.

[0014] In one embodiment, the modified hyperactive PiggyBac comprises the double mutations N347S and D450N, where the position numbers correspond to the amino acid numbering of the unmodified hyperactive PiggyBac in SEQ ID NO:9.In one embodiment, the modified hyperactive PiggyBac mutation comprises one of the following amino acid substitutions or combinations of amino acid substitutions: R372A / K375A / R376A / D450N, K375A / R376A / E377A / E380A / D450N, R372A / K375A / R376A / E377A / E380A / D450N, M194V, R376A, E377A, E380A, M194V / R372A / K375A, S351A / R372A / K375A / R388A / D450N / W465A / S573A / M589V / S5 92G / F594L, R245A / R275A / R277A / R372A / W465A / M589V, R275A / 325A / R372 A / T560A, N347A / D450N, N347S / D450N / T560A / S573A / F594L, R202K / R275A / N347S / R372A / D450N / T560A / F594L, R275A / N347S / K375A / D450N / S592G, R275A / N347S / R372A / D450N / T560A / F594L, R275A / R277A / N347S / R372A / D4 50N / T560A / S564P / F594L, R245A / N347S / R372A / D450N / T560A / S564P / S57 3A / S592G, R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592 G / F594L, V34M / R275A / G325A / N347S / S351A / R372A / K375A / D450N / T560A / S564P, G325A / N347S / K375A / D450N / S573A / M589V / S592G, S230N / R277A / N3 47S / K375A / D450N, T43I / R372A / K375A / A411T / D450N, G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G; the position numbers correspond to the amino acid numbers of the hyperactive PiggyBac of SEQ ID NO: 9, and typically the modified transposase has an amino acid sequence selected from any of SEQ ID NOs: 2-8, 10-18, and 135-149.

[0015] In one embodiment, the composition further comprises a third protein comprising or consisting of a second transposase, or a nucleic acid construct encoding the third protein; wherein the second transposase is either a hyperactive PiggyBac having SEQ ID NO: 9 or a modified hyperactive PiggyBac comprising one or more amino acid mutations compared to the hyperactive PiggyBac having SEQ ID NO: 9. In one embodiment, the first, second, and third proteins are fused to each other, optionally via a linker, to form a triple fusion protein.

[0016] In one embodiment, the first protein comprises or consists of an RNA-guided nuclease or nickase, or a zinc finger nuclease. In one embodiment, the first protein is a nuclease protein that comprises an active DNA cleavage domain and a guide RNA binding domain and has at least 80%, 90%, 95%, 99%, or at least 100% identity to Streptococcus pyogenes Cas9 (SpCas9) of SEQ ID NO: 31, Staphylococcus aureus Cas9 (SaCas9) of SEQ ID NO: 72, Cpf1 of SEQ ID NO: 74, Campylobacter jejuni Cas9 (CjCas9) of SEQ ID NO: 29, Streptococcus pyogenes Cas9 nickase (nCas9) of SEQ ID NO: 70, CasX of SEQ ID NO: 75, or Staphylococcus aureus Cas9 nickase of SEQ ID NO: 76; preferably, the first protein is a Cas9 protein selected from the group consisting of Staphylococcus aureus Cas9 (SaCas9) of SEQ ID NO: 72 and Streptococcus pyogenes Cas9 (SpCas9) of SEQ ID NO: 31.

[0017] In one embodiment, the composition further comprises a guide RNA and an exogenous nucleic acid for insertion into the genome.

[0018] In one embodiment, the transposase is fused to an RNA binding protein capable of binding to at least one specific RNA sequence contained in the guide RNA, optionally the RNA binding protein is MS2 bacteriophage coat protein (MCP), and the guide RNA preferably comprises an MS2 RNA tetraloop binding sequence that shares at least 75% identity with SEQ ID NO: 153.

[0019] In one embodiment, the exogenous nucleic acid is a large DNA fragment, typically having a size of 5 kb to 25 kb, more preferably 8 kb to 20 kb.

[0020] In one embodiment, the composition is contained in nanoparticles.

[0021] The present invention also relates to nucleic acids, typically in the form of messenger RNA (mRNA), encoding any one of the fusion proteins disclosed herein.

[0022] The present invention also relates to an in vitro method for site-specific integration of an exogenous nucleic acid sequence into the genome of a cell, the method comprising delivering the composition of the present invention, a guide RNA, and the exogenous nucleic acid to the cell.

[0023] The present invention also relates to the compositions, guide RNAs, and exogenous nucleic acids of the present invention for use in treating disease by site-specific integration of an exogenous nucleic acid sequence into the genome of a cell. [Brief explanation of the drawings]

[0024] [Figure 1] Programmable transposase technology: Cas9 (red) is combined with an engineered PB transposase domain (pink). A table of the mutants used in the experiment and their corresponding positions within the PB core model (position 563 is on the Ct and is not included in this model) is shown. [Figure 2]Figure 2A shows the dependency of programmable transposase on Cas9 variants. Fusion of nuclease Cas9 with PB, in contrast to fusions with dead Cas9 (dCas9) or nickase Cas9 (nCas9), shows superior results in both targeted and total insertions. Blue indicates targeted insertions, and yellow indicates off-target insertions. Figure 2B shows the dependency of programmable transposase on PB variants. Mutants with enhanced excision and reduced DNA binding exhibit the best on-target:off-target ratios (orange). On-target insertions were performed at AAV (green) and TRAC (blue) sites. Figure 2C shows testing of various linkers. Linker length and topology do not significantly affect the on-target activity of SpCas9 and PB fusions. [Figure 3] Hershey reporter cell line: HEK293T cell line was engineered to contain a C-terminal fragment of GFP preceded by one splice acceptor and gRNA target site. PB transposons were generated by combining a CAG promoter, an N-terminal fragment of GFP, followed by a splice donor. Grey triangles indicate PB ITRs; SA: splice acceptor; SD: splice donor; target: target insertion site; *ITR disrupted by the insertion process. [Figure 4] Figure 4A shows the programmable transposase dependence of PB variants. Mutant 450, which exhibits enhanced excision in conjunction with various mutations to reduce DNA binding, exhibits the best on-target activity. Simultaneous mutation of R372 and R376 to A is not well tolerated. E377 is not involved in DNA binding, but mutation to A may be beneficial to avoid the accumulation of negative charges in that region when K375 and R376 are mutated to A. In Figure 4B, R372A / K375A reduces PB integration activity as a result of reduced binding to target DNA (similarly observed with D450N). Off-target integration studies are ongoing. [Figure 5]Double-strand breaks and programmable DNA-binding domains influence target insertion. Efficient on-target insertion requires co-localization of double-strand breaks and PBs at the insertion site. [Figure 6] Sanger sequencing validation of multiple insertions (as measured by NGS, see Figure 2a for a more comprehensive distribution). The ITR TTAA is lost during the targeted insertion. The NGG Pam is highlighted in red. [Figure 7] Insertional activity of hyPB K375A_R376A_E377A_E380A_D450N without cas9. To further investigate the targeted insertion mechanism, hyPB K375A_R376A_E377A_E380A_D450N was cloned without cas9 and its insertion efficiency was tested compared to hyPB WT using the RFP transposon in hek293T cells. The results show that without the fused cas9, this mutant has no insertional activity. [Figure 8] A. Characterization of the targeted insertion site using Guide-seq. The programmable transposase inactivates the ITR site with multiple indels, resulting in irreversible insertions. B. Characterization of the entire insertion site using Guide-seq. Only on-target insertions were detected in the TCR locus (top panel). Sanger sequencing is shown for four clones (bottom panel). [Figure 9] Characterization of the programmable transposase by Guide-seq insertion profiling showed that the combination of the hyPB mutant with Cas9 performed precise transposon insertion. [Figure 10] Benchmarking of Cas9-hyPB R372A-K375A-D450N against other targeted insertion platforms such as Cas9-induced HDR (using 300 bp homology arms) is shown. [Figure 11] Figure 1 shows the in vivo spread of Cas9-hyPB R372A-K375A-D450N in mouse liver. Relative copy numbers measured by qPCR are reported. [Figure 12] Programmable transposases can be engineered using various Cas variants, including CasX, CjCas9, Cpf1, and SaCas9, some of which achieved similar results to SpCas9 in terms of programmable insertion at target sites. Each of the tested Cas variants used three independent gRNAs to target specific target regions in a split-GFP reporter cell line. [Figure 13] Targeted insertion is achieved by double-strand breaks mediated by Cas9 and a single gRNA (gRNA-TCR1 or AAVS1-3), or by nickase-Cas9 and two gRNAs targeting nearby locations (gRNA-TCR1 and AAVS1-3), as well as a programmable DNA-binding domain (ZnF) fused to a modified hyPB (mutant R372A-K375A-D405N). Efficient on-target insertion requires colocalization of the double-strand break and PB at the insertion site. This can be achieved by dual cleavage with nuclease-Cas9 or nickase-Cas9. [Figure 14] A programmable transposase can be engineered as a dimeric polypeptide of two hyPB domains and a Cas9 nuclease, resulting in superior programmable insertion compared to Cas9-hyPB. A split-GFP reporter cell line was used for programmable insertion of the split-GFP transposon into target sites. The hyPB R372A-K375A-D450N mutant has been used for monomeric or dimeric fusion to Cas9. Conditions: 1 - negative control using only hyPB as insertion mechanism; 2: positive control of Cas9-hyPB R372A-K375A-D450N in pcDNA expression vector; 3: positive control of Cas9-hyPB R372A-K375A-D450N in lentiviral expression vector; 4: Cas9 nuclease fused to two units of hyPB R372A-K375A-D450N at the C-terminus; 5: Cas9 nuclease fused to two units of hyPB R372A-K375A-D450N, one at the C-terminus and the other at the N-terminus. [Figure 15]By performing several cycles of selection for cells that had undergone programmable transposition, we were able to select optimal mutant combinations from the library and identified several mutants that, when fused to Cas9, exhibited higher enrichment and programmable insertion capacity than Cas9-hyPB R372A-K375A-D450N. [Figure 16] As the selection cycles progressed, on-target efficiency improved. Bulk variants selected from each cycle were co-transfected into a reporter cell line with a gRNA targeting AAVS1 and a 1 / 2 GFP transposon. The amount of plasmid was corrected by the PB copy number to normalize the cloning efficiency. [Figure 17] (A) On-target efficiency of the top selected candidates is shown. Six individual candidates were selected based on the highest on-target activity among 96 random clones selected from the last cycle. Individual on-target activity was compared to Cas9-hyPB R372A-K375A-D450N. (B) Logos depicting key PB residues in the top on-target active variants are shown. [Figure 18] Figure 1 shows benchmarking of Cas9-hyPB R372A-K375A-D450N (FiCAT) for homology-independent targeted insertion (HITI). [Figure 19] Programmable insertion activity of FiCAT R372A-K375A-D450N using four different nuclease proteins. SpCas9 is used as a control for programmable insertion with gRNA-TRAC-1 alone (left). Each nuclease was used with three independent gRNAs (1-3) for targeted insertion into a 1 / 2GFP reporter cell line. [Figure 20] Figure 1 shows the integration of a minicircle luciferase transposon into the liver. The minicircle luciferase transposon, sgRNA targeting the Rosa26 locus, and FiCAT (Cas9-hyPB R372A-K375A-D450N) mRNA were delivered by hydrodynamic injection, and the luciferase signal was monitored. [Figure 21] (a) Editing activity by CasX (left) and Cpf1 (center). (b) Editing activity by SaCas9 (left) and CjCas9 (center). The mean % + / - SD of indel-containing reads is shown for two technical repeats. Representative images of N=3 biological replicates are shown. SpCas9 targeting the TRAC-1 site was used as a reference (right). [Figure 22] The improvement in on-target efficiency across selection cycles is shown. (A) Selected bulk variants from each cycle were co-transfected into a reporter cell line with a gRNA targeting AAVS1 and a 1 / 2 GFP transposon. The amount of plasmid was corrected by the PB copy number to normalize cloning efficiency. (B) Lentiviruses expressing bulk variants from each cycle were generated and used to infect reporter cell lines. [Figure 23] Specific targeted integration is shown compared to FiCAT (hyPB R372A-K375A-D450N) of one mutant isolated from bulk variants after 4 and 5 cycles of cas9_PB library enrichment co-transfected with gRNA tcr1 and 1 / 2GFP MC transposon. [Figure 24] 1 shows the programmable insertion activity of dimeric hyPB R372A-K375A-D450N fused to either SpCas9 or SaCas9 for targeted insertion in a ½ GFP reporter cell line. [Figure 25]Relative comparison of programmable insertion activity for targeted insertion in a 1 / 2 GFP reporter cell line. (A) Comparison of hyPB R372A-K375A-D450N fused to SpCas9 protein (left) and hyPB R372A-K375A-D450N fused to MCP protein (right) with separate addition of SpCas9. (B) Comparison of three hyPB mutants (R372A-K375A-D450N; R202K-R275A-N347S-R372A-D450N-T560A-F594L; and R275A-N347S-R372A-D450N-T560A-F594L) fused to MCP protein (right) with separate addition of SpCas9. [Figure 26] Comparison of programmable insertion activity for targeted insertion in a 1 / 2 GFP reporter cell line. (A) Coexpression of hyPB R372A-K375A-D450N with SpCas9 protein (left) and a fusion protein containing hyPB R372A-K375A-D450N and SpCas9 protein (right). (B) Relative comparison between three hyPB mutants (R372A-K375A-D450N; R202K-R275A-N347S-R372A-D450N-T560A-F594L; and R275A-N347S-R372A-D450N-T560A-F594L) coexpressed with SpCas9. [Figure 27] Figure 1 shows a relative comparison of programmable insertion activity for targeted insertion in a 1 / 2 GFP reporter cell line by coexpression of a first fusion protein containing SpCas and hyPB R372A-K375A-D450N with a second fusion protein containing MCP protein and hyPB mutants (R372A-K375A-D450N; R202K-R275A-N347S-R372A-D450N-T560A-F594L; and R275A-N347S-R372A-D450N-T560A-F594L). [Figure 28]Figure 1 shows a relative comparison of programmable insertion activity for targeted insertion in a 1 / 2GFP reporter cell line by coexpression of a fusion protein containing SpCas and hyPB R372A-K375A-D450N with three hyPB mutants (R372A-K375A-D450N; R202K-R275A-N347S-R372A-D450N-T560A-F594L; and R275A-N347S-R372A-D450N-T560A-F594L). [Figure 29] Comparison of programmable insertion activity for targeted insertion in a 1 / 2 GFP reporter cell line between SpCas9 fused to a dimer of hyPB R272A-K275A-D450N (left) and SpCas9 fused to the first hyPB R272A-K275A-D450N and the second hyPB mutant (right).

[0025] definition As used herein, the singular forms "a," "an," and "the" include singular and plural references unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes a single agent as well as a plurality of such agents.

[0026] The terms "nucleic acid sequence" and "nucleotide sequence" can be used interchangeably to refer to any molecule composed of or containing monomeric nucleotides. A nucleic acid can be an oligonucleotide or a polynucleotide. A nucleotide sequence can be DNA, RNA, or a mixture thereof. A nucleotide sequence can be chemically modified or artificial. Nucleic acid sequences include peptide nucleic acids (PNAs), morpholinos, and locked nucleic acids (LNAs), as well as glycol nucleic acids (GNAs) and threose nucleic acids (TNAs). Each of these sequences is distinguished from naturally occurring DNA or RNA by changes in the molecular backbone. Phosphorothioate nucleotides can also be used. Other deoxynucleotide analogs include, but are not limited to, methyl phosphonates, phosphoramidates, phosphorodithioates, N3'P5'-phosphoramidates, and oligoribonucleotide phosphorothioates, as well as their 2'-O-allyl analogs and 2'-O-methylribonucleotide methylphosphonates, which can be used in the nucleotides of the present disclosure.

[0027] The term "transgene" refers to an exogenous nucleic acid sequence, particularly an exogenous DNA or cDNA encoding a gene product. The gene product can be RNA, a peptide, or a protein. In addition to the coding region (CDS) for the gene product, the transgene may contain or be associated with one or more engineered sequences to facilitate or enhance expression, such as, for example, a promoter, enhancer(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s), and / or other functional element(s). Embodiments of the present disclosure may utilize any known suitable promoter(s), enhancer(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s), and / or other functional element(s), unless otherwise specified. Suitable elements and sequences are well known to those of skill in the art.

[0028] The terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of a corresponding naturally occurring amino acid.

[0029] The term "binding protein" refers to a protein that can non-covalently bind to another molecule. A binding protein can bind, for example, to a DNA molecule (DNA-binding protein), an RNA molecule (RNA-binding protein), and / or a protein molecule (protein-binding protein). In the case of a protein-binding protein, it can bind to one or more molecules of the same protein to form homodimers, homotrimers, etc., and / or it can bind to one or more molecules of different protein(s). A binding protein can have two or more types of binding activity. For example, a zinc finger protein has DNA-binding activity, RNA-binding activity, and protein-binding activity.

[0030] The term "Cas9" or "Cas9 nuclease" refers to an RNA-guided nuclease comprising the Cas9 protein or a fragment thereof (e.g., a protein comprising the active or inactive DNA cleavage domain of Cas9 and / or the gRNA-binding domain of Cas9). Cas9 nuclease is also sometimes referred to as casn1 nuclease or CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease. CRISPR is an adaptive immune system that provides defense against mobile genetic elements (viruses, transposable elements, and conjugated plasmids). CRISPR clusters contain a spacer, a sequence complementary to the preceding mobile element, and a target invading nucleic acid. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In the type II CRISPR system, proper processing of the pre-crRNA requires a transcoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. The tracrRNA functions as a guide for processing of the pre-crRNA using RNase 3. Cas9 / crRNA / tracrRNA then endonucleolytically cleaves linear or circular dsDNA targets complementary to the spacer. The target strand that is not complementary to the crRNA is first endonucleolytically cleaved and then trimmed by a 3'-5' exonuclease. In nature, DNA binding and cleavage typically require a protein and both RNAs. However, single-stranded guide RNAs ("sgRNAs" or simply "gNRAs") can be engineered to incorporate aspects of both crRNA and tracrRNA into a single RNA species.

[0031] Cas9 recognizes a short motif (PAM or protospacer adjacent motif) within the CRISPR repeat sequence, which helps distinguish self from non-self. The sequence and structure of Cas9 nuclease are well known to those skilled in the art. Cas9 orthologs have been described in various species, including, but not limited to, Streptococcus pyogenes (S. pyogenes) and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those skilled in the art based on this disclosure, including the Cas9 sequences from the organisms and loci disclosed in Chylinski et al., 2013 (RNABiol. 10(5):726-37), the entire contents of which are incorporated herein by reference.

[0032] In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain. Nuclease-inactivated Cas9 proteins may be interchangeably referred to as "dCas9" proteins (short for nuclease "dead" Cas9). Methods for generating Cas9 proteins (or fragments thereof) with inactive DNA cleavage domains are known in the art (see, e.g., Jinek et al., 2012. Science. 337(6096):816-821; Qi et al., 2013. Cell. 152(5):1173-83, the entire contents of each of which are incorporated herein by reference).

[0033] The term "zinc finger protein" refers to a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner via one or more zinc fingers, which are regions of amino acid sequence within the binding domain of a zinc finger protein whose structure is stabilized by the coordination of a zinc ion. The term "zinc finger protein" is often abbreviated as "ZFP."

[0034] The term "zinc finger nuclease" refers to an artificial restriction enzyme created by fusing a zinc finger DNA binding domain to a DNA cleavage domain. The zinc finger domain can be engineered to target specific desired DNA sequences, allowing the zinc finger nuclease to target unique sequences within complex genomes. "Zinc finger nuclease" is often abbreviated as "ZFN" or "ZNP."

[0035] As used herein, the term "amino acid sequence" or "polypeptide" or "protein" refers to a polymer of amino acid residues. Unless otherwise specified, the polymer of amino acid residues may be of any length.

[0036] As used herein, the term "exogenous" refers to a molecule that does not naturally occur in a cell but can be introduced into the cell by one or more genetic, biochemical, or other methods. The natural presence in a cell can also be determined based on the specific developmental stage and environmental conditions of the cell. Thus, for example, a molecule that only exists during early muscle development is an exogenous molecule for adult muscle cells. Similarly, a molecule induced by heat shock is an exogenous molecule for non-heat-shocked cells. An exogenous molecule can include, for example, a functional version of a dysfunctional endogenous molecule or a dysfunctional version of a normally functioning endogenous molecule.

[0037] 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, endogenous nucleic acids can include chromosomes, the genome of mitochondria, chloroplasts, or other organelles, or naturally occurring episomal nucleic acids. Additional endogenous molecules can include, for example, proteins such as transcription factors and enzymes.

[0038] A "target sequence" or "target nucleic acid sequence" or "target site" is a sequence that defines a portion of a nucleic acid, e.g., in a genome, to which a binding molecule will bind, provided sufficient conditions for binding exist. For example, the sequence 5'-GAATTC-3' is the target site for the EcoRI restriction endonuclease.

[0039] The term "fusion" refers to a molecule in which two or more subunit molecules are linked. In some embodiments, the link between the two is a covalent bond; alternatively, the link between the two can be non-covalent, e.g., relying on intermolecular interactions. The subunit molecules can be of the same chemical type or of different chemical types.

[0040] The term "fusion protein" refers to a hybrid polypeptide containing protein domains from at least two different proteins. For example, one protein domain may be located at the amino-terminal (N-terminal) portion or the carboxy-terminal (C-terminal) portion of the fusion protein, thus forming an "amino-terminal fusion protein" or a "carboxy-terminal fusion protein," respectively. In a preferred embodiment, a fusion protein is a single-chain polypeptide that may be fully encoded by a nucleic acid sequence and contains at least two protein domains covalently linked by peptide bonds directly or, optionally, via a peptide linker.

[0041] As used herein, the term "gene" or "genome" includes a DNA region that encodes a gene product, as well as all DNA regions that regulate the production of that gene product, regardless of whether such regulatory sequences are adjacent to the coding and / or transcribed sequence. Thus, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions.

[0042] The term "eukaryotic" cells includes, but is not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells and human cells (eg, T cells).

[0043] As used herein, the term "linked" refers to the juxtaposition of two or more components (such as sequence elements) where the components are positioned so as to allow both components to function normally and for at least one of the components to mediate a function exerted by at least one of the other components.

[0044] A "functional fragment" of a protein, polypeptide, or nucleic acid is a protein, polypeptide, or nucleic acid, respectively, whose sequence is not identical to the full-length protein, polypeptide, or nucleic acid, but which retains the same function as the full-length protein, polypeptide, or nucleic acid. A functional fragment can have more, fewer, or the same number of residues as the corresponding native molecule, and / or can contain one or more amino acid or nucleotide substitutions.

[0045] As used herein, the term "transfect" refers to the introduction of nucleic acid (either DNA or RNA) into a eukaryotic or prokaryotic cell or organism.

[0046] The term "cleavage" refers to the breakage of the covalent backbone of a DNA molecule. Cleavage can be initiated by various methods, including, but not limited to, enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-strand and double-strand cleavage are possible, and double-strand cleavage can occur as a result of two different single-strand cleavage events. DNA cleavage can produce either blunt ends or staggered ends. In certain embodiments, fusion polypeptides are used to target double-stranded DNA cleavage.

[0047] The term "specificity" refers to the ability to selectively bind sequences that share a certain degree of sequence identity to a selected sequence.

[0048] The terms "insertion" and "integration" refer to the addition of a nucleic acid sequence to a second nucleic acid sequence or a genome or a part thereof. The terms "specific," "site-specific," "targeted," and "on-targeted" with respect to insertion or integration are used interchangeably herein and refer to the insertion of a nucleic acid into a specific site of a second nucleic acid or a specific site of a genome or a part thereof. Conversely, the terms "random," "non-targeted," and "off-targeted" refer to the non-specific and unintended insertion of a nucleic acid into an undesired site. The term "total" or "total" refers to the total number of insertions.

[0049] The term "mutation" refers to the substitution of a residue in a sequence, e.g., a nucleic acid or amino acid sequence, with another residue; and / or the deletion or insertion of one or more residues in a nucleic acid or amino acid sequence. Mutations are typically described herein by identifying the original residue, then specifying the position of the residue in the sequence, and then specifying 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 can be found, for example, in Green & Sambrook, 2012 (Molecular cloning: a laboratory manual (4 th Ed.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). In a preferred embodiment, the term mutation in a protein refers to an amino acid substitution.

[0050] The term "transposase" refers to an enzyme that binds to the ends of a transposon and catalyzes its movement to another part of the genome by a cut-and-paste or replicative transposition mechanism.

[0051] The term "modified" refers to a protein or nucleic acid sequence that differs from the corresponding unmodified protein or nucleic acid sequence.

[0052] The term "linker" refers to a chemical group or molecule that connects two adjacent molecules or moieties.

[0053] As used herein, the terms "vector" and "plasmid" refer to any polynucleotide that can carry, for example, a second polynucleotide of interest, for example, a gene sequence that can be transferred into a target cell. Thus, this term includes not only integrating vectors, but also cloning and expression vehicles. In particular, the term "expression vector" as used herein refers to any polynucleotide that can direct the expression of a nucleic acid. In some embodiments, the terms "vector" and "plasmid" are used interchangeably with the term "nucleic acid construct."

[0054] As used herein, the percent identity between two sequences corresponds to the number of identical positions shared by the sequences, taking into account the number of gaps (i.e., % identity = number of identical positions / total number of positions x 100), and the length of each gap that must be introduced to optimally align the two sequences. The comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm, as described below.

[0055] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17, 1988) as incorporated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453, 1970) algorithm as incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using either a Blossom62 matrix or a PAM250 matrix, with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0056] The percent identity between two nucleotide amino acid sequences can also be determined using an algorithm such as the BLASTN program for nucleic acid sequences, which uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of both strands.

[0057] As used herein, the term "recombinant" or "engineered" refers to a protein or nucleic acid sequence that has been made artificially.

[0058] The term "subject," as used herein, refers to an individual organism, e.g., an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, goat, cow, 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.

[0059] The terms "treatment," "treat," and "treating," as described herein, refer to a clinical intervention aimed at reversing, alleviating, delaying the onset of, or inhibiting the progression of a disease or disorder, or one or more symptoms thereof. As used herein, the terms "treatment," "treat," and "treating," as described herein, refer to a clinical intervention aimed at reversing, alleviating, delaying the onset of, or inhibiting the progression of a disease or disorder, or one or more symptoms thereof. In some embodiments, treatment may be administered after one or more symptoms have developed and / or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent, reduce the likelihood of, or delay the onset of, symptoms, or inhibit the onset or progression of a disease. For example, treatment can be administered to a susceptible individual prior to the onset of symptoms (e.g., in terms of a history of symptoms and / or in terms of genetic or other susceptibility factors). Treatment may continue after symptoms have resolved, e.g., to prevent or delay their recurrence. DETAILED DESCRIPTION OF THE INVENTION

[0060] The present invention provides (i) a first protein comprising or consisting of a site-specific DNA-binding protein capable of binding and cleaving a target nucleic acid sequence; or a nucleic acid construct encoding the first protein; (ii) a second protein comprising or consisting of a transposase; or a nucleic acid construct encoding the second protein; Here, the transposase is a modified hyperactive PiggyBac that contains one or more amino acid mutations compared to the hyperactive PiggyBac of SEQ ID NO:9.

[0061] Current genome engineering tools, such as engineered zinc finger proteins (ZFPs), transcription activator-like effector nucleases (TALENs), and more recently RNA-guided DNA nucleases such as Cas9, affect sequence-specific DNA cleavage within the genome. This programmable cleavage can result in mutation of the DNA at the cleavage site by non-homologous end joining (NHEJ) or replacement of the DNA surrounding the cleavage site by homology-directed repair (HDR).

[0062] In one embodiment, the site-specific DNA binding protein is selected from the group comprising or consisting of an RNA-guided DNA nuclease, a zinc finger protein, and a transcription activator-like effector nuclease.

[0063] In one embodiment, the site-specific DNA binding protein is selected from the group comprising or consisting of an RNA-guided DNA nuclease and a zinc finger protein.

[0064] In one embodiment, the site-specific DNA binding protein is an RNA-guided nuclease.

[0065] In one embodiment, the site-specific DNA-binding protein is a Cas9 protein (e.g., without limitation, Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), or Campylobacter jejuni Cas9 (CjCas9); several other suitable examples are described below), or a variant thereof (e.g., nickase Cas9 (nCas9) or dead Cas9 (dCas9)), a Cas12a protein, a Cas12b protein, a Cpf1 protein, or a CasX protein (including variants and functional fragments thereof).

[0066] In one embodiment, the site-specific DNA binding protein is a Cas9 protein, including variants and functional fragments thereof.

[0067] The CRISPR-Cas9 system is a highly effective tool for inactivating or modifying genes through sequence-specific double-strand breaks (DSBs). These DSBs are recognized by the cell's DNA damage response machinery and can be repaired by endogenous DSB repair pathways. The primary repair pathway is non-homologous end joining (NHEJ), which often results in frameshift mutations and small insertions and / or deletions that can disrupt gene function. This pathway can be utilized to generate gene knockout mutations. Alternatively, in the presence of a repair template (e.g., a nucleic acid construct containing or consisting of a transgene encoding laminin-α2 protein, its functional variants, or fragments), the damage can be seamlessly repaired by homology-directed repair (HDR). However, despite significant advances, HDR-mediated genome editing for introducing precise gene modifications is much less efficient than NHEJ-mediated gene disruption. Furthermore, large, multi-kb replacements by the HDR pathway pose challenges, necessitating selection and / or sorting of large cell populations. Therefore, HDR pathways are currently primarily limited to local replacement of key regions within genes, but not large full-length genes. As explained above, the present invention overcomes this drawback.

[0068] In one embodiment, the Cas9 protein comprises (i) an active DNA cleavage domain and (ii) a guide RNA binding domain.

[0069] Among the known Cas9 proteins, the Streptococcus pyogenes Cas9 protein is widely used as a tool for genome engineering. This Cas9 protein is a large multidomain protein containing two distinct nuclease domains.

[0070] In one embodiment, the Cas9 protein is selected from the group consisting 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 baltica (NCBI Ref:NC_021314.1) SEQ ID NO: 25; 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 (CjCas9) (NCBI Ref:YP_002344900.1) SEQ ID NO:29 (encoded by SEQ ID NO:81); Neisseria meningitidis (NCBI Ref:YP_002342100.1) SEQ ID NO:30; Staphylococcus aureus (SaCas9) SEQ ID NO:72 (encoded by SEQ ID NO:77); and Streptococcus pyogenes (SpCas9) (NCBI Ref:YP_002342100.1) SEQ ID NO:30 Ref:NC_017053.1) The Cas9 protein is selected from the group comprising or consisting of SEQ ID NO: 31.

[0071] In one embodiment, when a wild-type Cas9 protein is referred to herein, the wild-type Cas9 protein corresponds to Cas9 from Streptococcus pyogenes (spCas9) having SEQ ID NO: 31, unless otherwise specified.

[0072] In one embodiment, the Cas9 protein may be a "Cas9 variant." As used herein, a "Cas9 variant" is a protein that shares homology with the Cas9 proteins described herein, including fragments thereof.

[0073] In one embodiment, the Cas9 variant can be at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the wild-type Cas9 protein having SEQ ID NO: 31 or any other Cas9 protein having SEQ ID NOs: 19-30 or 72.

[0074] In one embodiment, Cas9 variants comprise the amino acid sequence of Cas9 protein with one or several amino acid substitutions.For example, it is known that the DNA cleavage domain of Cas9 comprises two subdomains: HNH nuclease subdomain and RuvC1 subdomain.The HNH subdomain cleaves the strand complementary to gRNA, while the RuvC1 subdomain cleaves the non-complementary strand.

[0075] Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the substitutions D10A and H841A are known to completely inactivate the nuclease activity of the Streptococcus pyogenes Cas9 protein having SEQ ID NO: 31, resulting in a dead Cas9 (dCas9) that still retains the ability to bind DNA in a manner programmed by an sgRNA. In principle, when dCas9 is fused to another protein or domain, it can be targeted to virtually any DNA sequence simply by co-expressing it with an appropriate sgRNA. In one embodiment, the dCas9 protein is encoded by a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 66. In one embodiment, the dCas9 protein comprises or consists of an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO:71.

[0076] Cas9 nickase (nCas9) is a variant of Cas9 nuclease that differs by a point mutation (D10A) in the RuvC nuclease domain and is capable of nicking but not cleaving DNA. In one embodiment, the nCas9 protein is encoded by a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 65. In one embodiment, the nCas9 protein comprises or consists of an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 70. In some embodiments, the SaCas9 nickase (SanCas9) is encoded by a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 80. In some embodiments, the SaCas9 nickase (SanCas9) comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 76.

[0077] In one embodiment, the Cas9 variant comprises a fragment of Cas9 such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of the wild-type Cas9 protein having SEQ ID NO: 31 or any other Cas9 protein having SEQ ID NOs: 19-30 or 72.

[0078] In one embodiment, the Cas9 variant comprises only one of the DNA cleavage domain or the guide RNA binding domain.

[0079] In one embodiment, an exemplary Cas9 variant is humanized Cas9 (hCas9) or a variant or functional fragment thereof. As used herein, the term "humanized Cas9" or "hCas9" refers to a Cas9 protein that has been sequence-optimized for use in human cells.

[0080] In one embodiment, the hCas9 protein is encoded by a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 64. In one embodiment, the hCas9 protein comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 69.

[0081] In one embodiment, the site-specific DNA-binding protein is a cpf1 protein. In one embodiment, the cpf1 protein is encoded by a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 78. In one embodiment, the cpf1 protein comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 74.

[0082] In one embodiment, the site-specific DNA-binding protein is a CasX protein. In one embodiment, CasX is encoded by a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 79. In one embodiment, CasX comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 75.

[0083] As described in more detail below, certain aspects of the present disclosure also relate to vectors or plasmids (e.g., expression vectors, packaging vectors, etc.) comprising a nucleic acid construct encoding a site-specific DNA-binding protein, particularly an RNA-guided nuclease, particularly any of the Cas9 proteins described herein; the vector or plasmid is preferably suitable for expression in a host cell, e.g., a mammalian cell, a yeast cell, an insect cell, a plant cell, a fungal cell, or an algal cell.

[0084] In one embodiment, the site-specific DNA binding protein is a zinc finger protein (ZFP).

[0085] Zinc finger proteins are proteins that can bind to DNA in a sequence-specific manner. ZFPs are heterogeneously distributed in eukaryotes. ZFPs have been identified as being involved in DNA recognition, RNA binding, and protein binding. The specific classification of zinc finger proteins is based on "fold groups," which consider the overall shape of the protein backbone of the folded domain. The most common "fold groups" of zinc fingers are C2H2 or Cys2His2-like ("classical zinc finger"), treble clef, and zinc ribbon. Representative motifs that characterize these proteins are disclosed in Table 1 of Li & Liu, 2020 (Int J Mol Sci. 21(4):1361), which is incorporated herein by reference.

[0086] The ZFP can be any ZFP, its variant or functional fragment that can bind to a specific genomic DNA sequence in the genome.Non-limiting examples of ZFP include ZFPs that contain a fold group or zinc finger motif selected from C2H2, gag knuckle, treble clef, zinc ribbon, Zn2 / Cys6-like, or TAZ2 domain-like, or any combination thereof.In one embodiment, the ZFP is a C2H2 zinc finger protein.

[0087] In one embodiment, the ZFP is an engineered ZFP. Engineered zinc finger arrays can be fused to a DNA cleavage domain (usually the cleavage domain of FokI) to generate zinc finger nucleases. Such zinc finger-FokI fusions have become useful reagents for genome engineering.

[0088] A ZFP can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more zinc finger domains. A ZFP can contain 2 to 12, 2 to 10, 2 to 8, 3 to 8, 4 to 8, or 5 to 8 zinc finger domains. In one embodiment, a ZFP contains 6 zinc finger domains.

[0089] A common modular assembly process involves combining individual zinc fingers, each capable of recognizing a 3-base pair DNA sequence, to generate 3-, 4-, 5-, or 6-finger arrays that recognize target sites ranging in length from 9 to 18 base pairs. Another method uses 2-finger modules to generate zinc finger arrays containing up to 6 individual zinc fingers.

[0090] In one embodiment, the binding domain of a ZFP can be engineered to bind to a sequence of interest. The engineered zinc finger binding domain can have improved binding specificity compared to a naturally occurring ZFP.

[0091] In one embodiment, exemplary nucleic acid sequences encoding a ZFP comprise or consist of SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, or SEQ ID NO: 38. In one embodiment, exemplary amino acid sequences encoded by these sequences comprise or consist of SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, or SEQ ID NO: 39.

[0092] In one embodiment, the ZFP comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to any one of SEQ ID NOs: 33, 35, 37, or 39.

[0093] In one embodiment, the ZFP does not have a Gal4 DNA binding domain.11 Gal4 binds to -CCG, where N can be any base. This protein is a positive regulator of gene expression of galactose-inducible genes such as GAL1, GAL2, GAL7, GAL10, and MEL1, which encode enzymes used to convert galactose to glucose. It recognizes a 17-base pair sequence in the upstream activating sequence (UAS-G) of these genes. Therefore, Gal4 is not site-specific because it recognizes short sequences that are very frequent in the genome. In one embodiment, the ZFP has a Gal4 DNA-binding domain engineered to be site-specific.

[0094] As described in further detail below, certain embodiments of the present disclosure are directed to vectors or plasmids (e.g., expression vectors, packaging vectors, etc.) that contain nucleic acid constructs encoding site-specific DNA-binding proteins, particularly the ZFPs described herein; the vectors or plasmids are preferably suitable for expression in a host cell, e.g., a mammalian cell, a yeast cell, an insect cell, a plant cell, a fungal cell, or an algal cell.

[0095] According to the present invention, the second protein comprises or consists of a transposase.

[0096] A transposon is a chromosomal segment capable of transposition, e.g., DNA that can translocate as a whole even in the absence of a complementary sequence in the host DNA. Transposons can be used to manipulate DNA over long distances in human cells. Common transposon systems used in mammalian cells include, but are not limited to, Sleeping Beauty (SB), which was reconstructed from an inactive transposon, and PiggyBac (PB), isolated from the moth Trichoplusia. PiggyBac has higher transposition activity than SB and can be excised without scarring.

[0097] Native DNA transposons typically contain a single gene encoding a transposase protein flanked by inverted terminal repeats (ITRs) containing transposase binding sites. During their transposition, the transposase protein recognizes these ITRs and catalyzes the excision of the element in a random manner and its subsequent reintegration elsewhere. Furthermore, some of these transposons can be adapted for use in gene therapy protocols, where they are used as two-component systems: a plasmid contains an expression cassette in which a DNA sequence of interest is placed between the transposon ITRs and can be introduced into the host genome by induction with a co-transfected plasmid containing a sequence encoding the transposase enzyme or its in vitro-synthesized mRNA. According to the present disclosure, transposon-based systems are used to efficiently mediate stable integration and sustained expression of transgenes as therapeutic genes in cells.

[0098] The transposase or modified transposase of the present disclosure can be any transposase capable of inserting an exogenous nucleic acid into a specific site in a genome. Some aspects of the present disclosure provide transposase fusion proteins designed using the methods and strategies described herein. Some embodiments of the present disclosure provide nucleic acids encoding such transposases or modified transposases and / or fusion proteins comprising them. Some embodiments of the present disclosure provide plasmids or expression vectors comprising such nucleic acid constructs encoding transposases or modified transposases and / or fusion proteins comprising them.

[0099] Non-limiting examples of transposases include Frog Prince, Sleeping Beauty, hyperactive Sleeping Beauty, PiggyBac, and hyperactive PiggyBac.

[0100] In one embodiment, the transposase is a hyperactive PiggyBac transposase. In some embodiments, the transposase is a hyperactive PiggyBac transposase corresponding to SEQ ID NO: 9 or encoded by SEQ ID NO: 67 (also referred to in the present disclosure as hyPB or simply PB).

[0101] In one embodiment, the transposase is a modified hyperactive PiggyBac transposase.

[0102] As used herein, a "modified hyperactive PiggyBac transposase" refers to a transposase that contains one or more amino acid substitutions, typically no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, compared to the wild-type hyperactive PiggyBac transposase of SEQ ID NO: 9. More specifically, the modified hyperactive PiggyBac contains (i) one or more amino acid substitutions to increase excision activity compared to the wild-type hyperactive PiggyBac transposase, and / or (ii) one or more amino acid substitutions to decrease DNA-binding activity compared to the wild-type hyperactive PiggyBac transposase. In one embodiment, the modified hyperactive PiggyBac transposase contains an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 9.

[0103] In some embodiments, the one or more mutations to the hyperactive PiggyBac transposase do not consist of the triple mutation R372A / K375A / D450N, where the position numbers correspond to the amino acid numbering of unmodified hyperactive PiggyBac in SEQ ID NO:9.

[0104] In some embodiments, the modified hyperactive PiggyBac comprises one or more amino acid mutations to increase excision activity.

[0105] In some embodiments, the modified hyperactive Piggybac comprises one or more amino acid mutations to increase excision activity selected from among amino acid mutations within the region defined by amino acid position numbers [194-200], [214-222], [434-442] or [446-456], e.g., amino acid substitutions at positions D198, D201, R202, M212 and / or S213 (position numbers correspond to the amino acid numbering of the unmodified hyperactive Piggybac in SEQ ID NO: 9).

[0106] In some embodiments, the modified hyperactive Piggybac comprises one or more amino acid mutations to increase excision activity selected from among amino acid mutations at positions 450, 560, 564, 573, 589, 592, and / or 594, where the position numbers correspond to the amino acid numbers of the unmodified hyperactive Piggybac in SEQ ID NO:9.

[0107] In some embodiments, the modified hyperactive PiggyBac comprises one or more amino acid mutations to increase excision activity selected from among amino acid mutations at positions M194 and / or D450, where the position numbers correspond to the amino acid numbers of the unmodified hyperactive Piggybac in SEQ ID NO: 9, and preferably the amino acid substitutions are selected from among M194V and / or D450N.

[0108] In some embodiments, the modified hyperactive PiggyBac comprises one or more amino acid mutations to reduce DNA-binding activity.

[0109] In some embodiments, the modified hyperactive PiggyBac comprises one or more amino acid mutations to reduce DNA binding activity selected from among amino acid mutations at positions 254, 275, 277, 347, 372, 375, and / or 465, where the position numbers correspond to the amino acid numbers of the unmodified hyperactive PiggyBac in SEQ ID NO:9.

[0110] In some embodiments, the modified hyperactive PiggyBac comprises one or more amino acid mutations to reduce DNA binding activity selected from R275, N347, R372, K375, R376, E377, and E380, where the position numbers correspond to the amino acid numbers of the unmodified hyperactive PiggyBac in SEQ ID NO: 9.

[0111] In some embodiments, the modified hyperactive PiggyBac comprises one or more amino acid mutations that reduce DNA binding activity selected from R372, K375, R376, E377, and E380, where the position numbers correspond to the amino acid numbering of the unmodified hyperactive Piggybac in SEQ ID NO: 9, and preferably selected from among the amino acid substitutions R372A, K375A, R376A, E377A, and / or E380A.

[0112] In some embodiments, the modified hyperactive PiggyBac comprises one or more amino acid mutations to reduce DNA binding activity selected from N347, R372, and K375, where the position numbers correspond to the amino acid numbers of the unmodified hyperactive PiggyBac in SEQ ID NO: 9, and are preferably selected from among the amino acid substitutions N347S, N347A, R372A, K375A, and more preferably selected from among the amino acid substitutions N347S, N347A.

[0113] In some embodiments, the modified hyperactive Piggybac comprises one or more amino acid mutations for increasing excision activity, as defined above; and one or more amino acid mutations for decreasing DNA binding activity, as defined above.

[0114] In some embodiments, the modified hyperactive Piggybac comprises at least one amino acid substitution at position D450 to increase excision activity and at least two amino acid substitutions at positions N347, R372, and K375 to decrease DNA-binding activity, preferably, the modified transposase of hyperactive Piggybac comprises the double mutation N347S and D450N, or the triple mutation D450N, R372A, and K375A, where the position numbers correspond to the amino acid numbering of unmodified hyperactive Piggybac in SEQ ID NO: 9. In a more preferred embodiment, the modified transposase of hyperactive Piggybac comprises the double mutation N347S and D450N, where the position numbers correspond to the amino acid numbering of unmodified hyperactive Piggybac in SEQ ID NO: 9.

[0115] In some embodiments, the modified hyperactive Piggybac disclosed in the preceding embodiments further comprises at least one mutation in the region defined by amino acid position numbers [158-169], e.g., A166S and / or at least one mutation at positions Y527, R518, K525, N463.

[0116] Typically, the modified hyperactive Piggybac comprises an amino acid sequence that has at least 85%, at least 90%, at least 95% identity, or 100% identity to the modified hyperactive Piggybac of SEQ ID NO:1.

[0117] In some embodiments, the modified hyperactive Piggybac is a variant of the hyperactive Piggybac of SEQ ID NO: 9 having one or more amino acid substitutions, typically no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions compared to SEQ ID NO: 9.

[0118] In some embodiments, the modified hyperactive Piggybac further comprises one or more of the following amino acid mutations at positions 34, 43, 117, 202, 230, 245, 268, 275, 277, 287, 290, 315, 325, 341, 346, 347, 350, 351, 356, 357, 388, 409, 411, 412, 432, 447, 460, 461, 465, 517, 560, 564, 571, 573, 576, 586, 587, 589, 592, and / or 594. The position numbers correspond to the amino acid numbering of the hyperactive PiggyBac sequence (SEQ ID NO: 9).

[0119] In some embodiments, the modified hyperactive PiggyBac comprises the following mutations or combinations of mutations: V34M, T43I, Y177H, R202K, S230N, R245A, D268N, K287A, K290A, K287A / K290A, R315A, G325A, R341A, D346N, N347A, N347S, T350A, S351E, S351P, S351A, K356E, N357A, R388A, K409A, A411T, K412A, K432A, D447A, D447N , D450N, R460A, K461A, W465A, S517A, T560A, S564P, S571N, S573A, K576A, H586A, I587A, M589V, S592G, or F594L, D450N / R372A / K375A, R275A / R277A, K409A / K412A, R460A / K461A, R275A / R277A / N347S / K375A / T560A / S573A / M589V / S592G and R245A / R275A / R277A / R372A / W465A.

[0120] In some embodiments, the modified hyperactive PiggyBac comprises the following amino acid substitutions or combinations of amino acid substitutions: R372A / K375A / D450N, R372A / K375A / R376A / D450N, K375A / R376A / E377A / E380A / D450N, R372A / K375A / R376A / E377A / E380A / D450N, M194V, M194V / R372A / K375A, S351A / R372A / K375A / R388A / D450N / W465A / S 573A / M589V / S592G / F594L, R245A / R275A / R277A / R372A / W465A / M589V, R275A / 325A / R372A / T560A, N347A / D450N, N347S / D450N / T560A / S5 73A / F594L, R202K / R275A / N347S / R372A / D450N / T560A / F594L, R275A / N347S / K375A / D450N / S592G, R275A / N347S / R372A / D450N / T560A / F5 94L, R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L, R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G, R277A / G325A / N3 47A / K375A / D450N / T560A / S564P / S573A / S592G / F594L, V34M / R275A / G325A / N347S / S351A / R372A / K375A / D450N / T560A / S564P, G325A / N34 7S / K375A / D450N / S573A / M589V / S592G, S230N / R277A / N347S / K375A / D450N, T43I / R372A / K375A / A411T / D450N, G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G; position numbers correspond to the amino acid numbers of the hyperactive PiggyBac sequence (SEQ ID NO: 9).

[0121] Highly preferred modified hyperactive PiggyBac transposases for use in accordance with the present disclosure include modified hyperactive PiggyBacs containing the following combinations of amino acid substitutions: R372A / K375A / D450N, S351A / R372A / K375A / R388A / D450N / W465A / S573A / M589V / S592G / F594L, R245A / R275A / R277A / R372A / W465A / M589V, N 347A / D450N, N347S / D450N / T560A / S573A / F594L, R202K / R275A / N347S / R372A / D450N / T560A / F594L, R275A / N347S / K375A / D45 0N / S592G, R275A / N347S / R372A / D450N / T560A / F594L, R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L, R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G, R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L, V34M / R27 5A / G325A / N347S / S351A / R372A / K375A / D450N / T560A / S564P, G325A / N347S / K375A / D450N / S573A / M589V / S592G, S230N / R277A / N347S / K375A / D450N, T43I / R372A / K375A / A411T / D450N, G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G and R275A / 325A / R372A / T560A; position numbers correspond to the amino acid numbers of the hyperactive PiggyBac sequence (SEQ ID NO: 9).

[0122] In some embodiments, the modified hyperactive PiggyBac comprises the following amino acid substitutions or combinations of amino acid substitutions: R245A / R275A / R277A / R372A / W465A / M589V, R275A / 325A / R372A / T560A, N347A / D450N, N347S / D450N / T560A / S573A / F594 L, R202K / R275A / N347S / R372A / D450N / T560A / F594L, R275A / N347S / K375A / D450N / S592G, R275A / N347S / R372A / D450N / T560A / F594L, R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L, R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G, R277A / G325A / N347A / K375A / D450N / T 560A / S564P / S573A / S592G / F594L, G325A / N347S / K375A / D450N / S573A / M589V / S592G, S230N / R2 77A / N347S / K375A / D450N, G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G; position numbers correspond to the amino acid numbers of the hyperactive PiggyBac sequence (SEQ ID NO: 9).

[0123] In a more preferred embodiment, the modified hyperactive PiggyBac contains the following combinations of amino acid substitutions: N347A / D450N, N347S / D450N / T560A / S573A / F594L, R202K / R275A / N347S / R372A / D450N / T560A / F594L, R275A / N347S / K375A / D450N / S592G, R275A / N347S / R372A / D450N / T560A / F594L, R275A / R277 A / N347S / R372A / D450N / T560A / S564P / F594L, R245A / N347S / R372A / D450N / T560A / S56 4P / S573A / S592G, R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F 594L, G325A / N347S / K375A / D450N / S573A / M589V / S592G, S230N / R277A / N347S / K375A / D450N, G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G; position numbers correspond to the amino acid numbers of the hyperactive PiggyBac sequence (SEQ ID NO: 9).

[0124] In some embodiments, the modified transposase has an amino acid sequence selected from any of SEQ ID NOs: 1-8, 10-18, and 135-149.

[0125] In some embodiments, the modified transposase has an amino acid sequence selected from any of SEQ ID NOs: 1-8 and 10-18.

[0126] In some embodiments, the modified transposase has an amino acid sequence selected from any of SEQ ID NOs: 90-99.

[0127] In some embodiments, the modified transposase has an amino acid sequence selected from any of SEQ ID NOs: 135-149. In some embodiments, the modified transposase has an amino acid sequence selected from any of SEQ ID NOs: 135-140. In some embodiments, the modified transposase has an amino acid sequence selected from any of SEQ ID NOs: 141-149.

[0128] In some embodiments, the modified transposase may include one or more mutations at amino acids 268 and / or 346 (e.g., D268N and / or D346N), corresponding to the amino acid numbers of SEQ ID NO:9 or SEQ ID NO:11, compared to hyPB, which is involved in the conserved catalytic triad.

[0129] In some embodiments, the modified transposase may include one or more mutations at amino acids 287, 287 / 290 and / or 460 / 461 (e.g., K287A, K287A / K290A, and / or R460A / K461A), corresponding to the amino acid numbers of SEQ ID NO:9 or SEQ ID NO:12, relative to hyPB that are important for excision.

[0130] In some embodiments, the modified transposase may include one or more mutations, e.g., at amino acids 351, 356, and / or 379 (e.g., S351E, S351P, S351A, and / or K356E), corresponding to the amino acid numbers of SEQ ID NO:9 or SEQ ID NO:13, compared to the hyPB involved in target binding.

[0131] In some embodiments, the modified transposase may include one or more mutations at, for example, amino acids 560, 564, 571, 573, 589, 592, and / or 594 (e.g., T560A, S564P, S571N, S573A, M589V, S592G, and / or F594L) corresponding to the amino acid numbers of SEQ ID NO:9 or SEQ ID NO:14, relative to hyPB that is important for integration.

[0132] In some embodiments, the modified transposase may include one or more mutations, e.g., at amino acids 325, 347, 350, 357 and / or 465 (e.g., G325A, N347A, N347S, T350A and / or W465A), corresponding to the amino acid numbers of SEQ ID NO:9 or SEQ ID NO:15, compared to the hyPB involved in the alignment.

[0133] In some embodiments, the modified transposase can include one or more mutations compared to the well-conserved hyPB, for example, at amino acids 576 and / or 587 (e.g., K576A and / or I587A), corresponding to the amino acid numbers of SEQ ID NO:9 or SEQ ID NO:16.

[0134] In some embodiments, the modified transposase may include one or more mutations, e.g., at amino acid number 586 (e.g., H586A), corresponding to SEQ ID NO: 9 or SEQ ID NO: 17, compared to hyPB, which is involved in Zn2+ binding.

[0135] In some embodiments, the programmable transposase may contain one or more mutations compared to the hyPB involved in integration, e.g., at amino acid positions 315, 341, 372, and / or 375 (e.g., R315A, R341A, R372A, and / or K375A), corresponding to amino acid numbers 315, 341, 372, and / or 375 of SEQ ID NO:9 or SEQ ID NO:18.

[0136] In some embodiments, the modified hyperactive PiggyBac comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 9. In some embodiments, the modified hyperactive PiggyBac is selected because of its increased specificity for integration of DNA into the genome compared to hyperactive PiggyBac. In some embodiments, the modified hyperactive PiggyBac comprises an amino acid sequence having one or more of the modifications disclosed herein with respect to SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18, and retains at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence represented by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18, respectively.

[0137] In some embodiments, the hyperactive PiggyBac transposase is encoded by a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 67. In some embodiments, the SB100 transposase is encoded by a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 68.

[0138] In some embodiments, the SB100 transposase comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:73.

[0139] In some embodiments, the modified transposase is a modified Sleeping Beauty transposase containing 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, I212K, N245S, K252A, and Q271L of SEQ ID NO:9 or SEQ ID NO:73.

[0140] In certain embodiments, the modified transposase is not a Himar1C9 mutant.

[0141] Certain aspects of the present disclosure are directed to vectors or plasmids (e.g., expression vectors or packaging vectors) comprising a nucleic acid construct comprising a transposase or modified transposase of the present disclosure suitable for expression in a host cell, e.g., a mammalian cell, a yeast cell, an insect cell, a plant cell, a fungal cell, or an algae cell. In some embodiments, the modified transposase is expressed as a fusion protein with Cas9. In some embodiments, the modified transposase is co-expressed with Cas9 from a separate vector but delivered to the same cell. In some embodiments, the modified transposase or a fusion protein comprising it is packaged into a lentiviral particle for delivery to the cell.

[0142] As shown in the Examples, a newly developed hyperactive PiggyBac transposase mutant library was used to identify modified hyperactive PiggyBacs that perform specific targeted transpositions. Such libraries were used to identify modified hyperactive PiggyBacs with positive targeted transpositions.

[0143] In some embodiments, the modified hyperactive PiggyBac transposase can include mutations in one or more amino acids selected from amino acids 245, 275, 277, 325, 347, 351, 372, 375, 388, 450, 465, 560, 564, 573, 589, 592, and 594, which correspond to the amino acid numbers of SEQ ID NO:9.

[0144] In some embodiments, the modified hyperactive PiggyBac transposase mutations can comprise one or more amino acid modifications selected from the following: 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 numbers in SEQ ID NO:9.

[0145] In one embodiment, the modified hyperactive PiggyBac transposase comprises the amino acid modification D450N, which corresponds to the amino acid numbering of SEQ ID NO:9.

[0146] In one embodiment, the modified hyperactive PiggyBac transposase corresponds to SEQ ID NO: 1 and includes the amino acid modifications R372A, K375A, and D450.

[0147] In one embodiment, the modified hyperactive PiggyBac transposase comprises amino acid modifications R245A and D450, which correspond to the amino acid numbering of SEQ ID NO:9.

[0148] 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.

[0149] In one embodiment, the modified hyperactive PiggyBac transposase comprises amino acid modifications R245A, G325A, D450, and S573P, corresponding to the amino acid numbering of SEQ ID NO:9.

[0150] In one embodiment, the modified hyperactive PiggyBac transposase comprises the amino acid modification N347S or N347A, which corresponds to the amino acid numbering of SEQ ID NO:9.

[0151] In one embodiment, the modified hyperactive PiggyBac transposase comprises the amino acid modifications N347S and D450N, which correspond to the amino acid numbering of SEQ ID NO:9.

[0152] In other cases, the modified hyperactive PiggyBac transposase comprises the amino acid modifications N347A and D450N, which correspond to the amino acid numbering of SEQ ID NO: 9. In one embodiment, the modified hyperactive PiggyBac transposase comprises the amino acid sequence of SEQ ID NO: 137.

[0153] As previously mentioned, the present specification provides a modified hyperactive PiggyBac transposase that can be fused to the elements disclosed herein, but can also be used alone or in combination with different elements. The transposase has been generated by the present inventors. Thus, a modified hyperactive PiggyBac transposase is provided that comprises the amino acid sequence of SEQ ID NO: 9, the amino acid at position 34 is V or M, the amino acid at position 43 is T or I, the amino acid at position 177 is Y or H, the amino acid at position 202 is R or K, the amino acid at position 230 is S or N, the amino acid at position 245 is A, the amino acid at position 268 is D or N, the amino acid at position 277 is R or 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 S or A, the amino acid at position 351 is E, P, or A; the amino acid at position 372 is R or A, the amino acid at position 375 is K or A, the amino acid at position 388 is R or A, the amino acid at position 409 is K or A, the amino acid at position 411 is A or T, the amino acid at position 412 is K or A, the amino acid at position 450 is D or N, the amino acid at position 460 is R or A, the amino acid at position 465 is W or A, the amino acid at position 517 is S 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 571 is S or N, the amino acid at position 573 is S or A, the amino acid at position 576 is K or A, the amino acid at position 586 is H or A, the amino acid at position 587 is I or A, the amino acid at position 589 is M or V, the amino acid at position 592 is G or S, and / or The amino acid at position 594 is L or F.

[0154] The present disclosure also relates to the modified hyperactive PiggyBac transposases provided herein for use as pharmaceuticals, particularly in ex vivo or in vivo gene therapy.

[0155] In one embodiment, the first protein and the second protein are fused to each other directly or indirectly via a linker to form a fusion protein, wherein the first protein comprises or consists of a site-specific DNA-binding protein (as described above) capable of binding and cleaving a target nucleic acid sequence, and the second protein comprises or consists of a transposase (as described above).

[0156] Any embodiment relating to site-specific DNA binding proteins on the one hand and transposases on the other hand applies mutatis mutandis in the case of the fusion proteins described herein.

[0157] Thus, in one embodiment, the fusion protein comprises or consists of: (i) a first protein comprising or consisting of an RNA-guided DNA nuclease, a zinc finger protein, or a transcription activator-like effector nuclease, as described above; and (ii) A second protein comprising or consisting of a transposase that is a modified hyperactive PiggyBac that comprises one or more amino acid mutations compared to the hyperactive PiggyBac of SEQ ID NO: 9, as described above.

[0158] In one embodiment, the fusion protein comprises or consists of: (i) a first protein comprising or consisting of an RNA-guided DNA nuclease or a zinc finger protein, as described above; and (ii) A second protein comprising or consisting of a transposase that is a modified hyperactive PiggyBac that comprises one or more amino acid mutations compared to the hyperactive PiggyBac of SEQ ID NO: 9, as described above.

[0159] In one embodiment, the fusion protein comprises or consists of: (i) a first protein comprising or consisting of an RNA-guided DNA nuclease, as described above; and (ii) A second protein comprising or consisting of a transposase that is a modified hyperactive PiggyBac that comprises one or more amino acid mutations compared to the hyperactive PiggyBac of SEQ ID NO: 9, as described above.

[0160] In one embodiment, the fusion protein comprises or consists of: (i) a first protein comprising or consisting of a Cas9 protein or a variant thereof, as described above; and (ii) A second protein comprising or consisting of a transposase that is a modified hyperactive PiggyBac that comprises one or more amino acid mutations compared to the hyperactive PiggyBac of SEQ ID NO: 9, as described above.

[0161] In one embodiment, the first protein and the second protein can be oriented in any order in the fusion protein.

[0162] In one embodiment, the fusion protein comprises or consists of a first protein fused, directly or indirectly via a linker, to the C-terminal end of a second protein. In other words, the fusion protein comprises or consists of, from N-terminus to C-terminus: (i) a second protein (i.e., a transposase); (ii) optionally, a linker; and (iii) a first protein (i.e., a site-specific DNA-binding protein, preferably an RNA-guided DNA nuclease; more preferably, a Cas9 protein or a variant thereof).

[0163] In one embodiment, the fusion protein comprises or consists of a first protein fused to the N-terminal end of a second protein, either directly or indirectly via a linker. In other words, the fusion protein comprises or consists of, from N-terminus to C-terminus: (i) a first protein (i.e., a site-specific DNA-binding protein, preferably an RNA-guided DNA nuclease; more preferably a Cas9 protein or a variant thereof); (ii) optionally, a linker; and (iii) a second protein (i.e., a transposase).

[0164] In one embodiment, the fusion protein comprises a linker.

[0165] Suitable examples of linkers include a peptide linker between the first protein and the second protein (in any order).

[0166] In one embodiment, the peptide linker is (GGS) n , having SEQ ID NO: 133 (GGGGS) n , (G) n , having SEQ ID NO: 134 (EAAAK) n , XTEN linker, and (XP) n motif, and any combination thereof, wherein n is independently an integer from 1 to 50.

[0167] In one embodiment, the linker is 12 to 24 amino acids in length or is encoded by a nucleic acid sequence 36 to 72 nucleotides in length.

[0168] In one embodiment, the linker is an XTEN linker or (GGS) n It is a linker.

[0169] In one embodiment, the linker is selected from among the linkers shown in Table 1. [Table 1]

[0170] In one embodiment, the linker comprises an amino acid sequence comprising or 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, SEQ ID NO:63, or a combination thereof, and is encoded by the exemplary nucleic acid sequences of 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, respectively.

[0171] In one embodiment, the linker comprises or consists of the amino acid sequence of SEQ ID NO:49; encoded by the exemplary nucleic acid sequence of SEQ ID NO:48.

[0172] Also provided herein are fusion proteins obtained from expression of any of the nucleic acid constructs provided in this disclosure.

[0173] In one embodiment, the fusion protein is a triple fusion protein.

[0174] Such a triple fusion protein may comprise or consist of: - one first protein (i.e., one site-specific DNA-binding protein) and two second proteins (i.e., two transposases); or - two first proteins (i.e., two site-specific DNA-binding proteins) and one second protein (i.e., one transposase).

[0175] In one embodiment, the triple fusion comprises or consists of one first protein (i.e., one site-specific DNA-binding protein) and two second proteins (i.e., two transposases), and the triple fusion comprises, from N-terminus to C-terminus: (i) a site-specific DNA-binding protein, (ii) a first transposase, and (iii) a second transposase; or (i) a first transposase; (ii) a site-specific DNA-binding protein; and (iii) a second transposase; or - (i) a first transposase; (ii) a second transposase; and (iii) a site-specific DNA-binding protein.

[0176] In one embodiment, the first and second transposases are the same. In one embodiment, the first and second transposases are different. For example, the first transposase can be a hyperactive PiggyBac transposase, and the second transposase can be a modified hyperactive PiggyBac transposase selected from any of the modified hyperactive PiggyBac transposases described herein. Alternatively, the first and second transposases can both be modified hyperactive PiggyBac transposases, but each having a different substitution or a different combination of substitutions described herein.

[0177] In one embodiment, the first and second transposases are capable of forming a functional dimer.

[0178] In one embodiment, the triple fusion comprises or consists of two first proteins (i.e., two site-specific DNA binding proteins) and one second protein (i.e., one transposase), and the triple fusion comprises, from N-terminus to C-terminus: (i) a first site-specific DNA-binding protein, (ii) a second site-specific DNA-binding protein; (iii) a transposase; or (i) a first site-specific DNA-binding protein; (ii) a transposase; (iii) a second site-specific DNA-binding protein; or (i) a transposase; (ii) a first site-specific DNA-binding protein; and (iii) a second site-specific DNA-binding protein.

[0179] In one embodiment, the first and second site-specific DNA binding proteins are the same. In one embodiment, the first and second site-specific DNA binding proteins are different. For example, the first site-specific DNA binding protein can be a Cas9 protein, and the second site-specific DNA binding protein can be a variant of the Cas9 protein selected from any of the Cas9 protein variants described herein. Alternatively, both the first and second site-specific DNA binding proteins can be Cas9 protein variants, but each is a different variant.

[0180] In one embodiment, the triple fusion protein optionally includes a linker between two of the proteins or between the three proteins.

[0181] Also disclosed herein is a fusion protein comprising: (i) the second protein comprises or consists of a transposase; or a nucleic acid construct encoding the second protein; and (ii) an RNA-binding protein capable of binding to at least one specific RNA sequence; or a nucleic acid construct encoding the RNA-binding protein.

[0182] In one embodiment, the fusion protein comprises a linker as described above.

[0183] In one embodiment, the second protein comprises or consists of a transposase, wherein the transposase is a hyperactive PiggyBac having SEQ ID NO: 9. In one embodiment, the second protein comprises or consists of a transposase, wherein the transposase is a modified hyperactive PiggyBac comprising one or more amino acid mutations compared to the hyperactive PiggyBac having SEQ ID NO: 9. In particular, the modified hyperactive PiggyBac can be any of those disclosed herein.

[0184] In one embodiment, the transposase / RNA binding protein fusion can be further fused to a first protein that comprises or consists of a site-specific DNA binding protein, as described above.

[0185] In some embodiments, the RNA binding protein is MS2 bacteriophage coat protein (MCP) or a fragment thereof.

[0186] In some embodiments, the MCP has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 151 (e.g., encoded by the nucleic acid sequence of SEQ ID NO: 150).

[0187] In some embodiments, the RNA binding protein can bind to at least one specific RNA sequence, and the RNA sequence comprises a tetraloop. The term "tetraloop" is used interchangeably with the terms "stem loop" and "hairpin loop."

[0188] In some embodiments, at least one tetraloop is an MS2 RNA tetraloop binding sequence.

[0189] In some embodiments, the tetraloop is contained within a guide RNA (gRNA). In certain embodiments, the gRNA is complexed with a Cas9 protein, as described above.

[0190] In some embodiments, the gRNA comprises at least one MS2 RNA tetraloop binding sequence, hi some embodiments, the gRNA comprises two or more MS2 RNA tetraloop binding sequences.

[0191] In some embodiments, a gRNA comprising at least one MS2 RNA tetraloop binding sequence has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 153 (e.g., encoded by the DNA sequence of SEQ ID NO: 152).

[0192] In some embodiments, the MCP in the fusion protein non-covalently binds to at least one MS2 RNA tetraloop-binding sequence contained in the gRNA itself, which is non-covalently bound to the Cas9 protein; in particular, binding of the fusion protein to the Cas9 / gRNA complex directs the excision activity of the modified hyperactive PiggyBac transposase to the site specifically recognized by the Cas9 / gRNA complex.

[0193] As described in further detail below, certain aspects of the present disclosure are also directed to vectors or plasmids (e.g., expression vectors, packaging vectors, etc.) comprising nucleic acid constructs encoding the fusion proteins described herein; the vectors or plasmids are preferably suitable for expression in a host cell, e.g., a mammalian cell, a yeast cell, an insect cell, a plant cell, a fungal cell, or an algal cell.

[0194] According to the present invention, the composition may comprise the first protein and / or the second protein (or a fusion protein comprising both) either as proteins as described above or as nucleic acid constructs encoding these proteins.

[0195] Targeted editing of nucleic acid sequences, for example, the introduction of specific modifications into genomic DNA (e.g., the insertion of exogenous nucleic acids), is a promising approach for treating human genetic diseases.To achieve this goal, the present inventors aimed to provide an improved nucleic acid construct for genome editing that is highly efficient in incorporating desired modifications; has minimal off-target activity; and can be programmed to precisely edit sites within the human genome.

[0196] Therefore, certain aspects of the present application are directed to nucleic acid constructs for use in improving the site-specific insertion of exogenous nucleic acid, for example, gene of interest (GOI), into genome.In some embodiments, GOI is a therapeutic gene, for example, a gene encoding a therapeutic protein.Examples of therapeutic genes of interest include the CFTR gene (cystic fibrosis transmembrane conductance regulator) for treating cystic fibrosis disease; the SMN1 gene (survival of motor neuron 1) for treating spinal muscular atrophy (SMA); the LRP5 gene (LDL receptor-related protein 5) variant G171V for preventing osteoporosis and fractures; and the APP gene (amyloid beta precursor protein) variant A673T for reducing the predisposition to Alzheimer's disease.

[0197] In some embodiments, the exogenous nucleic acid for insertion (e.g., 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 about 30 kb in length, up to about 35 kb in length, or up to about 40 kb in length.

[0198] In some embodiments, the exogenous nucleic acid to be inserted can 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 about 1 kb to about 35 kb, for example, more preferably 5 to 25 kb, and typically 8 to 20 kb.

[0199] In one embodiment, the composition of the present invention comprises or consists of: a. a nucleic acid construct encoding the first protein described above, comprising or consisting of the site-specific DNA-binding protein described above; b. A nucleic acid construct encoding a second protein comprising or consisting of a transposase that is a modified hyperactive PiggyBac that comprises one or more amino acid mutations compared to the hyperactive PiggyBac of SEQ ID NO: 9, as described above.

[0200] In another embodiment, the composition of the present invention comprises or consists of a nucleic acid construct encoding the above-mentioned fusion protein comprising or consisting of (i) a first protein comprising or consisting of a site-specific DNA-binding protein, as described above, and (ii) a second protein comprising or consisting of a transposase that is a modified hyperactive PiggyBac comprising one or more amino acid mutations compared to the hyperactive PiggyBac of SEQ ID NO: 9, as described above.

[0201] In one embodiment, the nucleic acid construct encoding the fusion protein further comprises a nucleic acid sequence encoding a linker between the first and second proteins, or in the case of a triple fusion protein, between two of the proteins, or between three proteins, as described above.

[0202] According to the present disclosure, the first and second proteins, or a fusion protein comprising or consisting of the first and second proteins, allow for and / or facilitate site-specific insertion of exogenous nucleic acid.

[0203] Some embodiments are directed to a plasmid or vector (e.g., an expression vector) that includes any of the following: a nucleic acid construct encoding a first protein; or a nucleic acid construct encoding a second protein; or a nucleic acid construct encoding a first protein and a nucleic acid construct encoding a second protein; or - a nucleic acid construct encoding a fusion protein or a triple fusion protein.

[0204] In some embodiments, the plasmid is a packaging plasmid. In some embodiments, the plasmid further comprises a polynucleotide encoding a capsid protein, e.g., gag and pol. In some embodiments, the plasmid is combined with a second plasmid (envelope plasmid) comprising a polynucleotide encoding a viral envelope protein and a third plasmid comprising a nucleic acid construct comprising an exogenous nucleic acid transgene, wherein, when the combination is introduced into a production cell line (e.g., a eukaryotic cell, a prokaryotic cell and / or cell line), viral particles comprising the nucleic acid construct encoding the exogenous nucleic acid transgene and the nucleic acid construct encoding either a first protein, a second protein, both the first protein and the second protein, or a fusion protein are produced.

[0205] In some embodiments, the plasmid is combined with a second plasmid comprising polynucleotides encoding capsid proteins, e.g., gag and pol (a packaging plasmid, where the packaging plasmid lacks a functional integrase), a third plasmid (an envelope plasmid) comprising polynucleotides encoding proteins of the viral envelope, and a fourth plasmid comprising a nucleic acid construct comprising an exogenous nucleic acid transgene, and when this combination is introduced into a production cell line (e.g., eukaryotic and prokaryotic cells and / or cell lines), viral particles are produced comprising the nucleic acid construct comprising the exogenous nucleic acid transgene and the nucleic acid construct encoding either a first protein, a second protein, both the first protein and the second protein, or a fusion protein.

[0206] In one embodiment, the first protein, the second protein, both the first and second proteins or the fusion protein, and / or the exogenous transgene are delivered to the cell using lentiviral particles.

[0207] In one embodiment, the nucleic acid construct comprises a first polynucleotide sequence encoding a first protein comprising or consisting of a site-specific DNA-binding protein engineered to bind a target nucleic acid sequence, a second polynucleotide sequence encoding a second protein comprising or consisting of a transposase that enables insertion of an exogenous nucleic acid transgene into a genome, and optionally a third polynucleotide sequence comprising a nucleic acid sequence encoding a linker between the first and second polynucleotides. In some embodiments, the first protein is a zinc finger protein or a Cas9 protein or a variant thereof, as described above, and / or the second protein is a modified hyperactive PiggyBac transposase, as described above.

[0208] Examples of suitable linkers for producing fusion proteins are described above.

[0209] In some embodiments, a linker is not required because the first protein is expressed from a separate plasmid from the second protein.

[0210] In one embodiment, instead of using a linker, the first and / or second polynucleotide sequences comprise nucleic acids encoding the first and second proteins, respectively, and further comprise additional nucleotides at at least one of their termini that serve as a linker.

[0211] In one embodiment, the nucleic acid construct is in the form of DNA or RNA.

[0212] Also provided herein are vectors comprising any of the nucleic acid constructs provided herein. 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 herein.

[0213] In some embodiments, the nucleic acid constructs of the present disclosure are expressed in host cells. 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, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NS0, SP2 / 0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), and perC6 cells, as well as insect cells such as Spodoptera fugiperda (Sf), or fungal cells such as Saccharomyces, Pichia, and Schizosaccharomyces.

[0214] In some embodiments, the host cell is derived from a microorganism. Microorganisms useful for certain methods disclosed herein include, for example, bacteria (e.g., E. coli), yeast (e.g., Saccharomyces cerevisiae), and plants. The host cell can be a prokaryotic or eukaryotic cell. In some embodiments, the host cell is a eukaryotic cell. Suitable eukaryotic host cells include, but are not limited to, yeast cells, insect cells, plant cells, fungal cells, and algal cells.

[0215] In some embodiments, the host cells are competent host cells. In some embodiments, the host cells are naturally competent. In some embodiments, the host cells are made competent by a process using, for example, calcium chloride and heat shock. The cells used can be any competent cell, particularly eukaryotic cells, particularly mammalian, e.g., human or animal. They can be somatic stem or embryonic stem or differentiated. In some aspects, the cells include 293T cells, fibroblasts, hepatocytes, muscle cells (skeletal, cardiac, smooth, vascular, etc.), epithelial cells, neural cells (neurons, glial cells, astrocytes), kidney cells, ocular cells, etc. This can also include insect, plant, yeast, or prokaryotic cells. Furthermore, primary cells can be isolated and used ex vivo for reintroduction into a subject after treatment with a nuclease (e.g., ZFN or TALEN) or nuclease system (e.g., CRISPR / Cas). Suitable primary cells include peripheral blood mononuclear cells (PBMCs) and other blood cell subsets, including, but not limited to, T lymphocytes, such as CD4+ T cells or CD8+ T cells. Suitable cells also include stem cells, such as, by way of example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells (CD34+), neural stem cells, and mesenchymal stem cells.

[0216] In some embodiments, host cells are 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 a capsid protein, such as gag and pol. In some embodiments, host cells are transfected with (i) a plasmid comprising the nucleic acid construct and, combined therewith in the host cell, (ii) a plasmid comprising a polynucleotide encoding a viral envelope protein (envelope plasmid); or (iii) a plasmid comprising an exogenous nucleic acid sequence (e.g., a GOI), and the viral particles produced comprise the exogenous nucleic acid, e.g., the GOI, and the first and second proteins (separately or as part of the fusion protein described above).

[0217] In some embodiments, host cells are transfected with (i) a plasmid containing a nucleic acid construct combined with (ii) a plasmid containing a nucleic acid construct further comprising polynucleotides encoding capsid proteins, e.g., gag and pol (packaging plasmid, where the packaging plasmid lacks a functional integrase); (iii) a plasmid containing polynucleotides encoding proteins of the viral envelope (envelope plasmid); and (iv) a plasmid containing an exogenous nucleic acid sequence (e.g., a GOI), and the viral particles produced contain the exogenous nucleic acid, e.g., the GOI, and the first and second proteins (separately or as part of a fusion protein as described above).

[0218] In further embodiments, vectors, e.g., lentiviral vectors according to the present disclosure, can be used to deliver the nucleic acid construct of the present disclosure and the first and second proteins (either separately or as part of a fusion protein as described above) encoded by the exogenous nucleic acid to an organism, e.g., a mammal, more specifically to a mammalian target cell of interest. Lentiviral vectors containing the first and second proteins (either separately or as part of a fusion protein as described above) can transduce various cell types, such as 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 that do not normally divide, such as neural cells. However, it is not intended that the present disclosure be limited to non-dividing cells, such as muscle cells, leukocytes, spleen cells, liver cells, eye cells, epithelial cells, and the like.

[0219] In certain embodiments, the packaged first and second proteins (separately or as part of a fusion protein as described above) are administered to an organism, e.g., for gene editing of the organism's DNA. In some embodiments, the organism is a human. 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, cow, 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 engineered, e.g., a genetically engineered non-human subject. The organism may be of either sex and at any stage of development.

[0220] Methods for inserting nucleic acids, such as exogenous nucleic acids, into a genome have been described. 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. al.20(92):1-23(2013);Li et al.PNAS 25:E2279-E2287(2013);Mates et al.Nature Genetics 41(6):753-761(2009);Mali et al.Nat.Methods 10(10):957-963;Vargas et al. al.J.Trans.Med.14(288):1-15(2016);Gersbach et al. al. Acc. Chem. Res. 47:2309-2318 (2014); Chandrasegaran et al. Cell Gene Ther. 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.

[0221] The present disclosure provides nucleic acid constructs encoding a first protein and a second protein (separately or as part of a fusion protein as described above) for inserting a nucleic acid (typically an exogenous nucleic acid) into a specific site in a genome. The present invention also provides first and second proteins (separately or as part of a fusion protein as described above) for inserting an exogenous nucleic acid into a specific site in a genome. In some embodiments, the exogenous nucleic acid for insertion can be up to 5 kb in length, 10 kb in length, 15 kb in length, 20 kb in length, 25 kb in length, 30 kb in length, 35 kb in length, or 40 kb in length, with particularly long nucleic acids being, for example, 5 kb to 25 kb, typically 8 kb to 20 kb.

[0222] In another embodiment, a method is provided for site-specific insertion of a nucleic acid into a genome.

[0223] Accordingly, the present disclosure relates to a method for site-specific integration of an exogenous nucleic acid sequence into the genome of a cell, the method comprising: (i) a first and second protein disclosed herein (separately or as part of a fusion protein as described above), or a nucleic acid construct disclosed herein; (ii) an exogenous nucleic acid that is integrated into the genome of the cell, and (iii) delivering to the cell a composition comprising a guide RNA for determining site-specific integration of the exogenous nucleic acid into the genome of the cell; Binding of the first and second proteins (separately or as part of the fusion protein described above) to specific genomic DNA sequences within the genome of the cell results in cleavage of the genome and site-specific integration of the exogenous nucleic acid sequence into the genome of the cell, as determined by the guide RNA.

[0224] In certain embodiments of the method, the exogenous nucleic acid is a nucleic acid fragment having a size of at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, typically 5 to 25 kb, preferably 8 to 20 kb.

[0225] In certain embodiments of the method, the exogenous nucleic acid is a therapeutic transgene that is inserted into the genome of a subject in need thereof to correct a defect in a genetic disorder.

[0226] In certain embodiments of the method, the composition is delivered in vitro or ex vivo, typically in mammalian cells, preferably human cells, more preferably human cells obtained from a human subject suffering from a genetic disorder.

[0227] In certain embodiments of the method, the composition is delivered in vivo to a mammal, eg, a human subject, in need thereof, typically for therapeutic treatment of a genetic disorder.

[0228] In some embodiments, the method comprises contacting the target DNA with any of the fusion proteins comprising Cas9 and a transposase described herein. For example, in some embodiments, the method comprises contacting the DNA with a fusion protein comprising two linked polypeptides: (i) Cas9; and (ii) a transposase, wherein the active Cas9 binds a gRNA that hybridizes to a region of DNA, e.g., genomic DNA.

[0229] In some embodiments, the method comprises contacting the target DNA with any of the fusion proteins comprising a ZFP and an integrase described herein. For example, in some embodiments, the method comprises contacting the DNA with a fusion protein comprising two linked polypeptides: (i) a ZFP; and (ii) an integrase, wherein the active ZFP hybridizes to a region of DNA, e.g., genomic DNA.

[0230] In some embodiments, the first and second proteins (separately or as part of a fusion protein as described above) are delivered to organisms and / or cells containing target DNA, e.g., genomic DNA, using a viral vector, e.g., a lentiviral particle.

[0231] Methods for packaging lentiviruses 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; Li et al. 9(8):1-9(2014); Mates et al. Nature Genetics 41(6):753-761(2009); and Robert H Kutner1, et al. NATURE PROTOCOLS 4(4):495(2009), each of which is incorporated herein by reference.

[0232] Typically, lentiviral delivery systems use a partitioned system with different lentiviral genes on separate plasmids, which are used to produce complete viruses without the genetic components required to cause viral disease. For example, one plasmid (envelope plasmid) can encode the proteins of the viral envelope (env); another plasmid (packaging plasmid) can encode capsid proteins (e.g., gag and pol) and enzymes such as reverse transcriptase and / or integrase; and an additional plasmid (transfer plasmid) contains a gene of interest (GOI) flanked by long terminal repeats (for genome integration) and psi sequences (which indicate a signal for packaging the gene into the virus). When these plasmids are introduced into cells simultaneously, viruses containing the gene of interest without the viral genes required to cause disease are produced.

[0233] In certain aspects of the present disclosure, lentiviral vectors (or particles) of the present invention can be obtained in vitro by a partitioning system, e.g., a trans-complementation system (vector / packaging system), by transfecting permissive cells (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 GAG, POL, and envelope protein(s) or sufficient portions of these polypeptides to allow the formation of retroviral particles.

[0234] As an example, a host cell is transfected with a) a packaging plasmid containing lentiviral gag and pol sequences, b) a second plasmid (envelope expression plasmid or pseudotyping env plasmid) containing genes encoding envelope protein(s) (such as VSV-G), c) a plasmid vector containing the 5'-3' LTR sequence, the psi encapsidation sequence, and a transgene, and d) a plasmid vector containing a nucleic acid construct encoding the first and second proteins disclosed herein (separately or as part of a fusion protein as described above). In some embodiments, the nucleic acid constructs encoding the first and second proteins disclosed herein (separately or as part of a fusion protein as described above) are on the packaging plasmid rather than on separate plasmids. Nucleic acids encoding gag, pol, and env cDNAs can be advantageously prepared according to conventional techniques from viral gene sequences available in the art and databases.

[0235] In some embodiments, the lentiviral vector comprises a nucleic acid construct described herein. In some embodiments, the lentiviral vector comprises a first and second protein described herein (separately or as part of a fusion protein as described above).

[0236] The promoters used in the plasmids may be the same or different. In some embodiments, in a plasmid transcomplementation system, the promoters for driving the expression of the coat proteins gag and pol in the envelope plasmid and the plasmid vector, respectively, the mRNA and transgene of the vector genome may be the same or different. Such promoters can be advantageously selected from ubiquitous promoters or specific promoters, such as viral promoters CMV, TK, RSV LTR promoters and RNA polymerase III promoters such as U6 or H1, or promoters of helper viruses (i.e., adenovirus, baculovirus, herpesvirus) encoding env, gag, and pol.

[0237] To produce the lentiviral vectors of the present disclosure, the plasmids described herein can be introduced into host cells, and viruses can be produced and recovered. 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 generated from such cells include, for example, COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NS0, SP2 / 0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), and perC6 cells, as well as insect cells such as Spodoptera fugiperda (Sf), or fungal cells such as Saccharomyces, Pichia, and Schizosaccharomyces.

[0238] Once host cells are transfected with the plasmid and lentiviral vectors (or particles) of the present disclosure are produced, the lentiviral vectors (or particles) of the present disclosure can be purified from the cell supernatant. Purification of lentiviral vectors to increase their concentration can be achieved by any suitable method, such as density gradient purification (e.g., cesium chloride (CsCl)), chromatography techniques (e.g., column or batch chromatography), or ultracentrifugation. For example, vectors of the present invention can be subjected to two or three CsCl density gradient purification steps. The vectors are desirably purified from infected cells using a method comprising lysing the cells, applying the lysate to a chromatography resin, eluting the virus from the chromatography resin, and collecting fractions containing the lentiviral vectors of the present disclosure.

[0239] Lentiviral vector delivery methods have been described. See, for example, Vargas et al. J. Trans. Med. 14(288):1-15(2016); Mali et al. Nat. Methods 10(10):957-963; Mates et al. Nature Genetics 41(6):753-761(2009); Skipper et al. 20(92):1-23(2013).

[0240] The lentiviral vector containing the first and second proteins (separately or as part of the above-described fusion protein) or the nucleic acid construct encoding them can be administered to a subject by any route. In some embodiments, the lentiviral vector of the present disclosure can be delivered to a subject's cells either in vivo or ex vivo.

[0241] In some embodiments, the lentiviral vector of the present disclosure can be delivered in vivo. In some embodiments, a lentiviral vector containing the first and second proteins (separately or as part of the above-mentioned fusion protein) encoded by the nucleic acid construct of the present disclosure can be used to deliver a gene of interest and / or target a genetic defect in the DNA of a subject. In some embodiments, the lentiviral vector is administered to a subject parenterally, preferably intravascularly (including intravenously). When administered parenterally, the vector is preferably provided in a pharmaceutical vehicle suitable for injection, such as a sterile aqueous solution or dispersion.

[0242] In some embodiments, the lentiviral vectors of the present disclosure can be used ex vivo.

[0243] In some embodiments, lentiviral vectors containing the first and second proteins encoded by the nucleic acid constructs of the present disclosure (separately or as part of a fusion protein as described above) can be used to deliver a gene of interest and / or target a genetic defect in a subject's DNA. In some embodiments, cells are removed from a subject, and a lentiviral vector containing the first and second proteins encoded by the nucleic acid constructs of the present disclosure (separately or as part of a fusion protein as described above) is administered to the cells ex vivo to modify the cells' DNA. The cells harboring the modified DNA are then expanded and reinfused into the subject. In certain embodiments, lentiviral vectors containing the first and second proteins encoded by the nucleic acid constructs of the present disclosure (separately or as part of a fusion protein as described above) can be used in chimeric antigen receptor (CAR) T-cell therapy to genetically modify a patient's autologous T cells to express a CAR specific for a tumor antigen. In further embodiments, the modified CAR-T cells are expanded ex vivo and reinfused into the patient. In some embodiments, the modified T cells more specifically target cancer cells. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, resulting in long-term persistence.

[0244] After administering the lentiviral vector of the present disclosure or the cells modified ex vivo using the lentiviral vector of the present disclosure, the subject can be monitored to detect the expression of the transgene.The dosage and duration of treatment are determined individually according to the condition or disease to be treated.Various conditions or diseases can be treated based on the gene expression produced by administering the gene of interest in the vector of the present invention.The dosage of the vector delivered using the method of the present invention will vary depending on the desired response by the host and the vector used.

[0245] In some gene therapy applications, it is desirable for the gene therapy vector to be delivered with high specificity to a particular tissue type. Therefore, viral vectors can be modified to have specificity for a given cell type by expressing a ligand as a fusion protein with the viral coat protein on the outer surface of the virus. The ligand is selected to have affinity for a receptor known to be present in the target cell type.

[0246] Certain aspects of the present disclosure relate to a method for inserting an exogenous nucleic acid sequence into the genomic DNA of an organism, the method comprising identifying a specific genomic DNA sequence within the genome of the organism; and administering to the organism a lentiviral particle comprising a nucleic acid construct of the present disclosure, such that the particle binds to the specific genomic DNA sequence and inserts the exogenous nucleic acid into the genomic DNA, wherein the exogenous nucleic acid is integrated at the specific genomic DNA sequence.

[0247] Certain embodiments of the present disclosure relate to methods for the controlled, site-specific integration of single or multiple copies of an exogenous nucleic acid sequence into a cell, the method comprising: a) delivering to the cell a nucleic acid construct, vector, or first and second proteins of the present disclosure (separately or as part of a fusion protein as described above); and b) delivering to the cell an exogenous nucleic acid, wherein the first and second proteins (separately or as part of a fusion protein as described above) bind to specific genomic DNA sequences within the genome of the cell, resulting 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 embodiments, delivery to the cell is carried out by a lentiviral particle.

[0248] Several strategies can be used to test integration sites to screen for the optimal mechanism for directional integration.

[0249] For the analysis of the modified transposon disclosed herein, a reporter cell line can be used that has a promoter, half of the GFP coding sequence, and a splice site donor downstream of the target insertion site in the genome.For example, the lentivirus payload can have a fusion integrase variant, followed by an inverted splice site acceptor and the remaining half of the GFP.GFP expression occurs when direct insertion occurs, and GFP-containing mRNA is produced from the insertion site and the integrated payload, and the splicing of this mRNA results in the complete GFP CDS.

[0250] The VPR transcomplementation system can also be used to screen and compare integration mutants. This transcomplementation system can be used to target the insertion of lentiviral payloads containing fusion integrase variants that, when expressed and loaded into particles, promote their own integration. These fusion integrase variants would then be loaded into viral particles using VPR fusions. This transcomplements the integration-defective integrase encoded within the packaging vector used to produce the particles. Other methods that can be used for integration mapping include IC or FISH probes. Targeted insertion can also be screened by TCRa or RFP targeted disruption, or GFP activation upon targeted splice site integration.

[0251] In a FISH approach, which simultaneously stains the insertion region and the target region within chromatin, fluorescent in situ hybridization can be performed to identify the location of the transposon gene of interest within the Hek293T genome. Hek293T can be transfected with 1) the GOI-transposon, 2) a programmable transposase, and 3) gRNA to target PPP1R12. Probes designed to target the PPP1R12 gene, CD46 (as a negative control), and the GOI can be synthesized from PCR-amplified DNA using Nick Translation Mix (Sigma).

[0252] In some embodiments, the first and second proteins comprising the modified transposase disclosed herein (separately or as part of a fusion protein described above) improve the specificity of insertion of exogenous nucleic acids into the genome compared to wild-type transposases (or fusion proteins comprising the corresponding wild-type transposases), as determined, for example, by a Genetrap assay. In some embodiments, HEK293T cells or any other permissive cells are transfected or transduced with lentiviral particles carrying the following plasmids or payloads: (i) a plasmid comprising a gRNA targeting a specific region of DNA, (ii) a plasmid comprising a nucleic acid construct of the present disclosure encoding the first and second proteins (separately or as part of a fusion protein described above), where the second protein is a modified transposase, and (iii) a genetrap plasmid comprising a nucleic acid sequence encoding a promoterless reporter protein, e.g., GFP. In some embodiments, the genetrap plasmid further comprises a transposon with an inverted repeat.

[0253] In some embodiments, the percentage of cells containing GFP insertions can be determined by flow cytometry. In some embodiments, the first and second proteins (separately or as part of a fusion protein described above), where the second protein is a modified transposase, increase the percentage of cells containing GFP insertions by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% compared to the corresponding wild-type proteins. In some embodiments, the first and second proteins (separately or as part of a fusion protein described above), where the second protein is a modified transposase, increase the percentage of cells containing GFP insertions by about 15-30%.

[0254] In some embodiments, the percent insertion at the target site and the coverage at the target site (number of reads per insertion site) can be determined by genomic DNA extraction and targeted sequencing using oligonucleotides specific for the viral LTR. In some embodiments, the first and second proteins (separately or as part of a fusion protein described above), where the second protein is a modified transposase, increase the percent insertion at the target site by at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, 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 percent insertion at the target site is increased by about 10- to 100-fold. In some embodiments, the first and second proteins (separately or as part of a fusion protein as described above), where the second protein is a modified transposase, increase coverage (reads per insertion site) at the target site by at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 110-fold, at least 120-fold, at least 130-fold, at least 140-fold, at least 150-fold, at least 160-fold, at least 170-fold, at least 180-fold, at least 190-fold, or at least 200-fold compared to the corresponding wild-type protein. In some embodiments, the coverage (reads per insertion site) at the target site is at least 100-fold.

[0255] In some embodiments, the percent insertion at the target site and coverage at the target site (number of reads per insertion site) can be determined by genomic DNA extraction and targeted sequencing using oligonucleotides specific for the viral inserted LTR.

[0256] Potential uses of lentiviral vectors containing the first and second proteins of the present disclosure (separately or as part of the above-mentioned fusion protein) include gene therapy, i.e., gene transfer in any mammalian cell, particularly human cells. These may be dividing or quiescent cells, cells belonging to central or peripheral organs such as the liver, pancreas, muscle, or heart. Gene therapy may enable the expression of proteins such as neurotrophic factors, enzymes, transcription factors, and receptors. Lentiviral vectors according to the present invention may also be particularly suitable for research purposes.

[0257] In some embodiments, the nucleic acid construct, the first and second proteins (separately or as part of the above-described fusion protein), and / or the lentiviral vector of the present disclosure are administered to a subject to treat a disease. In some embodiments, the disease is a genetic disorder that can benefit from gene therapy.

[0258] In some embodiments, the first and second proteins (separately or as part of a fusion protein as described above), or nucleic acid constructs encoding them, or kits or compositions as disclosed below, or lentiviral vectors comprising the first and second proteins (separately or as part of a fusion protein as described above), or nucleic acid constructs according to the present disclosure can be used as a pharmaceutical.

[0259] Lentiviral vectors according to the present disclosure may be particularly suitable for treating genetic diseases in a subject.

[0260] The present invention provides (i) RNA-guided nucleases or zinc finger nucleases; (ii) transposase, (iii) a guide RNA, and (iv) compositions comprising a nucleic acid or gene of interest, e.g., an exogenous nucleic acid for insertion into a genome, Here, the transposase is a modified hyperactive Piggybac that contains one or more amino acid mutations compared to the hyperactive Piggybac of SEQ ID NO:9.

[0261] In one embodiment, the modified hyperactive PiggyBac mutation comprises the amino acid substitutions R372A / K375A / D450N.

[0262] In a preferred embodiment, the modified hyperactive PiggyBac mutation does not include the amino acid substitutions R372A / K375A / D450N.

[0263] In some embodiments, the modified hyperactive PiggyBac mutations include the following amino acid substitutions or combinations of amino acid substitutions: S351A / R372A / K375A / R388A / D450N / W465A / S573A / M589V / S592G / F594L, R245A / R275A / R277A / R372A / W465A / M589V, R275A / R325A / R372A / T560A, N347A / D450N ... 7S / D450N / T560A / S573A / F594L, R202K / R275A / N347S / R372A / D450N / T560A / F594L, R275A / N347S / K375A / D450N / S592G , R275A / N347S / R372A / D450N / T560A / F594L, R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L, R245A / N347S / R 372A / D450N / T560A / S564P / S573A / S592G, R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L, V3 4M / R275A / G325A / N347S / S351A / R372A / K375A / D450N / T560A / S564P, G325A / N347S / K375A / D450N / S573A / M589V / S592G , S230N / R277A / N347S / K375A / D450N, T43I / R372A / K375A / A411T / D450N, G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G, and the position numbers correspond to the amino acid numbers of the highly active PiggyBac sequence (sequence number 9).

[0264] In a preferred embodiment, the modified hyperactive PiggyBac mutation comprises the following amino acid substitutions or combinations of amino acid substitutions: R245A / R275A / R277A / R372A / W465A / M589V, R275A / 325A / R372A / T560A, N347A / D450N, N347S / D450N / T560A / S573A / F59 4L, R202K / R275A / N347S / R372A / D450N / T560A / F594L, R275A / N347S / K375A / D450N / S592G, R275 A / N347S / R372A / D450N / T560A / F594L, R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L , R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G, R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L, G325A / N347S / K375A / D450N / S573A / M589V / S592G, S230N / R 277A / N347S / K375A / D450N, G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G, position numbers correspond to the amino acid numbers of the hyperactive PiggyBac sequence (SEQ ID NO: 9).

[0265] In some embodiments, the RNA-guided nuclease is a Cas9 protein. In some embodiments, the RNA-guided nuclease is a SpCas9 protein. In some embodiments, the RNA-guided nuclease is a SaCas9 protein.

[0266] The present invention also relates to compositions comprising nucleic acids encoding: (i) the RNA-guided nuclease or zinc finger nuclease described above; (ii) the transposase described above; (iii) a guide RNA, and (iv) A nucleic acid or gene of interest, e.g., an exogenous nucleic acid for insertion into a genome.

[0267] In some embodiments, the nucleic acid of the composition is expressed in cells via a suitable expression vector. As used herein, the term "expression vector" refers to a vector containing a polynucleotide comprising an expression control sequence operably linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression. Other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate recombinant polynucleotides.

[0268] In a preferred embodiment, the two nucleic acids are co-expressed in the same cell or cell population. In some embodiments, the two nucleic acids are co-expressed simultaneously. In another embodiment, the nucleic acid encoding the RNA-guided nuclease or zinc finger nuclease is expressed first. In another embodiment, the nucleic acid encoding the transposase is expressed first.

[0269] The present invention further relates to a composition comprising: (i) a fusion protein comprising an RNA-guided nuclease and a transposase disclosed herein, or a nucleic acid encoding the same; (ii) a transposase disclosed herein, or a nucleic acid encoding same; (iii) a guide RNA, and (iv) A nucleic acid or gene of interest, e.g., an exogenous nucleic acid for insertion into a genome.

[0270] The present invention further relates to a composition comprising: (i) a first fusion protein comprising an RNA-guided nuclease and a transposase disclosed herein, or a nucleic acid encoding the same; (ii) a second fusion protein comprising an RNA-binding protein engineered to bind to at least one specific RNA sequence and a transposase disclosed herein, or a nucleic acid encoding same; (iii) a guide RNA, and (iv) A nucleic acid or gene of interest, e.g., an exogenous nucleic acid for insertion into a genome.

[0271] The present disclosure also provides compositions for carrying out the disclosed methods described herein. In some embodiments, the compositions include a nucleic acid construct or vector as defined in this disclosure and a polynucleotide sequence encoding an exogenous nucleic acid for insertion into a genome, the polynucleotide sequence being contained in or linked to a packaging vector.

[0272] The present disclosure further relates to a composition comprising: (i) a fusion protein comprising an RNA-guided nuclease and a transposase disclosed herein, or a nucleic acid encoding this fusion protein; (ii) a guide RNA, and (iii) A nucleic acid or gene of interest, e.g., an exogenous nucleic acid for insertion into a genome.

[0273] In certain embodiments, the nucleic acid or gene of interest is a large DNA fragment, typically having a size of 5 kb to 25 kb, more preferably 8 kb to 20 kb.

[0274] The present disclosure also provides kits for carrying out the disclosed methods described herein. The kits can include a nucleic acid construct or a fusion protein described herein. In some aspects, the kits can include a lentiviral particle containing a nucleic acid construct or a fusion protein described herein.

[0275] The kit may further include instructions for using the components of the kit to practice the method of the present invention. The instructions for practicing the method of the present invention are typically recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. Thus, the instructions may be present in the kit as a package insert, on the label of the kit's container or its components (i.e., associated with the packaging or subpackaging). In other embodiments, the instructions are present as an electronic storage data file residing on a suitable computer-readable storage medium. In yet other embodiments, the actual instructions are not present in the kit, but a means for obtaining the instructions from a remote source, for example, via the Internet, is provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or downloaded. Like the instructions, this means for obtaining the instructions is also recorded on a suitable substrate.

[0276] The present disclosure typically relates to kits comprising: A first composition comprising: (i) a first fusion protein, or a nucleic acid encoding a first fusion protein, as defined herein, wherein the first fusion protein comprises the amino acid sequence of a first inducible RNA nickase Cas9, typically SpCas9 nickase of SEQ ID NO: 70, fused to a modified hyperactive Piggybac; and (ii) a first composition comprising a first guided RNA nucleic acid; A second composition comprising: (iii) a second fusion protein as defined herein, or a nucleic acid encoding the second fusion protein, wherein the second fusion protein comprises the amino acid sequence of a second inducible RNA nickase Cas9, typically SaCas9 nickase of SEQ ID NO: 76, fused to a modified hyperactive Piggybac; and (iv) a second composition comprising a second guided RNA nucleic acid; and Optionally, a nucleic acid for insertion into the genome, for example a nucleic acid having a size of 5 kb to 25 kb, more preferably 8 kb to 20 kb; Here, the first and second fusion proteins can heterodimerize and form double cleavages determined by the first and second guided RNAs at adjacent sites in the genomic DNA region, and can optionally insert nucleic acid between the adjacent sites.

[0277] In certain embodiments, the composition or kit comprises the exogenous nucleic acid in a minicircle, a plasmid or a viral vector, particularly a non-integrating viral vector, such as a non-integrating lentiviral vector.

[0278] In certain embodiments, the compositions or kits disclosed herein are contained in nanoparticles.

[0279] In certain embodiments, the composition is a nucleic acid composition comprising: (i) a nucleic acid construct encoding a fusion protein disclosed herein; (ii) a guide RNA, and (iii) A nucleic acid or gene of interest, e.g., an exogenous nucleic acid for insertion into a genome.

[0280] In certain embodiments, the kit comprises: (i) a nucleic acid construct encoding a first fusion protein disclosed herein, wherein the first fusion protein comprises a first inducible RNA nickase Cas9, typically the amino acid sequence of SpCas9 nickase of SEQ ID NO: 70, fused to a modified hyperactive Piggybac; and (ii) a first composition comprising a first guided RNA nucleic acid; (iii) a nucleic acid construct encoding a second fusion protein disclosed herein, wherein the second fusion protein comprises the amino acid sequence of a second inducible RNA nickase Cas9, typically SaCas9 nickase of SEQ ID NO: 76, fused to a modified hyperactive Piggybac; and (iv) a second composition comprising a second guided RNA nucleic acid.

[0281] In certain embodiments, the kit or composition is for use as a medicament, particularly in the treatment of a disorder in a human, for example, to treat a genetic defect in a human subject in need thereof.

[0282] In some embodiments, the nucleic acid construct is in the form of RNA, DNA, or protein, depending on the delivery method, and the polynucleotide sequence encoding the exogenous nucleic acid is in the form of RNA or DNA. In particular, the polynucleotide sequence encoding the exogenous nucleic acid is in the form of RNA.

[0283] In some embodiments, the composition or kit does not include a virus, and the packaging vector is a nanoparticle, such as a polymer or lipid nanoparticle. The packaging vector may be a carrier that binds to elements of the composition. In some embodiments, the composition is contained in a viral vector, particularly a lentiviral particle.

[0284] In some embodiments, the compositions or kits comprise: (a) a nucleic acid construct described herein in the form of RNA (e.g., comprising Cas9 and a transposase); (b) optionally a guide RNA (e.g., as a separate linear single-stranded RNA molecule); and (c) a polynucleotide comprising an exogenous gene for insertion in DNA form (e.g., a vector) contained in or linked to a packaging vector.

[0285] In some embodiments, the composition comprises: (a) a fusion protein described herein in protein form (e.g., comprising Cas9 and a transposase); (b) optionally a guide RNA (e.g., as a separate linear single-stranded RNA molecule), wherein the fusion protein and the guide RNA form a ribonucleoprotein complex (RNP); and (c) a polynucleotide comprising an exogenous gene for insertion in DNA form (e.g., a vector) contained in or linked to a packaging vector.

[0286] In some embodiments, the composition comprises (a) a nucleic acid construct described herein in the form of DNA (e.g., comprising Cas9 and a transposase), (b) optionally a guide RNA (e.g., as a separate linear RNA molecule or as DNA within a vector), and (c) a polynucleotide comprising an exogenous gene for insertion in DNA form (e.g., a vector) contained in or linked to a packaging vector.

[0287] In some embodiments, the composition comprises (a) a fusion protein described herein in the form of a protein (e.g., comprising Cas9 and integrase), (b) optionally a guide RNA (e.g., as a separate RNA molecule complexed with the fusion protein), and (c) a polynucleotide comprising an exogenous gene to be inserted, contained in or linked to a packaging vector. In certain embodiments, the packaging vector is a lentiviral particle. In some embodiments, the (a) fusion protein is linked to the lentiviral capsid by gag-pol or VPR (viral protein R). In some embodiments, the (c) polynucleotide is in the form of RNA as the payload of the integrase.

[0288] In certain embodiments, when a ZFP is used, (b) a guide RNA may not be required.

[0289] The present invention provides (i) a fusion protein comprising an RNA-binding protein engineered to bind to at least one specific RNA sequence, a DNA-binding protein that allows for the insertion of an exogenous nucleic acid into a genome, and a linker connecting the first protein and the second protein; (ii) a Cas9 protein, and (iii) a guide RNA comprising at least one specific RNA sequence for binding of the fusion protein; (iv) a composition comprising a nucleic acid or gene of interest, e.g., an exogenous nucleic acid for insertion into a genome, The present invention further relates to compositions wherein the DNA-binding protein is a modified transposase of the present disclosure, typically a modified hyperactive Piggybac comprising one or more amino acid mutations to increase excision activity compared to unmodified hyperactive Piggybac, and one or more amino acid mutations to decrease DNA-binding activity compared to unmodified hyperactive Piggybac according to SEQ ID NO: 9.

[0290] In some embodiments, the RNA binding protein is MS2 bacteriophage coat protein (MCP).

[0291] In some embodiments, at least one RNA sequence recognized by the MCP of the fusion protein is a tetraloop. As used herein, the term "tetraloop" is used interchangeably with the terms "stem loop" and "hairpin loop." In some embodiments, at least one RNA tetraloop is an MS2 RNA tetraloop binding sequence.

[0292] In some embodiments, the guide RNA comprises at least one MS2 RNA tetraloop binding sequence. In some embodiments, the gRNA comprises two or more MS2 RNA tetraloop binding sequences. As used herein, the term "two or more" means 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more.

[0293] Various embodiments are set forth in the claims. Some additional embodiments are disclosed below.

[0294] E1. (i) a first protein comprising an RNA-guided nuclease or a zinc finger nuclease; (ii) a second protein comprising a transposase; and (iii) optionally a linker connecting the first protein and the second protein; wherein the transposase is a modified hyperactive Piggybac comprising one or more amino acid mutations compared to the hyperactive Piggybac of SEQ ID NO: 9; A fusion protein, in which the first protein is fused directly or indirectly via a linker to the C-terminal end of the second protein.

[0295] E2. The fusion protein of embodiment 1, wherein the transposase is a modified hyperactive Piggybac comprising one or more amino acid mutations to increase excision activity compared to unmodified hyperactive Piggybac and one or more amino acid mutations to decrease DNA-binding activity compared to unmodified hyperactive Piggybac.

[0296] E3. The fusion protein of embodiment 2, wherein the one or more amino acid mutations for increasing excision activity are selected from among amino acid mutations within the region defined by amino acid position numbers [194-200], [214-222], [434-442] or [446-456], for example, amino acid substitutions at positions D198, D201, R202, M212 or S213 (position numbers correspond to the amino acid numbering of the unmodified hyperactive Piggybac of SEQ ID NO: 9).

[0297] E4. The fusion protein of any one of embodiments 1 to 3, wherein the one or more amino acid mutations are selected from among the amino acid substitutions that increase excision activity at positions M194 or D450, where the position numbers correspond to the amino acid numbers of the unmodified hyperactive Piggybac of SEQ ID NO: 9, and are preferably selected from among the amino acid substitutions M194V and / or D450N.

[0298] E5. The fusion protein of any one of embodiments 1 to 4, wherein the one or more amino acid mutations are selected from among amino acid substitutions that reduce DNA binding activity at positions R372, K375, R376, E377, and / or E380, where the position numbers correspond to the amino acid numbers of the unmodified, highly active Piggybac of SEQ ID NO: 9, and preferably selected from among the amino acid substitutions R372A, K375A, R376A, E377A, and / or E380A.

[0299] E6. The fusion protein of any one of embodiments 1 to 5, wherein said modified hyperactive Piggybac comprises at least one amino acid substitution at position D450 to increase excision activity and at least two amino acid substitutions at positions R372 and K375 to decrease DNA-binding activity, preferably wherein said hyperactive Piggybac modified transposase comprises the triple mutation D450N, R372A and K375A, wherein the position numbers correspond to the amino acid numbers of unmodified hyperactive Piggybac in SEQ ID NO: 9.

[0300] E7. A fusion protein described in any one of embodiments 1 to 6, wherein the modified hyperactive Piggybac further comprises at least one mutation in the region defined by amino acid position numbers [158-169], such as A166S and / or at least one mutation at positions Y527, R518, K525, N463.

[0301] E8. The fusion protein of any one of embodiments 1-7, wherein the modified hyperactive Piggybac comprises an amino acid sequence having at least 85%, at least 90%, at least 95% identity, or 100% identity to the modified hyperactive Piggybac of SEQ ID NO: 1.

[0302] E9. The fusion protein of any one of embodiments 1-8, wherein the modified hyperactive Piggybac is a variant of the hyperactive Piggybac of SEQ ID NO: 1 having one or more amino acid substitutions, typically no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions.

[0303] E10. The fusion protein of any one of embodiments 1 to 9, wherein the modified hyperactive PiggyBac further comprises one or more amino acid substitutions at positions 245, 268, 275, 277, 287, 290, 315, 325, 341, 346, 347, 350, 351, 356, 357, 388, 409, 412, 432, 447, 460, 461, 465, 517, 560, 564, 571, 573, 576, 586, 587, 589, 592, and / or 594, wherein the position numbers correspond to the amino acid numbers of the hyperactive PiggyBac sequence (SEQ ID NO: 9).

[0304] E11. The modified hyperactive PiggyBac mutation comprises any of the following amino acid substitutions or combinations of amino acid substitutions: R245A, D268N, R275A, R277A, K287A, K290A, K287A / K290A, R315A, G325A, R341A, D346N, N347A, N347S, T350A, S351E, S351P, S351A, K356E, N357A, R388A, K409A, K412A, K432A, D447A, D447N, D450N, R460A, K461A, W465A, S517A, T560A, S 564P, S571N, S573A, K576A, H586A, I587A, M589V, S592G, or F594L, D450N / R372A / K375A, R275A / R277A, K409A / K412A, R460A / K461A, R275A / R277A / N347S / K375A / T560A / S573A / M589V / S592G, and R245A / R275A / R277A / R372A / W465A, where the position numbers correspond to the amino acid numbers of the hyperactive PiggyBac sequence (SEQ ID NO: 9).

[0305] E12. The modified hyperactive PiggyBac mutation comprises the following amino acid substitutions or combinations of amino acid substitutions: R372A / K375A / D450N, R372A / K375A / R376A / D450N, K375A / R376A / E377A / E380A / D450N, R372A / K375A / R376A / E377A / E380A / D45 0N, M194V, R376A, E377A, E380A, M194V / R372A / K375A, S351A / R372A / K375A / R388A / D450N / W465A / S573A / M589V / S592G / F594L, R245A / R275A / R277A / R372A / W465A / M589V, R275A / 325A / R372A / T5 60A, N347A / D450N, N347S / D450N / T560A / S573A / F594L, R202K / R275A / N347S / R372A / D450N / T560 A / F594L, R275A / N347S / K375A / D450N / S592G, R275A / N347S / R372A / D450N / T560A / F594L, R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L; the position numbers correspond to the amino acid numbers of the highly active PiggyBac sequence (SEQ ID NO: 9), and typically the modified transposase has an amino acid sequence selected from any of SEQ ID NOs: 1 to 8 and 10 to 18.

[0306] E13. The fusion protein of any one of embodiments 1-12, wherein said linker is a peptide linker comprising an XTEN sequence or a GGS sequence, preferably an XTEN sequence.

[0307] E14. The fusion protein of any one of embodiments 1 to 13, wherein the linker is a peptide linker having a length of 3 to 50 amino acids, typically selected from any 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.

[0308] E15. The fusion protein of any one of embodiments 1-14, wherein said first protein is a Cas9 protein comprising an active DNA cleavage domain and a guide RNA binding domain.

[0309] E16. The fusion protein of any one of embodiments 1 to 15, wherein the first protein is a nuclease protein comprising an active DNA cleavage domain and a guide RNA binding domain, and has at least 80%, 90%, 95%, 99%, or at least 100% identity to Streptococcus pyogenes Cas9 of SEQ ID NO: 31, SaCas9 of SEQ ID NO: 72, Cpf1 of SEQ ID NO: 74, CjCas9 of SEQ ID NO: 29, SpCas9 nickase of SEQ ID NO: 70, CasX of SEQ ID NO: 75, or SaCas9 nickase of SEQ ID NO: 76.

[0310] E17. The fusion protein of any one of embodiments 1-16, wherein the first protein is a Cas9 protein selected from the group consisting of SaCas9 of SEQ ID NO: 72 or Streptococcus pyogenes Cas9 of SEQ ID NO: 31.

[0311] E18. (i) a first protein comprising an RNA-guided nuclease or nickase; (ii) a second protein consisting of the first transposase; (iii) a third protein consisting of a second transposase; and (iv) optionally, a peptide linker between the first and second proteins and the second and third proteins, 18. The fusion protein of any one of embodiments 1 to 17, wherein the first and second transposases have the same or different modified Piggybac transposase sequences, for example as described in any one of embodiments 3 to 12.

[0312] E19. The fusion protein of any one of embodiments 1-14, wherein said first protein is a zinc finger protein of SEQ ID NO: 33.

[0313] E20. (i) a fusion protein according to any one of embodiments 1 to 19, or a nucleic acid encoding said fusion protein; (ii) a guide RNA; (iii) an exogenous nucleic acid for insertion into a genome.

[0314] E21. The composition of embodiment 20, wherein the exogenous nucleic acid is a large DNA fragment, typically having a size of 5 kb to 25 kb, more preferably 8 kb to 20 kb.

[0315] E22. A kit comprising: (i) - a first fusion protein or a nucleic acid encoding said first fusion protein according to any one of embodiments 1 or 18, wherein said first fusion protein comprises a first inducible RNA nickase Cas9 fused to a modified hyperactive Piggybac, typically the amino acid sequence of SpCas9 nickase of SEQ ID NO: 70; and a first composition comprising a first guided RNA nucleic acid; (ii) - a second fusion protein or a nucleic acid encoding a second fusion protein according to any one of embodiments 1 to 18, wherein the second fusion protein comprises a second inducible RNA nickase Cas9 fused to a modified hyperactive Piggybac, typically the amino acid sequence of the SaCas9 nickase of SEQ ID NO: 76; and a second composition comprising a second guided RNA nucleic acid; and (iii) optionally, an exogenous nucleic acid for insertion into the genome, for example, an exogenous nucleic acid having a size of 5 kb to 25 kb, more preferably 8 kb to 20 kb; wherein the first and second fusion proteins are capable of heterodimerizing to form double cleavage determined by the first and second guided RNAs at adjacent sites in a genomic DNA region, and optionally inserting the exogenous nucleic acid between the adjacent sites.

[0316] E23. The composition or kit of any one of embodiments 20-22, wherein the exogenous nucleic acid is comprised in a minicircle, a plasmid or a viral vector, in particular a non-integrating viral vector, such as a non-integrating lentiviral vector.

[0317] E24. The composition or kit of any one of embodiments 20-23, wherein the composition is contained in nanoparticles.

[0318] E25. A modified hyperactive Piggybac transposase, comprising at least one amino acid mutation to increase excision activity compared to an unmodified hyperactive Piggybac, and / or at least one amino acid mutation to decrease DNA binding activity compared to an unmodified hyperactive Piggybac; wherein the at least one mutation for increasing excision activity is an amino acid substitution of M at position 194, typically M194V, and / or the at least one amino acid mutation for decreasing DNA binding activity is selected from among the amino acid substitutions at positions R376, E377, and E380, typically R376A, E377A, and / or E380A.

[0319] E26. A modified hyperactive Piggybac transposase comprising the following combinations of mutations when compared to the unmodified hyperactive Piggybac of SEQ ID NO: 9: R372A / K375A / D450N, R372A / K375A / R376A / D450N, K375A / R376A / E377A / E380A / D450N, R372A / K375A / R376A / E377A / E380A / D450N, M194V, R376A, E377A, E380A, M194V / R372A / K375A, S351A / R372A / K375A / R388A / D450N / W465A / S573A / M58 9V / S592G / F594L, R245A / R275A / R277A / R372A / W465A / M589V, R275A / 325A / R372A / T560A, N347A / D450N, N347S / D450N / T560A / S573A / F594L, R202K / R275A / N347S / R3 72A / D450N / T560A / F594L, R275A / N347S / K375A / D450N / S592G, R275A / N347S / R372 A / D450N / T560A / F594L, R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L.

[0320] E27. The modified hyperactive Piggybac transposase of embodiment 25 or 26, further comprising one or more amino acid mutations selected from the group consisting of: an amino acid at position 245 that is A, an amino acid at position 275 that is R or A, an amino acid at position 277 that is R or A, an amino acid at position 325 that is A or G, an amino acid at position 347 that is N or A, an amino acid at position 351 that is E, P, or A, an amino acid at position 372 that is R, an amino acid at position 375 that is A, an amino acid at position 450 that is D or N, an amino acid at position 465 that is W or A, an amino acid at position 560 that is T or A, an amino acid that is P or S at position 564, an amino acid that is S or A at position 573, an amino acid that is G or S at position 592, and an amino acid that is L or F at position 594.

[0321] E28. The modified hyperactive PiggyBac transposase of embodiment 25 or 26, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional modifications compared to an unmodified hyperactive PiggyBac of SEQ ID NO: 9, wherein said modified hyperactive PiggyBac exhibits decreased DNA binding activity and / or increased excision activity compared to the hyperactive PiggyBac of SEQ ID NO: 9.

[0322] E29. A nucleic acid, typically a messenger RNA (mRNA), encoding the fusion protein of any one of embodiments 1-18.

[0323] E30. A nucleic acid encoding the fusion protein of embodiment 28, comprising a sequence selected from the group consisting of SEQ ID NOs: 110-112 or their corresponding mRNA sequences.

[0324] E31. A nucleic acid encoding a modified hyperactive Piggybac according to any one of embodiments 25-28.

[0325] E32. An expression vector comprising the nucleic acid of any one of embodiments 29-31.

[0326] E33. A host cell comprising a nucleic acid according to any one of embodiments 29-31, or an expression vector according to embodiment 32.

[0327] E34. A method for site-specific integration of an exogenous nucleic acid sequence into the genome of a cell, the method comprising: (i) a fusion protein according to any one of embodiments 1 to 18, or a nucleic acid according to any one of embodiments 29 to 31; (ii) an exogenous nucleic acid to be integrated into the genome of the cell; and (iii) delivering to the cell a composition comprising a guide RNA for directing site-specific integration of the exogenous nucleic acid into the genome of the cell; wherein the fusion protein binds to the specific genomic DNA sequence within the genome of the cell, resulting in cleavage of the genome and site-specific integration of the exogenous nucleic acid sequence into the genome of the cell as determined by the guide RNA.

[0328] E35. The method of embodiment 34, wherein the exogenous nucleic acid is a nucleic acid fragment having a size of at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, typically 5 to 25 kb, preferably 8 to 20 kb.

[0329] E36. The method of embodiment 34 or 35, wherein said exogenous nucleic acid is a therapeutic transgene inserted into the genome of a subject in need thereof to correct a defect in a genetic disorder.

[0330] E37. The method of any one of embodiments 34-36, wherein the composition is delivered in vitro or ex vivo, typically in mammalian cells, preferably human cells, more preferably human cells obtained from a human subject suffering from a genetic disorder.

[0331] E38. The method of any one of embodiments 34-37, wherein the composition is delivered in vivo to a mammal, e.g., a human subject, in need thereof, typically for therapeutic treatment of a genetic disorder.

[0332] E39. A method for inserting an exogenous nucleic acid sequence into the genomic DNA of an organism, comprising administering to the organism one or more compositions or kits according to any one of embodiments 20-24, whereby the fusion protein contained in the one or more compositions or kits binds to a specific genomic DNA sequence, allowing the exogenous nucleic acid contained in the composition to be inserted into the genomic DNA; wherein said exogenous nucleic acid is integrated into a specific site in the genome of a cell of said organism, e.g., a non-human organism or a human subject in need thereof. Example

[0333] To realize a programmable transposase, our design principle was to combine the precise genome targeting of the CRISPR system with a PB variant that exhibits enhanced insertion and excision activity and reduced target DNA binding activity. We started by fusing the nuclease SpCas9 with the PB transposase (Figure 1). We constructed a diverse library of PB and SpCas9 variants (Figure 2a, b, c). Here, we tested a library of PB variants, three SpCas9 variants (nuclease SpCas9 (cas9), dead SpCas9 (dcas9), and nickase SpCas9 (ncas9)), and six linkers (4xGGS, 5xGGS, 7xGGS, 8xGGS, XTEN, and FOKI).

[0334] We diversified various parts of the PB transposase, with particular emphasis on the catalytic core domain, formed by a catalytic triad of three aspartic acid residues (D268, D346, D447) surrounded by 15 arginine and lysine residues that may be involved in the interaction of the transposase with target DNA. 14 Highly active type previously investigated 9 In addition to the above, additional residues that may affect the excision and integration activity of PB 15We diversified the cas9-PB chimeric proteins (SEQ ID NOS: 1-N). To isolate the best-performing mutants, we developed a highly sensitive reporter system for target gene insertion based on emerald GFP (emGFP) reconstitution upon on-target integration. A reporter cell line (abbreviated as Hershey) was constructed by inserting the promoterless C-terminal (Ct) half of emGFP preceded by a splicing acceptor into the genome of Hek293T cells. A complementary "insertion trap reporter" was constructed, encoding the upstream promoter and the first N-terminal (Nt) half of emGFP, followed by a splice donor, all flanked by PB inverted repeats. A specific gRNA-guided cas9 gene directs PB to insert the Nt half adjacent to the Ct half, and splicing of the resulting transcript results in the production of green fluorescence (Figure 3). A library of cas9-PB chimeric proteins was constructed and transfected, along with the "insertion trap reporter" and guide RNA (gRNA), into cells containing the Hershey reporter. Variants exhibiting more highly programmable insertions were tested separately using the same reporter cell line (Figures 2a, 2b, 4a, and 4b). In this assay, a double transposon containing the N-half of emGFP and the entire RFP sequence upstream was used to test for on-target activity (emGFP-positive cells) and total transposition activity (RFP-positive cells). The on-target to off-target ratio was calculated by dividing the percentage of emGFP-positive cells (on-target activity) by the total percentage of RFP-positive cells (total insertion activity).

[0335] We tested various combinations of Cas9 variants fused to PB variants for on-target and off-target transposition in Hershey cell lines. The highest levels of programmable insertion were observed with Nter-Cas9-PB-Cter fusions containing i) Cas9 with intact nuclease activity (Figure 2A) and ii) PB variants with enhanced excision activity and iii) reduced t-DNA binding activity (Figure 2B; Figure 4A, Figure 4B). We observed that all three parameters are critical for achieving accurate and efficient targeted insertion. First, the requirement for Cas9 nuclease activity was demonstrated by the significantly lower efficiency of targeted insertion with nCas9(D10A) and dCas9(D10A and H840A) fused to PB (Figure 2A). To further explore the role of Cas9 double-strand break (DSB) activity in promoting targeted integration, we separated on-site targeting and DSB activity by using zinc finger-PB fusions for directional transposon localization and complemented the on-site DSB with an independent Cas9 nuclease. Znf-PB fusions exhibited no or very low targeted insertion activity, which was restored by combining gRNA-guided Cas9 with the introduction of a DSB near the Znf binding site (Figure 5). These results are consistent with a mechanism in which Cas9-mediated DSB generation near the PB promotes PB insertion activity and bypasses the requirement for a standard TTAA at the insertion site. Indeed, analysis of Cas9-PB insertion sites showed that inverted terminal repeat (ITR) sequences were disrupted by the presence of small indels near the target site (Figure 6). A key consequence of this disruption is the irreversibility of the PB-mediated integration mechanism. Disruption of the ITR or TTAA eliminates or reduces transposon mobility. 16This mechanism likely contributes to the efficiency of programmable insertion by cas9-PB, where the combination of the "search" and "cut" activities of cas9 and the "paste" activity of the modified PB contributes to the high level of precision. Second, the highly excising PB mutant appears to contribute to a higher degree of programmable insertion (D450N, M194V; Figure 2B, Figure 4A, Figure 4B). This enhanced excision may be the result of an increased donor DNA substrate primed for integration. Furthermore, disruption of the preferred excision substrate may prevent sufficient excision even in PB mutants with higher excision activity (Figure 6). Third, PB mutants with reduced t-DNA binding resulted in the lowest off-target levels (Figure 2B, Figure 4A, Figure 4B). This result is consistent with the reduction of intrinsic nonspecific DNA binding by PB, complemented by the sequence-specific DNA binding of cas9 in the cas9-PB fusion, which inactivates the insertion activity of PB alone (Figure 7).

[0336] Using a genome-guided approach, Guide-seq 17 Using a modified version of the original protocol, we were able to characterize the programmable transposase insertion sites and off-target levels (Figure 8A). None of the detected on-target insertions occurred at TTAA sites, and integration into DSB sites generated by Cas9 was further revealed, resulting in the loss of the preferred excision substrate (Figure 8B). Furthermore, we performed Guide-seq analysis on cells modified with programmable transposase technology targeting the TRAC locus. We detected all on-target insertions with a sensitivity of 1-10% (Figure 9).

[0337] We benchmarked programmable transposase technology with current methods for precise gene delivery, such as Cas9-based HDR (Figure 10). Programmable transposases exhibit higher efficiency, with the gap widening for larger payloads. The best variants achieved insertions (up to 8 kb) with twice the efficiency and precision compared to HDR. We also compared the programmable transposase with a HITI variant in which Cas9 was fused to a catalytically dead form of PB. This may aid in mobilizing DNA to the insertion site, as recently suggested by a similar approach using the DNA-binding domain of SB100 transposase. The programmable transposase exhibited twofold higher efficiency compared to alternative assisted HITI methods (Figure 18). Aiming to demonstrate programmable transposase activity in an in vivo mouse model, we constructed an mRNA version of the programmable transposase. Using the in vivo JetPEI reagent, we delivered a programmable transposase targeted to a safe harbor in the Rosa26 genome to mouse liver and observed higher transgene copy numbers compared to the endogenous gene (Figure 11) and sustained transgene expression over time (Figure 20).

[0338] In summary, we have combined the recognition and DNA cut-and-paste activity of CRISPR molecules with modified PBs to generate an efficient tool for precise and efficient gene delivery. This technology scales well with payload sizes. We demonstrated its efficacy in Hek293T and mouse liver. We envision programmable transposase technology as a versatile platform for therapeutic gene delivery for advanced therapies and other applications.

[0339] Results of Cas variants fused to hyPB To further characterize the ability of engineered hyPB to perform programmable transposition, the SpCas9 module of the programmable transposase being tested was substituted with other Cas proteins from different organisms that possess nuclease activity (i.e., SaCas9 of SEQ ID NO:72, cpf1 of SEQ ID NO:74, CasX of SEQ ID NO:75, and CjCas9 of SEQ ID NO:29). Specific gRNAs targeting the upstream region of the split GFP reporter were designed and cloned for Hershey cell line transfection (see Table 2 below). Targeted transposition was measured by GFP expression (Figure 12).

[0340] These results were confirmed in another series of experiments. While good programmable insertion activity was obtained for CjCas9 and LbCpf1, CasX did not achieve any programmable integration in this assay. Notably, SaCas9 had the highest level of programmable insertion among the Cas proteins tested, similar to that of SpCas9 fused to modified hyPB (Figure 19). Indels were determined by Ilumina NGS for the different Cas proteins used and three different gRNAs designed against each protein (Figure 21) and are shown for normalization purposes.

[0341] These positive results confirm that hyPB engineered for programmable transposition is useful for any sequence-specific nuclease module.

[0342] Consequences of adding Cas9 fused to a dimer of PB mutants Given the tendency of PB to act as a dimer during transposition, we attempted to generate fusion proteins between Cas9 and the hyPB R372A-K375A-D450N mutant. The on-target activity of these fusions was compared to that of the Cas9-PB mutant alone. We observed superior performance of the constitutive Cas9-PB-PB. Meanwhile, the constitutive PB-Cas9-PB did not outperform the Cas9-PB monomeric fusion (Figure 14). For dimeric fusions to Cas9, we used the documented version of the hyPB R372A-K375A-D450N mutant for ease of cloning and expression.

[0343] Interestingly, when Cas9 fused to dimeric hyPB R372A-K375A-D450N is SaCas9 rather than SpCas9, activity is further enhanced (Figure 24). The improved performance of SaCas9 over SpCas9 with dimeric hyPB is consistent with the results obtained with monomeric hyPB (Figures 12 and 19).

[0344] Results of ZNF-PB mutant recovery We further investigated the role of double-strand break (DSB) activity induced near (4 nucleotides) the target sites of two gRNAs in promoting targeted integration, promoting single-strand breaks by the SpCas9 nickase variant (D10A) while reducing off-target activity due to non-induced DSBs at off-target sites. We used zinc finger-PB fusions for directed localization of the transposon by fusion with the D450N and R372A-K375A-D450N mutants, complementing it with two on-site single-strand breaks by independent nickase Cas9s or one DSB by Cas9 nuclease.

[0345] Znf-PB fusions exhibited no or very low target insertion activity, which was restored when combined with the introduction of DSBs near the Znf binding site using single or dual gRNA-guided cas9, either a nuclease or a nickase (Figure 13).

[0346] Results of Cas9-hyPB mutant variants To further explore mutant combinations that could achieve programmable transposition with higher efficiency, we performed several selection cycles on cells in which GFP was reconstituted by programmable insertion of the split-GFP reporter system. Interestingly, several combinations were observed that outperformed Cas9-hyPB R372A-K375A-D450N (Figure 15). Of particular note are variants of hyPB fused to Cas9 that are mutated in hyPB at the following AA: A351-A372-A375-A388-N450-A465-A573-V589-G592-L594 (also identified as SEQ ID NO: 2) (several-fold enriched in the positive cell population compared to R372A-K375A-D450N (SEQ ID NO: 1)), and, to a lesser extent, A245-A275-A277-A372-A465-V589 (SEQ ID NO: 3) and A275-A325-A372-A560 (SEQ ID NO: 4).

[0347] In another set of experiments, a PiggyBac DNA library was generated by Twist Bioscience, fused to Cas9, cloned into a lentiviral vector, and transformed into stb4-competent cells, ensuring a 100x variant complexity. The plasmids were purified by maxiprep and co-transfected with lentiviral packaging plasmids into Hek293T cells. Lentivirus was used to infect a 1 / 2GFP reporter cell line. Infected cells were transfected with the 1 / 2GFP transposon and a gRNA targeting the AAVS1 sequence. GFP-positive cells were selected by flow cytometry sorting, and genomic DNA was extracted. PBs were amplified from the extracted gDNA and recloned into a lentiviral vector to initiate a new cycle. The best-performing programmable transposase variants were selected and individually transfected with the AAVS1 gRNA and MC1 / 2GFP.

[0348] First, a random selection of 96 variants was performed, and the best-performing variants were individually screened (Figure 16). An overview of the best PB amino acid variants for high on-target insertion confirms the importance of the mutations D450N, R372A, and K375A; however, it also highlights other important residues that contribute to increased targeting efficiency (Figure 17B). Six PB variants with the best on-target efficiency were selected (Figure 17A). Individual on-target activity was assessed by comparing the FiCAT (Cas9-hyPB) and FiCAT-hyPB (Cas9-hyPB) mutations. Compared to R372A-K375A-D450N, the following variants were significantly improved: N347A-D450N; N347S-D450N-T560A-S573A-F594L; R202K-R275A-N347S-R372A-D450N-T560A-F594L; R275A-N347S-K375A-D450N-S592G; R275A-N347S-R372A-D450N-T560A-F594L; and R275A-R277A-N347S-R372A-D450N-T560A-S564P-F594L (two-tailed t-test).

[0349] This experiment was confirmed by repeating it (Figure 22A). We also produced lentiviruses expressing bulk variants from each cycle and infected reporter cell lines with titers corrected by the PB variant CN, demonstrating a similar increase in on-target efficiency across all cycles (Figure 22B). Single mutants were isolated from bulk variants after 4 and 5 cycles of cas9_PB library enrichment. Mutants were individually tested by transfecting FiCAT mutants, gRNA tcr1, and 1 / 2GFP MC transposons into on-target reporter cell lines. The best FiCAT mutant is shown compared to FiCAT R372A_K375A_D450N (Figure 23). Individual on-target activity was significantly higher for FiCAT (Cas9-hyPB). Compared to R372A-K375A-D450N, the following variants showed significant improvements: R202K-R275A-N347S-R372A-D450N-T560A-F594L; R245A-N347S-R372A-D450N-T560A-S564P-S573A-S592G; R275A-N347S-R372A-D450N-T560A-F594L;N347A-D450N;R277A-G325A-N347A -K375A-D450N-T560A-S564P-S573A-S592G-F594L;N347S-D450N-T560A-S573A -F594L;V34M-R275A-G325A-N347S-S351A-R372A-K375A-D450N-T560A-S564P ;G325A-N347S-K375A-D450N-S573A-M589V-S592G;S230N-R277A-N347S-K375A -D450N;T43I-R372A-K375A-A411T-D450N;G325A-N347S-S351A-K375A-D450N- S573A-M589V-S592G;Y177H-R275A-G325A-K375A-D450N-T560A-S564P-S592G.

[0350] The superiority of the mutants R202K-R275A-N347S-R372A-D450N-T560A-F594L, R275A-R277A-N347S-R372A-D450N-T560A-S564P-F594L, and R275A-N347S-R372A-D450N-T560A-F594L compared to the mutant R372A-K375A-D450N was further demonstrated in triple fusion proteins containing SpCas9 and two hyPBs (Figure 29).

[0351] Results of non-covalent ligation of Cas9 and hyPB In addition to the covalent linkage between Cas9 and hyPB R372A-K375A-D450N via a linker, the MS2-MCP system is used to link Cas9 to a fusion protein consisting of MCP protein and hyPB R372A-K375A-D450N via a modified gRNA containing a tetraloop of the MS2 sequence that binds the MCP protein.

[0352] The combination of the MCP-hyPB R372A-K375A-D450N fusion protein with Cas9 showed increased programmable insertion activity compared to the Cas9-hyPB R372A-K375A-D450N fusion protein (Figure 25A). Furthermore, we fused the MCP protein to other variants of hyPB and combined them with SpCas9 to perform programmable transposition. Both variants used (R202K-R275A-N347S-R372A-D450N-T560A-F594L and R275A-N347S-R372A-D450N-T560A-F594L) outperformed R372A-K375A-D450N (Figure 25B).

[0353] Consequences of uncoupling Cas9 and hyPB for programmable transposition We also tested the performance of SpCas9 using hyPB R372A-K375A-D450N without the linker or MS2-MCP system. SpCas9 and hyPB R372A-K375A-D450N were coexpressed in the same cells, and increased programmable insertion activity was recorded compared to the Cas9-hyPB R372A-K375A-D450N fusion protein (Figure 26A). We expanded the number of hyPB mutant variants tested that were not fused to Cas9 but were coexpressed and interacted with each other to achieve programmable transposition activity (Figure 26B).

[0354] Co-expression of Cas9-hyPB and MCP-hyPB fusion proteins To obtain a dimeric form of the fusion in which one of the monomers is noncovalently linked, we co-transfected the hyPB R372A-K375A-D450N mutant fused to an MCP protein with a hyPB mutant fused to SpCas9. Several hyPB mutants fused to SpCas9 were compared for specific targeted integration (Figure 27).

[0355] Results of co-expression of Cas9-hyPB and hyPB variants In a similar manner, the SpCas9 hyPB R372A-K375A-D450N fusion protein and the hyPB mutant were independently co-transfected to obtain a dimeric form of the fusion in which one of the monomers was unlinked (Figure 28).

[0356] method Cloning and Plasmids. RFP transposon PB512-B for random insertion monitoring was purchased from System Biosciences Inc. The hyPB vector was obtained from the Wellcome Trust Sanger Institute (pCMV_hyPBase). 9The plasmid vector pCRTM-Blunt II-TOPO® was obtained from Invitrogen, and cas9, ncas9, and SP-dcas9-VPR were obtained from Addgene (Addgene plasmids #41815, #41816, #63798). Finally, SB100X and pT4-HB were kindly donated by Dr. Zsuzsana Zizsvak. gRNAs were generated using the Zero Blunt TOPO PCR Cloning Kit (Invitrogen). The gblock gene fragment (Integrated DNA Technologies) contains a U6 promoter, a 20-nt target site, a gRNA scaffold, and a terminator. gRNA TRAC was designed and validated in our laboratory, and the gRNA aavs1-3 sequence has been previously described. 18 .

[0357] Fusion of nuclease, nickase, and dead cas9 to the hyPB and PB RFP1 / 2<em>GFP SMN1 transposons was performed by Golden Gate assembly using BspQI enzyme and standard methods.

[0358] Various mutations were introduced into the hyPB sequence fused to cas9 (cas9_PB plasmid) by site-directed mutagenesis according to the QuickChange Lightning Mutagenesis Kit (Agilent) instructions. Primers were designed using QuickChange Primer Design to achieve the following mutations in the hyPB sequence: M194V, R245A, G325A, R372A, K375A, R376A, E377A, E380A, D450N, and S564P (see SEQ ID NOs: 90–99). All plasmids are available upon request. PB1 / 2emGFP SMN1 was obtained by introducing the first half of the emGFP sequence and the SMN1 intron 6 sequence into a piggyBac acceptor vector. pT4 SMN1 2 / 2emGFP was obtained by adding the second half of the SMN1 intron 6 and a partial emGFP sequence into the SB100X transposon vector. The SMN1 containing emGFP sequence was a gift from Sri Kosuri, DYP004reporter 19 Obtained from.

[0359] Transposons of different sizes and HDR templates were inserted upstream of a split emGFP reporter system into a partial cDNA (NC_000006. 12 ) fragment.

[0360] Cell culture, transfection, and electroporation. Hek293T cell lines (Thermo Fisher Scientific) and C2C12 cell lines (ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with high glucose (Gibco, Thermo Fisher Scientific), 10% fetal bovine serum (FBS), 2 mM glutamine, and 100 U penicillin / 0.1 mg / mL streptomycin at 37°C in a 5% CO2 incubator. Jurkat cell lines were cultured in Roswell Park Memorial Institute 1640 medium (RPMI) supplemented with Glutamax and HEPES (Gibco, Thermo Fisher Scientific) and 10% FBS at 37°C in a 5% CO2 incubator. This cell line was a gift from Manel Juan (Hospital Clinic, Barcelona). Hek293T cell transfection experiments were performed using Lipofectamine 3000 reagent according to the manufacturer's instructions or polyethyleneimine (PEI, Thermo Fisher Scientific) in OptiMem at a DNA-PEI ratio of 1:3. Cells were seeded the day before to reach 70% confluency on the day of transfection (typically 290,000 cells in an adherent p12-well plate). The ratio of plasmid DNA was 1:2.5:2.5 transposase:gRNA:transposon or 1:2.5:2.5 Cas9:gRNA:HDR template, using 0.076 pmol of either programmable transposase or Cas9 in a p12-well plate.

[0361] emGFP splicing-based reconstitution assay. Hek293T cell lines containing pT4 SMN1 2 / 2emGFP were generated by PEI-mediated transfection of SB100X and pT4 SMN1 2 / 2emGFP DNA constructs, followed by single-clone expansion and PCR genotyping (Supplementary Table 3). Positive clones were selected and expanded for subsequent assays. For the emGFP reconstitution assay, 12-well plates were transfected with 0.076 pmol of programmable transposase or hyPB and 0.19 pmol of transposon and gRNA. The programmable transposase, gRNA, and transposon plasmids were transfected at a ratio of 1:2.5:2.5 programmable transposase:gRNA:transposon. On-target insertion was measured by emGFP fluorescence 5 days after transfection. Off-target rearrangement was measured by RFP fluorescence 15 days after transfection. On-target insertion was measured by cotransfecting PB variant, gRNA, and PB1 / 2SMN1 RFP emGFP (insert final construct name) expression plasmids and by measuring emGFP expression (BD LSR Fortessa; BD Biosciences. Blue 488 nm laser with a 530 / 30 filter and yellow-green 561 nm laser with a 610 / 20 filter) by determining emGFP and RFP fluorescence after 14 days (setting n as the mean number of days for episome breakdown).

[0362] Junction PCR for insertion site sequencing. Junction PCR was performed on cells sorted for emGFP using a BD FACSAria (Biosciences). The selected cells contained an on-target insertion of the PB1 / 2emGFP SMN1 transposon targeting the TRAC target site on the reporter cell line. Genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen). Primers were designed to target the 3' ITR of the transposon (forward) and the intron of either the 2 / 2emGFP or endogenous T cell receptor (TRAC) (reverse) of the reporter cell line (Supplementary Table 4).

[0363] Bioinformatics analysis of Guide-SEQ experiments. Illumina reads were analyzed using usech 20 Using clustering and bwa-mem 21 For characterization of on-target insertions, a Python script and Samtools were used. 22 The reads covering the 5' and 3' junctions of the target insertion site were selected using CRISPR-GA. 23 For on-target and off-target experiments, clustered reads mapped to the vector were selected and mapped to the reference genome using bwa-mem. The significance of insertion peaks was assessed using macs2. 24 The algorithm was used to evaluate.

[0364] Preparation of a Guide-seq library adapted for targeted insertion. 15The protocol was implemented by extracting genomic DNA using the DNeasy Blood and Tissue Kit (Qiagen) and fragmenting it into 500-bp fragments using a Q800R3 Sonicator. End repair, A-tailing, and Y-adapter ligation were performed using the KAPA Hyper Prep Kit (KR0961-v5.16) and 3 μg of fragmented genomic DNA, followed by AMPure XP SPRI bead purification at a 1X ratio. After adapter ligation, each sample was split in two and amplified with GSP5' or GSP3' to capture the 5' and 3' junctions, respectively. To capture 5' and 3' transposon-genome junctions, two nested PCRs were performed using KAPA HiFi DNA polymerase according to the manufacturer's protocol: PCR1 containing P5_1 and PB_5_GSP1 or PB_3_GSP1 in a final volume of 25 μl; and PCR2 containing P5_2, PB_5_GSP2, or PB_3_GSP2 in a final volume of 25 μl. The 5' and 3' PCR products were purified using AMPure XP SPRI beads, mixed in equimolar ratios, and sequenced using the Illumina Miseq Reagent Kit V2 for 500 cycles (2x 250 bp paired-end). 3 μl of 100 μM custom primers Index 1 and Read 2 were added to the sequencing reaction.

[0365] Targeted insertion into mouse liver in vivo. Animal experimental procedures were approved by the Animal Experimentation Ethic Committee of Barcelona Biomedical Research Park. Eight to ten week old C57BL / 6J mice were used for this study. Animals were purchased from Jackson Laboratories and were used without distinction between males and females. Programmable transposase mRNA was generated using the RiboMAX Large Scale RNA Production Systems-T7 (Promega) according to the manufacturer's instructions. Rosa26 gRNA 25The transposon was purchased from IDT. Programmable transposase mRNA, gRNA targeting Rosa26, and PB512-B transposon were injected retroorbitally at a ratio of 1:1:2.5. A total of 60 μg of nucleic acid was complexed with in vivo-JetPEI (Polyplus Transfection) at an NP ratio of 7. Ten days after injection, animals were euthanized, and livers were isolated and homogenized. Genomic DNA was extracted from liver samples using the DNeasy Blood and Tissue Kit (Qiagen). The relative copy number of the transposon relative to the Tfrc endogenous gene was obtained by qPCR (primers are listed in Supplementary Table 1). Luciferase expression imaging was performed at various time points after administration of FiCAT-gRNA-transposon or transposon control using an IVIS Spectrum Imaging System (Caliper Life Sciences). Images were taken 5 minutes after intraperitoneal injection of D-luciferin potassium salt (Gold Biotechnology) according to the manufacturer's instructions.

[0366] PB structure modeling. The 3D structure of the piggyBac transposase protein from Trichoplusia ni was obtained using the Robetta Web protein structure prediction server (http: / / robetta.bakerlab.org). The core domain (131–550 aa) was predicted by the Rosetta comparative modeling method, which is based on the Monte Carlo algorithm with embedded Cartesian space minimization and all-atom optimization. 26The tertiary structure folding was analyzed and verified using knowledge-based methods in SPServer and ProSa-Web (Supplementary Figure 2). The secondary structure was analyzed using machine learning-based methods PSIPRED and HHPred. The PB core was then refined using PyMOL with comparative protein modeling. The refinement process was guided by superposition of the piggyBac model with a cryo-EM HIV-1 strand transfer complex intasome (PDB ID: 5U1C), consisting of the HIV integrase tetramer bound to viral DNA and target host DNA, and the X-ray diffraction Tn5 transposase complex structure (PDB ID: 1MUS27). The strand transfer DNA and donor DNA were extrapolated from the superposition of the HIV-1 intasome with Tn5, respectively. Nucleotides at the interface contacting the protein were analyzed using X3DNA as double-stranded DNA. Statistical potentials were used to score the interactions between the protein and DNA, and theoretical PWM was used. 28 The theoretical PWM is obtained by testing all potential double-stranded DNA sequences within this interface, ranking them by statistical potential, and selecting the top ones to create a multiple sequence alignment. During the submission of this paper, a cryo-EM structure became available, which shows significant agreement with the modeling performed. 29 The cryo-EM structure of the piggyBac transposase strand transfer complex (PDB ID: 6X67) confirmed the general fold of the model and identified the hypothesized domains involved in contacts with donor and target DNA.

[0367] [Table 2]

[0368] References 1. Porteus, MH & Carroll, D. Nat. Biotechnol. 23, 967-973 (2005). 2. Sander, J.D. & Joung, J.K. Nat. Biotechnol. 32, 347-355 (2014). 3. Rees, H.A. & Liu, D.R. Nat. Rev. Genet. doi:10.1038 / s41576-018-0059-1 4. Anzalone, A.V. et al. Nature 576, 149-157 (2019). 5. He, X. et al. Nucleic Acids Res. 44, e85 (2016). 6. Suzuki, K. et al. Nature 540, 144-149 (2016). 7. Klompe, S.E., Vo, P.L.H., Halpin-Healy, T.S. & Sternberg, S.H. Nature 1 (2019). 8. Strecker, J. et al. Science (2019).doi:10.1126 / science.aax9181 9. Yusa, K., Zhou, L., Li, M.A., Bradley, A. & Craig, N.L. Proc. Natl. Acad. Sci. U. S. A. 108, 1531-1536 (2011). 10. Hew, B.E., Sato, R., Mauro, D., Stoytchev, I. & Owens, J.B. Synth. Biol. 4, ysz018 (2019). 11. Kovac, A. et al. Elife 9, (2020). 12. Loperfido, M. et al. Nucleic Acids Research 44, 744-760 (2016). 13. Passos, D.O. et al. Science 355, 89-92 (2017). 14. Li, X. et al.doi:10.1073 / pnas.1305987110 15. Morellet, N. et al. Nucleic Acids Res. 46, 2660-2677 (2018). 16. Li, M.A. et al. Mol. Cell. Biol. 33, 1317-1330 (2013). 17. Tsai, S.Q. et al. Nat. Biotechnol. 33, 187-197 (2015). 18. Mali, P. et al. Science 339, 823-826 (2013). 19. Cheung, R. et al. Molecular Cell 73, 183-194.e8 (2019). 20. Edgar, R.C. Bioinformatics 26, 2460-2461 (2010). 21. Li, H. arXiv [q-bio.GN] (2013).at <http: / / arxiv.org / abs / 1303.3997> 22. Li, H. et al. Bioinformatics 25, 2078-2079 (2009). 23. Guell, M., Yang, L. & Church, G.M. Bioinformatics 30, 2968-2970 (2014). 24. Gaspar, J.M.496521 (2018).doi:10.1101 / 496521 25. Chu, V.T. et al. BMC Biotechnol. 16, 4 (2016). 26. Fu, D.Y. (2018) at <https: / / etd.library.vanderbilt.edu / available / etd-08012018-164524 / unrestricted / DarwinYFu_Thesis_Submit.pdf> 27. Steiniger-White, M., Rayment, I. & Reznikoff, WS Curr. Opin. Struct. Biol. 14, 50-57 (2004). 28. Meseguer, A. et al. NAR Genome Bioinform 2, (2020). 29. Chen, Q. et al. Nat. commun. 11, 3446 (2020).

Claims

1. (i) a first protein comprising or consisting of a site-specific DNA-binding protein capable of binding and cleaving a target nucleic acid sequence, or a nucleic acid construct encoding said first protein; and (ii) a second protein comprising or consisting of a transposase; or a nucleic acid construct encoding said second protein, wherein the transposase is a modified hyperactive PiggyBac comprising one or more amino acid mutations compared to the hyperactive PiggyBac of SEQ ID NO:

9.

2. The composition of claim 1 , wherein the first protein and the second protein are fused to each other to form a fusion protein.

3. The composition of claim 2 , wherein the first protein is fused to the C-terminal end of the second protein.

4. The composition described in claim 2, wherein the first protein is fused to the C-terminal portion of the second protein via a linker.

5. 2. The composition of claim 1, wherein the transposase is a modified hyperactive PiggyBac comprising one or more amino acid mutations to increase excision activity compared to unmodified hyperactive PiggyBac and / or one or more amino acid mutations to decrease DNA binding activity compared to unmodified hyperactive PiggyBac.

6. The composition of claim 1 , wherein the one or more amino acid mutations do not consist of R372A, K375A, and D450N.

7. 2. The composition of claim 1, wherein the one or more amino acid mutations are selected from among amino acid substitutions that increase excision activity at positions M194, D450, T560, S564, S573, S592, or F594, where the position numbers correspond to the amino acid numbers of unmodified hyperactive PiggyBac in SEQ ID NO:

9.

8. The composition described in claim 1, wherein the one or more amino acid mutations are selected from among amino acid substitutions M194V and / or D450N, and the position numbers correspond to the amino acid numbers of unmodified highly active PiggyBac of SEQ ID NO:

9.

9. The composition of claim 1, wherein the one or more amino acid mutations are selected from among amino acid substitutions that increase excision activity at positions M194 or D450, the position numbers corresponding to the amino acid numbers of unmodified hyperactive PiggyBac in SEQ ID NO:

9.

10. 2. The composition of claim 1, wherein the one or more amino acid mutations are selected from among amino acid substitutions that reduce DNA binding activity at positions R275, R277, R347, R372, K375, R376, E377, and / or E380, where the position numbers correspond to the amino acid numbers of unmodified, highly active PiggyBac in SEQ ID NO:

9.

11. The composition of claim 1, wherein the one or more amino acid mutations are selected from among amino acid substitutions R275A, R277, R347S, R372A, K375A, R376A, E377A, and / or E380A, and the position numbers correspond to the amino acid numbers of unmodified highly active PiggyBac of SEQ ID NO:

9.

12. The composition of claim 1, wherein the one or more amino acid mutations are selected from among amino acid substitutions that reduce DNA binding activity at positions R372, K375, R376, E377, and / or E380, the position numbers corresponding to the amino acid numbers of unmodified, highly active PiggyBac in SEQ ID NO:

9.

13. The composition of claim 1, wherein the one or more amino acid mutations are selected from among amino acid substitutions R372A, K375A, R376A, E377A, and / or E380A, and the position numbers correspond to the amino acid numbers of unmodified highly active PiggyBac of SEQ ID NO:

9.

14. 2. The composition of claim 1, wherein the modified hyperactive PiggyBac comprises the double mutation N347S and D450N, the position numbers corresponding to the amino acid numbers of the unmodified hyperactive PiggyBac of SEQ ID NO:

9.

15. The modified hyperactive PiggyBac mutations are selected from the group consisting of the following amino acid substitutions or combinations of amino acid substitutions: R372A / K375A / R376A / D450N, K375A / R376A / E377A / E380A / D450N, R372A / K375A / R376A / E377A / E380A / D450N, M194V, R376A, E377A, E380A, M194V / R372A / K375A, S351A / R372A / K375A / R388A / D450N / W465A / S573A / M589V / S592G / F594L, R245A / R 275A / R277A / R372A / W465A / M589V, R275A / 325A / R372A / T560A, N347A / D45 0N, N347S / D450N / T560A / S573A / F594L, R202K / R275A / N347S / R372A / D450N / T560A / F594L, R275A / N347S / K375A / D450N / S592G, R275A / N347S / R372A / D 450N / T560A / F594L, R275A / R277A / N347S / R372A / D450N / T560A / S564P / F59 4L, R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G, R277A / G325 A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L, V34M / R275A / G 325A / N347S / S351A / R372A / K375A / D450N / T560A / S564P, G325A / N347S / K37 5A / D450N / S573A / M589V / S592G, S230N / R277A / N347S / K375A / D450N, T43I / 2. The composition of claim 1, comprising one of the following amino acid sequences: R372A / K375A / A411T / D450N, G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G, wherein the position numbers correspond to the amino acid numbers of hyperactive PiggyBac in SEQ ID NO:9, and typically the modified transposase has an amino acid sequence selected from any of SEQ ID NOs:2-8, 10-18, and 135-149.

16. 2. The composition of claim 1, further comprising a third protein comprising or consisting of a second transposase, or a nucleic acid construct encoding said third protein, wherein said second transposase is either a hyperactive PiggyBac having SEQ ID NO:9, or a modified hyperactive PiggyBac comprising one or more amino acid mutations compared to said hyperactive PiggyBac having SEQ ID NO:

9.

17. 17. The composition of claim 16, wherein the first, second and third proteins are fused to each other to form a triple fusion protein.

18. 2. The composition of claim 1, wherein the first protein comprises or consists of an RNA-guided nuclease or nickase, or a zinc finger nuclease.

19. 2. The composition of claim 1, wherein the first protein is a nuclease protein comprising an active DNA cleavage domain and a guide RNA binding domain and having at least 80%, 90%, 95%, 99% or at least 100% identity to Streptococcus pyogenes Cas9 (SpCas9) of SEQ ID NO: 31, Staphylococcus aureus Cas9 (SaCas9) of SEQ ID NO: 72, Cpf1 of SEQ ID NO: 74, Campylobacter jejuni Cas9 (CjCas9) of SEQ ID NO: 29, Streptococcus pyogenes Cas9 nickase (nCas9) of SEQ ID NO: 70, CasX of SEQ ID NO: 75, or Staphylococcus aureus Cas9 nickase of SEQ ID NO:

76.

20. The composition of claim 1, wherein the first protein is a Cas9 protein selected from the group consisting of Staphylococcus aureus Cas9 (SaCas9) of SEQ ID NO: 72 and Streptococcus pyogenes Cas9 (SpCas9) of SEQ ID NO:

31.

21. 10. The composition of claim 1, further comprising a guide RNA and an exogenous nucleic acid for insertion into a genome.

22. 22. The composition of claim 21, wherein the transposase is fused to an RNA binding protein capable of binding to at least one specific RNA sequence contained in the guide RNA.

23. The composition of claim 21, wherein the transposase is fused to an MS2 bacteriophage coat protein (MCP) and the guide RNA comprises an MS2 RNA tetraloop binding sequence that shares at least 75% identity with SEQ ID NO:

153.

24. 22. The composition of claim 21, wherein the exogenous nucleic acid is a large DNA fragment, typically having a size of 5 kb to 25 kb.

25. The composition of claim 1 , wherein the composition is contained in a nanoparticle.

26. A nucleic acid encoding a fusion protein according to any one of claims 2 to 25, typically a messenger RNA (mRNA).

27. An in vitro method for site-specific integration of an exogenous nucleic acid sequence into the genome of a cell, comprising a composition according to any one of claims 1 to 25, a guide RNA and delivering said exogenous nucleic acid to said cell.

28. The composition, guide RNA and exogenous nucleic acid according to any one of claims 1 to 25 for use in the treatment of a disease by site-specific integration of said exogenous nucleic acid sequence into the genome of a cell.

29. Use of a composition, guide RNA, and exogenous nucleic acid described in any one of claims 1 to 25 for preparing a medicament for the treatment of a disease by site-specific integration of the exogenous nucleic acid sequence into the genome of a cell.

30. A composition according to any one of claims 1 to 25, or a nucleic acid according to claim 26, or (i) a first protein comprising or consisting of a site-specific DNA-binding protein capable of binding and cleaving a target nucleic acid sequence, or a nucleic acid construct encoding said first protein; and (ii) a second protein comprising or consisting of a transposase; or a nucleic acid construct encoding said second protein. A kit comprising: The kit, wherein the transposase is a modified hyperactive PiggyBac comprising one or more amino acid mutations compared to the hyperactive PiggyBac of SEQ ID NO:9.

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