Myotis lucifugus transposase engineering

Abstract

Compositions and methods related to recombinant mobile element systems and uses thereof are provided.

Description

[0001] MYOTIS LUCIFUGUS TRANSPOSASE ENGINEERING

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 644,337, filed on May 8, 2024 which is incorporated by reference herein in its entirety.

[0004] SEQUENCE LISTING

[0005] The instant application contains a sequence listing, which has been submitted electronically in XML format. The contents of the XML copy named "SAL-047PC_126933-5047_Sequence Listing,” which was created on May 2, 2025 and is 925,696 bytes in size, are incorporated herein by reference in their entirety.

[0006] FIELD

[0007] The present disclosure relates to recombinant mobile element systems and uses thereof.

[0008] BACKGROUND

[0009] Mobile elements transposon systems are tools for genome engineering but unselected integration into the genome carries genotoxic risks in therapeutic applications. A nucleic acid movement to a new location in the human genome is performed by the action of a helper second enzyme that binds to an “end sequence" and inserts a donor DNA sequence at a specific DNA sequence by a “cut and paste” mechanism. The donor DNA is flanked by end sequences in living organisms such as insects (e.g. , Trichnoplusia ni). Genomic DNA is excised by double strand cleavage at the hosts’ donor site and the donor DNA is integrated or inserted into a specific DNA sequence. Mobilization of the DNA sequences permits the intervening nucleic acid, or a transgene, to be inserted at the specific nucleotide sequence (i.e., TTAA) without a DNA footprint. Site-specific mutagenesis can also be improved by binding various DNA binding domains to transposon systems.

[0010] There is a need for compositions and methods for targeted site-directed integration.

[0011] SUMMARY

[0012] In aspects, there is provided a composition, and method of use of a composition, comprising a helper enzyme or a nucleic acid encoding the helper enzyme, an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1 and a non-polar aliphatic amino acid at position 2 of SEQ ID NO: 1, or a position corresponding thereto, one or more amino acid substitutions selected from S8X, C13X, Y281X, N335X, and D416X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, and one or more amino acid substitutions selected from T331X, I332X, R333X, K334X, R336X, G337X, and I338X of SEQ ID NO: 1 or a substitution at a position corresponding thereto. In aspects, there is provided a composition, and method of use of a composition, comprising a helper enzyme or a nucleic acid encoding the helper enzyme, an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1 and a non-polar aliphatic amino acid at position 2 of SEQ ID NO: 1, or a position corresponding thereto, and S8X, C13X, N335X, I338X, and D416X of SEQ ID NO: 1 or a substitution at a position corresponding thereto.

[0013] In aspects, there is provided a composition, and method of use of a composition, comprising a helper enzyme or a nucleic acid encoding the helper enzyme, an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1 and a non-polar aliphatic amino acid at position 2 of SEQ ID NO: 1, or a position corresponding thereto, S8X, C13X, R333X, N335X, and D416X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, and / or one or more amino acid substitutions selected from K16X, N19X, N28X, L48X, S56X, N128X, K137X, D208X, N226X, N241X, K286X, N317X, T331X, I332X, K334X, R336X, G337X, I338X, K349X, K369X, E381X, I388X, D480X, M481X, L486X, K525X, K541 R, F552X, Y572X of SEQ ID NO: 1 or a substitution at a position corresponding thereto.

[0014] In embodiments, the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), and proline (P).

[0015] In embodiments, X is selected from a non-polar amino acid, optionally selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), methionine (M), tryptophan (W), and phenylalanine (F), or a polar amino acid, optionally selected from serine (S), cysteine (C), asparagine (N), glutamine (Q), threonine (T), and tyrosine (Y), or a basic amino acid, optionally selected from arginine (R), histidine (H), or an acidic amino acid, optionally selected from aspartic acid (D), and glutamic acid (E), or a positively charged hydrophilic amino acid, optionally selected from lysine (K), arginine (R), and histidine (H).

[0016] In embodiments, the helper enzyme comprises a substitution at S8X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, optionally wherein X is a non-polar residue, optionally selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), methionine (M), tryptophan (W), and phenylalanine (F), or a polar and neutral hydrophilic residue, optionally proline (P).

[0017] In embodiments, the helper enzyme comprises a substitution at C13X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, optionally wherein X is a positively charged hydrophilic amino acid, optionally selected from arginine (R), lysine (K), and histidine (H).

[0018] In embodiments, the helper enzyme comprises a substitution at Y281X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, wherein X is a non-polar amino acid, optionally selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), methionine (M), tryptophan (W), and phenylalanine (F). In embodiments, the helper enzyme comprises a substitution at R333X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, optionally wherein X is a non-polar residue, optionally selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I).

[0019] In embodiments, the helper enzyme comprises a substitution at N335X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, wherein X is a non-polar amino acid, optionally selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), methionine (M), tryptophan (W), and phenylalanine (F).

[0020] In embodiments, the helper enzyme comprises a substitution at I338X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, wherein X is a non-polar amino acid, optionally selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), methionine (M), tryptophan (W), and phenylalanine (F), or a polar amino acid, optionally selected from serine (S), cysteine (C), asparagine (N), glutamine (Q), threonine (T), and tyrosine (Y).

[0021] In embodiments, the helper enzyme comprises a substitution at D416X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, wherein X is a polar and positively charged hydrophilic residue, optionally selected from arginine (R) and lysine (K), or a polar and neutral of charge hydrophilic residue, optionally selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C).

[0022] In embodiments, the helper enzyme comprises one or two, or three, or four, or five substitutions selected from: S8P, C13R, Y281A, N335A, and D416N or substitutions corresponding thereto relative to SEQ ID NO: 1.

[0023] In embodiments, the helper enzyme comprises one, or two, or three, or four, substitutions selected from T331 A, I332A, I332S, I332M, R333A, R333S, R333K, K334A, R336A, R336S, R336K, G337A, I338A, I338M, and I338S or substitutions corresponding thereto relative to SEQ ID NO: 1.

[0024] In embodiments, the helper enzyme comprises one or two, or three, or four, or five substitutions selected from: S8P, C13R, Y281A, N335A, and D416N or substitutions corresponding thereto relative to SEQ ID NO: 1, and one, or two, or three, or four substitutions selected from T331A, I332A, I332S, I332M, R333A, R333S, R333K, K334A, R336A, R336S, R336K, G337A, I338A, I338M, and I338S or substitutions corresponding thereto relative to SEQ ID NO: 1.

[0025] In embodiments, the helper enzyme comprises one, or two, or three, or four, or five, or six, or seven, or eight substitutions selected from: S8P, C13R, Y281A, T331A, I332A, I332S, I332M, R333A, R333S, R333K, K334A, N335A, R336A, R336S, R336K, G337A, I338A, I338M, I338S and D416N or substitutions corresponding thereto relative to SEQ ID NO: 1.

[0026] In embodiments, the helper enzyme comprises one or two, or three, or four, or five substitutions selected from: S8P, C13R, N335A, I338S, and D416N or substitutions corresponding thereto relative to SEQ ID NO: 1. In embodiments, the helper enzyme comprises one or two, or three, or four, or five, or six substitutions selected from: S8P, C13R, C13H, R333A, R333S, R333K, N335A, and D416N or substitutions corresponding thereto relative to SEQ ID NO: 1.

[0027] In embodiments, the composition comprises one or two, or three, or four, or five substitutions selected from: K16E, N19D, N28T, L48W, S56A, N128D, K137R, D208G, N226S, N241 I, K286R, N317T, T331A, I332A, I332M, I332S, K334A, K334R, K334S, R336A, R336S, R336K, G337A, I338A, I338M, I338S, K349R, K369R, E381G, I388T, D480G, M481 R, L486V, K525I, K541 R, F552I, Y572C, or substitutions corresponding thereto relative to SEQ ID NO: 1.

[0028] In embodiments, the composition comprises one or two, or three, or four, or five substitutions selected from: S8P, C13R, C13H, R333A, R333S, R333K, N335A, and D416N or substitutions corresponding thereto relative to SEQ ID NO: 1, and one, or two, or three, or four substitutions selected from K16E, N19D, N28T, L48W, S56A, N128D, K137R, D208G, N226S, N241 I, K286R, N317T, T331A, I332A, I332M, I332S, K334A, K334R, K334S, R336A, R336S, R336K, G337A, I338A, I338M, I338S, K349R, K369R, E381 G, I388T, D480G, M481 R, L486V, K525I, K541 R, F552I, Y572C, or substitutions corresponding thereto relative to SEQ ID NO: 1.

[0029] In embodiments, the helper enzyme comprises one, or two, or three, or four, or five, or six, or seven, or eight substitutions selected from: S8P, C13R, C13H, K16E, N19D, N28T, L48W, S56A, N128D, K137R, D208G, N226S, N241 I, K286R, N317T, T331A, I332A, I332M, I332S, R333A, R333S, R333K, K334A, K334R, K334S, N335A, R336A, R336S, R336K, G337A, I338A, I338M, I338S, K349R, K369R, E381 G, I388T, D416N, D480G, M481 R, L486V, K525I, K541R, F552I, and Y572C or substitutions corresponding thereto relative to SEQ ID NO: 1.

[0030] In embodiments, the helper enzyme comprises or consists of 6 amino acid substitutions.

[0031] In embodiments, the helper enzyme comprises or consists of 7 amino acid substitutions.

[0032] In embodiments, the helper enzyme comprises or consists of 8 amino acid substitutions.

[0033] In embodiments, the helper enzyme comprises a substitution selected from TABLE 1 or Table 2 or a substitution at positions corresponding thereto relative to SEQ ID NO: 1.

[0034] In embodiments, the helper enzyme comprises a deletion of about 30, or about 40, or about 50, or about 60, or about 70, or about 80, or about 90, or about 100 amino acids from an N-terminus or a C-terminus.

[0035] In embodiments, the helper enzyme comprises a deletion at positions about 1-35, or about 1-45, or about 1-55, or about 1-65, or about 1-75, or about 1-85, or about 1-95, or about 1-105, or about 2-35, or about 2-45, or about 2-68 or positions corresponding thereto, wherein the positions are relative to SEQ ID NO: 1.

[0036] In embodiments, the N terminal deletion yields reduced or ablated off-target effects of the enzyme compared to the enzyme without the N terminal deletion. In embodiments, the helper enzyme comprising the N terminal deletion has a sequence selected from SEQ ID NO: 504, 506, or 508, or a sequence having at least about 95%, or at least about 96%, at least about 97%, at least about 98%, at least about 99% identity thereto.

[0037] In embodiments, the helper enzyme comprising an N terminal deletion is fused to a DNA binding domain, optionally wherein the DNA binding domain comprises TALEs, and / or ZnF. In embodiments, the helper enzyme further comprises a targeting element. In embodiments, the helper enzyme is suitable of inserting a donor nucleic acid comprising a transgene in a genomic safe harbor site (GSHS). In embodiments, the GSHS is in an open chromatin location in a chromosome. In embodiments, the GSHS is selected from adeno-associated virus site 1 (AAVS1), chemokine (C-C motif) receptor 5 (CCR5) gene, HIV-1 coreceptor, and human Rosa26 locus. In embodiments, the GSHS comprises one or more TTAA integration sites.

[0038] In embodiments, the targeting element directs the helper enzyme to either one or more nucleic acid binding sites that are upstream and / or downstream of the TTAA integration sites or to the TTAA integration sites. In embodiments, the targeting element directs the helper enzyme to one or more nucleic acid binding sites that are upstream and / or downstream of the TTAA integration sites and within about 5 to about 30 base pairs of the TTAA integration sites or within about 15 to about 19 base pairs of the TTAA integration sites. In embodiments, the targeting element directs the helper enzyme to two nucleic acid binding sites of the TTAA integration sites, wherein a first site is upstream of TTAA and within about 5 to about 30 base pairs or about 15 to about 19 base pairs of the TTAA and a second site is downstream of TTAA and within about 5 to about 30 base pairs or about 15 to about 19 base pairs of the TTAA.

[0039] In embodiments, the targeting element is or comprises one or more of a Cas enzyme, which is optionally catalytically inactive and which is optionally associated with a guide RNA (gRNA), a transcription activator-like effector (TALE) DNA binding domain (DBD), a Zinc finger (ZnF), a catalytically inactive transcription factor, catalytically inactive nickase, a transcriptional activator, a transcriptional repressor, a recombinase, a DNA methyltransferase, a histone methyltransferase, a paternally expressed gene 10 (PEG10), and a transposon-encoded polypeptide D (TnsD) or a variant thereof.

[0040] In embodiments, the targeting element is or comprises a TALE DBD. In embodiments, the TALE DBD comprises one or more repeat sequences. In embodiments, the TALE DBD comprises about 14, or about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences. In embodiments, the repeat sequences each independently comprises about 33 or 34 amino acids In embodiments, the repeat sequences each independently comprises a repeat variable di-residue (RVD) at residue 12 or 13 of the 33 or 34 amino acids, respectively. In embodiments, the RVD recognizes one base pair in a target nucleic acid sequence.

[0041] In embodiments, the RVD recognizes a C residue in the target nucleic acid sequence and is selected from HD, N(gap), HA, ND, and HI. In embodiments, the RVD recognizes a G residue in the target nucleic acid sequence and is selected from NN, NH, NK, HN, and NA. In embodiments, the RVD recognizes an A residue in the target nucleic acid sequence and is selected from Nl and NS. In embodiments, the RVD recognizes a T residue in the target nucleic acid sequence and is selected from NG, HG, H(gap), and IG.

[0042] In embodiments, the targeting element is or comprises a Cas9 enzyme associated with a gRNA or a CasX enzyme associated with a gRNA. In embodiments, the Cas9 enzyme associated with a gRNA comprises a catalytically inactive dCas9 associated with a gRNA or an inactive dCasX associated with a gRNA. In embodiments, catalytically inactive dCas9 comprises at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity to an amino acid sequence of SEQ ID NO: 6 or a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 5 or a codon-optimized form thereof.

[0043] In embodiments, the target element is or comprises a CasX enzyme associated with a gRNA, optionally wherein the catalytic inactive dCasX. In embodiments, the targeting element is or comprises a Cas12 enzyme associated with a gRNA. In embodiments, the targeting element comprises a catalytically inactive Cas12 associated with a gRNA, optionally wherein the catalytically inactive Cas12 is dCas12j or dCas12a. In embodiments, the targeting element is or comprises a TnsC, TnsB, TnsA, TniQ, Cas6, Cas7, Cas8 enzyme associated with a gRNA. In embodiments, the targeting element comprises a TniQ subdomain of TnsD.

[0044] In embodiments, the composition comprises a linker connecting the helper enzyme and the targeting element.

[0045] In embodiments, the linker comprises less than about 25 amino acids or 75 nucleotides. In embodiments, the linker comprises about 10 amino acids to about 20 amino acids or about 12 amino acids to about 15 amino acids, or about 30 nucleotides to about 60 nucleotides or about 36 nucleotides to about 45 nucleotides. In embodiments, the linker is substantially comprised of glycine (G) and serine (S) residues. In embodiments, the linker is or comprises (GSS)4 or the linker is GS flanked on either side of a DNA binding domain, optionally TALE and ZnF. In embodiments, the linker comprises an amino acid sequence of AKLAGGAPAVGGGPKAADKFAATGGS (SEQ ID NO: 8) or a variant thereof having a substitution or deletion. In embodiments, the linker connects the targeting element to the N-terminus of the helper enzyme or connects the targeting element within the helper enzyme.

[0046] In embodiments, the composition is a nucleic acid, optionally an RNA. In embodiments, the RNA is or comprises messenger RNA (mRNA). In embodiments, the mRNA is or comprises modified mRNA (mmRNA). In embodiments, the mmRNA comprises one or more of a 5’-m7G cap (capO, cap1, or cap2), a pseudouridine or n-methyl-pseudouridine substitution, and a poly-A tail of about 30, or of about 50, or of about 100, or of about 150 nucleotides in length.

[0047] In embodiments, the composition further comprises a donor nucleic acid or is suitable for insertion of a donor nucleic acid, optionally wherein the donor nucleic acid is a transposon. In embodiments, the helper enzyme is suitable of inserting a donor nucleic acid comprising a transgene in a genomic safe harbor site (GSHS) and / or wherein the targeting element is suitable for directing the helper enzyme to a GSHS. In embodiments, a donor DNA and a helper RNA are suitable for transfection at a donor DNA to helper RNA ratio of about 1 to about 4, or about 1 to about 2, or about 1 to about 1.

[0048] In embodiments, the composition further comprises a nucleic acid encoding a donor comprising a transgene to be integrated, optionally wherein the transgene is defective or substantially absent in a disease state. In embodiments,

[0049] In embodiments, the transgene comprises a cargo nucleic acid sequence and a first and a second donor end sequences.

[0050] In embodiments, the cargo nucleic acid sequence is flanked by the first and the second donor end sequences. In embodiments, the enzyme or variant thereof is incorporated into a vector or a vector-like particle. In embodiments, the vector or a vector-like particle comprises one or more expression cassettes. In embodiments, the vector or a vectorlike particle comprises one expression cassette. In embodiments, the expression cassette further comprises the enzyme or variant thereof, the transgene, the donor end sequences, or a combination thereof. In embodiments, the enzyme or variant thereof, the transgene, the donor end sequences, or a combination thereof are incorporated into one or more vectors or vector-like particles. In embodiments, the enzyme or variant thereof, the transgene, the donor end sequences, or combination thereof are incorporated into a same vector or vector-like particle. In embodiments, the enzyme or variant thereof, the transgene, the donor end sequences, or combination thereof is incorporated into different vectors or vector-like particles. In embodiments, the vector or vector-like particle is nonviral.

[0051] In embodiments, the donor is under the control of at least one tissue-specific promoter. In embodiments, at least one tissue-specific promoter is a single promoter. In embodiments, at least one tissue-specific promoter is under the control of a dual promoter or a tandem promoter. In embodiments, the transgene to be integrated comprises at least one gene of interest. In embodiments, the transgene to be integrated comprises one gene of interest. In embodiments, the transgene to be integrated comprises two or more genes of interest. In embodiments, there is provide a host cell comprising the composition of the present disclosure. In embodiments, the composition is encapsulated in a lipid nanoparticle (LNP). In embodiments, the polynucleotide encoding the enzyme orvariant thereof and the polynucleotide encoding the donor are in the form of the same LNP, optionally in a co-formulation.

[0052] In embodiments, the LNP comprises one or more lipids selected from 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), a cationic cholesterol derivative mixed with dimethylaminoethane-carbamoyl (DC-Chol), phosphatidylcholine (PC), triolein (glyceryl trioleate), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG), 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol - 2000 (DMG-PEG 2K), and 1 ,2 distearol -sn-glycerol-3phosphocholine (DSPC) and / or comprising of one or more molecules selected from polyethylenimine (PEI) and poly(lactic-co-glycolic acid) (PLGA), and N-Acetylgalactosamine (GalNAc).

[0053] In embodiments, there is provided a method for inserting a gene into the genome of a cell, comprising contacting a cell with the composition of the present disclosure. In embodiments, there is provided a method for treating a disease or disorder ex vivo, comprising contacting a cell with the composition of the present disclosure and administering the cell to a subject in need thereof. In embodiments, there is provided a method for treating a disease or disorder in vivo, comprising administering the composition of the present disclosure to a subject in need thereof.

[0054] In embodiments, there is provided a composition comprising a helper enzyme or a nucleic acid encoding the helper enzyme comprising: an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1 and a nonpolar aliphatic amino acid at position 2 of SEQ ID NO: 1 , or a position corresponding thereto, and a plurality of amino acid substitutions of any row of TABLE A.

[0055] In embodiments, there is provided a composition comprising a helper enzyme or a nucleic acid encoding the helper enzyme comprising: an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1 and a nonpolar aliphatic amino acid at position 2 of SEQ ID NO: 1 , or a position corresponding thereto, and a plurality of amino acid substitutions of any row of TABLE B.

[0056] BRIEF DESCRIPTION OF DRAWINGS

[0057] FIG. 1 shows a schematic of mutations generated.

[0058] FIG. 2 shows a schematic of utilizing an engineered HEK293 cell line containing a landing pad that has been genomically integrated in multiple copies with Sleeping beauty transposase.

[0059] FIG. 3 shows a graph demonstrating on and off-target integration of mutants.

[0060] DETAILED DESCRIPTION

[0061] In aspects, there is provided a composition, and method of use of a composition, comprising a helper enzyme or a nucleic acid encoding the helper enzyme, an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1 and a non-polar aliphatic amino acid at position 2 of SEQ ID NO: 1, or a position corresponding thereto, one or more amino acid substitutions selected from S8X, C13X, Y281X, N335X, and D416X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, and one or more amino acid substitutions selected from T331X, I332X, R333X, K334X, R336X, G337X, and I338X of SEQ ID NO: 1 or a substitution at a position corresponding thereto.

[0062] In aspects, there is provided a composition, and method of use of a composition, comprising a helper enzyme or a nucleic acid encoding the helper enzyme, an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1 and a non-polar aliphatic amino acid at position 2 of SEQ ID NO: 1, or a position corresponding thereto, and S8X, C13X, N335X, I338X, and D416X of SEQ ID NO: 1 or a substitution at a position corresponding thereto.

[0063] In aspects, there is provided a composition, and method of use of a composition, comprising a helper enzyme or a nucleic acid encoding the helper enzyme, an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1 and a non-polar aliphatic amino acid at position 2 of SEQ ID NO: 1, or a position corresponding thereto, S8X, C13X, R333X, N335X, and D416X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, and / or one or more amino acid substitutions selected from K16X, N19X, N28X, L48X, S56X, N128X, K137X, D208X, N226X, N241X, K286X, N317X, T331X, I332X, K334X, R336X, G337X, I338X, K349X, K369X, E381X, I388X, D480X, M481X, L486X, K525X, K541 R, F552X, Y572X of SEQ ID NO: 1 or a substitution at a position corresponding thereto.

[0064] In embodiments, the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), and proline (P).

[0065] In embodiments, X is selected from a non-polar amino acid, optionally selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), methionine (M), tryptophan (W), and phenylalanine (F), or a polar amino acid, optionally selected from serine (S), cysteine (C), asparagine (N), glutamine (Q), threonine (T), and tyrosine (Y), or a basic amino acid, optionally selected from arginine (R), histidine (H), or an acidic amino acid, optionally selected from aspartic acid (D), and glutamic acid (E), or a positively charged hydrophilic amino acid, optionally selected from lysine (K), arginine (R), and histidine (H).

[0066] In embodiments, the helper enzyme comprises a substitution selected from TABLE 1 or TABLE 2 or a substitution at positions corresponding thereto relative to SEQ ID NO: 1.

[0067] In embodiments, the helper enzyme is an engineered form of an enzyme reconstructed from Myotis lucifugus. In embodiments, the helper enzyme includes but is not limited to an engineered version that is a monomer, dimer, tetramer (or another multimer), hyperactive (Exc+), and / or has a reduced interaction with non-TTAA recognitions sites (I nt-), of a helper enzyme reconstructed from Myotis lucifugus or a predecessor thereof.

[0068] In embodiments, the helper enzyme has deletions which confer hyperactivity and Exc+ / lnt-. In some embodiments, the helper enzyme has an amino acid sequence having deletions at N-terminus positions, 2-35, 2-45 or 2-68 relative to the amino acid sequence of SEQ ID NO: 1 and optionally fused to the amino acid sequence of the DNA binders, or a functional equivalent thereof.

[0069] In embodiments, the helper enzyme further comprises a targeting element.

[0070] In embodiments, the helper enzyme is linked to a targeting element comprising one or more of a Cas enzyme, which is optionally catalytically inactive and which is optionally associated with a guide RNA (gRNA), a transcription activatorlike effector (TALE) DNA binding domain (DBD), a Zinc finger (ZF) or a variant thereof; wherein the linker comprises less than about 25 amino acids or 75 nucleotides. In embodiments, the linker is or comprises AKLAGGAPAVGGGPKAADKFAATGGS (SEQ ID NO: 8), or a variant thereof having a substitution or deletion.

[0071] In embodiments, the linker comprises about 10 amino acids to about 20 amino acids or about 12 amino acids to about 15 amino acids, or about 30 nucleotides to about 60 nucleotides or about 36 nucleotides to about 45 nucleotides. In embodiments, there is substantially comprised of glycine (G) and serine (S) residues. In embodiments, the linker is or comprises (GSS)4 or the linker is (GS)i on either side of the DNA binder (TALE, ZnF). In embodiments, the linker connects the targeting element to the N-terminus of the helper enzyme or connects the targeting element within the helper enzyme.

[0072] In embodiments, the linker is a non-covalent linker. In embodiments, the non-covalent linker comprises an epitope tag, optionally wherein the epitope tag comprises an ALFA tag. In embodiments, the ALFA tag further comprises recombinant variable domains of heavy-chain-only antibodies (VHH) (NANOBODY). In embodiments, the targeting elements are oriented on opposite sides of the transposase.

[0073] In embodiments, the helper enzyme is suitable of inserting a donor nucleic acid comprising a transgene in a genomic safe harbor site (GSHS) or intron 1 of a disease gene (e.g., HPRT, CFTR) and / or wherein the targeting element is suitable for directing the helper enzyme to a GSHS or intron 1 of a disease gene (e.g., HPRT, CFTR). In embodiments, the GSHS is in an open chromatin location in a chromosome. In embodiments, the GSHS is selected from the human Rosa26 locus, adeno-associated virus site 1 (AAVS1), chromosome 4 GSHS, chromosome 22 GSHS. In embodiments, the GSHS comprises one or more TTAA integration sites. In embodiments, the targeting element directs the helper enzyme to one or more nucleic acid binding sites that are upstream and / or downstream of the TTAA integration sites. In embodiments, the targeting element directs the helper enzyme to either one or more nucleic acid binding sites that are upstream and / or downstream of the TTAA integration sites or to the TTAA integration sites and within about 9 to about 30 base pairs of the TTAA integration sites or within about 15 to about 25 base pairs of the TTAA integration sites. In embodiments, the targeting element directs the helper enzyme to two nucleic acid binding sites of the TTAA integration sites, wherein a first site is upstream of TTAA and within about 9 to about 30 base pairs or about 15 to about 25 base pairs of the TTAA and a second site is downstream of TTAA and within about 9 to about 30 base pairs or about 15 to about 25 base pairs of the TTAA. In embodiments, the TTAA integration sites comprise ttTTAAaa (SEQ ID NO: 880) or taTTAAta (SEQ ID NO: 881) sites.

[0074] The present disclosure is based, in part, on the discovery of DNA binding proteins (e.g., without limitations, ZnF, TALE, dCas), linkers, and fusion sites that target specific TTAA integration sites. In embodiments, the present disclosure provides a developed landing pad assay that can show site- and sequence-specific targeting. In embodiments, the landing pad assay enables Amplicon-seq to show high efficiency targeting using covalent and non-covalent linkers and flanking DNA binding recognition sites. In embodiments, the high efficiency targeting is up to about 10%, or up to about 20%, or up to about 30%, or up to about 40%, or up to about 50%, or up to about 60%, or up to about 70%, or up to about 80%, or up to about 90%, or up to about 100%. In embodiments, the flanking DNA binding recognition sites are within about 9 to about 30 base pairs of the target TTAA integration sites. In embodiments the flanking DNA binding recognition sites are within about 15 to about 25 base pairs of the target TTAA integration sites. In embodiments, the present disclosure provides MLT transposase N-terminus deletion mutants. In embodiments the MLT transposase N- terminus deletion mutants show favorable integration or epigenetic profile and promotes recruitment to intergenic target TTAA. The present disclosure is based, in part, on the discovery of an engineered helper enzyme capable of gene insertion that finds uses in multiple applications, including, without limitation, in gene therapy. In aspects, there is provided an engineered enzyme, e.g., having an amino acid sequence of SEQ ID NO: 1 or a variant thereof, inclusive of all variants disclosed herein (e.g., SEQ ID NO: 1 and TABLES 1-2) (occasionally referred to as "engineered”, or variants thereof “MLT”, as used herein, refers to Myotis lucifugus helper, as engineered herein.

[0075] In embodiments, the illustrative bioengineered RNA helper constructs that are contained in a replication backbone (e.g., plasmid, miniplasmid, nanoplasmid, doggybone, or close-ended linear DNA) with a T7 promoter (cap dependent), betaglobin 5’-UTR, and a helper enzyme with 2 or more mutations in the Myotis lucifugus helper followed by a beta-globin 3'-UTR, and a poly-alanine tail. In embodiments, minicircle DNA is a novel, synthetic DNA vector and enzymatic DNA manufacturing process enabling rapid DNA production.

[0076] The present disclosure is based, in part, on the discovery that an enzyme capable of targeted genomic integration by transposition (e.g., an unengineered recombinase, integrase, or helper enzyme), as a monomer or a dimer, can be fused with a transcription activator-like effector proteins (TALE) DNA binding domain (DBD), a dCas / gRNA, or a zinc finger (ZnF) sequence to thereby create a chimeric enzyme capable of a site- or locus-specific transposition. For instance, in the case of a fusion to a ZnF DBD, the enzyme (e.g., without limitation, a chimeric helper) utilizes the specificity of ZnF DBD to certain sites within a host genome, which allows using DBDs to target any desired location in the genome. In this way, the chimeric helper in accordance with the present disclosure allows achieving targeted integration of a transgene.

[0077] In embodiments, the helper has one or more mutations that confer hyperactivity. In embodiments, the helper is a mammal-derived helper, optionally a helper RNA helper. Thus, the present compositions and methods for gene transfer utilize a dual donor / helper system. Transposable elements are non-viral gene delivery vehicles found ubiquitously in nature. Donor-based vectors have the capacity of stable genomic integration and long-lasting expression of transgene constructs in cells. Generally, dual donor and helper systems work via a cut-and-paste mechanism whereby donor DNA containing a transgene(s) of interest is integrated into chromosomal DNA by a helper enzyme at a repetitive sequence site. Dual donor / helper (or “donor / helper”) plasmid systems insert a transgene flanked by inverted terminal ends (“ends”), such as TTAA tetranucleotide sites, ttTTAAaa or taTTAAta octanucleotide sites, without leaving a DNA footprint in the human genome. The helper enzyme is transiently expressed (on the same or a different vector from a vector encoding the donor) and it catalyzes the insertion events from the donor plasmid to the host genome. Genomic insertions primarily target introns but may target other TTAA sites.

[0078] In embodiments, the disclosure describes a DNA integration system, which is highly active in mammals, and is derived from a mammalian mobile DNA element. In embodiments, this mammal-derived mobile genetic element is engineered to insert donor DNA at specific TTAA insertion “hotspots” that are frequently favored insertion sites for the unengineered enzyme. In embodiments, this technology exploits a helper RNA encoding enzyme with engineered DNA binding proteins and a donor DNA contained between the ends of a mobile element of the gene to be inserted into the genome. In embodiments, the mammal-derived enzyme is fused to a protein domain at its N-terminus, or within internal DNA binding loops (without loss of activity and “engineered” by fusing DNA binding domains (DBD) that can target almost any location in the genome). In embodiments, excision competent / target binding defective enzymes (Exc+ / lnt) mutants are described, that when combined with programmable, synthetic DBDs only insert at a TTAAs at a single target site. The engineered enzymes described in this disclosure displays several highly desirable features that are of great advantage for transgene integration. In embodiments, no DNA double strand breaks are introduced into the target genome. Furthermore, upon enzyme-mediated excision containing a gene of interest from its donor DNA, the flanking donor backbone ends are very efficiently rejoined, leaving no double strand break in the donor DNA to signal DNA damage. The helper enzyme inserts the excised element at high frequency selectively into a TTAA target site. Notably, because excision from the donor site results in the covalent linkage of a TTAA segment to each 5’ donor end, the joining of the 3’ donor ends to staggered positions on the top and bottom strands of the DNA flanking the target TTAA, a simple ligation restores intact duplex DNA, and no DNA synthesis is required for repair. Finally, the helper enzyme delivers a large cargo size as compared to other mobile genetic elements or integrating viral systems to date. See Liang, et al. (2009). Chromosomal mobilization and reintegration of Sleeping Beauty and PiggyBac donors. Genesis, 47(6), 404-408; Mitra, et al. (2013). Functional characterization of piggy Bat from the bat Myotis lucifugus unveils an active mammalian DNA donor. Proc Natl Acad Sci U S A, 110(1), 234-239; Ray, et al. (2008). Multiple waves of recent DNA donor activity in the bat, Myotis lucifugus. Genome Res, 78(5), 717-728.

[0079] In embodiments, the helper enzyme is delivered as an RNA instead of as a DNA. Other mobile genetic elements including helpers such as hyperactive piggyBac (pB) and SB100X, when delivered as RNA, have significantly less activity when compared to DNA. See Bire, et al. (2013). Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition. BMC Biotechnol, 13, 75; Bire, et al. (2013). Optimization of the piggyBac donor using mRNA and insulators: toward a more reliable gene delivery system. PLoS One, 8(12), e82559; Wilber, et al. (2006). RNA as a source of helper for Sleeping Beauty-mediated gene insertion and expression in somatic cells and tissues. Mol Then, 13(3), 625-630. The helper enzyme described herein has the same or better activity when delivered as RNA. The use of helper RNA offers several advantages over delivery of a DNA molecule. Wilber, et al. (2006). RNA as a source of helper for Sleeping Beauty-mediated gene insertion and expression in somatic cells and tissues. Mol Ther, 13(3), 625-630. For instance, without wishing to be bound by theory, there is improved control with respect to the duration of helper enzyme expression, minimizing persistence in the tissue, and there is potential for transgene remobilization and re-insertion following the initial transposition event. Furthermore, in embodiments, the helper-encoding RNA sequence is incapable of integrating into the host genome, thereby eliminating concerns about long-term helper expression and destabilizing effects with respect to the gene of interest. This safety feature, in embodiments, prevents the integration of the helper enzyme gene into the human genome and circumvents potential oncogenic and mutagenic effects. In embodiments, the present disclosure provides a dual DNA donor and RNA helper system. The donor DNA plasmid contains helper-specific inverted terminal repeats (ITRs) flanking the transgene while the helper-RNA transiently expresses a synthetic helper enzyme that catalyzes the insertion events from the donor plasmid to the host genome. This two component DNA / RNA system is, in embodiments, co-encapsulated in a single lipid nanoparticle using microfluidic technology and the lipid nanoparticles protect the RNA from extracellular degradation by in vivo injection.

[0080] In embodiments, the helper enzyme described herein is amenable to be fused to protein domain at the N-terminus and internal loops without loss of activity. Deletions of the C-terminus, in embodiments, cause a loss of helper enzyme excision and integration activity that may be restored when fused to binding ligands (e.g., rapamycin-induced FRB- FKBP fusion, SH3 plus high affinity ligand). This feature permits, inter alia, the synthesis of an “engineered” helper enzyme that target specific genomic regions of interest by fusing to the helper enzyme particular DNA binding domains that can target almost any location in the genome.

[0081] Helper Enzyme

[0082] In aspects, there is provided a composition, and method of use of a composition, comprising a helper enzyme or a nucleic acid encoding the helper enzyme, an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1 and a non-polar aliphatic amino acid at position 2 of SEQ ID NO: 1, or a position corresponding thereto, one or more amino acid substitutions selected from S8X, C13X, Y281X, N335X, and D416X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, and one or more amino acid substitutions selected from T331X, I332X, R333X, K334X, R336X, G337X, and I338X of SEQ ID NO: 1 or a substitution at a position corresponding thereto.

[0083] In aspects, there is provided a composition, and method of use of a composition, comprising a helper enzyme or a nucleic acid encoding the helper enzyme, an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1 and a non-polar aliphatic amino acid at position 2 of SEQ ID NO: 1, or a position corresponding thereto, and S8X, C13X, N335X, I338X, and D416X of SEQ ID NO: 1 or a substitution at a position corresponding thereto.

[0084] In aspects, there is provided a composition, and method of use of a composition, comprising a helper enzyme or a nucleic acid encoding the helper enzyme, an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1 and a non-polar aliphatic amino acid at position 2 of SEQ ID NO: 1, or a position corresponding thereto, S8X, C13X, R333X, N335X, and D416X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, and / or one or more amino acid substitutions selected from K16X, N19X, N28X, L48X, S56X, N128X, K137X, D208X, N226X, N241X, K286X, N317X, T331X, I332X, K334X, R336X, G337X, I338X, K349X, K369X, E381X, I388X, D480X, M481X, L486X, K525X, K541 R, F552X, Y572X, of SEQ ID NO: 1 or a substitution at a position corresponding thereto.

[0085] In embodiments, the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), and proline (P) In embodiments, X is selected from a non-polar amino acid, optionally selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), methionine (M), tryptophan (W), and phenylalanine (F), or a polar amino acid, optionally selected from serine (S), cysteine (0), asparagine (N), glutamine (Q), threonine (T), and tyrosine (Y), or a basic amino acid, optionally selected from arginine (R), histidine (H), or an acidic amino acid, optionally selected from aspartic acid (D), and glutamic acid (E), or a positively charged hydrophilic amino acid, optionally selected from lysine (K), arginine (R), and histidine (H).

[0086] In embodiments, the helper enzyme comprises an amino acid sequence of at least about 90% identity to SEQ ID NO: 1. In embodiments, the helper enzyme comprises an amino acid sequence of at least about 93% identity to SEQ ID NO: 1. In embodiments, the helper enzyme comprises an amino acid sequence of at least about 95% identity to SEQ ID NO: 1. In embodiments, the helper enzyme comprises an amino acid sequence of at least about 98% identity to SEQ ID NO: 1 . In embodiments, the helper enzyme comprises an amino acid sequence of at least about 99% identity to SEQ ID NO: 1.

[0087] In embodiments, the nucleic acid that encodes the helper enzyme has a nucleotide sequence of SEQ ID NO: 10 or a codon-optimized form thereof.

[0088] SEQ ID NO: 2: amino acid sequence of a variant of a hyperactive helper with P at position 8, R at position 13, and N at position 416 (S8P / C13R / 416N)(572 amino acids)

[0089] In embodiments, the helper enzyme comprises an amino acid sequence of at least about 90% identity to SEQ ID NO: 2. In embodiments, the helper enzyme comprises an amino acid sequence of at least about 93% identity to SEQ ID NO: 2. In embodiments, the helper enzyme comprises an amino acid sequence of at least about 95% identity to SEQ ID NO: 2. In embodiments, the helper enzyme comprises an amino acid sequence of at least about 98% identity to SEQ ID NO: 2. In embodiments, the helper enzyme comprises an amino acid sequence of at least about 99% identity to SEQ ID NO: 2.

[0090] In embodiments, the helper enzyme comprises a substitution at S8X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, optionally wherein X is a non-polar residue, optionally selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), methionine (M), tryptophan (W), and phenylalanine (F), or a polar and neutral hydrophilic residue, optionally proline (P).

[0091] In embodiments, the helper enzyme comprises a substitution at C13X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, optionally wherein X is a positively charged hydrophilic amino acid, optionally selected from arginine (R), lysine (K), and histidine (H).

[0092] In embodiments, the helper enzyme comprises a substitution at Y281X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, wherein X is a non-polar amino acid, optionally selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), methionine (M), tryptophan (W), and phenylalanine (F). In embodiments, the helper enzyme comprises a substitution at R333X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, optionally wherein X is a non-polar residue, optionally selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I).

[0093] In embodiments, the helper enzyme comprises a substitution at N335X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, wherein X is a non-polar amino acid, optionally selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), methionine (M), tryptophan (W), and phenylalanine (F).

[0094] In embodiments, the helper enzyme comprises a substitution at I338X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, wherein X is a non-polar amino acid, optionally selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), methionine (M), tryptophan (W), and phenylalanine (F), or a polar amino acid, optionally selected from serine (S), cysteine (C), asparagine (N), glutamine (Q), threonine (T), and tyrosine (Y).

[0095] In embodiments, the helper enzyme comprises a substitution at D416X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, wherein X is a polar and positively charged hydrophilic residue, optionally selected from arginine (R) and lysine (K), or a polar and neutral of charge hydrophilic residue, optionally selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C).

[0096] In embodiments, the helper enzyme comprises one or two, or three, or four, or five substitutions selected from: S8P, C13R, Y281A, N335A, and D416N or substitutions corresponding thereto relative to SEQ ID NO: 1.

[0097] In embodiments, the helper enzyme comprises one, or two, or three, or four, substitutions selected from T331A, I332A, I332S, I332M, R333A, R333S, R333K, K334A, R336A, R336S, R336K, G337A, I338A, I338M, and I338S or substitutions corresponding thereto relative to SEQ ID NO: 1.

[0098] In embodiments, the helper enzyme comprises one or two, or three, or four, or five substitutions selected from: S8P, C13R, Y281A, N335A, and D416N or substitutions corresponding thereto relative to SEQ ID NO: 1 , and one, or two, or three, or four substitutions selected from T331A, I332A, I332S, I332M, R333A, R333S, R333K, K334A, R336A, R336S, R336K, G337A, I338A, I338M, and I338S or substitutions corresponding thereto relative to SEQ ID NO: 1.

[0099] In embodiments, the helper enzyme comprises one, or two, or three, or four, or five, or six, or seven, or eight substitutions selected from: S8P, C13R, Y281A, T331A, I332A, I332S, I332M, R333A, R333S, R333K, K334A, N335A, R336A, R336S, R336K, G337A, I338A, I338M, I338S and D416N or substitutions corresponding thereto relative to SEQ ID NO: 1.

[0100] In embodiments, the helper enzyme comprises one or two, or three, or four, or five substitutions selected from: S8P, C13R, N335A, I338S, and D416N or substitutions corresponding thereto relative to SEQ ID NO: 1. In embodiments, the helper enzyme comprises one or two, or three, or four, or five, or six substitutions selected from: S8P, C13R, C13H, R333A, R333S, R333K, N335A, and D416N or substitutions corresponding thereto relative to SEQ ID NO: 1.

[0101] In embodiments, the composition comprises one or two, or three, or four, or five substitutions selected from: K16E, N19D, N28T, L48W, S56A, N128D, K137R, D208G, N226S, N241 I, K286R, N317T, T331A, I332A, I332M, I332S, K334A, K334R, K334S, R336A, R336S, R336K, G337A, I338A, I338M, I338S, K349R, K369R, E381 G, I388T, D480G, M481R, L486V, K525I, K541R, F552I, Y572C, or substitutions corresponding thereto relative to SEQ ID NO: 1.

[0102] In embodiments, the composition comprises one or two, or three, or four, or five substitutions selected from: S8P, C13R, C13H, R333A, R333S, R333K, N335A, and D416N or substitutions corresponding thereto relative to SEQ ID NO: 1 , and one, or two, or three, or four substitutions selected from K16E, N19D, N28T, L48W, S56A, N128D, K137R, D208G, N226S, N241 I, K286R, N317T, T331A, I332A, I332M, I332S, K334A, K334R, K334S, R336A, R336S, R336K, G337A, I338A, I338M, I338S, K349R, K369R, E381G, I388T, D480G, M481 R, L486V, K525I, K541R, F552I, Y572C, or substitutions corresponding thereto relative to SEQ ID NO: 1.

[0103] In embodiments, the helper enzyme comprises one, or two, or three, or four, or five, or six, or seven, or eight substitutions selected from: S8P, C13R, C13H, K16E, N19D, N28T, L48W, S56A, N128D, K137R, D208G, N226S, N241 I, K286R, N317T, T331A, I332A, I332M, I332S, R333A, R333S, R333K, K334A, K334R, K334S, N335A, R336A, R336S, R336K, G337A, I338A, I338M, I338S, K349R, K369R, E381G, I388T, D416N, D480G, M481 R, L486V, K525I, K541R, F552I, and Y572C or substitutions corresponding thereto relative to SEQ ID NO: 1.

[0104] In embodiments, the helper enzyme comprises or consists of 6 amino acid substitutions.

[0105] In embodiments, the helper enzyme comprises or consists of 7 amino acid substitutions.

[0106] In embodiments, the helper enzyme comprises or consists of 8 amino acid substitutions.

[0107] In embodiments, the helper enzyme comprises a substitution selected from TABLE 1 or a substitution at positions corresponding thereto relative to SEQ ID NO: 1.

[0108] Table 1 : Exemplary Mutants

[0109] In embodiments, the helper enzyme comprises:

[0110] S8P_C13R_Y281 A_I332A_K334A_N335A_D416 N , S8P_C13R_Y281A_I332A_K334A_N335A_G337A_D416N,

[0111] S8P_C 13R_Y281 A_I332A_K334A_N335A_I338A_D416N ,

[0112] S8P_C13R_Y281A_I332A_K334A_N335A_I338S_D416N,

[0113] S8P_C13R_Y281A_I332A_K334A_N335A_R336A_D416N,

[0114] S8P_C13R_Y281A_I332A_K334A_N335A_R336K_D416N,

[0115] S8P_C13R_Y281A_I332A_K334A_N335A_R336S_D416N,

[0116] S8P_C13R_Y281 A_I332A_R333A_K334A_N335A_D416N,

[0117] S8P_C13R_Y281A_I332A_R333K_K334A_N335A_D416N,

[0118] S8P_C13R_Y281A_I332A_R333S_K334A_N335A_D416N,

[0119] S8P_C13R_Y281A_I332M_K334A_N335A_D416N,

[0120] S8P_C13R_Y281 A_I332M_K334A_N335A_G337A_D416N,

[0121] S8P_C13R_Y281A_I332M_K334A_N335A_I338M_D416N,

[0122] S8P_C13R_Y281A_I332M_K334A_N335A_R336A_D416N,

[0123] S8P_C13R_Y281A_I332M_K334A_N335A_R336K_D416N,

[0124] S8P_C13R_Y281A_I332M_K334A_N335A_R336S_D416N,

[0125] S8P_C13R_Y281A_I332M_R333A_K334A_N335A_D416N,

[0126] S8P_C13R_Y281A_I332M_R333K_K334A_N335A_D416N,

[0127] S8P_C13R_Y281A_I332M_R333S_K334A_N335A_D416N,

[0128] S8P_C13R_Y281 A_I332S_K334A_N335A_D416 N,

[0129] S8P_C13R_Y281A_I332S_K334A_N335A_G337A_D416N,

[0130] S8P_C13R_Y281A_I332S_K334A_N335A_I338A_D416N,

[0131] S8P_C13R_Y281A_I332S_K334A_N335A_I338S_D416N,

[0132] S8P_C13R_Y281A_I332S_K334A_N335A_R336A_D416N,

[0133] S8P_C13R_Y281A_I332S_K334A_N335A_R336K_D416N,

[0134] S8P_C13R_Y281A_I332S_K334A_N335A_R336S_D416N,

[0135] S8P_C13R_Y281A_I332S_R333A_K334A_N335A_D416N, S8P_O13R_Y281A_I332S_R333K_K334A_N335A_D416N,

[0136] S8P_C13R_Y281A_I332S_R333S_K334A_N335A_D416N,

[0137] S8P_C13R_Y281 A_K334A_N335A_D416N,

[0138] S8P_C13R_Y281 A_K334A_N335A_G337A_D416 N ,

[0139] S8P_C13R_Y281A_K334A_N335A_G337A_I338M_D416N,

[0140] S8P_C13R_Y281 A_K334A_N335A_I338A_D416 N ,

[0141] S8P_C13R_Y281 A_K334A_N335A_I338M_D416 N ,

[0142] S8P_C13R_Y281 A_K334A_N335A_I338S_D416 N ,

[0143] S8P_C13R_Y281 A_K334A_N335A_R336A_D416 N ,

[0144] S8P_C13R_Y281 A_K334A_N335A_R336A_G337A_D416 N ,

[0145] S8P_C13R_Y281 A_K334A_N335A_R336A_I338A_D416N,

[0146] S8P_C13R_Y281A_K334A_N335A_R336A_I338M_D416N,

[0147] S8P_C13R_Y281A_K334A_N335A_R336A_I338S_D416N,

[0148] S8P_C13R_Y281A_K334A_N335A_R336K_D416N,

[0149] S8P_C13R_Y281A_K334A_N335A_R336K_G337A_D416N,

[0150] S8P_C13R_Y281A_K334A_N335A_R336K_I338A_D416N,

[0151] S8P_C13R_Y281A_K334A_N335A_R336K_I338M_D416N,

[0152] S8P_C13R_Y281A_K334A_N335A_R336K_I338S_D416N,

[0153] S8P_C13R_Y281A_K334A_N335A_R336S_D416N,

[0154] S8P_C13R_Y281A_K334A_N335A_R336S_G337A_D416N,

[0155] S8P_C13R_Y281A_K334A_N335A_R336S_I338A_D416N,

[0156] S8P_C13R_Y281A_K334A_N335A_R336S_I338M_D416N,

[0157] S8P_C13R_Y281A_K334A_N335A_R336S_I338S_D416N,

[0158] S8P_C13R_Y281 A_R333A_K334A_N335A_D416 N ,

[0159] S8P_C13R_Y281 A_R333A_K334A_N335A_G337A_D416 N ,

[0160] S8P_C13R_Y281 A_R333A_K334A_N335A_I338A_D416N, S8P_O13R_Y281A_R333A_K334A_N335A_I338M_D416N,

[0161] S8P_C13R_Y281A_R333A_K334A_N335A_I338S_D416N,

[0162] S8P_C13R_Y281 A_R333A_K334A_N335A_R336A_D416 N ,

[0163] S8P_C13R_Y281 A_R333A_K334A_N335A_R336K_D416 N ,

[0164] S8P_C13R_Y281 A_R333A_K334A_N335A_R336S_D416 N ,

[0165] S8P_C13R_Y281A_R333K_K334A_N335A_D416N,

[0166] S8P_C13R_Y281A_R333K_K334A_N335A_G337A_D416N,

[0167] S8P_C13R_Y281A_R333K_K334A_N335A_I338A_D416N,

[0168] S8P_C13R_Y281A_R333K_K334A_N335A_I338M_D416N,

[0169] S8P_C13R_Y281A_R333K_K334A_N335A_I338S_D416N, S8P_C13R_Y281 A_R333K_K334A_N335A_R336A_D416 N , S8P_C13R_Y281A_R333K_K334A_N335A_R336K_D416N,

[0170] S8P_C13R_Y281A_R333K_K334A_N335A_R336S_D416N,

[0171] S8P_C13R_Y281A_R333S_K334A_N335A_D416N,

[0172] S8P_C13R_Y281A_R333S_K334A_N335A_G337A_D416N,

[0173] S8P_C13R_Y281 A_R333S_K334A_N335A_I338A_D416N,

[0174] S8P_C13R_Y281A_R333S_K334A_N335A_I338M_D416N,

[0175] S8P_C13R_Y281A_R333S_K334A_N335A_I338S_D416N, S8P_C13R_Y281 A_R333S_K334A_N335A_R336A_D416 N ,

[0176] S8P_C13R_Y281A_R333S_K334A_N335A_R336K_D416N,

[0177] S8P_C13R_Y281A_R333S_K334A_N335A_R336S_D416N,

[0178] S8P_C13R_Y281 A_T331 A_I332A_K334A_N335A_D416N,

[0179] S8P_C13R_Y281A_T331A_I332M_K334A_N335A_D416N,

[0180] S8P_C13R_Y281A_T331A_I332S_K334A_N335A_D416N,

[0181] S8P_C13R_Y281 A_T331 A_K334A_N335A_D416N,

[0182] S8P_C13R_Y281 A_T331 A_K334A_N335A_G337A_D416 N , S8P_C 13R_Y281 A_T331 A_K334A_N335A_I338A_D416N,

[0183] S8P_C13R_Y281 A_T331 A_K334A_N335A_I338M_D416N,

[0184] S8P_C13R_Y281 A_T331 A_K334A_N335AJ338S_D416N,

[0185] S8P_C13R_Y281 A_T331 A_K334A_N335A_R336A_D416N,

[0186] S8P_C13R_Y281 A_T331 A_K334A_N335A_R336K_D416N,

[0187] S8P_C13R_Y281 A_T331 A_K334A_N335A_R336S_D416N,

[0188] S8P_C13R_Y281 A_T331 A_R333A_K334A_N335A_D416 N ,

[0189] S8P_C13R_Y281 A_T331 A_R333K_K334A_N335A_D416 N ,

[0190] S8P_O13R_Y281A_T331A_R333S_K334A_N335A_D416N, of SEQ ID NO: 1 or a substitution at a position corresponding thereto.

[0191] In embodiments, the helper enzyme comprises S8P_O13R_N335A_I338S_D416N of SEQ ID NO: 1 or a substitution at a position corresponding thereto.

[0192] In embodiments, the helper enzyme comprises a substitution selected from TABLE 2 or a substitution at positions corresponding thereto relative to SEQ ID NO: 1.

[0193] Table 2: Exemplary mutants

[0194] In embodiments, the helper enzyme comprises:

[0195] S8P_C13R_I332A_R333A_K334A_N335A_D416N , S8P_C13R_I332A_R333A_K334A_N335A_D416N, S8P_C13R_I332A_R333A_K334S_N335A_D416N , S8P_C13R_I332A_R333A_N335A_D416 N , S8P_C13R_I332A_R333A_N335A_G337A_D416N, S8P_C13R_I332A_R333A_N335A_I338A_D416N, S8P_C13R_I332A_R333A_N335A_I338S_D416N, S8P_C13R_I332A_R333A_N335A_R336A_D416N, S8P_C13R_I332A_R333A_N335A_R336K_D416N, S8P_C13R_I332A_R333A_N335A_R336S_D416N,

[0196] S8P_C13R_I332M_R333A_K334A_N335A_D416N, S8P_C13R_I332M_R333A_K334R_N335A_D416N, S8P_C13R_I332M_R333A_K334S_N335A_D416N, S8P_C13R_I332M_R333A_N335A_D416N, S8P_C13R_I332M_R333A_N335A_G337A_D416N, S8P_C13R_I332M_R333A_N335A_I338M_D416N, S8P_C13R_I332M_R333A_N335A_R336A_D416N, S8P_C13R_I332M_R333A_N335A_R336K_D416N, S8P_C13R_I332M_R333A_N335A_R336S_D416N, S8P_C13R_I332S_R333A_K334A_N335A_D416N , S8P_C13R_I332S_R333A_K334R_N335A_D416N, S8P_C13R_I332S_R333A_K334S_N335A_D416N , S8P_C13R_I332S_R333A_N335A_D416N, S8P_C13R_I332S_R333A_N335A_G337A_D416N, S8P_C13R_I332S_R333A_N335A_I338A_D416N, S8P_C13R_I332S_R333A_N335A_I338S_D416N, S8P_C13R_I332S_R333A_N335A_R336K_D416N, S8P_C13R_I332S_R333A_N335A_R336K_D416N, S8P_C13R_I332S_R333A_N335A_R336S_D416N, S8P_C13R_R333A_K334A_N335A_D416N , S8P_C13R_R333A_K334A_N335A_G337A_D416N, S8P_C13R_R333A_K334A_N335A_I338A_D416N, S8P_C13R_R333A_K334A_N335A_I338M_D416 N , S8P_C13R_R333A_K334A_N335A_I338S_D416N, S8P_C13R_R333A_K334A_N335A_R336A_D416 N , S8P_C13R_R333A_K334A_N335A_R336K_D416 N, S8P_C13R_R333A_K334A_N335A_R336S_D416N, S8P_C13R_R333A_K334A_N335A_R336S_D416 N, S8P_C13R_R333A_K334R_N335A_D416N,

[0197] S8P_C13R_R333A_K334R_N335A_G337A_D416N, S8P_C13R_R333A_K334R_N335A_I338A_D416N, S8P_C13R_R333A_K334R_N335A_I338M_D416N, S8P_C13R_R333A_K334R_N335A_I338S_D416N, S8P_C13R_R333A_K334R_N335A_R336A_D416N, S8P_C13R_R333A_K334R_N335A_R336S_D416N, S8P_C 13R_R333A_K334S_N335A_D416N,

[0198] S8P_C13R_R333A_K334S_N335A_G337A_D416N, S8P_C13R_R333A_K334S_N335A_I338A_D416N, S8P_C13R_R333A_K334S_N335A_I338M_D416N, S8P_C13R_R333A_K334S_N335A_I338S_D416N, S8P_C13R_R333A_K334S_N335A_R336A_D416 N , S8P_C13R_R333A_K334S_N335A_R336K_D416 N, S8P_C13R_R333A_K334S_N335A_R336S_D416 N,

[0199] S8P_C13R_R333A_N335A_D416N,

[0200] S8P_C13R_R333A_N335A_G337A_D416N,

[0201] S8P_C13R_R333A_N335A_G337A_I338M_D416N,

[0202] S8P_C13R_R333A_N335A_I338A_D416N, S8P_C13R_R333A_N335A_I338M_D416N,

[0203] S8P_C13R_R333A_N335A_R336A_D416N,

[0204] S8P_C13R_R333A_N335A_R336A_G337A_D416N, S8P_C13R_R333A_N335A_R336A_I338A_D416N, S8P_C13R_R333A_N335A_R336A_I338S_D416N, S8P_C13R_R333A_N335A_R336K_D416N,

[0205] S8P_C13R_R333A_N335A_R336K_G337A_D416N, S8P_C13R_R333A_N335A_R336K_I338A_D416N, S8P_C13R_R333A_N335A_R336K_I338M_D416N, S8P_C13R_R333A_N335A_R336K_I338M_D416N, S8P_C13R_R333A_N335A_R336K_I338S_D416N, S8P_C13R_R333A_N335A_R336S_D416N,

[0206] S8P_C13R_R333A_N335A_R336S_G337A_D416N, S8P_C13R_R333A_N335A_R336S_I338A_D416N, S8P_C13R_R333A_N335A_R336S_I338M_D416N, S8P_C13R_R333A_N335A_R336S_I338S_D416N, S8P_C13R_T331 A_I332A_R333A_N335A_D416N, S8P_C13R_T331A_I332M_R333A_N335A_D416N, S8P_C13R_T331 A_I332S_R333A_N335A_D416N ,

[0207] S8P_C13R_T331 A_R333A_K334A_N335A_D416 N , S8P_C13R_T331 A_R333A_K334R_N335A_D416 N , S8P_C13R_T331 A_R333A_K334S_N335A_D416 N , S8P_C 13R_T331 A_R333A_N335A_D416 N , S8P_C13R_T331 A_R333A_N335A_G337A_D416 N , S8P_C13R_T331 A_R333A_N335A_I338A_D416N, S8P_C13R_T331A_R333A_N335A_I338M_D416N, S8P_C13R_T331 A_R333A_N335A_I338S_D416N, S8P_C13R_T331 A_R333A_N335A_R336A_D416 N , S8P_C13R_T331 A_R333A_N335A_R336K_D416 N , S8P_C13R_T331 A_R333A_N335A_R336S_D416 N ,

[0208] S8P_C13R.Y281 A_I332A_K334A_N335A_D416N, S8P_C13R_Y281A_I332A_K334A_N335A_G337A_D416N, S8P_C 13R_Y281 A_l 332A_K334A_N335A J 338A_D 416 N , S8P_C 13R_Y281 A_l 332A_K334A_N335A J 338S_D 416 N , S8P_C13R_Y281A_I332A_K334A_N335A_R336A_D416N, S8P_C13R_Y281A_I332A_K334A_N335A_R336K_D416N, S8P_C13R_Y281A_I332A_K334A_N335A_R336S_D416N, S8P_C13R_Y281A_I332A_R333A_K334A_N335A_D416N, S8P_C13R_Y281A_I332A_R333K_K334A_N335A_D416N, S8P_C13R_Y281A_I332A_R333S_K334A_N335A_D416N,

[0209] S8P_C13R_Y281A_I332M_K334A_N335A_D416N, S8P_C13R_Y281 AJ332M_K334A_N335A_G337A_D416N, S8P_C13R_Y281A_I332M_K334A_N335A_I338M_D416N, S8P_C13R_Y281AJ332M_K334A_N335A_R336A_D416N, S8P_C13R_Y281AJ332M_K334A_N335A_R336K_D416N, S8P_C13R_Y281A_I332M_K334A_N335A_R336S_D416N, S8P_C13R_Y281A_I332M_R333A_K334A_N335A_D416N, S8P_C13R_Y281AJ332M_R333K_K334A_N335A_D416N, S8P_C13R_Y281A_I332M_R333S_K334A_N335A_D416N, S8P_C13R_Y281 A_I332S_K334A_N335A_D416N , S8P_C13R_Y281A_I332S_K334A_N335A_G337A_D416N, S8P_C 13R_Y281 A_l 332S_K334A_N335A J 338A_D 416 N , S8P_C 13R_Y281 A_l 332S_K334A_N335A J 338S_D 416 N , S8P_C13R_Y281A_I332S_K334A_N335A_R336A_D416N, S8P_C13R_Y281A_I332S_K334A_N335A_R336K_D416N, S8P_C13R_Y281A_I332S_K334A_N335A_R336S_D416N, S8P_O13R_Y281A_I332S_R333A_K334A_N335A_D416N, S8P_C13R_Y281A_I332S_R333K_K334A_N335A_D416N, S8P_C13R_Y281A_I332S_R333S_K334A_N335A_D416N, S8P_C13R_Y281 A_K334A_N335A_D416 N , S8P_C13R_Y281 A_K334A_N335A_G337A_D416 N , S8P_C13R_Y281A_K334A_N335A_G337A_I338M_D416N, S8P_C13R_Y281 A_K334A_N335A_I338A_D416N , S8P_C13R_Y281 A_K334A_N335A_I338M_D416 N , S8P_C13R_Y281 A_K334A_N335A_I338S_D416 N , S8P_C13R_Y281 A_K334A_N335A_R336A_D416 N , S8P_C13R_Y281A_K334A_N335A_R336A_G337A_D416N, S8P_C13R_Y281A_K334A_N335A_R336A_I338A_D416N, S8P_O13R_Y281A_K334A_N335A_R336A_I338M_D416N, S8P_O13R_Y281A_K334A_N335A_R336A_I338S_D416N, S8P_C13R_Y281 A_K334A_N335A_R336K_D416 N , S8P_C13R_Y281A_K334A_N335A_R336K_G337A_D416N, S8P_C13R_Y281A_K334A_N335A_R336K_I338A_D416N, S8P_C13R_Y281A_K334A_N335A_R336K_I338M_D416N, S8P_O13R_Y281A_K334A_N335A_R336K_I338S_D416N, S8P_C13R_Y281 A_K334A_N335A_R336S_D416 N ,

[0210] S8P_C13R_Y281A_K334A_N335A_R336S_G337A_D416N, S8P_C13R_Y281A_K334A_N335A_R336S_I338A_D416N, S8P_C13R_Y281A_K334A_N335A_R336S_I338M_D416N, S8P_C13R_Y281A_K334A_N335A_R336S_I338S_D416N, S8P 013R Y281 A_R333A_K334A_N335A_D416 N , S8P_C13R_Y281A_R333A_K334A_N335A_G337A_D416N, S8P_C13R_Y281A_R333A_K334A_N335A_I338A_D416N, S8P_C13R_Y281A_R333A_K334A_N335A_I338M_D416N, S8P_C13R_Y281A_R333A_K334A_N335A_I338S_D416N, S8P_C13R_Y281A_R333A_K334A_N335A_R336A_D416N, S8P_C13R_Y281A_R333A_K334A_N335A_R336K_D416N, S8P_C13R_Y281A_R333A_K334A_N335A_R336S_D416N, S8P_C13R_Y281 A_R333K_K334A_N335A_D416 N , S8P_C13R_Y281A_R333K_K334A_N335A_G337A_D416N, S8P_C13R_Y281A_R333K_K334A_N335A_I338A_D416N, S8P_C13R_Y281A_R333K_K334A_N335A_I338M_D416N, S8P_C13R_Y281A_R333K_K334A_N335A_I338S_D416N, S8P_C13R_Y281A_R333K_K334A_N335A_R336A_D416N, S8P_C13R_Y281A_R333K_K334A_N335A_R336K_D416N, S8P_C13R_Y281A_R333K_K334A_N335A_R336S_D416N, S8P_C13R_Y281 A_R333S_K334A_N335A_D416 N , S8P_C13R_Y281A_R333S_K334A_N335A_G337A_D416N, S8P_C13R_Y281A_R333S_K334A_N335A_I338A_D416N, S8P_C13R_Y281A_R333S_K334A_N335A_I338M_D416N, S8P_C13R_Y281A_R333S_K334A_N335A_I338S_D416N, S8P_C13R_Y281A_R333S_K334A_N335A_R336A_D416N, S8P_C13R_Y281A_R333S_K334A_N335A_R336K_D416N, S8P_C13R_Y281A_R333S_K334A_N335A_R336S_D416N, S8P_C13R_Y281 A_T331 AJ332A_K334A_N335A_D416N, S8P_C13R_Y281A_T331A_I332M_K334A_N335A_D416N, S8P_C13R_Y281 A_T331 AJ332S_K334A_N335A_D416N, S8P_C13R_Y281A_T331A_K334A_N335A_D416N,

[0211] S8P_C 13R_Y281 A_T331 A_K334A_N335A_G337A_D416N , S8P_C 13R_Y281 A_T331 A_K334A_N335AJ 338A_D416N, S8P_C13R_Y281A_T331A_K334A_N335AJ338M_D416N, S8P_C13R_Y281A_T331A_K334A_N335A_I338S_D416N, S8P_C 13R_Y281 A_T331 A_K334A_N335A_R336A_D416N , S8P_C13R_Y281 A_T331 A_K334A_N335A_R336K_D416N , S8P_C13R_Y281 A_T331 A_K334A_N335A_R336S_D416N , S8P_C 13R_Y281 A_T331 A_R333A_K334A_N335A_D416N , S8P_C 13R_Y281 A_T331 A_R333K_K334A_N335A_D416N , S8P_C 13R_Y281 A_T331 A_R333S_K334A_N335A_D416N , S8P_C13R_R333A_N335A_D416N_K369R, S8P_C13R_R333A_N335A_D416N_M481 R, S8P_C13R_R333A_N335A_D416N_S56A, S8P_C13R_R333A_N335A_D416N_F552I, S8P_C13R_R333A_N335A_D416N_K16E,

[0212] S8P_C13R_R333A_N335A_D416N_N317T_K525I, S8P_C 13R_R333A_N335A_D416 N_N317T_K525I_K369R ,

[0213] S8P_C13R_R333A_N335A_D416N_N 128D,

[0214] S8P_C13R_R333A_N335A_D416N_1388T,

[0215] S8P_C13R_R333A_N335A_D416N_K349R,

[0216] S8P_C13R_R333A_N335A_D416 N_K 16E_N 19D,

[0217] S8P_C13R_R333A_N335A_D416N_L48W,

[0218] S8P_C13R_R333A_N335A_D416N_N 19D_K369R,

[0219] S8P_C13R_R333A_N335A_D416N_K 16E_K369R,

[0220] S8P_C13R_R333A_N335A_D416 N_K 16E_N 19D_K369R,

[0221] S8P_C13R_R333A_N335A_D416N_D208G_K286R,

[0222] S8P_C13R_R333A_N335A_D416N_K541 R,

[0223] S8P_C13R_R333A_N335A_D416N_N28T,

[0224] S8P_C13R_R333A_N335A_D416N_L486V,

[0225] S8P_C13H_R333A_N335A_D416N_D480G,

[0226] S8P_C 13R_R333A_N335A_D416N_N 19D_K 137R_N226S,

[0227] S8P_C13R_R333A_N335A_D416N_K16E_N241 I,

[0228] S8P_C13R_R333A_N335A_D416N_N19D_E381 G,

[0229] S8P_C13R_R333A_N335A_D416N_Y572C, S8P_C13R_R333A_N335A_D416N_N19D, of SEQ ID NO: 1 or a substitution at a position corresponding thereto.

[0230] In embodiments, there is provided a composition comprising a helper enzyme or a nucleic acid encoding the helper enzyme comprising: an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1 and a nonpolar aliphatic amino acid at position 2 of SEQ ID NO: 1 , or a position corresponding thereto, and a plurality of amino acid substitutions of any row of TABLE A.

[0231] Table A: Exemplary mutants

[0232] In embodiments, there is provided a composition comprising a helper enzyme or a nucleic acid encoding the helper enzyme comprising: an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1 and a nonpolar aliphatic amino acid at position 2 of SEQ ID NO: 1 , or a position corresponding thereto, and a plurality of amino acid substitutions of any row of TABLE B.

[0233] Table B: Exemplary mutants

[0234] In embodiments, the helper enzyme has one or more mutations which confer hyperactivity.

[0235] Engineered Helper Enzyme

[0236] In embodiments, the helper enzyme further comprises a zinc finger (E2C) fused to the N-terminus and / or C-terminus. In embodiments, the helper enzyme further comprises a zinc finger (E2C) fused to the N-terminus. In embodiments, the helper enzyme further comprises a zinc finger (E2C) inserted after the glycine at amino acid position 66 relative to SEQ ID NO: 1 within the N-terminus. In embodiments, the helper enzyme is linked to a zinc finger (E2C) using recombinant variable domains of heavy-chain-only antibodies (VHH) (NANOBODY) bound to an ALFA-tag peptide.

[0237] In embodiments, the helper enzyme further comprises a zinc finger inserted after a residue selected from S60-E70 relative to SEQ ID NO: 1. In embodiments, the helper enzyme further comprises a zinc finger inserted after the G66 residue relative to SEQ ID NO: 1 . In embodiments, the helper enzyme further comprises a zinc finger inserted after the S60 residue relative to SEQ ID NO: 1. In embodiments, the helper enzyme further comprises a zinc finger inserted after the E70 residue relative to SEQ ID NO: 1. In embodiments, the helper enzyme further comprises a zinc finger inserted after the L80 residue relative to SEQ ID NO: 1. In embodiments, the helper enzyme further comprises a zinc finger inserted after the N90 residue relative to SEQ ID NO: 1. In embodiments, the helper enzyme further comprises a zinc finger inserted after the D100 residue relative to SEQ ID NO: 1.

[0238] In embodiments, the helper enzyme further comprises a zinc finger inserted after the G66 residue relative to SEQ ID NO: 1 with mutations selected from one or more mutations selected from Table 1 or Table 2 relative to SEQ ID NO: 1 In embodiments, the helper enzyme further comprises a zinc finger inserted immediately after the G66 residue relative to SEQ ID NO: 1 with mutations selected from one or more mutations selected from Table 1 or Table 2 relative to SEQ ID NO: 1. In embodiments, the helper enzyme further comprises a TALE fused to the N-terminus and / or C-terminus. In embodiments, the helper enzyme further comprises a TALE fused to the N-terminus. In embodiments, the helper enzyme further comprises a TALE inserted after the glycine at amino acid position 66 relative to SEQ ID NO: 1 within the N-terminus. In embodiments, the helper enzyme is linked to a TALE using recombinant variable domains of heavy- chain-only antibodies (VHH) (NANOBODY) bound to an ALFA-tag peptide (NbALFA). In embodiments, the helper enzyme tethered to a TALE using recombinant variable domains of heavy-chain-only antibodies (VHH) (NANOBODY) bound to an ALFA-tag peptide (NbALFA) can bind different recognition sequences spaced about 15, or about 16, or about 17, or about 18, or about 19, or about 20 base pairs on either side of the target.

[0239] In embodiments, the helper enzyme further comprises a TALE inserted after the G66 residue relative to SEQ ID NO: 1.

[0240] In embodiments, the helper enzyme further comprises a TALE inserted immediately after the G66 residue relative to SEQ ID NO: 1.

[0241] In embodiments, the helper enzyme further comprises an ALFA tag inserted after the G66 residue relative to SEQ ID NO: 1 . In embodiments, the helper enzyme further comprises an ALFA tag inserted immediately after the G66 residue relative to SEQ ID NO: 1. In embodiments, the helper enzyme further comprises an ALFA tag. In embodiments, the ALFA tag comprises an amino acid sequence of SRLEEELRRRLTE (SEQ ID NO: 892), or a variant thereof having a substitution or deletion.

[0242] In embodiments, the ALFA tag inserted after the G66 residue of MLT comprises an amino acid sequence of at least about 90% identity, or at least about 93% identity, or at least about 95% identity, or at least about 98% identity, or at least about 99% identity to SEQ ID NO: 885. In embodiments, the ALFA tag inserted immediately after the G66 residue of MLT comprises an amino acid sequence of at least about 90% identity, or at least about 93% identity, or at least about 95% identity, or at least about 98% identity, or at least about 99% identity to SEQ ID NO: 885.

[0243] SEQ ID NO: 885: ALFA tag inserted after the G66 residue relative to SEQ ID NO: 1 of MLT

[0244] ATGCCGAAAAAAAAACGAAAGGTGTACCCCTACGATGTACCGGACTATGCAGGAAGCGCCCAGCACAGCGACTACCC CGACGACGAGTTCAGAGCCGATAAGCTGAGTAACTACAGCTGCGACAGCGACCTGGAAAACGCCAGCACATCCGACG AGGACAGCTCTGACGACGAGGTGATGGTGCGGCCCAGAACCCTGAGACGGAGAAGAATCAGCAGCTCTAGCAGCGAC TCTGAATCCGACATCGAGGGCTCCAGACTGGAAGAGGAACTGAGAAGAAGGCTCACAGAAGGCCGGGAAGAGTGGAG CCACGTGGACAACCCTCCTGTTCTGGAAGATTTTCTGGGCCATCAGGGCCTGAACACCGACGCCGTGATCAACAACA TCGAGGATGCCGTGAAGCTGTTCATAGGAGATGATTTCTTTGAGTTCCTGGTCGAGGAATCCAACCGCTATTACAAC CAGAATAGAAACAACTTCAAGCTGAGCAAGAAAAGCCTGAAGTGGAAGGACATCACCCCTCAGGAGATGAAAAAGTT CCTGGGACTGATCGTTCTGATGGGACAGGTGCGGAAGGACAGAAGGGATGATTACTGGACAACCGAACCTTGGACCG AGACCCCTTACTTTGGCAAGACCATGACCAGAGACAGATTCAGACAGATCTGGAAAGCCTGGCACTTCAACAACAAT GCTGATATCGTGAACGAGTCTGATAGACTGTGTAAAGTGCGGCCAGTGTTGGATTACTTCGTGCCTAAGTTCATCAA CATCTATAAGCCTCACCAGCAGCTGAGCCTGGATGAAGGCATCGTGCCCTGGCGGGGCAGACTGTTCTTCAGAGTGT ACAATGCTGGCAAGATCGTCAAATACGGCATCCTGGTGCGCCTTCTGTGCGAGAGCGATACAGGCTACATCTGTAAT ATGGAAATCTACTGCGGCGAGGGCAAAAGACTGCTGGAAACCATCCAGACCGTCGTTTCCCCTTATACCGACAGCTG GTACCACATCTACATGGACAACTACTACAATTCTGTGGCCAACTGCGAGGCCCTGATGAAGAACAAGTTTAGAATCT GCGGCACAATCAGAAAAAACAGAGGCATCCCTAAGGACTTCCAGACCATCTCTCTGAAGAAGGGCGAAACCAAGTTC

[0245] In embodiments, the DNA binder of the present disclosure further comprises recombinant variable domains of heavy- chain-only antibodies (VHH) (NANOBODY) bound to an ALFA-tag peptide (NbALFA). In embodiments, the DNA binder further comprising recombinant variable domains of heavy-chain-only antibodies (VHH) (NANOBODY) bound to an ALFA-tag peptide (NbALFA) comprises an amino acid sequence of at least about 90% identity, or at least about 93% identity, or at least about 95% identity, or at least about 98% identity, or at least about 99% identity to SEQ ID NO: 886.

[0246] SEQ ID NO: 886: HoD4 TALE fused to the recombinant variable domains of heavy-chain-only antibodies (VHH) (NANOBODY) bound to an ALFA-tag peptide (NbALFA)

[0247] In embodiments, the DNA binder of the present disclosure further comprises recombinant variable domains of heavychain-only antibodies (VHH) (NANOBODY) bound to an ALFA-tag peptide (NbALFA). In embodiments, the DNA binder further comprising recombinant variable domains of heavy-chain-only antibodies (VHH) (NANOBODY) bound to an ALFA-tag peptide (NbALFA) comprises an amino acid sequence of at least about 90% identity, or at least about 93% identity, or at least about 95% identity, or at least about 98% identity, or at least about 99% identity to SEQ ID NO: 888. In embodiments, the DNA binder further comprising recombinant variable domains of heavy-chain-only antibodies (VHH) (NANOBODY) bound to an ALFA-tag peptide (NbALFA) comprises an amino acid sequence of at least about 90% identity, or at least about 93% identity, or at least about 95% identity, or at least about 98% identity, or at least about 99% identity to SEQ ID NO: 889.

[0248] SEQ ID NO: 888: Ch6L left TALE fused to the recombinant variable domains of heavy-chain-only antibodies (VHH) (NANOBODY) bound to an ALFA-tag peptide (NbALFA)

[0249] SEQ ID NO: 889: Ch6R right TALE fused to the recombinant variable domains of heavy-chain-only antibodies (VHH) (NANOBODY) bound to an ALFA-tag peptide (NbALFA)

[0250] In embodiments, the helper enzyme comprises a deletion of about 30, or about 40, or about 50, or about 60, or about 70, or about 80, or about 90, or about 100 amino acids from an N-terminus or a C-terminus.

[0251] In embodiments, the helper enzyme comprises a deletion of about 30, or about 40, or about 50, or about 60, or about 70 amino acids from an N-terminus of the polypeptide having an amino acid sequence of SEQ ID NO: 1. In embodiments, the helper enzyme comprises a deletion at positions about 1-35, or about 1-45, or about 1-55, or about 1-65, or about 1-75, or about 1-85, or about 1-95, or about 1-105, or about 2-35, or about 2-45, or about 2-68 or positions corresponding thereto, wherein the positions are relative to SEQ ID NO: 1.

[0252] In embodiments, the helper enzyme comprises a deletion at positions about 2-35, or about 2-45, or about 2-68, or positions corresponding thereto, wherein the positions are relative to SEQ ID NO: 1. In embodiments, the N terminal deletion yields reduced or ablated off-target effects of the enzyme compared to the enzyme without the N terminal deletion.

[0253] In embodiments, the helper enzyme has increased activity relative to an enzyme comprising an amino acid sequence of SEQ ID NO: 1 , or functional equivalent thereof.

[0254] In embodiments, the helper enzyme is excision positive. In embodiments, the helper enzyme is integration deficient. In embodiments, the helper enzyme has decreased integration activity relative to a helper enzyme comprising an amino acid sequence of SEQ ID NO: 1 , or functional equivalent thereof. In embodiments, the helper enzyme has increased excision activity relative to a helper enzyme comprising an amino acid sequence of SEQ ID NO: 1 , or functional equivalent thereof.

[0255] In embodiments, the helper enzyme of the present disclosure comprises a substitution selected from TABLE 1 and / or TABLE 2, a deletion at positions about 1-35, or about 1-45, or about 1-68, or substitutions and positions corresponding thereto relative to SEQ ID NO: 1 , and a DNA binding domain selected from TABLE 8-17. In embodiments, the enzyme is MLT. In embodiments, the helper enzyme comprising an N terminal deletion is further fused to a DNA binding domain. In embodiments, the DNA binder comprises TALEs, ZnF, and / or dCas (TABLES 8- 17). In embodiments, the helper enzyme further comprises a targeting element. In embodiments, the helper enzyme is capable of inserting a donor comprising a transgene in a genomic safe harbor site (GSHS) and / or wherein the targeting element is suitable for directing the helper enzyme to a GSHS.

[0256] In embodiments, the binding of a GSHS of a nucleic acid molecule in a mammalian cell is with high target specificity, relative to a control. In embodiments, the control is a composition comprising a helper enzyme comprising an amino acid sequence of SEQ ID NO: 1 or a nucleic acid comprising one or more of the Exc+lnt- mutations and DNA binders in TABLES 1-17 and 19-20 or a codon-optimized forms thereof.

[0257] In embodiments, the targeting element is able to direct a transposition machinery to the GSHS of a nucleic acid molecule in a mammalian cell. In embodiments, the targeting element is able to direct a transposition machinery to a GSHS or or intron 1 of a disease gene (e.g., HPRT, CFTR) of a nucleic acid molecule in a mammalian cell. In embodiments, the GSHS is in an open chromatin location in a chromosome.

[0258] In embodiments, the GSHS is selected from the human Rosa26 locus, adeno-associated virus site 1 (AAVS1), chromosome 4 GSHS, chromosome 22 GSHS, chemokine (C-C motif) receptor 5 (CCR5) gene, HIV-1 coreceptor. In embodiments, the GSHS is a located at the human Rosa26 locus. In embodiments, the GSHS is an adeno-associated virus site 1 (AAVS1). In embodiments, the GSHS is located on human chromosome 2, 3, 4, 6, 10, 11, 17, 19, 22, or X.

[0259] In embodiments, the DNA binding element that directs the transposase to GSHS is selected from TABLES 3-17 and TABLES 19-20. In embodiments, the guide RNA for targeting human genomic safe harbor sites using any of the gRNA based targeting elements, is selected from GUIDE 44, GUIDE 45-C, GUIDE 46-C, SPG GUIDE1-C, SPG GUIDE2-C, SPG GUIDE3-C, SPG GUIDE4-C, SPG GUIDE5-C, SPG GUIDE6-C, SPG GUIDE7-C, SPG GUIDES, SPG GUIDE9, SPG GUIDE10, SPG GUIDE11, SPG GUIDE12, SPG GUIDE13, SPG GUIDE14, GUIDE N1 , GUIDE N2, GUIDE N3- C, GUIDE 012, GUIDE 013, GUIDE 014-C, AAV GUIDE 12, AAV GUIDE 13c, AAV GUIDE 14, AAV GUIDE 14c, AAV GUIDE 15, AAV GUIDE 16, AAV GUIDE 17, AAV GUIDE 18, AAV GUIDE 19, AAV GUIDE 20, AAV GUIDE 21 , AAV GUIDE 22, AAV GUIDE 23, AAV GUIDE 24, AAV GUIDE 25, AAV GUIDE 26, AAV GUIDE 27, AAV GUIDE 28, AAV GUIDE 29, AAV GUIDE 30c, AAV GUIDE 31 , AAV GUIDE 32c, AAV GUIDE 33c, AAV GUIDE 34, AAV GUIDE 35, Guide C4-1 , Guide C4-2, Guide C4-3, Guide C4-4, Guide C4-5, Guide C4-6, Guide C4-7, Guide C4-8, Guide C4-9, Guide 04-10, Guide 04-11 , Guide 04-12, Guide 04-13, Guide 04-14, Guide 04-15, Guide 04-16, Guide 04-17, Guide 04-18, Guide 04-19, Guide 04-20, Guide C4A1 , Guide C4A2, Guide C4A3, Guide C4A4, Guide C4A5, Guide C4A6, Guide C4A7, Guide C4A8, Guide C4A9, Guide C4A10, Guide C4A11, Guide C4A12, Guide C4A13, Guide C4A14, Guide C4A15, Guide C4A16, Guide C4A17, Guide C4A18, Guide C4A19, Guide C4A20, Guide C22-1 , Guide C22-2, Guide 022-3, Guide C22-4, Guide C22-5, Guide 022-6, Guide C22-7, Guide C22-8, Guide 022-9, Guide 022-10, Guide 022-11 , Guide 022-12, Guide 022-13, Guide 022-14, Guide 022-15, Guide 022-16, Guide C22-17, Guide C22- 18, Guide C22-19, Guide C22-20, Guide C22A1 , Guide C22A2, Guide C22A3, Guide C22A4, Guide C22A5, Guide C22A6, Guide C22A7, Guide C22A8, Guide C22A9, Guide C22A10, Guide C22A11, Guide C22A12, Guide C22A13, Guide C22A14, Guide C22A15, Guide C22A16, Guide C22A17, Guide C22A18, Guide C22A19, Guide C22A20, Guide CX-1 , Guide CX-2, Guide CX-3, Guide CX-4, Guide CX-5, Guide CX-6, Guide CX-7, Guide CX-8, Guide CX-9, Guide CX-10, Guide CX-11, Guide CX-12, Guide CX-13, Guide CX-14, Guide CX-15, Guide CX-16, Guide CX-17, Guide CX- 18, Guide CX-19, Guide CX-20, TALES: R1 , R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 , R12, R13, R14, AAV1c, AAV2c, AAV3c, AAV4c, AAV5c, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV13c, AAV14c, AAV15c, AAV16c, AAV17c, TALE4-R001 , TALE4-R002, TALE4-F003, TALE4-F004, TALE4-F005, TALE4-F006, TALE4-R007, TALE4-F008, TALE4-R009, TALE4-F010, TALE4-F011, TALE4-F012, TALE4-R013, TALE4-F014, TALE4-F015, TALE4-R016, TALE4-F017, TALE4-F018, TALE4-F019, TALE4-F020, TALE22F-R001, TALE22-F002, TALE22-F003, TALE22-F004, TALE22-F005, TALE22-F006, TALE22-F007, TALE22-F008, TALE22-R009, TALE22-F010, TALE22- F001A, TALE22-F002A, TALE22-R03A, TALE22-F004A, TALE22-F005A, TALE22-F006A, TALE22-R007A, TALE22- R008A, TALE22-R009A, TALE22-F010A, TALE F002, TALE F003, TALE F004, TALE FOOT, TALE F008, TALE F009, TALE R002, TALE R003, TALE R004, TALE R005, TALE R007, TALE F020, TALE F021 , TALE F030, TALE F031 , TALE F034, TALE F036, TALE F037, TALE F040, TALE R022, TALE R033, TALE R035, TALE R038, TALE R039, Zinc Fingers: ZnF3a, ZnF5a, ZnF5b, ZnF5c, ZnF5d, ZnF5e, ZnF5f, ZnF5g, ZnF5h, ZnF12a, ZnF13a, ZnF13b, ZnF13c, ZnF11a, ZnF10a, ZnF12b, ZnF13b, ZnF14a, ZnF15a, ZnF16a, ZnF17a, ZnF18a, ZnF19a, ZnF20b, ZnF21 b, ZnF22a, ZnF23a, ZnF24a, ZnF31 F, ZnF32F, ZnF33F, ZnF34F, ZnF35F, ZnF36F, ZnF37R, ZnF38R, ZnF39R, ZnF1a, ZnF1 b, ZnF2a, ZnF3a, ZnF3b, ZnF5aR, ZnF5bR, ZnF6aR, ZnF6bR, ZnF1OF, ZnF11 F, ZnF12F, ZnF13F, ZnF14R, ZnF15R, ZnF14R, ZnF15R, ZnF41 F, ZnF42F, ZnF43F, ZnF44R, ZnF45R, ZnF46R, ZnF47R, ZnF48R.

[0260] In embodiments, the GSHS is selected from TALC1 , TALC2, TALC3, TALC4, TALC5, TALC7, TALC8, AVS1 , AVS2, AVS3, ROSA1 , ROSA2, TALER1 , TALER2, TALER3, TALER4, TA-LER5, SHCHR2-1 , SHCHR2-2, SHCHR2-3, SHCHR2-4, SHCHR4-1 , SHCHR4-2, SHCHR4-3, SHCHR6-1, SHCHR6-2, SHCHR6-3, SHCHR6-4, SHCHR10-1, SHCHR10-2, SHCHR10-3, SHCHR10-4, SHCHR10-5, SHCHR11-1 , SHCHR11-2, SHCHR11-3, SHCHR17-1, SHCHR17-2, SHCHR17-3, and SHCHR17-4.

[0261] In embodiments, the GSHS comprises one or more TTAA integration sites. In embodiments, the targeting element directs the helper enzyme to either one or more nucleic acid binding sites that are upstream and / or downstream of the TTAA integration sites or to the TTAA integration sites. In embodiments, the targeting element directs the helper enzyme to one or more nucleic acid binding sites that are upstream and / or downstream of the TTAA integration sites and within about 5 to about 30 base pairs of the TTAA integration sites or within about 15 to about 19 base pairs of the TTAA integration sites. In embodiments, the targeting element directs the helper enzyme to two nucleic acid binding sites of the TTAA integration sites, wherein a first site is upstream of TTAA and within about 5 to about 30 base pairs or about 15 to about 19 base pairs of the TTAA and a second site is downstream of TTAA and within about 5 to about 30 base pairs or about 15 to about 19 base pairs of the TTAA.

[0262] In embodiments, the targeting element is or comprises one or more of a Cas enzyme, which is optionally catalytically inactive and which is optionally associated with a guide RNA (gRNA), transcription activator-like effector (TALE) DNA binding domain (DBD), Zinc finger, catalytically inactive transcription factor, catalytically inactive nickase, a transcriptional activator, a transcriptional repressor, a recombinase, a DNA methyltransferase, a histone methyltransferase, a paternally expressed gene 10 (PEG10), and a transposon-encoded polypeptide D (TnsD) or a variant thereof. In embodiments, the targeting element comprises a TALE DBD. In embodiments, the TALE DBD comprises one or more repeat sequences. In embodiments, the TALE DBD comprises about 14, or about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences. In embodiments, the repeat sequences each independently comprises about 33 or 34 amino acids. In embodiments, the repeat sequences each independently comprises a repeat variable di-residue (RVD) at residue 12 or 13 of the 33 or 34 amino acids, respectively. In embodiments, the RVD recognizes one base pair in a target nucleic acid sequence. In embodiments, the RVD recognizes a C residue in the target nucleic acid sequence and is selected from HD, N(gap), HA, ND, and HI. In embodiments, the RVD recognizes a G residue in the target nucleic acid sequence and is selected from NN, NH, NK, HN, and NA. In embodiments, the RVD recognizes an A residue in the target nucleic acid sequence and is selected from Nl and NS. In embodiments, the RVD recognizes a T residue in the target nucleic acid sequence and is selected from NG, HG, H(gap), and IG.

[0263] In embodiments, the TALE DBD targets one or more of GSHS sites selected from TABLES 8-12 and TABLE 20.

[0264] In embodiments, the TALE DBD comprises one or more of RVD selected from TABLES 8-12 and TABLE 20, or variants thereof comprising about 1 , about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 mutations.

[0265] In embodiments, the gRNA targets one or more of GSHS sites selected from TABLES 3-7 and TABLE 19. In embodiments, the targeting element comprises a Cas9 enzyme associated with a gRNA. In embodiments, the Cas9 enzyme associated with a gRNA comprises a catalytically inactive dCas9 associated with a gRNA.

[0266] In embodiments, the targeting element is or comprises a Cas9 enzyme associated with a gRNA or a CasX enzyme associated with a gRNA.

[0267] In embodiments, the target element is or comprises a CasX enzyme associated with a gRNA, optionally wherein the catalytic inactive dCasX.

[0268] In embodiments, the catalytically inactive dCas9 comprises at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity to an amino acid sequence of SEQ ID NO: 6 or a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 5 or a codon-optimized form thereof.

[0269] In embodiments, the targeting element comprises a Cas12 enzyme associated with a gRNA. In embodiments, the targeting element comprises a catalytically inactive Cas12 associated with a gRNA, optionally wherein the catalytically inactive Cas12 is dCas12j, dCas12a or a small DCasX. In embodiments, the targeting element comprises a TnsC, TnsB, TnsA, TniQ, Cas6, Cas7, Cas8 enzyme associated with a gRNA.

[0270] In embodiments, the guide RNA is selected from TABLES 3-7 and TABLE 19, or variants thereof comprising about 1 , about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 mutations. In embodiments, the guide RNA targets one or more sites selected from TABLES 3-7 and TABLE 19. In embodiments, the zinc finger comprises one of the sequences selected from TABLES 13-17, or variants thereof comprising about 99, about 98, about 97, about 95, about 94, about 93, about 92, about 91 , about 90, about 89, about 88, about 87, about 86, about 85, about 84, about 83, about 82, about 81 , about 80 percent identity to the sequence. In embodiments, the zinc finger targets one or more sites selected from TABLES 13-17.

[0271] In embodiments, the targeting element comprises a nucleic acid binding component of a gene-editing system. In embodiments, the helper enzyme or variant thereof and the targeting element are connected. In embodiments, the helper enzyme and the targeting element are fused to one another or linked via a linker to one another. In embodiments, the composition comprises a linker connecting the helper enzyme and the targeting element. In embodiments, the linker comprises less than about 25 amino acids or 75 nucleotides. In embodiments, the flexible linker is of about 20, or about 30, or about 40, or about 50, or about 60 amino acid residues. In embodiments, the linker comprises about 10 amino acids to about 20 amino acids or about 12 amino acids to about 15 amino acids, or about 30 nucleotides to about 60 nucleotides or about 36 nucleotides to about 45 nucleotides. In embodiments, the linker is a covalent or non- covalent linker.

[0272] In embodiments, the linker is a flexible linker. In embodiments, the flexible linker is substantially comprised of glycine and serine residues, optionally wherein the flexible linker comprises (Gly4Ser)n, where n is an integer from 1-12. In embodiments, the linker is or comprises (GSS)4 or the linker is GS flanked on either side of a DNA binding domain, optionally TALE and ZnF. In embodiments, the linker comprises an amino acid sequence of AKLAGGAPAVGGGPKAADKFAATGGS (SEQ ID NO: 8) or a variant thereof having a substitution or deletion. In embodiments, the linker connects the targeting element to the N-terminus of the helper enzyme or connects the targeting element within the helper enzyme.

[0273] In embodiment, the linker is a non-covalent linker. In embodiments, the non-covalent linker comprises an epitope tag In embodiments, the non-covalent linker comprises an ALFA tag. In embodiments, the ALFA tag further comprises recombinant variable domains of heavy-chain-only antibodies (VHH) (NANOBODY). In embodiments, the non-covalent linker comprises recombinant variable domains of heavy-chain-only antibodies (VHH) (NANOBODY) bound to an ALFA-tag peptide (NbALFA). In embodiments, the non-covalent linker comprises recombinant variable domains of heavy-chain-only antibodies (VHH) (NANOBODY) bound to an ALFA-tag peptide (NbALFA). In embodiments, the linker comprisesis a monobody. In embodiments, the linker comprises anis an ALFAIfa tag and monobody.

[0274] In embodiments, the TnsD comprises a nucleic acid binding component of a gene-editing system. In embodiments, the enzyme or variant thereof (optionally, wherein the enzyme is a helper enzyme, optionally, wherein the helper enzyme is reconstructed from Myotis lucifugus) and the TnsD are connected. In embodiments, the targeting element comprises a TniQ subdomain of TnsD. In embodiments, the helper enzyme and the TniQ subdomain of TnsD are fused to one another or linked via a linker to one another. In embodiments, the linker is a flexible linker. In embodiments, the flexible linker is substantially comprised of glycine and serine residues, optionally wherein the flexible linker comprises (Gly4Ser)n, where n is an integer from 1-12. In embodiments, the flexible linker is of about 20, or about 30, or about 40, or about 50, or about 60 amino acid residues. In embodiments, the helper enzyme is directly fused to the N-terminus of the TniQ subdomain of TnsD.

[0275] In embodiments, the E. coll TnsD comprises at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity to an amino acid sequence of SEQ ID NO: 12. In embodiments, the TnsD comprises a truncated TnsD. In embodiments, the TnsD comprises the TniQ subdomain. In embodiments, the TnsD is truncated at its C-terminus. In embodiments, the TnsD is truncated at its N-terminus. In embodiments, the TnsD or variant thereof comprises a zinc finger motif. In embodiments, the zinc finger motif comprises a C3H-type motif (e.g., CCCH).

[0276] In embodiments, the TnsD binds at or near an attTn7 attachment site. In embodiments, the TnsD binds at or near a region downstream of the glmS gene. GlmS (L-glucosamine--fructose-6-phosphate aminotransferase) is highly conserved and found in a wide variety of organisms from bacteria to humans. In embodiments, the TnsD binding region of glmS encodes the active site region of GlmS. In embodiments, TnsD binds at or near the human homologs of glmS, e.g., gfpt-1 and gfpt-2. In embodiments, TnsD binds the human glmS homologs gfpt-1 and gfpt-2. In embodiments, the transgene is inserted into atiTn7.

[0277] In embodiments, the helper enzyme or variant thereof is able to directly or indirectly cause transposition of a target gene. In embodiments, the helper enzyme or variant thereof is able to directly or indirectly interact and / or form a complex with one or more proteins or nucleic acids.

[0278] Construct

[0279] In some embodiments, the composition (e.g., without limitation, a hyperactive helper of the present disclosure), system, or method further comprising a nucleic acid encoding a donor comprising a transgene to be integrated. In some embodiments, the transgene is defective or substantially absent in a disease state. In some embodiments, the transgene comprises a cargo nucleic acid sequence and a first and a second donor end sequences. In some embodiments, the cargo nucleic acid sequence is flanked by the first and the second donor end sequences.

[0280] In some embodiments, the donor end sequences are selected from nucleotide sequences of SEQ ID NO: 3 and / or SEQ ID NO: 4, or a nucleotide sequence having at least about 90% identity thereto.

[0281]

[0282] In some embodiments, the end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the at least one repeat from the nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 3 is positioned at the 5’ end of the donor. In some embodiments, the end sequences can further include at least one repeat from a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the at least one repeat from the nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 4 is positioned at the 3’ end of the donor.

[0283] In embodiments, the left ITR comprises a nucleotide sequence of ttaacacttggattgcgggaaacgagttaa gtcggctcgcgtgaattgcgcgtactccgcgggagccgtcttaactc (SEQ ID NO: 878), or a variant thereof comprising substitutions or deletions.

[0284] In embodiments, the right ITR comprises a nucleotide sequence of catttcaatc gaacccatac ttcaaaagat ataggcattt taaactaact ctgattttgc gcgggaaacc taaataattg cccgcgccat cttatatttt ggcgggaaat tcacccgaca ccgtagtgtt aa (SEQ ID NO: 879), or a variant thereof comprising substitutions or deletions.

[0285] In embodiments, the ITRs or the end sequences are selected from SEQ ID NO: 878 and SEQ ID NO: 879, or a nucleotide sequence having at least about 90% identity thereto, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

[0286] In embodiments, the end sequences include at least one repeat with a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 878. In embodiments, the at least one repeat from the nucleotide sequence having at least about 90% identity, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to the nucleotide sequence of SEQ ID NO: 878 is positioned at the 5' end of the donor. In embodiments, the end sequences can further include at least one repeat with a nucleotide sequence having at least about 90% identity, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to the nucleotide sequence of SEQ ID NO: 879. In embodiments, the at least one repeat from the nucleotide sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to the nucleotide sequence of SEQ ID NO: 879 is positioned at the 3' end of the donor.

[0287] In some embodiments, the helper enzyme or variant thereof is incorporated into a vector or a vector-like particle. In some embodiments, the vector or a vector-like particle comprises one or more expression cassettes. In some embodiments, the vector or a vector-like particle comprises one expression cassette. In some embodiments, the expression cassette further comprises the helper enzyme or variant thereof, the transgene, the donor end sequences, or a combination thereof.

[0288] In some embodiments, the helper enzyme or variant thereof, the transgene, the donor end sequences, or a combination thereof are incorporated into one or more vectors or vector-like particles. In some embodiments, the helper enzyme or variant thereof, the transgene, the donor end sequences, or combination thereof are incorporated into a same vector or vector-like particle. In some embodiments, the helper enzyme or variant thereof, the transgene, the donor end sequences, or combination thereof is incorporated into different vectors vector-like particles. In some embodiments, the vector or vector-like particle is nonviral. In some embodiments, the composition comprises DNA, RNA, or both. In some embodiments, the helper enzyme or variant thereof is in the form of RNA.

[0289] In embodiments, the composition is a nucleic acid, optionally an RNA. In embodiments, the RNA is or comprises messenger RNA (mRNA). In embodiments, the mRNA is or comprises modified mRNA (mmRNA). In embodiments, the mmRNA comprises one or more of a 5’-m7G cap (capO, cap1, or cap2), a pseudouridine or n-methyl-pseudouridine substitution, and a poly-A tail of about 30, or of about 50, or of about 100, or of about 150 nucleotides in length.

[0290] In embodiments, the donor is under the control of at least one tissue-specific promoter. In embodiments, the at least one tissue-specific promoter is a single promoter. In embodiments, the at least one tissue-specific promoter is under the control of a dual promoter or a tandem promoter.

[0291] In embodiments, the transgene to be integrated comprises at least one gene of interest. In embodiments, the transgene to be integrated comprises one gene of interest. In embodiments, the transgene to be integrated comprises two genes of interest.

[0292] In embodiments, the at least one gene of interest comprises peptides for linking genes of interest. In embodiments, the peptides are 2A self-cleaving peptides, or functional variants thereof, wherein the 2A self-cleaving peptide is optionally selected from P2A, E2A, F2A, and T2A, or derivative thereof.

[0293] In embodiments, the at least one gene of interest is linked to polynucleotide comprising a sequence comprising a 5'- miRNA, a sense and antisense miRNA pair, and / or a 3’-miRNA.

[0294] In embodiments, the donor is used in combination with a gene silencing construct. In embodiments, there is provided a method of gene therapy in a cell comprising contacting the cell with a construct comprising the helper enzyme and / or donor or transgene described herein and / or a gene silencing construct. In embodiments, there is provided a method of gene replacement and silencing comprising contacting the cell with a construct comprising the helper enzyme and / or donor or transgene described herein and / or a gene silencing construct. In embodiments, there is provided a method of gene therapy in a subject comprising administering a construct comprising the helper enzyme and / or donor or transgene described herein and / or a gene silencing construct. In embodiments, there is provided a method of gene replacement and silencing in a subject comprising administering a construct comprising the helper enzyme and / or donor or transgene described herein and / or a gene silencing construct. In embodiments, the donor or transgene described herein and the gene silencing construct are separate constructs. In embodiments, the donor or transgene described herein and the gene silencing construct are separate D NA constructs.

[0295] In embodiments, the donor is a dual gene construct. In embodiments, the donor is dual gene construct which comprises DNA. In embodiments, the donor is a bicistronic construct. In embodiments, the donor is a multicistrionic construct. In embodiments, the bicistronic construct allows for the contemporaneous expression of two proteins, e.g, separately from the same RNA transcript. In embodiments, the multicistrionic construct allows for the contemporaneous expression of multiple proteins, e.g., separately from the same RNA transcript.

[0296] In embodiments, the bicistronic and / or multicistronic construct comprises a gene of interest and a genetic silencing element. In embodiments, the genetic silencing element provides regulation of gene expression in a cell to prevent, reduce, or ablate the expression of a certain gene. In embodiments, the gene silencing element is capable of silencing during either transcription or translation. In embodiments, the gene silencing element is capable of gene knockdown or knockout. Accordingly, in embodiments, the donor is suitable for contemporaneous “knocking in” and “knocking out” of two or more genes. For example, in embodiments, a gene of interest is provided to a cell to have a beneficial effect and a deleterious gene is knocked out of a cell to reduce or eliminate a deleterious effect.

[0297] In embodiments, the gene silencing element is or comprises an RNA-based gene inhibitor or silencer. In embodiments, the gene silencing element is or comprises a short interfering RNA (siRNA), a microRNA (miRNA) and / or a short hairpin RNA (shRNA). embodiments, the donor is a bicistronic and / or multicistronic construct comprising one or more genes of interest, e.g., a transgene to be integrated, optionally wherein the transgene is defective or substantially absent in a disease state and one or more gene silencing element, e.g, one or more siRNA, miRNA, and shRNA. In embodiments, the donor is a bicistronic and / or multicistronic construct comprising one or more genes of interest, e.g, a transgene to be integrated, optionally wherein the transgene is defective or substantially absent in a disease state and one or more gene silencing element, e.g., one or more siRNA, miRNA, and shRNA and the donor is flanked by a first and a second donor end sequences.

[0298] In embodiments, the present compositions and methods provide for the helper enzyme or variant thereof excising and / or integrating both one or more one or more genes of interest, e.g, a transgene to be integrated, and one or more gene silencing element, e.g., one or more siRNA, miRNA, and shRNA. In embodiments, the present compositions and methods provide for gene replacement and silencing via a signal donor construct.

[0299] N or C Terminal Deletion Variants

[0300] In aspects, the present disclosure further provides a hyperactive helper enzyme with a deletion of various amino acids at either the N or C terminus. In embodiments, the hyperactive helper enzyme comprises a deletion in the N-terminus. In embodiments, the hyperactive helper enzyme comprises a deletion in the C-terminus. In embodiments, the deletion in the N or C termini begins at various positions. In embodiments, the deletion in the N or C termini comprises various lengths.

[0301] In embodiments, the helper enzyme of the present disclosure comprises a deletion at positions about 2-35, or about 2-45, or about 2-68, or positions corresponding thereto, wherein the positions are relative to SEQ ID NO: 1. In embodiments, the helper enzyme of the present disclosure comprises a deletion at positions about 2-35, or about 2- 45, or about 2-68, or positions corresponding thereto, wherein the positions are relative to SEQ ID NO: 2. In embodiments, the helper enzyme of the present disclosure comprises a deletion at positions about 2-35 (SEQ ID NO: 504), or about 2-45 (SEQ ID NO: 506), or about 2-68 (SEQ ID NO: 508), or positions corresponding thereto, wherein the positions are relative to SEQ ID NO: 1 including the mutations in TABLE 1 and TABLE 2 and DNA binders in TABLE 8-17, and 20. In embodiments, the helper enzyme is an MLT. In embodiments, the deletion comprises an N terminal deletion. In embodiments, the N terminal deletion yields reduced or ablated off-target effects of the helper enzyme compared to the helper enzyme without the N terminal deletion. In embodiments, the helper enzyme comprising the N terminal deletion is or comprises SEQ ID NO: 504. In embodiments, the helper enzyme comprising the N terminal is or comprises SEQ ID NO: 506. In embodiments, the helper enzyme comprising the N terminal is or comprises SEQ ID NO: 508. In embodiments, the mutant with an N or C terminal deletion is further fused to a DNA binder. In embodiments, the DNA binder comprises TALEs, ZnF, and / or dCas.

[0302] In embodiments, the hyperactive helper enzyme comprises a deletion from an N- or C-terminus of the polypeptide having an amino acid sequence of SEQ ID NO: 2.

[0303]

[0304] In embodiments, the hyperactive helper enzyme comprises a deletion of about 5, or about 10, or about 20, or about 30, or about 40, or about 50, or about 60, or about 70, or about 80, or about 90, or about 100, or about 110, or about 120, or about 130, or about 140, or about 150, or about 160 amino acids from an N-terminus of the polypeptide having an amino acid sequence of SEQ ID NO: 2, or a sequence having at least about 90% identity thereto.

[0305] In embodiments, the hyperactive helper enzyme comprises a deletion of about 5, or about 10, or about 20, or about 30, or about 40, or about 50, or about 60, or about 70, or about 80, or about 90, or about 100, or about 110, or about 120, or about 130, or about 140, or about 150, or about 160 amino acids from an N-terminus of the polypeptide having an amino acid sequence of SEQ ID NO: 1 , or a sequence having at least about 90% identity thereto.

[0306] In embodiments, the hyperactive helper enzyme with deletion from the N-terminus comprises SEQ ID NO: 504, SEQ ID NO: 506, SEQ ID NO: 508, or SEQ ID NO: 510, or a sequence having at least about 90% identity thereto.

[0307] SEQ ID NO: 503: N-terminal deletion Myositis lucifugus (hyperactive helper) nucleotide sequence (N1; nucleotide 4- 105 deletion). 1614 bp

[0308] 1 ATGAGCTCTG ACGACGAGGT GATGGTGCGG CCCAGAACCC TGAGACGGAG AAGAATCAGC In embodiments, the hyperactive helper enzyme comprises a deletion of about 5, or about 10, or about 20, or about 30, or about 40, or about 50, or about 60, or about 70, or about 80, or about 90, or about 100, or about 110, or about 120, or about 130, or about 140, or about 150, or about 160 amino acids from an C-terminus of the polypeptide having an amino acid sequence of SEQ ID NO: 2.

[0309] In embodiments, the hyperactive helper enzyme comprises a deletion of about 5, or about 10, or about 20, or about 30, or about 40, or about 50, or about 60, or about 70, or about 80, or about 90, or about 100, or about 110, or about 120, or about 130, or about 140, or about 150, or about 160 amino acids from an C-terminus of the polypeptide having an amino acid sequence of SEQ ID NO: 1.

[0310] In embodiments, the hyperactive helper enzyme with deletion from the C-terminus comprises SEQ ID NO: 512 or SEQ ID NO: 514.

[0311]

[0312] In embodiments, the hyperactive helper enzyme comprises a deletion at positions about 1-5, or about 1-15, or about 1-25, or about 1-35, or about 1-45, or about 1-55, or about 1-65, or about 1-75, or about 1-85 , or about 1-95, or about 1-105, or about 1-115, or about 1-125, or about 1-135, or about 1-145, or about 1-155 or about 2-35, or about 2-45, or about 2-68, or positions corresponding thereto, wherein the positions are relative to SEQ ID NO: 2.

[0313] In embodiments, the hyperactive helper enzyme comprises a deletion at positions about 1-5, or about 1-15, or about 1-25, or about 1-35, or about 1-45, or about 1-55, or about 1-65, or about 1-75, or about 1-85 , or about 1-95, or about 1-105, or about 1-115, or about 1-125, or about 1-135, or about 1-145, or about 1-155 or about 2-35, or about 2-45, or about 2-68, or positions corresponding thereto, wherein the positions are relative to SEQ ID NO: 1.

[0314] In aspects, the N terminal deletion variant is further fused one or more DNA binders. In embodiments, the DNA binder comprises, without limitation, dCasx, dCas9, dCas12j, TALEs, and ZnF. In embodiments, the DNA binder guides donor insertion to specific genomic sites. In embodiments, the C terminal deletion variant is further fused one or more DNA binders. In embodiments, the N terminal deletion variant is further fused one or more DNA binders at the N-terminus. In embodiments, the N terminal deletion variant is further fused one or more DNA binders at the C-terminus. In embodiments, the C terminal deletion variant is further fused one or more DNA binders at the N-terminus. In embodiments, the C terminal deletion variant is further fused one or more DNA binders at the C-terminus.

[0315] In embodiments, the hyperactive helper mutant exhibits improved excision frequencies compared to those without the terminal deletions and / or DNA binders. In embodiments, the hyperactive helper mutant exhibits improved integration frequencies compared to those without the terminal deletions and / or DNA binders. In embodiments, the hyperactive helper mutant exhibits improved excision and integration frequencies compared to those without the terminal deletions and / or DNA binders.

[0316] In embodiments, the N or C terminal mutant exhibit different Exc+ / lnt- frequencies. In embodiments, deletion of either N or C termini can result in MLT mutants with higher excision activity. In embodiments, N-terminal deletion yields a mutant with decreased integration compared to mutant without N-terminal deletion. In embodiments, C-terminal deletion yields a mutant with reduced excision and no integration.

[0317] In embodiments, the N or C terminal deletion yields reduced or ablated off-target effects of the helper enzyme compared to the helper enzyme without the N or C terminal deletion.

[0318] Host Cell

[0319] In some aspects, the present disclosure further provides a host cell comprising the composition in accordance with embodiments of the present disclosure.

[0320] Methods

[0321] In certain embodiments, the present disclosure provides a method for inserting a gene into the genome of a cell, comprising contacting a cell with the composition of the present disclosure or host cell of the present disclosure. In some embodiments, the method further comprises contacting the cell with a polynucleotide encoding a donor. In embodiments, the helper enzyme is suitable of inserting a donor nucleic acid comprising a transgene in a genomic safe harbor site (GSHS) and / or wherein the targeting element is suitable for directing the helper enzyme to a GSHS. In embodiments, a donor DNA and a helper RNA are suitable for transfection at a donor DNA to helper RNA ratio of about 1 to about 4, or about 1 to about 2, or about 1 to about 1. In embodiments, the composition of the present disclosure further comprises a nucleic acid encoding a donor comprising a transgene to be integrated, optionally wherein the transgene is defective or substantially absent in a disease state. In embodiments, the transgene comprises a cargo nucleic acid sequence and a first and a second donor end sequences. In embodiments, the cargo nucleic acid sequence is flanked by the first and the second donor end sequences.

[0322] In some embodiments, the donor comprises a gene encoding a complete polypeptide.

[0323] In some embodiments, the donor comprises a gene which is defective or substantially absent in a disease state.

[0324] In certain embodiments, the present disclosure provides a method for treating a disease or disorder ex vivo, comprising contacting a cell with the composition of the present disclosure or host cell of the present disclosure and administering the cell to a subject in need thereof.

[0325] In certain embodiments, the present disclosure provides a method for treating a disease or disorder in vivo, comprising administering the composition of the present disclosure or host cell of the present disclosure to a subject in need thereof.

[0326] Transgene

[0327] In embodiments, the transgene is an exogenous wild-type gene that, e.g., corrects a defective function of one or more mutations in a recipient. For instance, in embodiments, the recipient may have a mutation that provides a disease phenotype (e.g., a defective or absent gene product). In embodiments, the donor system or method of the present disclosure provides a correction that restores the gene product and diminishes the disease phenotype.

[0328] In embodiments, the transgene is a gene that replaces, inactivates, or provides suicide or helper functions.

[0329] In embodiments, the transgene and / or disease to be treated is one or more of:

[0330] • beta-thalassemia: BCL11a or p-globin or |3A-T87Q-globi n,

[0331] • LCA: RPE65,

[0332] • LHON: ND4,

[0333] • Achromatopsia: CNGA3 or CNGA3 / CNGB3,

[0334] • Choroideremia: REP1,

[0335] PKD: RPK (Red cell PK), • Hemophilia: F8,

[0336] • ADA-SCID: ADA,

[0337] • Fabry disease: GLA,

[0338] • MPS type I: IDUA, and

[0339] • MPS type II: / DS.

[0340] In embodiments, the donor comprises a gene encoding a complete polypeptide. In embodiments, the donor comprises a gene which is defective or substantially absent in a disease state.

[0341] In embodiments, the transfecting of the cell is carried out using electroporation or calcium phosphate precipitation.

[0342] In embodiments, the transfecting of the cell is carried out using a lipid vehicle, optionally N-[1 -(2,3-dioleoyloxy)propyl]- N,N,N-trimethylammonium chloride (DOTMA), 1,2-bis(oleoyloxy)-3-3-(trimethylammonia) propane (DOTAP), or 1 ,2- dioleoyl-3-dimethylammonium-propane (DODAP), dioleoylphosphatidylethanolamine (DOPE), cholesterol, LIPOFECTIN (cationic liposome formulation), LIPOFECTAMINE (cationic liposome formulation), LIPOFECTAMINE 2000 (cationic liposome formulation), LIPOFECTAMINE 3000 (cationic liposome formulation), TRANSFECTAM (cationic liposome formulation), a lipid nanoparticle, or a liposome and combinations thereof.

[0343] In embodiments, the transfecting of the cell is carried out using a lipid selected from one or more of the following categories: cationic lipids; anionic lipids; neutral lipids; multi-valent charged lipids; and zwitterionic lipids. In embodiments, a cationic lipid may be used to facilitate a charge-charge interaction with nucleic acids. In embodiments, the lipid is a neutral lipid. In embodiments, the neutral lipid is dioleoylphosphatidylethanolamine (DOPE), 1 ,2-Dioleoyl- sn-glycero-3-phosphocholine (DOPC), or cholesterol. In embodiments, cholesterol is derived from plant sources. In other embodiments, cholesterol is derived from animal, fungal, bacterial, or archaeal sources. In embodiments, the lipid is a cationic lipid. In embodiments, the cationic lipid is N-[1 -(2,3-dioleoyloxy)propyl]-N,N, N-trimethylammonium chloride (DOTMA), 1,2-bis(oleoyloxy)-3-3-(trimethylammonia) propane (DOTAP), or 1 ,2-dioleoyl-3- dimethylammonium-propane (DODAP). In embodiments, one or more of the phospholipids 18:0 PC, 18:1 PC, 18:2 PC, DMPC, DSPE, DOPE, 18:2 PE, DMPE, or a combination thereof are used as lipids. In embodiments, the lipid is DOTMA and DOPE, optionally in a ratio of about 1 : 1. In embodiments, the lipid is DHDOS and DOPE, optionally in a ratio of about 1 : 1. In embodiments, the lipid is a commercially available product (e.g, LIPOFECTIN (cationic liposome formulation), LIPOFECTAMINE (cationic liposome formulation), LIPOFECTAMINE 2000 (cationic liposome formulation), LIPOFECTAMINE 3000 (cationic liposome formulation) (Life Technologies)).

[0344] In embodiments, the transfecting of the cell is carried out using a cationic vehicle, optionally LIPOFECTIN or TRANSFECTAM.

[0345] In embodiments, the transfecting of the cell is carried out using a lipid nanoparticle or a liposome. In embodiments, the method is helper virus-free.

[0346] Epigenetic regulatory elements can be used to protect a transgene from unwanted epigenetic effects when placed near the transgene on a vector, including the transgene. See Ley et al., PloS One vol. 8,4 e62784. 30 Apr. 2013, doi:10.1371 / journaL pone.0062784. For example, MARs were shown to increase genomic integration and integration of a transgene while preventing heterochromatin silencing, as exemplified by the human MAR 1-68. See id.; see also Grandjean et al., Nucleic Acids Res. 2011 Aug; 39(15):e104. MARs can also act as insulators and thereby prevent the activation of neighboring cellular genes. Gaussin et al., Gene Then. 2012 Jan; 19(1 ): 15-24. It has been shown that a piggyBac donor containing human MARs in CHO cells mediated efficient and sustained expression from a few transgene copies, using cell populations generated without an antibiotic selection procedure. See Ley et al. (2013).

[0347] In embodiments, the cell is further transfected with a third nucleic acid having at least one chromatin element, wherein the at least one chromatin element is optionally a Matrix Attachment Region (MAR) element. MARs are expressionenhancing, epigenetic regulator elements which are used to enhance and / or facilitate transgene expression, as described, for example, in PCT / IB2010 / 002337 (WO2011033375), which is incorporated by reference herein in its entirety. A MAR element can be located in cis or trans to the transgene.

[0348] In embodiments, the transgene has a size of 100,000 bases or less, e.g, about 100,000 bases, or about 50,000 bases, or about 30,000 bases, or about 10,000 bases, or about 5,000 bases, or about 10,000 to about 100,000 bases, or about 30,000 to about 100,000 bases, or about 50,000 to about 100,000 bases, or about 10,000 to about 50,000 bases, or about 10,000 to about 30,000 bases, or about 30,000 to about 50,000 bases.

[0349] In embodiments, the transgene has a size of about 200,000 bases or less, e.g., about 200,000 bases, or about 10,000 to about 200,000 bases, or about 30,000 to about 200,000 bases, or about 50,000 to about 200,000 bases, or about 100,000 to about 200,000 bases, or about 150,000 to about 200,000 bases.

[0350] In embodiments, the insertion size of a gene is at least about 1,000 bases, or at least about 1 ,500 bases, or at least about 2,000 bases, or at least about 2,500 bases, or at least about 3,000 bases, or at least about 3,500 bases, or at least about 4,000 bases, or at least about 4,500 bases, or at least about 5,000 bases, or at least about 5,500 bases, or at least about 6,000 bases, or at least about 6,500 bases, or at least about 7,000 bases, or at least about 7,500 bases.

[0351] Targeting Chimeric Constructs

[0352] In aspects, the present disclosure provides for a donor system, e.g., in embodiments, a helper enzyme comprises a targeting element.

[0353] In embodiments, the helper enzyme associated with the targeting element, is capable of inserting the donor comprising a transgene, optionally at a TA dinucleotide site or a TTAA (SEQ ID NO: 440) tetranucleotide site in a genomic safe harbor site (GSHS). In embodiments, the helper enzyme associated with the targeting element, is capable of inserting the donor comprising a transgene, optionally at a TA dinucleotide site or a ttTTAAaa (SEQ ID NO: 880) octanucleotide site in a genomic safe harbor site (GSHS). In embodiments, the helper enzyme associated with the targeting element, is capable of inserting the donor comprising a transgene, optionally at a TA dinucleotide site or a taTTAAta (SEQ ID NO: 881) octanucleotide site in a genomic safe harbor site (GSHS).

[0354] In embodiments, the helper enzyme associated with the targeting element has one or more mutations which confer hyperactivity.

[0355] In embodiments, the helper enzyme associated with the targeting element has gene cleavage (Exc) and / or gene integration (lnt+) activity.

[0356] In embodiments, the helper enzyme associated with the targeting element has gene cleavage (Exc) and / or a lack of gene integration (Int-) activity.

[0357] In embodiments, the targeting element comprises one or more proteins or nucleic acids that are capable of binding to a nucleic acid.

[0358] In embodiments, the targeting element comprises one or more of a of a gRNA, optionally associated with a Cas enzyme, which is optionally catalytically inactive, transcription activator-like effector (TALE), Zinc finger, catalytically inactive transcription factor, nickase, a transcriptional activator, a transcriptional repressor, a recombinase, a DNA methyltransferase, a histone methyltransferase, and paternally expressed gene 10 (PEG10).

[0359] In embodiments, the targeting element comprises a transcription activator-like effector (TALE) DNA binding domain (DBD).

[0360] In embodiments, the TALE DBD comprises one or more repeat sequences. In embodiments, the TALE DBD comprises about 14, or about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences. In embodiments, the TALE DBD repeat sequences comprise 33 or 34 amino acids. In embodiments, the TALE DBD repeat sequences comprise a repeat variable di-residue (RVD) at residue 12 or 13 of the 33 or 34 amino acids. In embodiments, the RVD recognizes one base pair in the nucleic acid molecule. In embodiments, the RVD recognizes a C residue in the nucleic acid molecule and is selected from HD, N(gap), HA, ND, and HI. In embodiments, the RVD recognizes a G residue in the nucleic acid molecule and is selected from NN, NH, NK, HN, and NA. In embodiments, the RVD recognizes an A residue in the nucleic acid molecule and is selected from Nl and NS. In embodiments, the RVD recognizes a T residue in the nucleic acid molecule and is selected from NG, HG, H(gap), and IG. In embodiments, the GSHS is in an open chromatin location in a chromosome. In embodiments, the GSHS is selected from adeno-associated virus site 1 (AAVS1), chemokine (C-C motif) receptor 5 (CCR5) gene, HIV-1 coreceptor, chromosome 4 GSHS, chromosome 22 GSHS and human Rosa26 locus. In embodiments, the GSHS is a located at the human Rosa26 locus. In embodiments, the GSHS is an adeno-associated virus site 1 (AAVS1). In embodiments, the GSHS is located on human chromosome 2, 3, 4, 6, 10, 11, 17, 19 or 22. In embodiments, the GSHS is a located in intron 1 of a disease gene (e.g., HPRT, CFTR) or intron 1 of a disease gene (e.g., HPRT, CFTR). In embodiments, the targeting element is able to direct a transposition machinery to a GSHS or or intron 1 of a disease gene (e.g., HPRT, CFTR) of a nucleic acid molecule in a mammalian cell. In embodiments, the GSHS is selected from TALC1 , TALC2, TALC3, TALC4, TALC5, TALC7, TALC8, AVS1, AVS2, AVS3, ROSA1 , ROSA2, TALER1 , TALER2, TALER3, TALER4, TALER5, SHCHR2-1 , SHCHR2-2, SHCHR2-3, SHCHR2-4, SHCHR4-1, SHCHR4-2, SHCHR4-3, SHCHR6-1 , SHCHR6-2, SHCHR6-3, SHCHR6-4, SHCHR10-1 , SHCHR10-2, SHCHR10-3, SHCHR10-4, SHCHR10- 5, SHCHR11-1, SHCHR11-2, SHCHR11-3, SHCHR17-1 , SHCHR17-2, SHCHR17-3, and SHCHR17-4.

[0361] In embodiments, the targeting element comprises a Cas9 enzyme guide RNA complex. In embodiments, the Cas9 enzyme guide RNA complex comprises a nuclease-deficient dCas9 guide RNA complex. In embodiments, the targeting element comprises a Cas12 enzyme guide RNA complex. In embodiments, the targeting element comprises a nuclease-deficient dCas12 guide RNA complex, optionally dCas12j guide RNA complex or dCas12a guide RNA complex. In embodiments, the targeting element comprises a Cas12k enzyme guide RNA complex. In embodiments, the targeting element comprises a nuclease-deficient dCas12 guide RNA complex, optionally dCas12k guide RNA complex.

[0362] In embodiments, the targeting element comprises a CasX enzyme associated with a gRNA. In embodiments, the CasX enzyme associated with a gRNA comrprises a catalytically inactive dCasx associated with a gRNA. In embodiments, the targeting element is or comprises a a CasX enzyme associated with a gRNA, optionally wherein the catalytic inactive dCasX.

[0363] In embodiments, a targeting chimeric system or construct, having a DBD fused to the helper enzyme directs binding of the helper to a specific sequence (e.g., transcription activator-like effector proteins (TALE) repeat variable di-residues (RVD) or gRNA) near a helper enzyme recognition site. The helper enzyme is thus prevented from binding to random recognition sites. In embodiments, the targeting chimeric construct binds to human GSHS. In embodiments, dCas9 (i.e., deficientfor nuclease activity) is programmed with gRNAs directed to bind at a desired sequence of DNA in GSHS.

[0364] In embodiments, TALEs described herein can physically sequester the helper enzyme to GSHS and promote transposition to nearby TTAA (SEQ ID NO: 440) sequences in close proximity to the RVD TALE nucleotide sequences. GSHS in open chromatin sites are specifically targeted based on the predilection for helpers to insert into open chromatin.

[0365] In embodiments, the helper enzyme is capable of targeted genomic integration by transposition is linked to or fused with a TALE DNA binding domain (DBD) or a Cas-based gene-editing system, such as, e.g, Cas9 or a variant thereof.

[0366] In embodiments, the targeting element targets the helper enzyme to a locus of interest. In embodiments, the targeting element comprises CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) associated protein 9 (Cas9), or a variant thereof. A CRISPR / Cas9 tool only requires Cas9 nuclease for DNA cleavage and a single-guide RNA (sgRNA) for target specificity. See Jinek et al. (2012) Science 337, 816-821 ; Chylinski et al. (2014) Nucleic Acids Res 42, 6091-6105. The inactivated form of Cas9, which is a nuclease-deficient (or inactive, or “catalytically dead” Cas9, is typically denoted as “dCas9,” has no substantial nuclease activity. Qi, L. S. et al. (2013). Cell 152, 1173-1183. CRISPR / dCas9 binds precisely to specific genomic sequences through targeting of guide RNA (gRNA) sequences. See Dominguez etal., Nat Rev Mol Cell Biol. 2016;17:5-15; Wang et al., Annu Rev Biochem. 2016;85:227-64. dCas9 is utilized to edit gene expression when applied to the transcription binding site of a desired site and / or locus in a genome. When the dCas9 protein is coupled to guide RNA (gRNA) to create dCas9 guide RNA complex, dCas9 prevents the proliferation of repeating codons and DNA sequences that might be harmful to an organism's genome. Essentially, when multiple repeat codons are produced, it elicits a response, or recruits an abundance of dCas9 to combat the overproduction of those codons and results in the shut-down of transcription. Thus, dCas9 works synergistically with gRNA and directly affects the DNA polymerase II from continuing transcription.

[0367] In embodiments, the targeting element comprises a nuclease-deficient Cas enzyme guide RNA complex. In embodiments, the targeting element comprises a nuclease-deficient (or inactive, or “catalytically dead” Cas, e.g., Cas9, typically denoted as “dCas” or “dCas9”) guide RNA complex.

[0368] In embodiments, the dCas9 / gRNA complex comprises a guide RNA selected from: GTTTAGCTCACCCGTGAGCC (SEQ ID NO: 91), CCCAATATTATTGTTCTCTG (SEQ ID NO: 92), GGGGTGGGATAGGGGATACG (SEQ ID NO: 93), GGATCCCCCTCTACATTTAA (SEQ ID NO: 94), GTGATCTTGTACAAATCATT (SEQ ID NO: 95), CTACACAGAATCTGTTAGAA (SEQ ID NO: 96), TAAGCTAGAGAATAGATCTC (SEQ ID NO: 97), and TCAATACACTTAATGATTTA (SEQ ID NO: 98), wherein the guide RNA directs the helper enzyme to a chemokine (C- C motif) receptor 5 (CCR5) gene.

[0369] In embodiments, the dCas9 / gRNA complex comprises a guide RNA selected from:

[0370] CACCGGGAGCCACGAAAACAGATCC (SEQ ID NO: 99);CACCGCGAAAACAGATCCAGGGACA (SEQ ID NO: 100); CACCGAGATCCAGGGACACGGTGCT (SEQ ID NO: 101); CACCGGACACGGTGCTAGGACAGTG (SEQ ID NO: 102); CACCGGAAAATGACCCAACAGCCTC (SEQ ID NO: 103); CACCGGCCTGGCCGGCCTGACCACT (SEQ ID NO: 104); CACCGCTGAGCACTGAAGGCCTGGC (SEQ ID NO: 105); CACCGTGGTTTCCACTGAGCACTGA (SEQ ID NO: 106); CACCGGATAGCCAGGAGTCCTTTCG (SEQ ID NO: 107); CACCGGCGCTTCCAGTGCTCAGACT (SEQ ID NO: 108); CACCGCAGTGCTCAGACTAGGGAAG (SEQ ID NO: 109); CACCGGCCCCTCCTCCTTCAGAGCC (SEQ ID NO: 110); CACCGTCCTTCAGAGCCAGGAGTCC (SEQ ID NO: 111); CACCGTGGTTTCCGAGCTTGACCCT (SEQ ID NO: 112); CACCGCTGCAGAGTATCTGCTGGGG (SEQ ID NO: 113); CACCGCGTTCCTGCAGAGTATCTGC (SEQ ID NO: 114); AAACGGATCTGTTTTCGTGGCTCCC (SEQ ID NO: 115); AAACTGTCCCTGGATCTGTTTTCGC (SEQ ID NO: 116); AAACAGCACCGTGTCCCTGGATCTC (SEQ ID NO: 117); AAACCACTGTCCTAGCACCGTGTCC (SEQ ID NO: 118); AAACGAGGCTGTTGGGTCATTTTCC (SEQ ID NO: 119); AAACAGTGGTCAGGCCGGCCAGGCC (SEQ ID NO: 120); AAACGCCAGGCCTTCAGTGCTCAGC (SEQ ID NO: 121); AAACTCAGTGCTCAGTGGAAACCAC (SEQ ID NO: 122); AAACCGAAAGGACTCCTGGCTATCC (SEQ ID NO: 123); AAACAGTCTGAGCACTGGAAGCGCC (SEQ ID NO: 124); AAACCTTCCCTAGTCTGAGCACTGC (SEQ ID NO: 125); AAACGGCTCTGAAGGAGGAGGGGCC (SEQ ID NO: 126); AAACGGACTCCTGGCTCTGAAGGAC (SEQ ID NO: 127); AAACAGGGTCAAGCTCGGAAACCAC (SEQ ID NO: 128); AAACCCCCAGCAGATACTCTGCAGC (SEQ ID NO: 129); AAACGCAGATACTCTGCAGGAACGC (SEQ ID NO: 130); TCCCCTCCCAGAAAGACCTG (SEQ ID NO: 131); TGGGCTCCAAGCAATCCTGG (SEQ ID NO: 132);

[0371] GTGGCTCAGGAGGTACCTGG (SEQ ID NO: 133); GAGCCACGAAAACAGATCCA (SEQ ID NO: 134);

[0372] AAGTGAACGGGGAAGGGAGG (SEQ ID NO: 135); GACAAAAGCCGAAGTCCAGG (SEQ ID NO: 136);

[0373] GTGGTTGATAAACCCACGTG (SEQ ID NO: 137); TGGGAACAGCCACAGCAGGG (SEQ ID NO: 138);

[0374] GCAGGGGAACGGGGATGCAG (SEQ ID NO: 139); GAGATGGTGGACGAGGAAGG (SEQ ID NO: 140);

[0375] GAGATGGCTCCAGGAAATGG (SEQ ID NO: 141); TAAGGAATCTGCCTAACAGG (SEQ ID NO: 142);

[0376] TCAGGAGACTAGGAAGGAGG (SEQ ID NO: 143); TATAAGGTGGTCCCAGCTCG (SEQ ID NO: 144);

[0377] CTGGAAGATGCCATGACAGG (SEQ ID NO: 145); GCACAGACTAGAGAGGTAAG (SEQ ID NO: 146);

[0378] ACAGACTAGAGAGGTAAGGG (SEQ ID NO: 147); GAGAGGTGACCCGAATCCAC (SEQ ID NO: 148);

[0379] GCACAGGCCCCAGAAGGAGA (SEQ ID NO: 149); CCGGAGAGGACCCAGACACG (SEQ ID NO: 150);

[0380] GAGAGGACCCAGACACGGGG (SEQ ID NO: 151); GCAACACAGCAGAGAGCAAG (SEQ ID NO: 152);

[0381] GAAGAGGGAGTGGAGGAAGA (SEQ ID NO: 153); AAGACGGAACCTGAAGGAGG (SEQ ID NO: 154);

[0382] AGAAAGCGGCACAGGCCCAG (SEQ ID NO: 155); GGGAAACAGTGGGCCAGAGG (SEQ ID NO: 156);

[0383] GTCCGGACTCAGGAGAGAGA (SEQ ID NO: 157); GGCACAGCAAGGGCACTCGG (SEQ ID NO: 158);

[0384] GAAGAGGGGAAGTCGAGGGA (SEQ ID NO: 159); GGGAATGGTAAGGAGGCCTG (SEQ ID NO: 160);

[0385] GCAGAGTGGTCAGCACAGAG (SEQ ID NO: 161); GCACAGAGTGGCTAAGCCCA (SEQ ID NO: 162);

[0386] GACGGGGTGTCAGCATAGGG (SEQ ID NO: 163); GCCCAGGGCCAGGAACGACG (SEQ ID NO: 164);

[0387] GGTGGAGTCCAGCACGGCGC (SEQ ID NO: 165); ACAGGCCGCCAGGAACTCGG (SEQ ID NO: 166);

[0388] ACTAGGAAGTGTGTAGCACC (SEQ ID NO: 167); ATGAATAGCAGACTGCCCCG (SEQ ID NO: 168);

[0389] ACACCCCTAAAAGCACAGTG (SEQ ID NO: 169); CAAGGAGTTCCAGCAGGTGG (SEQ ID NO: 170);

[0390] AAGGAGTTCCAGCAGGTGGG (SEQ ID NO: 171); TGGAAAGAGGAGGGAAGAGG (SEQ ID NO: 172);

[0391] TCGAATTCCTAACTGCCCCG (SEQ ID NO: 173); GACCTGCCCAGCACACCCTG (SEQ ID NO: 174);

[0392] GGAGCAGCTGCGGCAGTGGG (SEQ ID NO: 175); GGGAGGGAGAGCTTGGCAGG (SEQ ID NO: 176);

[0393] GTTACGTGGCCAAGAAGCAG (SEQ ID NO: 177); GCTGAACAGAGAAGAGCTGG (SEQ ID NO: 178);

[0394] TCTGAGGGTGGAGGGACTGG (SEQ ID NO: 179); GGAGAGGTGAGGGACTTGGG (SEQ ID NO: 180);

[0395] GTGAACCAGGCAGACAACGA (SEQ ID NO: 181); CAGGTACCTCCTGAGCCACG (SEQ ID NO: 182);

[0396] GGGGGAGTAGGGGCATGCAG (SEQ ID NO: 183); GCAAATGGCCAGCAAGGGTG (SEQ ID NO: 184);

[0397] CAAATGGCCAGCAAGGGTGG (SEQ ID NO: 309); GCAGAACCTGAGGATATGGA (SEQ ID NO: 310); AATACACAGAATGAAAATAG (SEQ ID NO: 311); CTGGTGACTAGAATAGGCAG (SEQ ID NO: 312);

[0398] TGGTGACTAGAATAGGCAGT (SEQ ID NO: 313); TAAAAGAATGTGAAAAGATG (SEQ ID NO: 314);

[0399] TCAGGAGTTCAAGACCACCC (SEQ ID NO: 315); TGTAGTCCCAGTTATGCAGG (SEQ ID NO: 316);

[0400] GGGTTCACACCACAAATGCA (SEQ ID NO: 317); GGCAAATGGCCAGCAAGGGT (SEQ ID NO: 318);

[0401] AGAAACCAATCCCAAAGCAA (SEQ ID NO: 319); GCCAAGGACACCAAAACCCA (SEQ ID NO: 320);

[0402] AGTGGTGATAAGGCAACAGT (SEQ ID NO: 321); CCTGAGACAGAAGTATTAAG (SEQ ID NO: 322);

[0403] AAGGTCACACAATGAATAGG (SEQ ID NO: 323); CACCATACTAGGGAAGAAGA (SEQ ID NO: 324);

[0404] CAATACCCTGCCCTTAGTGG (SEQ ID NO: 327); AATACCCTGCCCTTAGTGGG (SEQ ID NO: 325);

[0405] TTAGTGGGGGGTGGAGTGGG (SEQ ID NO: 326); GTGGGGGGTGGAGTGGGGGG (SEQ ID NO: 328); GGGGGGTGGAGTGGGGGGTG (SEQ ID NO: 329); GGGGTGGAGTGGGGGGTGGG (SEQ ID NO: 330); GGGTGGAGTGGGGGGTGGGG (SEQ ID NO: 331); GGGGGTGGGGAAAGACATCG (SEQ ID NO: 332); GCAGCTGTGAATTCTGATAG (SEQ ID NO: 333); GAGATCAGAGAAACCAGATG (SEQ ID NO: 334); TCTATACTGATTGCAGCCAG (SEQ ID NO: 335); CACCGAATCGAGAAGCGACTCGACA (SEQ ID NO: 185); CACCGGTCCCTGGGCGTTGCCCTGC (SEQ ID NO: 186); CACCGCCCTGGGCGTTGCCCTGCAG (SEQ ID NO: 187); CACCGCCGTGGGAAGATAAACTAAT (SEQ ID NO: 188); CACCGTCCCCTGCAGGGCAACGCCC (SEQ ID NO: 189); CACCGGTCGAGTCGCTTCTCGATTA (SEQ ID NO: 190); CACCGCTGCTGCCTCCCGTCTTGTA (SEQ ID NO: 191); CACCGGAGTGCCGCAATACCTTTAT (SEQ ID NO: 192); CACCGACACTTTGGTGGTGCAGCAA (SEQ ID NO: 193); CACCGTCTCAAATGGTATAAAACTC (SEQ ID NO: 194); CACCGAATCCCGCCCATAATCGAGA (SEQ ID NO: 195); CACCGTCCCGCCCATAATCGAGAAG (SEQ ID NO: 196); CACCGCCCATAATCGAGAAGCGACT (SEQ ID NO: 197); CACCGGAGAAGCGACTCGACATGGA (SEQ ID NO: 198); CACCGGAAGCGACTCGACATGGAGG (SEQ ID NO: 199); CACCGGCGACTCGACATGGAGGCGA (SEQ ID NO: 200); AAACTGTCGAGTCGCTTCTCGATTC (SEQ ID NO: 201); AAACGCAGGGCAACGCCCAGGGACC (SEQ ID NO: 202); AAACCTGCAGGGCAACGCCCAGGGC (SEQ ID NO: 203); AAACATTAGTTTATCTTCCCACGGC (SEQ ID NO: 204); AAACGGGCGTTGCCCTGCAGGGGAC (SEQ ID NO: 205); AAACTAATCGAGAAGCGACTCGACC (SEQ ID NO: 206); AAACTACAAGACGGGAGGCAGCAGC (SEQ ID NO: 207); AAACATAAAGGTATTGCGGCACTCC (SEQ ID NO: 208); AAACTTGCTGCACCACCAAAGTGTC (SEQ ID NO: 209); AAACGAGTTTTATACCATTTGAGAC (SEQ ID NO: 210); AAACTCTCGATTATGGGCGGGATTC (SEQ ID NO: 211); AAACCTTCTCGATTATGGGCGGGAC (SEQ ID NO: 212); AAACAGTCGCTTCTCGATTATGGGC (SEQ ID NO: 213); AAACTCCATGTCGAGTCGCTTCTCC (SEQ ID NO: 214); AAACCCTCCATGTCGAGTCGCTTCC (SEQ ID NO: 215); AAACTCGCCTCCATGTCGAGTCGCC (SEQ ID NO: 216); CACCGACAGGGTTAATGTGAAGTCC (SEQ ID NO: 217); CACCGTCCCCCTCTACATTTAAAGT (SEQ ID NO: 218); CACCGCATTTAAAGTTGGTTTAAGT (SEQ ID NO: 219); CACCGTTAGAAAATATAAAGAATAA (SEQ ID NO: 220); CACCGTAAATGCTTACTGGTTTGAA (SEQ ID NO: 221); CACCGTCCTGGGTCCAGAAAAAGAT (SEQ ID NO: 222); CACCGTTGGGTGGTGAGCATCTGTG (SEQ ID NO: 223); CACCGCGGGGAGAGTGGAGAAAAAG (SEQ ID NO: 224); CACCGGTTAAAACTCTTTAGACAAC (SEQ ID NO: 225); CACCGGAAAATCCCCACTAAGATCC (SEQ ID NO: 226); AAACGGACTTCACATTAACCCTGTC (SEQ ID NO: 227); AAACACTTTAAATGTAGAGGGGGAC (SEQ ID NO: 228); AAACACTTAAACCAACTTTAAATGC (SEQ ID NO: 229); AAACTTATTCTTTATATTTTCTAAC (SEQ ID NO: 230); AAACTTCAAACCAGTAAGCATTTAC (SEQ ID NO: 231); AAACATCTTTTTCTGGACCCAGGAC (SEQ ID NO: 232); AAACCACAGATGCTCACCACCCAAC (SEQ ID NO: 233); AAACCTTTTTCTCCACTCTCCCCGC (SEQ ID NO: 234); AAACGTTGTCTAAAGAGTTTTAACC (SEQ ID NO: 235); AAACGGATCTTAGTGGGGATTTTCC (SEQ ID NO: 236); AGTAGCAGTAATGAAGCTGG (SEQ ID NO: 237); ATACCCAGACGAGAAAGCTG (SEQ ID NO: 238); TACCCAGACGAGAAAGCTGA (SEQ ID NO: 239);

[0406] GGTGGTGAGCATCTGTGTGG (SEQ ID NO: 240); AAATGAGAAGAAGAGGCACA (SEQ ID NO: 241);

[0407] CTTGTGGCCTGGGAGAGCTG (SEQ ID NO: 242); GCTGTAGAAGGAGACAGAGC (SEQ ID NO: 243);

[0408] GAGCTGGTTGGGAAGACATG (SEQ ID NO: 244); CTGGTTGGGAAGACATGGGG (SEQ ID NO: 245);

[0409] CGTGAGGATGGGAAGGAGGG (SEQ ID NO: 246); ATGCAGAGTCAGCAGAACTG (SEQ ID NO: 247);

[0410] AAGACATCAAGCACAGAAGG (SEQ ID NO: 248); TCAAGCACAGAAGGAGGAGG (SEQ ID NO: 249);

[0411] AACCGTCAATAGGCAAAGGG (SEQ ID NO: 250); CCGTATTTCAGACTGAATGG (SEQ ID NO: 251);

[0412] GAGAGGACAGGTGCTACAGG (SEQ ID NO: 252); AACCAAGGAAGGGCAGGAGG (SEQ ID NO: 253);

[0413] GACCTCTGGGTGGAGACAGA (SEQ ID NO: 254); CAGATGACCATGACAAGCAG (SEQ ID NO: 255);

[0414] AACACCAGTGAGTAGAGCGG (SEQ ID NO: 256); AGGACCTTGAAGCACAGAGA (SEQ ID NO: 257);

[0415] TACAGAGGCAGACTAACCCA (SEQ ID NO: 258); ACAGAGGCAGACTAACCCAG (SEQ ID NO: 259);

[0416] TAAATGACGTGCTAGACCTG (SEQ ID NO: 260); AGTAACCACTCAGGACAGGG (SEQ ID NO: 261);

[0417] ACCACAAAACAGAAACACCA (SEQ ID NO: 262); GTTTGAAGACAAGCCTGAGG (SEQ ID NO: 263);

[0418] GCTGAACCCCAAAAGACAGG (SEQ ID NO: 264); GCAGCTGAGACACACACCAG (SEQ ID NO: 265);

[0419] AGGACACCCCAAAGAAGCTG (SEQ ID NO: 266); GGACACCCCAAAGAAGCTGA (SEQ ID NO: 267);

[0420] CCAGTGCAATGGACAGAAGA (SEQ ID NO: 268); AGAAGAGGGAGCCTGCAAGT (SEQ ID NO: 269);

[0421] GTGTTTGGGCCCTAGAGCGA (SEQ ID NO: 270); CATGTGCCTGGTGCAATGCA (SEQ ID NO: 271);

[0422] TACAAAGAGGAAGATAAGTG (SEQ ID NO: 272); GTCACAGAATACACCACTAG (SEQ ID NO: 273);

[0423] GGGTTACCCTGGACATGGAA (SEQ ID NO: 274); CATGGAAGGGTATTCACTCG (SEQ ID NO: 275);

[0424] AGAGTGGCCTAGACAGGCTG (SEQ ID NO: 276); CATGCTGGACAGCTCGGCAG (SEQ ID NO: 277);

[0425] AGTGAAAGAAGAGAAAATTC (SEQ ID NO: 278); TGGTAAGTCTAAGAAACCTA (SEQ ID NO: 279);

[0426] CCCACAGCCTAACCACCCTA (SEQ ID NO: 280); AATATTTCAAAGCCCTAGGG (SEQ ID NO: 281);

[0427] GCACTCGGAACAGGGTCTGG (SEQ ID NO: 282); AGATAGGAGCTCCAACAGTG (SEQ ID NO: 283);

[0428] AAGTTAGAGCAGCCAGGAAA (SEQ ID NO: 284); TAGAGCAGCCAGGAAAGGGA (SEQ ID NO: 285);

[0429] TGAATACCCTTCCATGTCCA (SEQ ID NO: 286); CCTGCATTGCACCAGGCACA (SEQ ID NO: 287);

[0430] TCTAGGGCCCAAACACACCT (SEQ ID NO: 288); TCCCTCCATCTATCAAAAGG (SEQ ID NO: 289);

[0431] AGCCCTGAGACAGAAGCAGG (SEQ ID NO: 290); GCCCTGAGACAGAAGCAGGT (SEQ ID NO: 291);

[0432] AGGAGATGCAGTGATACGCA (SEQ ID NO: 292); ACAATACCAAGGGTATCCGG (SEQ ID NO: 293); TGATAAAGAAAACAAAGTGA (SEQ ID NO: 294); AAAGAAAACAAAGTGAGGGA (SEQ ID NO: 295); GTGGCAAGTGGAGAAATTGA (SEQ ID NO: 296); CAAGTGGAGAAATTGAGGGA (SEQ ID NO: 297);

[0433] GTGGTGATGATTGCAGCTGG (SEQ ID NO: 298); CTATGTGCCTGACACACAGG (SEQ ID NO: 299);

[0434] GGGTTGGACCAGGAAAGAGG (SEQ ID NO: 300); GATGCCTGGAAAAGGAAAGA (SEQ ID NO: 301);

[0435] TAGTATGCACCTGCAAGAGG (SEQ ID NO: 302); TATGCACCTGCAAGAGGCGG (SEQ ID NO: 303); AGGGGAAGAAGAGAAGCAGA (SEQ ID NO: 304); GCTGAATCAAGAGACAAGCG (SEQ ID NO: 305);

[0436] AAGCAAATAAATCTCCTGGG (SEQ ID NO: 306); AGATGAGTGCTAGAGACTGG (SEQ ID NO: 307); and CTGATGGTTGAGCACAGCAG (SEQ ID NO: 308)

[0437] In embodiments, the guide RNAs are: AATCGAGAAGCGACTCGACA (SEQ ID NO: 425), and tgccctgcaggggagtgagc (SEQ ID NO: 426). In embodiments, the guide RNAs are gaagcgactcgacatggagg (SEQ ID NO: 427) and cctgcaggggagtgagcagc (SEQ ID NO: 428).

[0438] In embodiments, guide RNAs (gRNAs) for targeting human genomic safe harbor sites using any of the gRNA-based targeting elements, e.g., without limitation dCas, in areas of open chromatin are as shown in TABLE 19.

[0439] TABLE 19

[0440] In embodiments, the DNA binder comprises TALEs, ZnF, and / or dCas (TABLES 8-17). In embodiments, the helper enzyme comprises a targeting element. In embodiments, the helper enzyme is capable of inserting a donor comprising a transgene in a genomic safe harbor site (GSHS). In embodiments, the binding of a GSHS of a nucleic acid molecule in a mammalian cell is with high target specificity, relative to a control. In embodiments, the control is a composition comprising a helper enzyme comprising an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 1 or a nucleic acid comprising one or more of the Exc+lnt- mutations and DNA binders in TABLES 1-17 and 19-20 or a codon-optimized forms thereof.

[0441] In embodiments, the DNA binding element that directs the transpoase to GSHS is selected from TABLES 3-17 and TABLES 19-20. In embodiments, the guide RNA for targeting human genomic safe harbor sites using any of the gRNA based targeting elements, is selected from GUIDE 44, GUIDE 45-C, GUIDE 46-C, SPG GUIDE1-C, SPG GUIDE2-C, SPG GUIDE3-C, SPG GUIDE4-C, SPG GUIDE5-C, SPG GUIDE6-C, SPG GUIDE7-C, SPG GUIDE8, SPG GUIDE9, SPG GUIDE10, SPG GUIDE11, SPG GUIDE12, SPG GUIDE13, SPG GUIDE14, GUIDE N1 , GUIDE N2, GUIDE N3- C, GUIDE 012, GUIDE 013, GUIDE 014-0, AAV GUIDE 12, AAV GUIDE 13c, AAV GUIDE 14, AAV GUIDE 14c, AAV GUIDE 15, AAV GUIDE 16, AAV GUIDE 17, AAV GUIDE 18, AAV GUIDE 19, AAV GUIDE 20, AAV GUIDE 21 , AAV GUIDE 22, AAV GUIDE 23, AAV GUIDE 24, AAV GUIDE 25, AAV GUIDE 26, AAV GUIDE 27, AAV GUIDE 28, AAV GUIDE 29, AAV GUIDE 30c, AAV GUIDE 31 , AAV GUIDE 32c, AAV GUIDE 33c, AAV GUIDE 34, AAV GUIDE 35, Guide C4-1 , Guide C4-2, Guide C4-3, Guide C4-4, Guide C4-5, Guide C4-6, Guide C4-7, Guide C4-8, Guide C4-9, Guide 04-10, Guide 04-11 , Guide 04-12, Guide 04-13, Guide 04-14, Guide 04-15, Guide 04-16, Guide 04-17, Guide 04-18, Guide 04-19, Guide 04-20, Guide C4A1 , Guide C4A2, Guide C4A3, Guide C4A4, Guide C4A5, Guide C4A6, Guide C4A7, Guide C4A8, Guide C4A9, Guide C4A10, Guide C4A11 , Guide C4A12, Guide C4A13, Guide C4A14, Guide C4A15, Guide C4A16, Guide C4A17, Guide C4A18, Guide C4A19, Guide C4A20, Guide C22-1 , Guide C22-2, Guide C22-3, Guide C22-4, Guide C22-5, Guide C22-6, Guide C22-7, Guide C22-8, Guide C22-9, Guide 022-10, Guide 022-11 , Guide 022-12, Guide 022-13, Guide 022-14, Guide 022-15, Guide 022-16, Guide 022-17, Guide C22- 18, Guide 022-19, Guide 022-20, Guide C22A1, Guide C22A2, Guide C22A3, Guide C22A4, Guide C22A5, Guide C22A6, Guide C22A7, Guide C22A8, Guide C22A9, Guide C22A10, Guide C22A11, Guide C22A12, Guide C22A13, Guide C22A14, Guide C22A15, Guide C22A16, Guide C22A17, Guide C22A18, Guide C22A19, Guide C22A20, Guide CX-1 , Guide CX-2, Guide CX-3, Guide CX-4, Guide CX-5, Guide CX-6, Guide CX-7, Guide CX-8, Guide CX-9, Guide CX-10, Guide CX-11 , Guide CX-12, Guide CX-13, Guide CX-14, Guide CX-15, Guide CX-16, Guide CX-17, Guide CX- 18, Guide CX-19, Guide CX-20, TALES: R1 , R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 , R12, R13, R14, AAV1c, AAV2c, AAV3c, AAV4c, AAV5c, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV13c, AAV14c, AAV15c, AAV16c, AAV17c, TALE4-R001 , TALE4-R002, TALE4-F003, TALE4-F004, TALE4-F005, TALE4-F006, TALE4-R007, TALE4-F008, TALE4-R009, TALE4-F010, TALE4-F011 , TALE4-F012, TALE4-R013, TALE4-F014, TALE4-F015, TALE4-R016, TALE4-F017, TALE4-F018, TALE4-F019, TALE4-F020, TALE22F-R001 , TALE22-F002, TALE22-F003, TALE22-F004, TALE22-F005, TALE22-F006, TALE22-F007, TALE22-F008, TALE22-R009, TALE22-F010, TALE22- F001A, TALE22-F002A, TALE22-R03A, TALE22-F004A, TALE22-F005A, TALE22-F006A, TALE22-R007A, TALE22- R008A, TALE22-R009A, TALE22-F010A, TALE F002, TALE F003, TALE F004, TALE F007, TALE F008, TALE F009, TALE R002, TALE R003, TALE R004, TALE R005, TALE R007, TALE F020, TALE F021, TALE F030, TALE F031 , TALE F034, TALE F036, TALE F037, TALE F040, TALE R022, TALE R033, TALE R035, TALE R038, TALE R039, Zinc Fingers: ZnF3a, ZnF5a, ZnF5b, ZnF5c, ZnF5d, ZnF5e, ZnF5f, ZnF5g, ZnF5h, ZnF12a, ZnF13a, ZnF13b, ZnF13c, ZnF11a, ZnF10a, ZnF12b, ZnF13b, ZnF14a, ZnF15a, ZnF16a, ZnF17a, ZnF18a, ZnF19a, ZnF20b, ZnF21 b, ZnF22a, ZnF23a, ZnF24a, ZnF31 F, ZnF32F, ZnF33F, ZnF34F, ZnF35F, ZnF36F, ZnF37R, ZnF38R, ZnF39R, ZnF1a, ZnF1 b, ZnF2a, ZnF3a, ZnF3b, ZnF5aR, ZnF5bR, ZnF6aR, ZnF6bR, ZnF10F, ZnF11 F, ZnF12F, ZnF13F, ZnF14R, ZnF15R, ZnF14R, ZnF15R, ZnF41 F, ZnF42F, ZnF43F, ZnF44R, ZnF45R, ZnF46R, ZnF47R, ZnF48R. In embodiments, gRNAs for targeting human genomic safe harbor sites using any of the gRNA-based targeting elements, e.g., without limitation, dCas, in areas of open chromatin are shown in TABLES 3-7.

[0442] TABLE 3. Guide RNA sequences targeting the genomic safe harbor site hROSA26 (hg38 chr3:9,396, 132-9,396,332)

[0443] TABLE 4. Guide RNA sequences targeting the genomic safe harbor site AAVS1 (hg38 chr19:55, 112,850-

[0444] TABLE 5. Guide RNA sequences targeting a chromosome 4 genomic safe harbor site (hg38 chr4:30, 793, 038- 30,793,980)

[0445] In embodiments, the gRNA comprises one or more of the sequences outlined herein or a variant sequence having at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.

[0446] In embodiments, a Cas-based targeting element comprises Cas12 or a variant thereof, e.g., without limitation, Cas12a (e.g., dCas12a), or Cas12j (e.g., dCas12j), or Cas12k (e.g., dCas12k). In embodiments, the targeting element comprises a Cas12 enzyme guide RNA complex. In embodiments, comprises a nuclease-deficient dCas12 guide RNA complex, optionally dCas12j guide RNA complex or dCas12a guide RNA complex.

[0447] In embodiments, the targeting element is selected from a zinc finger (ZF), transcription activator-like effector (TALE), meganuclease, and clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein, any of which are, in embodiments, catalytically inactive. In embodiments, the CRISPR-associated protein is selected from Cas9, CasX, CasY, Cas12a (Cpf1), and gRNA complexes thereof. In embodiments, the CRISPR-associated protein is selected from Cas9, xCas9, Cas 6, Cas7, Cas8, Cas12a (Cpf1 ), Cas13a, Cas14, CasX, CasY, a Class 1 Cas protein, a Class 2 Cas protein, MAD7, MG1 nuclease, MG2 nuclease, MG3 nuclease, or catalytically inactive forms thereof, and gRNA complexes thereof.

[0448] In embodiments, the helper enzyme of the present disclosure is capable of inserting a donor DNA at a TA dinucleotide site, or a TTAA tetranucleotide site, or a ttTTAAaa octanucleotide site, or a taTTAAta octanucleotide site, in a genomic safe harbor site (GSHS) of a nucleic acid molecule. The helper enzyme of the present disclosure is suitable for causing insertion of the donor DNA in a GSHS when contacted with a biological cell.

[0449] In embodiments, the targeting element is suitable for directing the helper enzyme of the present disclosure to the GSHS sequence.

[0450] In embodiments, the targeting element comprises transcription activator-like effector (TALE) DNA binding domain (DBD). The TALE DBD comprises one or more repeat sequences. For example, in embodiments, the TALE DBD comprises about 14, or about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences. In embodiments, the TALE DBD repeat sequences comprise 33 or 34 amino acids.

[0451] In embodiments, the one or more of the TALE DBD repeat sequences comprise a repeat variable di-residue (RVD) at residue 12 or 13 of the 33 or 34 amino acids.

[0452] In embodiments, the targeting element (e.g., TALE or Cas (e.g, Cas9 or Cas12, or variants thereof) DBDs cause the the helper enzyme of the present disclosure to bind specifically to human GSHS. In embodiments, the TALEs or Cas DBDs sequester the helper to GSHS and promote transposition to nearby TA dinucleotide, or TTAA tetranucleotide, or ttTTAAaa octanucleotide, or taTTAAta octanucleotide sites which can be located in proximity to the repeat variable diresidues (RVD) TALE or gRNA nucleotide sequences. The GSHS regions are located in open chromatin sites that are susceptible to helper activity. Accordingly, the helper enzyme of the present disclosure does not only operate based on its ability to recognize TA or TTAA sites, but it also directs a donor DNA (having a transgene) to specific locations in proximity to a TALE or Cas DBD. The helper enzyme of the present disclosure in accordance with embodiments of the present disclosure has negligible risk of genotoxicity and exhibits superior features as compared to existing gene therapies.

[0453] In embodiments, the helper enzyme of the present disclosure is mutated to be characterized by reduced or inhibited binding of off-target sequences and consequently reliant on a DBD fused thereto, such as a TALE or Cas DBD, for transposition.

[0454] The described cells, compositions, and methods allow reducing vector and transgene insertions that increase a mutagenic risk. The described cells and methods make use of a gene transfer system that reduces genotoxicity compared to viral- and nuclease-mediated gene therapies.

[0455] In embodiments, TALE or Cas DBDs are customizable, such as a TALE or Cas DBDs is selected for targeting a specific genomic location. In embodiments, the genomic location is in proximity to a TA dinucleotide site, or a TTAA (SEQ ID NO: 440) tetranucleotide site, or a ttTTAAaa octanucleotide site, or a taTTAAta octanucleotide site. In embodiments, the TALE or Cas DBDs binding site is different in the donor and the target site. In embodiments, the TALE or Cas DBDs binding site is the same in the donor and the target site.

[0456] In embodiments, Zinc fingers are customizable, such as a Zinc finger that is selected for targeting a specific genomic location. In embodiments, the genomic location is in proximity to a TA dinucleotide site, or a TTAA (SEQ ID NO: 440) tetranucleotide site, or a ttTTAAaa octanucleotide site, or a taTTAAta octanucleotide site. In embodiments, the Zinc finger binding site is different in the donor and the target site. In embodiments, the Zinc finger binding site is the same in the donor and the target site.

[0457] Embodiments of the present disclosure make use of the ability of TALE or Cas or dCas9 / gRNA DBDs to target specific sites in a host genome. The DNA targeting ability of a TALE or Cas DBD or dCas9 / gRNA DBD is provided by TALE repeat sequences (e.g., modular arrays) or gRNA which are linked together to recognize flanking DNA sequences. Each TALE or gRNA can recognize certain base pair(s) or residue(s).

[0458] TALE nucleases (TALENs) are a known tool for genome editing and introducing targeted double-stranded breaks. TALENs comprise endonucleases, such as Fokl nuclease domain, fused to a customizable DBD. This DBD is composed of highly conserved repeats from TALEs, which are proteins secreted by Xanthomonas bacteria to alter transcription of genes in host plant cells. The DBD includes a repeated highly conserved 33-34 amino acid sequence with divergent 12th and 13th amino acids. These two positions, referred to as the RVD, are highly variable and show a strong correlation with specific base pair or nucleotide recognition. This straightforward relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DBDs by selecting a combination of repeat segments containing the appropriate RVDs. Boch et al. Nature Biotechnology. 2011 ; 29 (2): 135-6. Accordingly, TALENs can be readily designed using a “protein-DNA code” that relates modular DNA-binding TALE repeat domains to individual bases in a target-binding site. See Joung et al. Nat Rev Mol Cell Biol. 2013; 14(1 ):49-55. doi: 10.1038 / nrm3486. The following table, for example, shows such code:

[0459] TABLE 18

[0460] It has been demonstrated that TALENs can be used to target essentially any DNA sequence of interest in human cell. Miller et al. Nat Biotechnol. 2011 ;29: 143-148. Guidelines for selection of potential target sites and for use of particular TALE repeat domains (harboring NH residues at the hypervariable positions) for recognition of G bases have been proposed. See Streubel et al. Nat Biotechnol. 2012;30:593-595.

[0461] Accordingly, in embodiments, the TALE DBD comprises one or more repeat sequences. In embodiments, the TALE DBD comprises about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11 , or about 12, or about 13, or about 14, or about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences. In embodiments, the TALE DBD comprises about 8.5 repeat sequences. In embodiments, the TALE DBD repeat sequences comprise 33 or 34 amino acids.

[0462] In embodiments, the one or more of the TALE DBD repeat sequences comprise an RVD at residue 12 or 13 of the 33 or 34 amino acids. The RVD can recognize certain base pair(s) or residue(s). In embodiments, the RVD recognizes one base pair in the nucleic acid molecule. In embodiments, the RVD recognizes a C residue in the nucleic acid molecule and is selected from HD, N(gap), HA, ND, and HI. In embodiments, the RVD recognizes a G residue in the nucleic acid molecule and is selected from NN, NH, NK, HN, and NA. In embodiments, the RVD recognizes an A residue in the nucleic acid molecule and is selected from Nl and NS. In embodiments, the RVD recognizes a T residue in the nucleic acid molecule and is selected from NG, HG, H(gap), and IG.

[0463] In embodiments, the GSHS is in an open chromatin location in a chromosome. In embodiments, the GSHS is selected from adeno-associated virus site 1 (AAVS1), chemokine (C-C motif) receptor 5 (CCR5) gene, HIV-1 coreceptor; and human Rosa26 locus. In embodiments, the GSHS is located on human chromosome 2, 3, 4, 6, 10, 11, 17, 19, or 22. In embodiments, the GSHS is selected from TALC1 , TALC2, TALC3, TALC4, TALC5, TALC7, TALC8, AVS1 , AVS2, AVS3, ROSA1, ROSA2, TALER1 , TALER2, TALER3, TALER4, TALER5, SHCHR2-1 , SHCHR2-2, SHCHR2-3, SHCHR2-4, SHCHR4-1 , SHCHR4-2, SHCHR4-3, SHCHR6-1 , SHCHR6-2, SHCHR6-3, SHCHR6-4, SHCHR10-1, SHCHR10-2, SHCHR10-3, SHCHR10-4, SHCHR10-5, SHCHR11-1, SHCHR11-2, SHCHR11-3, SHCHR17-1, SHCHR17-2, SHCHR17-3, and SHCHR17-4.

[0464] In embodiments, the GSHS comprises one or more of TGGCCGGCCTGACCACTGG (SEQ ID NO: 23), TGAAGGCCTGGCCGGCCTG (SEQ ID NO: 24), TGAGCACTGAAGGCCTGGC (SEQ ID NO: 25),

[0465] TCCACTGAGCACTGAAGGC (SEQ ID NO: 26), TGGTTTCCACTGAGCACTG (SEQ ID NO: 27),

[0466] TGGGGAAAATGACCCAACA (SEQ ID NO: 28), TAGGACAGTGGGGAAAATG (SEQ ID NO: 29),

[0467] TCCAGGGACACGGTGCTAG (SEQ ID NO: 30), TCAGAGCCAGGAGTCCTGG (SEQ ID NO: 31),

[0468] TCCTTCAGAGCCAGGAGTC (SEQ ID NO: 32), TCCTCCTTCAGAGCCAGGA (SEQ ID NO: 33),

[0469] TCCAGCCCCTCCTCCTTCA (SEQ ID NO: 34), TCCGAGCTTGACCCTTGGA (SEQ ID NO: 35),

[0470] TGGTTTCCGAGCTTGACCC (SEQ ID NO: 36), TGGGGTGGTTTCCGAGCTT (SEQ ID NO: 37),

[0471] TCTGCTGGGGTGGTTTCCG (SEQ ID NO: 38), TGCAGAGTATCTGCTGGGG (SEQ ID NO: 39),

[0472] CCAATCCCCTCAGT (SEQ ID NO: 40), CAGTGCTCAGTGGAA (SEQ ID NO: 41), GAAACATCCGGCGACTCA (SEQ ID NO: 42), TCGCCCCTCAAATCTTACA (SEQ ID NO: 43), TCAAATCTTACAGCTGCTC (SEQ ID NO: 44), TCTTACAGCTGCTCACTCC (SEQ ID NO: 45), TACAGCTGCTCACTCCCCT (SEQ ID NO: 46),

[0473] TGCTCACTCCCCTGCAGGG (SEQ ID NO: 47), TCCCCTGCAGGGCAACGCC (SEQ ID NO: 48),

[0474] TGCAGGGCAACGCCCAGGG (SEQ ID NO: 49), TCTCGATTATGGGCGGGAT (SEQ ID NO: 50), TCGCTTCTCGATTATGGGC (SEQ ID NO: 51), TGTCGAGTCGCTTCTCGAT (SEQ ID NO: 52),

[0475] TCCATGTCGAGTCGCTTCT (SEQ ID NO: 53), TCGCCTCCATGTCGAGTCG (SEQ ID NO: 54),

[0476] TCGTCATCGCCTCCATGTC (SEQ ID NO: 55), TGATCTCGTCATCGCCTCC (SEQ ID NO: 56), GCTTCAGCTTCCTA (SEQ ID NO: 57), CTGTGATCATGCCA (SEQ ID NO: 58), ACAGTGGTACACACCT (SEQ ID NO: 59), CCACCCCCCACTAAG (SEQ ID NO: 60), CATTGGCCGGGCAC (SEQ ID NO: 61), GCTTGAACCCAGGAGA (SEQ ID NO: 62), ACACCCGATCCACTGGG (SEQ ID NO: 63), GCTGCATCAACCCC (SEQ ID NO: 64), GCCACAAACAGAAATA (SEQ ID NO: 65), GGTGGCTCATGCCTG (SEQ ID NO: 66), GATTTGCACAGCTCAT (SEQ ID NO: 67), AAGCTCTGAGGAGCA (SEQ ID NO: 68), CCCTAGCTGTCCC (SEQ ID NO: 69), GCCTAGCATGCTAG (SEQ ID NO: 70), ATGGGCTTCACGGAT (SEQ ID NO: 71), GAAACTATGCCTGC (SEQ ID NO: 72), GCACCATTGCTCCC (SEQ ID NO: 73), GACATGCAACTCAG (SEQ ID NO: 74), ACACCACTAGGGGT (SEQ ID NO: 75), GTCTGCTAGACAGG (SEQ ID NO: 76), GGCCTAGACAGGCTG (SEQ ID NO: 77), GAGGCATTCTTATCG (SEQ ID NO: 78), GCCTGGAAACGTTCC (SEQ ID NO: 79), GTGCTCTGACAATA (SEQ ID NO: 80), GTTTTGCAGCCTCC (SEQ ID NO: 81), ACAGCTGTGGAACGT (SEQ ID NO: 82), GGCTCTCTTCCTCCT (SEQ ID NO: 83), CTATCCCAAAACTCT (SEQ ID NO: 84), GAAAAACTATGTAT (SEQ ID NO: 85), AGGCAGGCTGGTTGA (SEQ ID NO: 86), CAATACAACCACGC (SEQ ID NO: 87), ATGACGGACTCAACT (SEQ ID NO: 88), CACAACATTTGTAA (SEQ ID NO: 89), and ATTTCCAGTGCACA (SEQ ID NO: 90)

[0477] In embodiments, the TALE DBD binds to one of TGGCCGGCCTGACCACTGG (SEQ ID NO: 23), TGAAGGCCTGGCCGGCCTG (SEQ ID NO: 24), TGAGCACTGAAGGCCTGGC (SEQ ID NO: 25),

[0478] TCCACTGAGCACTGAAGGC (SEQ ID NO: 26), TGGTTTCCACTGAGCACTG (SEQ ID NO: 27),

[0479] TGGGGAAAATGACCCAACA (SEQ ID NO: 28), TAGGACAGTGGGGAAAATG (SEQ ID NO: 29),

[0480] TCCAGGGACACGGTGCTAG (SEQ ID NO: 30), TCAGAGCCAGGAGTCCTGG (SEQ ID NO: 31),

[0481] TCCTTCAGAGCCAGGAGTC (SEQ ID NO: 32), TCCTCCTTCAGAGCCAGGA (SEQ ID NO: 33),

[0482] TCCAGCCCCTCCTCCTTCA (SEQ ID NO: 34), TCCGAGCTTGACCCTTGGA (SEQ ID NO: 35),

[0483] TGGTTTCCGAGCTTGACCC (SEQ ID NO: 36), TGGGGTGGTTTCCGAGCTT (SEQ ID NO: 37),

[0484] TCTGCTGGGGTGGTTTCCG (SEQ ID NO: 38), TGCAGAGTATCTGCTGGGG (SEQ ID NO: 39),

[0485] CCAATCCCCTCAGT (SEQ ID NO: 40), CAGTGCTCAGTGGAA (SEQ ID NO: 41), GAAACATCCGGCGACTCA (SEQ ID NO: 42), TCGCCCCTCAAATCTTACA (SEQ ID NO: 43), TCAAATCTTACAGCTGCTC (SEQ ID NO: 44), TCTTACAGCTGCTCACTCC (SEQ ID NO: 45), TACAGCTGCTCACTCCCCT (SEQ ID NO: 46),

[0486] TGCTCACTCCCCTGCAGGG (SEQ ID NO: 47), TCCCCTGCAGGGCAACGCC (SEQ ID NO: 48),

[0487] TGCAGGGCAACGCCCAGGG (SEQ ID NO: 49), TCTCGATTATGGGCGGGAT (SEQ ID NO: 50), TCGCTTCTCGATTATGGGC (SEQ ID NO: 51), TGTCGAGTCGCTTCTCGAT (SEQ ID NO: 52),

[0488] TCCATGTCGAGTCGCTTCT (SEQ ID NO: 53), TCGCCTCCATGTCGAGTCG (SEQ ID NO: 54),

[0489] TCGTCATCGCCTCCATGTC (SEQ ID NO: 55), TGATCTCGTCATCGCCTCC (SEQ ID NO: 56), GCTTCAGCTTCCTA (SEQ ID NO: 57), CTGTGATCATGCCA (SEQ ID NO: 58), ACAGTGGTACACACCT (SEQ ID NO: 59), CCACCCCCCACTAAG (SEQ ID NO: 60), CATTGGCCGGGCAC (SEQ ID NO: 61), GCTTGAACCCAGGAGA (SEQ ID NO: 62), ACACCCGATCCACTGGG (SEQ ID NO: 63), GCTGCATCAACCCC (SEQ ID NO: 64), GCCACAAACAGAAATA (SEQ ID NO: 65), GGTGGCTCATGCCTG (SEQ ID NO: 66), GATTTGCACAGCTCAT (SEQ ID NO: 67), AAGCTCTGAGGAGCA (SEQ ID NO: 68), CCCTAGCTGTCCC (SEQ ID NO: 69), GCCTAGCATGCTAG (SEQ ID NO: 70), ATGGGCTTCACGGAT (SEQ ID NO: 71), GAAACTATGCCTGC (SEQ ID NO: 72), GCACCATTGCTCCC (SEQ ID NO: 73), GACATGCAACTCAG (SEQ ID NO: 74), ACACCACTAGGGGT (SEQ ID NO: 75), GTCTGCTAGACAGG (SEQ ID NO: 76), GGCCTAGACAGGCTG (SEQ ID NO: 77), GAGGCATTCTTATCG (SEQ ID NO: 78), GCCTGGAAACGTTCC (SEQ ID NO: 79), GTGCTCTGACAATA (SEQ ID NO: 80), GTTTTGCAGCCTCC (SEQ ID NO: 81), ACAGCTGTGGAACGT (SEQ ID NO: 82), GGCTCTCTTCCTCCT (SEQ ID NO: 83), CTATCCCAAAACTCT (SEQ ID NO: 84), GAAAAACTATGTAT (SEQ ID NO: 85), AGGCAGGCTGGTTGA (SEQ ID NO: 86), CAATACAACCACGC (SEQ ID NO: 87), ATGACGGACTCAACT (SEQ ID NO: 88), CACAACATTTGTAA (SEQ ID NO: 89), and ATTTCCAGTGCACA (SEQ ID NO: 90).

[0490] In embodiments, the TALE DBD comprises one or more of NH NH HD HD NH NH HD HD NG NH Nl HD HD Nl HD NG NH NH,

[0491] NH Nl Nl NH NH HD HD NG NH NH HD HD NH NH HD HD NG NH, NH Nl NH HD Nl HD NG NH Nl Nl NH NH HD HD NG NH NH HD, HD HD Nl HD NG NH Nl NH HD Nl HD NG NH Nl Nl NH NH HD, NH NH NG NG NG HD HD Nl HD NG NH Nl NH HD Nl HD NG NH, NH NH NH NH Nl Nl Nl Nl NG NH Nl HD HD HD Nl Nl HD Nl, Nl NH NH Nl HD Nl NH NG NH NH NH NH Nl Nl Nl Nl NG NH, HD HD Nl NH NH NH Nl HD Nl HD NH NH NG NH HD NG Nl NH, HD Nl NH Nl NH HD HD Nl NH NH Nl NH NG HD HD NG NH NH, HD HD NG NG HD Nl NH Nl NH HD HD Nl NH NH Nl NH NG HD, HD HD NG HD HD NG NG HD Nl NH Nl NH HD HD Nl NH NH Nl, HD HD Nl NH HD HD HD HD NG HD HD NG HD HD NG NG HD Nl, HD HD NH Nl NH HD NG NG NH Nl HD HD HD NG NG NH NH Nl, NH NH NG NG NG HD HD NH Nl NH HD NG NG NH Nl HD HD HD, NH NH NH NH NG NH NH NG NG NG HD HD NH Nl NH HD NG NG, HD NG NH HD NG NH NH NH NH NG NH NH NG NG NG HD HD NH, NH HD Nl NH Nl NH NG Nl NG HD NG NH HD NG NH NH NH NH, HD HD Nl Nl NG HD HD HD HD NG HD Nl NH NG, HD Nl NH NG NH HD NG HD Nl NH NG NH NH Nl Nl, NH Nl Nl Nl HD Nl NG HD HD NH NH HD NH Nl HD NG HD Nl, HD NH HD HD HD HD NG HD Nl Nl Nl NG HD NG NG Nl HD Nl, HD Nl Nl Nl NG HD NG NG Nl HD Nl NH HD NG NH HD NG HD, HD NG NG Nl HD Nl NH HD NG NH HD NG HD Nl HD NG HD HD, Nl HD Nl NH HD NG NH HD NG HD Nl HD NG HD HD HD HD NG, NH HD NG HD Nl HD NG HD HD HD HD NG NH HD Nl NH NH NH, HD HD HD HD NG NH HD Nl NH NH NH HD Nl Nl HD NH HD HD, NH HD Nl NH NH NH HD Nl Nl HD NH HD HD HD Nl NH NH NH,

[0492] HD NG HD NH Nl NG NG Nl NG NH NH NH HD NH NH NH Nl NG, HD NH HD NG NG HD NG HD NH Nl NG NG Nl NG NH NH NH HD, NH NG HD NH Nl NH NG HD NH HD NG NG HD NG HD NH Nl NG, HD HD Nl NG NH NG HD NH Nl NH NG HD NH HD NG NG HD NG, HD NH HD HD NG HD HD Nl NG NH NG HD NH Nl NH NG HD NH, HD NH NG HD Nl NG HD NH HD HD NG HD HD Nl NG NH NG HD, NH Nl NG HD NG HD NH NG HD Nl NG HD NH HD HD NG HD HD, NH HD NG NG HD Nl NH HD NG NG HD HD NG Nl, HD NG NK NG NH Nl NG HD Nl NG NH HD HD Nl, Nl HD Nl NN NG NN NN NG Nl HD Nl HD Nl HD HD NG, HD HD Nl HD HD HD HD HD HD Nl HD NG Nl Nl NN, HD Nl NG NG NN NN HD HD NN NN NN HD Nl HD, NN HD NG NG NN Nl Nl HD HD HD Nl NN NN Nl NN Nl, Nl HD Nl HD HD HD NN Nl NG HD HD Nl HD NG NN NN NN, NN HD NG NN HD Nl NG HD Nl Nl HD HD HD HD,

[0493] NN NN HD Nl HD NN Nl Nl Nl HD Nl HD HD HD NG HD HD, NN NN NG NN NN HD NG HD Nl NG NN HD HD NG NN, NN Nl NG NG NG NN HD Nl HD Nl NN HD NG HD Nl NG, Nl Nl NH HD NG HD NG NH Nl NH NH Nl NH HD, HD HD HD NG Nl NK HD NG NH NG HD HD HD HD, NH HD HD NG Nl NH HD Nl NG NH HD NG Nl NH, Nl NG NH NH NH HD NG NG HD Nl HD NH NH Nl NG, NH Nl Nl Nl HD NG Nl NG NH HD HD NG NH HD, NH HD Nl HD HD Nl NG NG NH HD NG HD HD HD, NH Nl HD Nl NG NH HD Nl Nl HD NG HD Nl NH, Nl HD Nl HD HD Nl HD NG Nl NH NH NH NH NG,

[0494] NH NG HD NG NH HD NG Nl NH Nl HD Nl NH NH,

[0495] NH NH HD HD NG Nl NH Nl HD Nl NH NH HD NG NH,

[0496] NH Nl NH NH HD Nl NG NG HD NG NG Nl NG HD NH,

[0497] NN HD HD NG NN NN Nl Nl Nl HD NN NG NG HD HD,

[0498] NN NG NN HD NG HD NG NN Nl HD Nl Nl NG Nl,

[0499] NN NG NG NG NG NN HD Nl NN HD HD NG HD HD,

[0500] Nl HD Nl NN HD NG NN NG NN NN Nl Nl HD NN NG,

[0501] HD Nl Nl NN Nl HD HD NN Nl NN HD Nl HD NG NN HD NG NN,

[0502] HD NG Nl NG HD HD HD Nl Nl Nl Nl HD NG HD NG,

[0503] NH Nl Nl Nl Nl Nl HD NG Nl NG NH NG Nl NG,

[0504] Nl NH NH HD Nl NH NH HD NG NH NH NG NG NH Nl,

[0505] HD Nl Nl NG Nl HD Nl Nl HD HD Nl HD NN HD,

[0506] Nl NG NN Nl HD NN NN Nl HD NG HD Nl Nl HD NG,

[0507] HD Nl HD Nl Nl HD Nl NG NG NG NN NG Nl Nl, and

[0508] Nl NG NG NG HD HD Nl NN NG NN HD Nl HD Nl.

[0509] In embodiments, the TALE DBD comprises one or more of the sequences outlined herein or a variant sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.

[0510] In embodiments, the GSHS and the TALE DBD sequences are selected from:

[0511] TGGCCGGCCTGACCACTGG (SEQ ID NO: 23) and NH NH HD HD NH NH HD HD NG NH Nl HD HD Nl HD NG NH NH;

[0512] TGAAGGCCTGGCCGGCCTG (SEQ ID NO: 24) and NH Nl Nl NH NH HD HD NG NH NH HD HD NH NH HD HD NG NH;

[0513] TGAGCACTGAAGGCCTGGC (SEQ ID NO: 25) and NH Nl NH HD Nl HD NG NH Nl Nl NH NH HD HD NG NH NH HD; TCCACTGAGCACTGAAGGC (SEQ ID NO: 26) and HD HD Nl HD NG NH Nl NH HD Nl HD NG NH Nl Nl NH NH HD;

[0514] TGGTTTCCACTGAGCACTG (SEQ ID NO: 27) and NH NH NG NG NG HD HD Nl HD NG NH Nl NH HD Nl HD NG NH;

[0515] TGGGGAAAATGACCCAACA (SEQ ID NO: 28) and NH NH NH NH Nl Nl Nl Nl NG NH Nl HD HD HD Nl Nl HD Nl;

[0516] TAGGACAGTGGGGAAAATG (SEQ ID NO: 29) and Nl NH NH Nl HD Nl NH NG NH NH NH NH Nl Nl Nl Nl NG NH;

[0517] TCCAGGGACACGGTGCTAG (SEQ ID NO: 30) and HD HD Nl NH NH NH Nl HD Nl HD NH NH NG NH HD NG Nl NH;

[0518] TCAGAGCCAGGAGTCCTGG (SEQ ID NO: 31) and HD Nl NH Nl NH HD HD Nl NH NH Nl NH NG HD HD NG NH NH;

[0519] TCCTTCAGAGCCAGGAGTC (SEQ ID NO: 32) and HD HD NG NG HD Nl NH Nl NH HD HD Nl NH NH Nl NH NG HD;

[0520] TCCTCCTTCAGAGCCAGGA (SEQ ID NO: 33) and HD HD NG HD HD NG NG HD Nl NH Nl NH HD HD Nl NH NH Nl;

[0521] TCCAGCCCCTCCTCCTTCA (SEQ ID NO: 34) and HD HD Nl NH HD HD HD HD NG HD HD NG HD HD NG NG HD Nl;

[0522] TCCGAGCTTGACCCTTGGA (SEQ ID NO: 35) and HD HD NH Nl NH HD NG NG NH Nl HD HD HD NG NG NH NH Nl;

[0523] TGGTTTCCGAGCTTGACCC (SEQ ID NO: 36) and NH NH NG NG NG HD HD NH Nl NH HD NG NG NH Nl HD HD HD;

[0524] TGGGGTGGTTTCCGAGCTT (SEQ ID NO: 37) and NH NH NH NH NG NH NH NG NG NG HD HD NH Nl NH HD NG NG;

[0525] TCTGCTGGGGTGGTTTCCG (SEQ ID NO: 38) and HD NG NH HD NG NH NH NH NH NG NH NH NG NG NG HD HD NH;

[0526] TGCAGAGTATCTGCTGGGG (SEQ ID NO: 39) and NH HD Nl NH Nl NH NG Nl NG HD NG NH HD NG NH NH NH NH;

[0527] CCAATCCCCTCAGT (SEQ ID NO: 40) and HD HD Nl Nl NG HD HD HD HD NG HD Nl NH NG;

[0528] CAGTGCTCAGTGGAA (SEQ ID NO: 41) and HD Nl NH NG NH HD NG HD Nl NH NG NH NH Nl Nl;

[0529] GAAACATCCGGCGACTCA (SEQ ID NO: 42) and NH Nl Nl Nl HD Nl NG HD HD NH NH HD NH Nl HD NG HD Nl;

[0530] TCGCCCCTCAAATCTTACA (SEQ ID NO: 43) and HD NH HD HD HD HD NG HD Nl Nl Nl NG HD NG NG Nl HD Nl; TCAAATCTTACAGCTGCTC (SEQ ID NO: 44) and HD Nl Nl Nl NG HD NG NG Nl HD Nl NH HD NG NH HD NG HD;

[0531] TCTTACAGCTGCTCACTCC (SEQ ID NO: 45) and HD NG NG Nl HD Nl NH HD NG NH HD NG HD Nl HD NG HD HD;

[0532] TACAGCTGCTCACTCCCCT (SEQ ID NO: 46) and Nl HD Nl NH HD NG NH HD NG HD Nl HD NG HD HD HD HD NG;

[0533] TGCTCACTCCCCTGCAGGG (SEQ ID NO: 47) and NH HD NG HD Nl HD NG HD HD HD HD NG NH HD Nl NH NH NH;

[0534] TCCCCTGCAGGGCAACGCC (SEQ ID NO: 48) and HD HD HD HD NG NH HD Nl NH NH NH HD Nl Nl HD NH HD HD;

[0535] TGCAGGGCAACGCCCAGGG (SEQ ID NO: 49) and NH HD Nl NH NH NH HD Nl Nl HD NH HD HD HD Nl NH NH NH;

[0536] TCTCGATTATGGGCGGGAT (SEQ ID NO: 50) and HD NG HD NH Nl NG NG Nl NG NH NH NH HD NH NH NH Nl NG;

[0537] TCGCTTCTCGATTATGGGC (SEQ ID NO: 51) and HD NH HD NG NG HD NG HD NH Nl NG NG Nl NG NH NH NH HD;

[0538] TGTCGAGTCGCTTCTCGAT (SEQ ID NO: 52) and NH NG HD NH Nl NH NG HD NH HD NG NG HD NG HD NH Nl NG;

[0539] TCCATGTCGAGTCGCTTCT (SEQ ID NO: 53) and HD HD Nl NG NH NG HD NH Nl NH NG HD NH HD NG NG HD NG;

[0540] TCGCCTCCATGTCGAGTCG (SEQ ID NO: 54) and HD NH HD HD NG HD HD Nl NG NH NG HD NH Nl NH NG HD NH;

[0541] TCGTCATCGCCTCCATGTC (SEQ ID NO: 55) and HD NH NG HD Nl NG HD NH HD HD NG HD HD Nl NG NH NG HD;

[0542] TGATCTCGTCATCGCCTCC (SEQ ID NO: 56) and NH Nl NG HD NG HD NH NG HD Nl NG HD NH HD HD NG HD HD;

[0543] GCTTCAGCTTCCTA (SEQ ID NO: 57) and NH HD NG NG HD Nl NH HD NG NG HD HD NG Nl;

[0544] CTGTGATCATGCCA (SEQ ID NO: 58) and HD NG NK NG NH Nl NG HD Nl NG NH HD HD Nl;

[0545] ACAGTGGTACACACCT (SEQ ID NO: 59) and Nl HD Nl NN NG NN NN NG Nl HD Nl HD Nl HD HD NG;

[0546] CCACCCCCCACTAAG (SEQ ID NO: 60) and HD HD Nl HD HD HD HD HD HD Nl HD NG Nl Nl NN; CATTGGCCGGGCAC (SEQ ID NO: 61) and HD Nl NG NG NN NN HD HD NN NN NN HD Nl HD;

[0547] GCTTGAACCCAGGAGA (SEQ ID NO: 62) and NN HD NG NG NN Nl Nl HD HD HD Nl NN NN Nl NN Nl;

[0548] ACACCCGATCCACTGGG (SEQ ID NO: 63) and Nl HD Nl HD HD HD NN Nl NG HD HD Nl HD NG NN NN NN;

[0549] GCTGCATCAACCCC (SEQ ID NO: 64) and NN HD NG NN HD Nl NG HD Nl Nl HD HD HD HD;

[0550] GCCACAAACAGAAATA (SEQ ID NO: 65) and NN NN HD Nl HD NN Nl Nl Nl HD Nl HD HD HD NG HD HD;

[0551] GGTGGCTCATGCCTG (SEQ ID NO: 66) and NN NN NG NN NN HD NG HD Nl NG NN HD HD NG NN;

[0552] GATTTGCACAGCTCAT (SEQ ID NO: 67) and NN Nl NG NG NG NN HD Nl HD Nl NN HD NG HD Nl NG;

[0553] AAGCTCTGAGGAGCA (SEQ ID NO: 68) and Nl Nl NH HD NG HD NG NH Nl NH NH Nl NH HD;

[0554] CCCTAGCTGTCCC (SEQ ID NO: 69) and HD HD HD NG Nl NK HD NG NH NG HD HD HD HD;

[0555] GCCTAGCATGCTAG (SEQ ID NO: 70) and NH HD HD NG Nl NH HD Nl NG NH HD NG Nl NH;

[0556] ATGGGCTTCACGGAT (SEQ ID NO: 71) and Nl NG NH NH NH HD NG NG HD Nl HD NH NH Nl NG;

[0557] GAAACTATGCCTGC (SEQ ID NO: 72) and NH Nl Nl Nl HD NG Nl NG NH HD HD NG NH HD;

[0558] GCACCATTGCTCCC (SEQ ID NO: 73) and NH HD Nl HD HD Nl NG NG NH HD NG HD HD HD;

[0559] GACATGCAACTCAG (SEQ ID NO: 74) and NH Nl HD Nl NG NH HD Nl Nl HD NG HD Nl NH;

[0560] ACACCACTAGGGGT (SEQ ID NO: 75) and Nl HD Nl HD HD Nl HD NG Nl NH NH NH NH NG;

[0561] GTCTGCTAGACAGG (SEQ ID NO: 76) and NH NG HD NG NH HD NG Nl NH Nl HD Nl NH NH;

[0562] GGCCTAGACAGGCTG (SEQ ID NO: 77) and NH NH HD HD NG Nl NH Nl HD Nl NH NH HD NG NH;

[0563] GAGGCATTCTTATCG (SEQ ID NO: 78) and NH Nl NH NH HD Nl NG NG HD NG NG Nl NG HD NH;

[0564] GCCTGGAAACGTTCC (SEQ ID NO: 79) and NN HD HD NG NN NN Nl Nl Nl HD NN NG NG HD HD;

[0565] GTGCTCTGACAATA (SEQ ID NO: 80) and NN NG NN HD NG HD NG NN Nl HD Nl Nl NG Nl;

[0566] GTTTTGCAGCCTCC (SEQ ID NO: 81) and NN NG NG NG NG NN HD Nl NN HD HD NG HD HD;

[0567] ACAGCTGTGGAACGT (SEQ ID NO: 82) and Nl HD Nl NN HD NG NN NG NN NN Nl Nl HD NN NG;

[0568] GGCTCTCTTCCTCCT (SEQ ID NO: 83) and HD Nl Nl NN Nl HD HD NN Nl NN HD Nl HD NG NN HD NG NN;

[0569] CTATCCCAAAACTCT (SEQ ID NO: 84) and HD NG Nl NG HD HD HD Nl Nl Nl Nl HD NG HD NG;

[0570] GAAAAACTATGTAT (SEQ ID NO: 85) and NH Nl Nl Nl Nl Nl HD NG Nl NG NH NG Nl NG;

[0571] AGGCAGGCTGGTTGA (SEQ ID NO: 86) and Nl NH NH HD Nl NH NH HD NG NH NH NG NG NH Nl; CAATACAACCACGC (SEQ ID NO: 87) and HD Nl Nl NG Nl HD Nl Nl HD HD Nl HD NN HD;

[0572] ATGACGGACTCAACT (SEQ ID NO: 88) and Nl NG NN Nl HD NN NN Nl HD NG HD Nl Nl HD NG; and

[0573] CACAACATTTGTAA (SEQ ID NO: 89) and HD Nl HD Nl Nl HD Nl NG NG NG NN NG Nl Nl.

[0574] In embodiments, the GSHS is within about 25, or about 50, or about 100, or about 150, or about 200, or about 300, or about 500 nucleotides of the TA dinucleotide site, or TTAA (SEQ ID NO: 440) tetranucleotide site or ttTTAAaa (SEQ ID NO: 880) octanucleotide site, or taTTAAta (SEQ ID NO: 881) octanucleotide site.

[0575] Illustrative DNA binding codes for targeting human genomic safe harbor in areas of open chromatin via TALES, encompassed by various embodiments are provided in TABLE 20.

[0576] TABLE 20

[0577] In embodiments, the GSHS is selected from TALES in TABLES 8-12. Further illustrative DNA binding codes for targeting human genomic safe harbor in areas of open chromatin via TALES, encompassed by embodiments are provided in TABLES 8-12. In embodiments, the helper enzyme of the present disclosure is capable of inserting a donor DNA at a TA dinucleotide site. In embodiments, the helper enzyme of the present disclosure is capable of inserting a donor DNA at a TTAA (SEQ ID NO: 440) tetranucleotide site, or a ttTTAAaa (SEQ ID NO: 880) octanucleotide site, or a taTTAAta (SEQ ID NO: 881 ) octanucleotide site.

[0578] TABLE 8. TALE sequences targeting the genomic safe harbor site hROSA26 (hg38 chr3:9,396, 132-9,396,332). >

[0579] TABLE 9. TALE sequences targeting the genomic safe harbor site AAVS1 (hg38 chr19:55,112,850-55,113,324).

[0580] TABLE 10. TALE sequences targeting a chromosome 4 genomic safe harbor site (hg38 chr4: 30, 793,038- 30,793,980). TABLE 11. TALE sequences targeting a chromosome 22 genomic safe harbor site (hg38 chr22:35,373,428- 35,380,000).

[0581] TABLE 12. TALE sequences targeting chromosome X (HPRT) (hg38 chrX: 134,475,808-134,476,794).

[0582] *TALES near hotspots with 85 and 51 hits .

[0583] In embodiments, the zinc finger comprises one of the sequences selected from TABLES 13-17, or variants thereof comprising about 99, about 98, about 97, about 95, about 94, about 93, about 92, about 91 , about 90, about 89, about 88, about 87, about 86, about 85, about 84, about 83, about 82, about 81 , about 80 percent identity to the sequence. In embodiments, the zinc finger targets one or more sites selected from TABLES 13-17.

[0584] TABLE 13. Zinc finger sequences targeting the genomic safe harbor site hROSA26 (hg38 chr3:9,396, 132- 9,396,332).

[0585] No Sequences have Target site overlap (TSO) . The f irst and last 4 amino acid residues may be omitted from the amino acid code .

[0586] Available on the world wide web at scripps . edu / barbas / zfdesign / searchsequence . php

[0587] TABLE 14. Zinc finger sequences targeting the genomic safe harbor site AAVS1 (hg38 chr19:55, 112,850- 55,113,324).

[0588] No Sequences have Target site overlap (TSO) . The f irst and last 4 amino acid residues may be omitted from the amino acid code .

[0589] Available on the world wide web at scripps . edu / barbas / zfdesign / searchsequence . php

[0590] TABLE 15. Zinc finger sequences targeting a chromosome 4 genomic safe harbor site (hg38 chr4:30,793,038- 30,793,980).

[0591] No Sequences have Target site overlap (TSO) . The f irst and last 4 amino acid residues may be omitted from the amino acid code .

[0592] Available on the world wide web at scripps . edu / barbas / zfdesign / searchsequence . php

[0593] TABLE 16. Zinc finger sequences targeting a chromosome 22 genomic safe harbor site (hg38 chr22: 35,373,428- 35,380,000).

[0594] No Sequences have Target site overlap (TSO) . The f irst and last 4 amino acid residues may be omitted from the amino acid code .

[0595] Available on the world wide web at scripps . edu / barbas / zfdesign / searchsequence . php

[0596] In embodiments, the present disclosure relates to a system having nucleic acids encoding the enzyme (e.g., without limitation, the helper enzyme) and the donor DNA, respectively.

[0597] Linkers

[0598] In some embodiments, the targeting element comprises a nucleic acid binding component of a gene-editing system. In some embodiments, the helper enzyme the targeting element are connected. Without wishing to be bound by a particular theory, the targeting element may refer to a nucleic acid binding component of the gene-editing system. In some embodiments, the helper enzyme and the targeting element are connected. For example, in embodiments, the the helper enzyme and the targeting element are fused to one another or linked via a linker to one another. In embodiments, the linker is a covalent or non-covalent linker In embodiments, the linker comprises an amino acid sequence of AKLAGGAPAVGGGPKAADKFAATGGS (SEQ ID NO: 8), or a variant thereof having a substitution or deletion.

[0599] In some embodiments, the linker is a flexible linker. In some embodiments, the flexible linker is substantially comprised of glycine and serine residues, optionally wherein the flexible linker comprises (Gly4Ser)n, where n is an integer from 1 to 12. In some embodiments, the flexible linker is of about 20, or about 30, or about 40, or about 50, or about 60 amino acid residues. In embodiments, the flexible linker is about 50, or about 100, or about 150, or about 200 amino acid residues in length. In embodiments, the flexible linker comprises at least about 150 nucleotides (nt), or at least about 200 nt, or at least about 250 nt, or at least about 300 nt, or at least about 350 nt, or at least about 400 nt, or at least about 450 nt, or at least about 500 nt, or at least about 500 nt, or at least about 600 nt. In embodiments, the flexible linker comprises from about 450 nt to about 500 nt.

[0600] In embodiments, the linker is or comprises (GSS)4 or the linker is GS flanked on either side of a DNA binding domain, optionally TALE and ZnF. In embodiments, the linker connects the targeting element of the N-terminus of the helper enzyme or connects the targeting element within the helper enzyme.

[0601] Inteins Inteins (INTervening protEINS) are mobile genetic elements that are protein domains, found in nature, with the capability to carry out the process of protein splicing. See Sarmiento & Camarero (2019) Current protein & peptide science, 20(5), 408-424, which is incorporated by reference herein in its entirety. Protein spicing is a post-translation biochemical modification which results in the cleavage and formation of peptide bonds between precursor polypeptide segments flanking the intein. Id. Inteins apply standard enzymatic strategies to excise themselves post-translationally from a precursor protein via protein splicing. Nanda et al., Microorganisms vol. 8,12 2004. 16 Dec. 2020, doi:10.3390 / microorganisms8122004. An intein can splice its flanking N- and C-terminal domains to become a mature protein and excise itself from a sequence. For example, split inteins have been used to control the delivery of heterologous genes into transgenic organisms. See Wood & Camarero (2014) J Biol Chem. 289(21): 14512-14519. This approach relies on splitting the target protein into two segments, which are then post-translationally reconstituted in vivo by protein trans-splicing (PTS). See Aboye & Camarero (2012) J. Biol. Chem. 287, 27026-27032. More recently, an intein-mediated split-Cas9 system has been developed to incorporate Cas9 into cells and reconstitute nuclease activity efficiently. Truong etal., Nucleic Acids Res. 2015, 43 (13), 6450-6458. The protein splicing excises the internal region of the precursor protein, which is then followed by the ligation of the N-extein and C-extein fragments, resulting in two polypeptides - the excised intein and the new polypeptide produced by joining the C- and N-exteins. Sarmiento & Camarero (2019).

[0602] In embodiments, intein-mediated incorporation of DNA binders such as, without limitation, dCas9, dCas12j, or TALEs, allows creation of a split-enzyme system such as, without limitation, split helper system, that permits reconstitution of the full-length enzyme, e.g., helper, from two smaller fragments. This allows avoiding the need to express DNA binders at the N- or C-terminus of an enzyme, e.g., helper. In this approach, the two portions of an enzyme, e.g., helper, are fused to the intein and, after co-expression, the intein allows producing a full-length enzyme, e.g, helper, by posttranslation modification. Thus, in embodiments, a nucleic acid encoding the enzyme capable of targeted genomic integration by transposition comprises an intein. In embodiments, the nucleic acid encodes the helper enzyme in the form of first and second portions with the intein encoded between the first and second portions, such that the first and second portions are fused into a functional helper enzyme upon post-translational excision of the intein from the helper enzyme.

[0603] In embodiments, an intein is a suitable ligand-dependent intein, for example, an intein selected from those described in U.S. Patent No. 9,200,045; Mootz et al., J. Am. Chem. Soc. 2002; 124, 9044-9045; Mootz et al., J. Am. Chem. Soc. 2003; 125, 10561-10569; Buskirk et al., Proc. Natl. Acad. Sci. USA. 2004; 101 , 10505-10510; Skretas & Wood. Protein Sci. 2005; 14, 523-532; Schwartz, et al., Nat. Chem. Biol. 2007; 3, 50-54; Peck et al., Chem. Biol. 2011 ; 18 (5), 619- 630; the entire contents of each of which are hereby incorporated by reference herein. In embodiments the intein is NpuN (Intein-N) (SEQ ID NO: 423) and / or NpuC (Intein-C) (SEQ ID NO: 424), or a variant thereof, e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

[0604] Dimerization Enhancers

[0605] In embodiments, a nucleic acid encoding the helper enzyme capable of targeted genomic integration by transposition comprises a dimerization enhancer. In embodiments, the nucleic acid encodes the helper enzyme in the form of first and second portions with the dimerization enhancer encoded between the first and second portions, such that the first and second portions are fused into a functional helper enzyme upon post-translational excision of the dimerization enhancer from the helper enzyme. In embodiments, the dimerization enhancer is suitable for linking the helper enzyme and the targeting element. In embodiments, the dimerization enhancer is selected from: a protein comprising a SH3 domain, biotin, avidin, or a rapamycin binder, optionally, wherein the rapamycin binder is FKBP12 or mTOR, or a variant thereof.

[0606] Nucleic Acids of the Disclosure

[0607] In embodiments, a nucleic acid encoding the enzyme (e.g., without limitation, the helper enzyme) is RNA. In embodiments, a nucleic acid encoding the transgene is DNA. In embodiments, the RNA is or comprises messenger RNA (mRNA). In embodiments, the mRNA is or comprises modified mRNA (mmRNA).

[0608] In embodiments, the enzyme (e.g., without limitation, the helper enzyme) is encoded by a recombinant or synthetic nucleic acid. In embodiments, the nucleic acid is RNA, optionally a helper RNA. In embodiments, the nucleic acid is RNA that has a 5’-m7G cap (capO, or cap1 , or cap2), optionally with pseudouridine substitution (e.g., without limitation n-methyl-pseudouridine), and optionally a poly-A tail of about 30, or about 50, or about 100, of about 150 nucleotides in length. In embodiments, the poly-A tail is of about 30 nucleotides in length, optionally 34 nucleotides in length. In embodiments, a nuclear localization signal is placed before the enzyme start codon at the N-terminus, optionally at the C-terminus.

[0609] In embodiments, the nucleic acid that is RNA has a 5’-m7G cap (cap 0, or cap 1, or cap 2). In embodiments, the nucleic acid comprises a 5’ cap structure, a 5’-UTR comprising a Kozak consensus sequence, a 5'-UTR comprising a sequence that increases RNA stability in vivo, a 3'-UTR comprising a sequence that increases RNA stability in vivo, and / or a 3' poly (A) tail.

[0610] In embodiments, the enzyme (e.g., without limitation, a helper) is incorporated into a vector or a vector-like particle. In embodiments, the vector is a non-viral vector.

[0611] In embodiments, a nucleic acid encoding the helper enzyme in accordance with embodiments of the present disclosure, is DNA.

[0612] In various embodiments, a construct comprising a donor is any suitable genetic construct, such as a nucleic acid construct, a plasmid, or a vector. In various embodiments, the construct is DNA, which is referred to herein as a donor DNA. In embodiments, sequences of a nucleic acid encoding the donor is codon optimized to provide improved mRNA stability and protein expression in mammalian systems.

[0613] In embodiments, the helper enzyme and the donor are included in different vectors. In embodiments, the helper enzyme and the donor are included in the same vector.

[0614] In various embodiments, a nucleic acid encoding the helper enzyme capable of targeted genomic integration by transposition (e.g., without limitation, the helper enzyme) is RNA (e.g., helper RNA), and a nucleic acid encoding a donor is DNA.

[0615] In embodiments, the one or more nucleic acid-based agents is in the form of DNA. In embodiments, the DNA is or comprises plasmid DNA. In embodiments, the plasmid DNA has a size of up to about 10 kb, or up to about 12 kb, up to about 15 kb, or about 7.5 to about 15 kb, or about 10 to about 15 kb.

[0616] In embodiments, the plasmid is conjugative, non-conjugative or mobilized. In embodiments, the plasmid is selected from fertility F-plasmids, resistance plasmids, Col plasmids, degradative plasmids, or virulence plasmids. In embodiments, the plasmid is an artificially construct plasmid that is used as vectors in genetic engineering. In embodiments, the plasmid is selected from miniplasmid, nanoplasmid, doggybone or close-ended linear DNA.

[0617] In embodiments, the composition further comprises a donor nucleic acid or is suitable for insertion of a donor nucleic acid, optionally wherein the donor nucleic acid is a transposon. In embodiments, the helper enzyme is suitable of inserting a donor nucleic acid comprising a transgene in a genomic safe harbor site (GSHS) and / or wherein the targeting element is suitable for directing the helper enzyme to a GSHS. In embodiments, a donor DNA and a helper RNA are suitable for transfection at a donor DNA to helper RNA ratio of about 1 to about 4, or about 1 to about 2, or about 1 to about 1. In embodiments, the composition further comprises a nucleic acid encoding a donor comprising a transgene to be integrated, optionally wherein the transgene is defective or substantially absent in a disease state. In embodiments, the transgene comprises a cargo nucleic acid sequence and a first and a second donor end sequences. In embodiments, transgene comprises a cargo nucleic acid sequence and a first and a second donor end sequences. As would be appreciated in the art, a donor often includes an open reading frame that encodes a transgene at the middle of donor and terminal repeat sequences at the 5' and 3' end of the donor. The translated helper (e.g., without limitation, the helper enzyme) binds to the 5' and 3’ sequence of the donor and carries out the transposition function.

[0618] In embodiments, a donor is used interchangeably with transposable elements, which are used to refer to polynucleotides capable of inserting copies of themselves into other polynucleotides. The term donor is well known to those skilled in the art and includes classes of donors that can be distinguished on the basis of sequence organization, for example inverted terminal sequences at each end, and / or directly repeated long terminal repeats (LTRs) at the ends. In embodiments, the donor as described herein may be described as a piggy Bac like element, e.g., a donor element that is characterized by its traceless excision, which recognizes TTAA (SEQ ID NO: 440) sequence and restores the sequence at the insert site back to the original TTAA (SEQ ID NO: 440) sequence after removal of the donor.

[0619] In embodiments, the donor is flanked by one or more end sequences or terminal ends. In embodiments, the donor is or comprises a gene encoding a complete polypeptide. In embodiments, the donor is or comprises a gene which is defective or substantially absent in a disease state.

[0620] In embodiments, a transgene is associated with various regulatory elements that are selected to ensure stable expression of a construct with the transgene. Thus, in embodiments, a transgene is encoded by a non-viral vector (e.g., without limitation, a DNA plasmid) that can comprise one or more insulator sequences that prevent or mitigate activation or inactivation of nearby genes. The insulators flank the donor (transgene cassette) to reduce transcriptional silencing and position effects imparted by chromosomal sequences. As an additional effect, the insulators can eliminate functional interactions of the transgene enhancer and promoter sequences with neighboring chromosomal sequences. In embodiments, the one or more insulator sequences comprise an HS4 insulator (1.2-kb 5'-HS4 chicken [3-globin (cHS4) insulator element) and an D4Z4 insulator (tandem macrosatellite repeats linked to Facio-Scapulo-Humeral Dystrophy (FSHD). In embodiments, the sequences of the HS4 insulator and the D4Z4 insulator are as described in Rival-Gervier et al. Mol Ther. 2013 Aug; 21 (8): 1536-50, which is incorporated herein by reference in its entirety.

[0621] In embodiments, the transgene is inserted into a GSHS location in a host genome. GSHSs is defined as loci well-suited for gene transfer, as integrations within these sites are not associated with adverse effects such as proto-oncogene activation, tumor suppressor inactivation, or insertional mutagenesis. GSHSs can defined by the following criteria: (1) distance of at least 50 kb from the 5 end of any gene, (2) distance of at least 300 kb from any cancer-related gene, (3) distance of at least 300 kb from any microRNA (miRNA), (4) location outside a transcription unit, and (5) location outside ultra-conserved regions (UCRs) of the human genome. See Papapetrou et al. Nat Biotechnol 2011;29:73-8; Bejerano et al. Science 2004;304: 1321-5. Furthermore, the use of GSHS locations can allow stable transgene expression across multiple cell types. One such site, chemokine C-C motif receptor 5 (CCR5) has been identified and used for integrative gene transfer. CCR5 is a member of the beta chemokine receptor family and is required for the entry of R5 tropic viral strains involved in primary infections. A homozygous 32 bp deletion in the CCR5 gene confers resistance to HIV-1 virus infections in humans. Disrupted CCR5 expression, naturally occurring in about 1 % of the Caucasian population, does not appear to result in any reduction in immunity. Lobritz at a / ., Viruses 2010;2:1069-105. A clinical trial has demonstrated safety and efficacy of disrupting CCR5 via targetable nucleases. Tebas at al., HIV. N Engl J Med 2014;370:901-10.

[0622] In embodiments, the donor is under control of a tissue-specific promoter. The tissue-specific promoter is, e.g, without limitation, a liver-specific promoter. In embodiments, the liver-specific promoter is an LP1 promoter that, in embodiments, is a human LP1 promoter. The LP1 promoter is described, e.g, in Nathwani ef al. Blood vol. 2006; 107(7): 2653-61 , and it is constructed, without limitation, as described in Nathawani et al.

[0623] It should be appreciated however that a variety of promoters can be used, including other tissue-specific promoters, inducible promoters, constitutive promoters, etc.

[0624] In embodiments, the present nucleic acids include polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or analogs or derivatives thereof. In embodiments, there is provided double- and singlestranded DNA, as well as double- and single-stranded RNA, and RNA-DNA hybrids. In embodiments, transcriptionally- activated polynucleotides such as methylated or capped polynucleotides are provided. In embodiments, the present compositions are mRNA or DNA.

[0625] In embodiments, the present non-viral vectors are linear or circular DNA molecules that comprise a polynucleotide encoding a polypeptide and is operably linked to control sequences, wherein the control sequences provide for expression of the polynucleotide encoding the polypeptide. In embodiments, the non-viral vector comprises a promoter sequence, and transcriptional and translational stop signal sequences. Such vectors may include, among others, chromosomal and episomal vectors, e.g, vectors bacterial plasmids, from donors, from yeast episomes, from insertion elements, from yeast chromosomal elements, and vectors from combinations thereof. The present constructs may contain control regions that regulate as well as engender expression.

[0626] In embodiments, the construct comprising the helper enzyme and / or transgene is codon optimized. Transgene codon optimization is used to optimize therapeutic potential of the transgene and its expression in the host organism. Codon optimization is performed to match the codon usage in the transgene with the abundance of transfer RNA (tRNA) for each codon in a host organism or cell. Codon optimization methods are known in the art and described in, for example, WO 2007 / 142954, which is incorporated by reference herein in its entirety. Optimization strategies can include, for example, the modification of translation initiation regions, alteration of mRNA structural elements, and the use of different codon biases. In embodiments, the construct comprising the helper enzyme and / or transgene includes several other regulatory elements that are selected to ensure stable expression of the construct. Thus, in embodiments, the non-viral vector is a DNA plasmid that can comprise one or more insulator sequences that prevent or mitigate activation or inactivation of nearby genes. In embodiments, the one or more insulator sequences comprise an HS4 insulator (1 ,2-kb 5'-H S4 chicken P-globin (cHS4) insulator element) and an D4Z4 insulator (tandem macrosatellite repeats linked to Facio-Scapulo- Humeral Dystrophy (FSHD). In embodiments, the sequences of the HS4 insulator and the D4Z4 insulator are as described in Rival-Gervier et al. Mol Ther. 2013 Aug; 21 (8): 1536-50, which is incorporated herein by reference in its entirety. In embodiments, the gene of the construct comprising the helper enzyme and / or transgene is capable of transposition in the presence of a helper. In embodiments, the non-viral vector in accordance with embodiments of the present disclosure comprises a nucleic acid construct encoding a helper. The helper (e.g., without limitation, the helper enzyme of the present disclosure) is an RNA helper plasmid. In embodiments, the non-viral vector further comprises a nucleic acid construct encoding a DNA helper plasmid. In embodiments, the helper is an in v / fro-transcribed mRNA helper. The helper (e.g., without limitation, the helper enzyme of the present disclosure) is capable of excising and / or transposing the gene from the construct comprising the helper enzyme and / or transgene to site- or locus-specific genomic regions.

[0627] In embodiments, the enzyme (e.g., without limitation, the helper enzyme) and the donor are included in the same vector.

[0628] In embodiments, the helper enzyme is disposed on the same (c / s) or different vector (trans) than a donor with a transgene. Accordingly, in embodiments, the helper enzyme and the donor encompassing a transgene are in cis configuration such that they are included in the same vector. In embodiments, the helper enzyme and the donor encompassing a transgene are in trans configuration such that they are included in different vectors. The vector is any non-viral vector in accordance with the present disclosure.

[0629] In some aspects, a nucleic acid encoding the donor system of the present disclosure capable of targeted genomic integration by transposition (e.g., a helper) in accordance with embodiments of the present disclosure is provided. The nucleic acid is or comprises DNA or RNA. In embodiments, the nucleic acid encoding the helper enzyme is DNA. In embodiments, the nucleic acid encoding the helper enzyme capable of targeted genomic integration by transposition (e.g., a helper of the present disclosure) is RNA such as, e.g., helper RNA. In embodiments, the helper is incorporated into a vector. In embodiments, the vector is a non-viral vector.

[0630] In embodiments, the enzyme or variant thereof is incorporated into a vector or a vector-like particle. In embodiments, the vector or a vector-like particle comprises one or more expression cassettes. In embodiments, the vector or a vectorlike particle comprises one expression cassette. In embodiments, the expression cassette further comprises the enzyme or variant thereof, the transgene, the donor end sequences, or a combination thereof. In embodiments, the enzyme or variant thereof, the transgene, the donor end sequences, or a combination thereof are incorporated into one or more vectors or vector-like particles. In embodiments, the enzyme or variant thereof, the transgene, the donor end sequences, or combination thereof are incorporated into a same vector or vector-like particle. In embodiments, the enzyme or variant thereof, the transgene, the donor end sequences, or combination thereof is incorporated into different vectors or vector-like particles. In embodiments, the vector or vector-like particle is nonviral.

[0631] In embodiments, the donor is under the control of at least one tissue-specific promoter. In embodiments, at least one tissue-specific promoter is a single promoter. In embodiments, at least one tissue-specific promoter is under the control of a dual promoter or a tandem promoter. In embodiments, the transgene to be integrated comprises at least one gene of interest. In embodiments, the transgene to be integrated comprises one gene of interest. In embodiments, the transgene to be integrated comprises two or more genes of interest.

[0632] In embodiments, the present helper enzyme can be in the form or an RNA or DNA and have one or two N-terminus nuclear localization signal (NLS) to shuttle the protein more efficiently into the nucleus. For example, in embodiments, the present helper enzyme further comprises one, two, three, four, five, or more NLSs. Examples of NLS are provided in Kosugi et al. (J. Biol. Chem. (2009) 284:478-485; incorporated by reference herein). In a particular embodiment, the NLS comprises the consensus sequence K(K / R)X(K / R) (SEQ ID NO: 348). In an embodiment, the NLS comprises the consensus sequence (K / R)(K / R)X10-12(K / R)3 / 5(SEQ ID NO: 349), where (K / R)3 / 5represents at least three of the five amino acids is either lysine or arginine. In an embodiment, the NLS comprises the c-myc NLS. In a particular embodiment, the c-myc NLS comprises the sequence PAAKRVKLD (SEQ ID NO: 350). In a particular embodiment, the NLS is the nucleoplasmin NLS. In embodiments, the nucleoplasmin NLS comprises the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 351). In embodiments, the NLS comprises the SV40 Large T-antigen NLS. In embodiments, the SV40 Large T-antigen NLS comprises the sequence PKKKRKV (SEQ ID NO: 352). In a particular embodiment, the NLS comprises three SV40 Large T-antigen NLSs (e.g., DPKKKRKVDPKKKRKVDPKKKRKV (SEQ ID NO: 353). In embodiments, the NLS may comprise mutations / variations in the above sequences such that they contain 1 or more substitutions, additions, or deletions (e.g., about 1 , or about 2, or about 3, or about 4, or about 5, or about 10 substitutions, additions, or deletions).

[0633] In some aspects, a host cell comprising the nucleic acid in accordance with embodiments of the present disclosure is provided.

[0634] Lipids and LNP Delivery

[0635] In embodiments, a composition or a nucleic acid in accordance with embodiments of the present disclosure is provided wherein the composition is in the form of a lipid nanoparticle (LNP). In embodiments, the composition is encapsulated in an LNP.

[0636] In embodiments, a nucleic acid encoding the helper enzyme and a nucleic acid encoding the transgene are contained within the same lipid nanoparticle (LNP). In embodiments, the nucleic acid encoding the helper enzyme and the nucleic acid encoding the donor are a mixture incorporated into or associated with the same LNP. In embodiments, the polynucleotide encoding the helper enzyme and the polynucleotide encoding the donor are in the form of the same LNP, optionally in a co-formulation.

[0637] In embodiments, the LNP comprises one or more lipids selected from 1 ,2-dioleoyl-3-trimethylammonium propane (DOTAP), a cationic cholesterol derivative mixed with dimethylaminoethane-carbamoyl (DC-Chol), phosphatidylcholine (PC), triolein (glyceryl trioleate), and 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol - 2000 (DMG-PEG 2K), and 1 ,2 distearol -sn-glycerol-3phosphocholine (DSPC) and / or comprising of one or more molecules selected from polyethylenimine (PEI) and poly(lactic-co-glycolic acid) (PLGA), and N-Acetylgalactosamine (GalNAc).

[0638] In embodiments, an LNP is as described, e.g., in Patel et al., J Control Release 2019; 303:91-100. The LNP can comprise one or more of a structural lipid (e.g., DSPC), a PEG-conjugated lipid (CDM-PEG), a cationic lipid (MC3), cholesterol, and a targeting ligand (e.g., GalNAc).

[0639] In embodiments, the one or more lipids comprise cationic lipids; anionic lipids; neutral lipids; multi-valent charged lipids; and zwitterionic lipids.

[0640] In embodiments, the one or more lipids include a delivery lipid. In embodiments, the delivery lipid is or comprises a cationic lipid. In embodiments, the cationic lipid is ionizable and / or hydrolysable. In embodiments, the delivery lipid encapsulates, in whole or in part, the one or more nucleic acid-based agents. In embodiments, the delivery lipid, e.g., when contacted with a cell, promotes or increases endosomal escape. In embodiments, the delivery lipid is or comprises MC3 (DLin-MC3-DMA), C12-200 (1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl) (2- hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol)), SM102 (9-Heptadecanyl 8-{(2- hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate), squaramide (3, 4-diaminocyclobut-3-ene-1 , 2-dione; SQAM), SS-EC (ssPalmE-P4C2), SS-OP (ssPalmO-Phe), [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2- hexyldecanoate), KC2 (DLin-KC2-DMA), DOTAP (Dioleoyl-3-trimethylammonium propane), cKK-E12 (3,6-bis({4- [bis(2-hydroxydodecyl)amino]butyl})piperazine-2, 5-dione), ATX-001 (di((Z)-non-2-en-1-yl) 8,8'-((2-((2-

[0641] (dimethylamino)ethyl)thio)acetyl)azanediyl)dioctanoate), ATX-002 (di((Z)-non-2-en-1-yl) 8,8'-((((2-

[0642] (dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate), ATX-012, ATX-61 , ATX-0081 , ATX-0095, Lipid 5 (8-[(2- hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]-octanoic acid, 1-octylnonyl ester), and / or ALC-0315 (2-hexyl-decanoic acid, 1,1'-[[(4-hydroxybutyl)imino]di-6, 1 -hexanediyl] ester, [(4-Hydroxybutyl)azanediyl]di(hexane-6, 1-diyl) bis(2- hexyldecanoate)). Various ATX lipids that are in embodiments delivery lipids of the disclosure are described in WO2018222890 and W02015074085, each of which is hereby incorporate by reference in its entirety. In embodiments, the delivery lipid is or comprises DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12-5, and C12-200. In embodiments, the one or more lipids include one type of delivery lipid. In embodiments, the one or more lipids include more than one type of delivery lipid. In embodiments, the one or more lipids include two types of delivery lipid. In embodiments, the one or more lipids include both MC3 and C12-200.

[0643] In embodiments, the one or more lipids include cholesterol. In embodiments, the cholesterol provides increased or enhanced LNP stability.

[0644] In embodiments, the one or more lipids include a helper lipid. In embodiments, the helper lipid encapsulates, in whole or in part, the one or more nucleic acid-based agents. In embodiments, the helper lipid, e.g, when contacted with a cell, promotes or increases endosomal escape. In embodiments, the helper lipid is a phospholipid. In embodiments the helper lipid is a neutral lipid. In embodiments, the neutral lipid is or comprises dioleoylphosphatidylethanolamine (DOPE) and / or 1 ,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC). In embodiments, the helper lipid is a cationic lipid. In embodiments, the cationic lipid is or comprises N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1,2-bis(oleoyloxy)-3-3-(trimethylammonia) propane (DOTAP), or 1 ,2-dioleoyl-3-dimethylammonium- propane (DODAP). In embodiments, the helper lipid is or comprises DOPE, DOPC, and / or Distearoylphosphatidylcholine (DSPC). In embodiments, the helper lipid is or comprises both DOPE and DSPC. In embodiments, the helper lipid is or comprises both DOPE and DOPC. In embodiments, the helper lipid is or comprises all of DOPE, DSPC, and DOPC.

[0645] In embodiments, the one or more lipids include a PEGylated lipid. In embodiments, the PEGylated lipid shields the LNP and reduces or prevents degradation of the LNP, e.g., in the bloodstream. In embodiments, the PEGylated lipid has a PEG molecule covalently attached to it, where the PEG has an average molecular weight of from about 1 kDa to about 50 kDa, or about 1 kDa to about 30 kDa, or from about 1 kDa to about 10 kDa, or about 1 kDa, or about 2 kDa, or about 3 kDa, or about 4 kDa, or about 5 kDa, or about 10 kDa. In embodiments, the PEG is a linear, a branched PEG, a star PEG, or a comb PEG. In embodiments the PEG is selected from PEG200, PEG300, PEG400, PEG600, PEG800, PEG1000, PEG1500, PEG2000, PEG3000, and PEG4000. In embodiments, the PEG is PEG2000. In embodiments the PEGylated lipid is or comprises 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG). In embodiments, the DMG-PEG is or comprises 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-2k- DMG) and / or a PEG conjugated to a ceramide, e.g., C8 PEG 2k Ceramide, C16 PEG 2k Ceramide. In embodiments the PEGylated lipid is or comprises PEG-dimyristyloxypropyl (PEG-DMA), a PEG- distearyloxypropyl (PEG-DSA), PEG-diacylglycerol (PEG-DAG), or PEG-dialkyloxypropyl (PEG-DAA).

[0646] In embodiments, the one or more lipids do not include a PEGylated lipid. In embodiments, the one or more lipids do not include a PEGylated lipid, e.g, when being formulated for local administration.

[0647] In embodiments, the composition comprises a mixture of PEGylated lipids and free PEG chains. In embodiments, a nanoparticle is a particle having a diameter of less than about 1000 nm. In embodiments, nanoparticles of the present disclosure have a greatest dimension (e.g., diameter) of about 500 nm or less, or about 400 nm or less, or about 300 nm or less, or about 200 nm or less, or about 100 nm or less. In embodiments, nanoparticles of the present disclosure have a greatest dimension ranging between about 50 nm and about 150 nm, or between about 70 nm and about 130 nm, or between about 80 nm and about 120 nm, or between about 90 nm and about 110 nm. In embodiments, the nanoparticles of the present disclosure have a greatest dimension (e.g., a diameter) of about 100 nm.

[0648] In some aspects, the cell in accordance with the present disclosure is prepared via an in vivo genetic modification method. In embodiments, a genetic modification in accordance with the present disclosure is performed via an ex vivo method.

[0649] In some aspects, the cell in accordance with the present disclosure is prepared by contacting a cell with a helper enzyme capable of targeted genomic integration by transposition (e.g., without limitation, the helper enzyme) in vivo. In embodiments, the cell is contacted with the helper enzyme ex vivo.

[0650] In embodiments, the present method provides high specific targeting as compared to a method that does not use the helper enzyme with a target selector.

[0651] Therapeutic Applications

[0652] In embodiments, the transgene of interest in accordance with embodiments of the present disclosure can encode various genes.

[0653] In embodiments, the helper enzyme and the donor are included in the same pharmaceutical composition.

[0654] In embodiments, the helper enzyme and the donor are included in different pharmaceutical compositions.

[0655] In embodiments, the helper enzyme and the donor are co-transfected.

[0656] In embodiments the helper enzyme and the donor are transfected separately.

[0657] In embodiments, a transfected cell for gene therapy is provided, wherein the transfected cell is generated using the helper enzyme in accordance with embodiments of the present disclosure.

[0658] In embodiments, a method of delivering a cell therapy is provided, comprising administering to a patient in need thereof the transfected cell generated using the helper enzyme in accordance with embodiments of the present disclosure.

[0659] In embodiments, a method of treating a disease or condition using a cell therapy, comprising administering to a patient in need thereof the transfected cell generated using the helper enzyme in accordance with embodiments of the present disclosure. In embodiments, there is provided a method for inserting a gene into the genome of a cell, comprising contacting a cell with the composition of the present disclosure.

[0660] In embodiments, there is provided a method for treating a disease or disorder ex vivo, comprising contacting a cell with the composition of the present disclosure and administering the cell to a subject in need thereof.

[0661] In embodiments, there is provided a method for treating a disease or disorder in vivo, comprising administering the composition of the present disclosure to a subject in need thereof.

[0662] In embodiments, the disease or condition may comprise cancer. In embodiments, the cancer is or comprises an adrenal cancer, a biliary track cancer, a bladder cancer, a bone / bone marrow cancer, a brain cancer, a breast cancer, a cervical cancer, a colorectal cancer, a cancer of the esophagus, a gastric cancer, a head / neck cancer, a hepatobiliary cancer, a kidney cancer, a liver cancer, a lung cancer, an ovarian cancer, a pancreatic cancer, a pelvis cancer, a pleura cancer, a prostate cancer, a renal cancer, a skin cancer, a stomach cancer, a testis cancer, a thymus cancer, a thyroid cancer, a uterine cancer, a lymphoma, a melanoma, a multiple myeloma, or a leukemia.

[0663] In embodiments, the cancer is selected from one or more of the basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer; glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer; melanoma; myeloma; neuroblastoma; oral cavity cancer; ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; Hodgkin's lymphoma; non-Hodgkin's lymphoma; B-cell lymphoma; small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); and Hairy cell leukemia.

[0664] In embodiments, the cancer is selected from one or more of basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade / foll icular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (e.g., that associated with brain tumors), and Meigs syndrome.

[0665] In embodiments, the disease or condition is or comprises an infectious disease. In embodiments, the infectious disease is a coronavirus infection, optionally selected from infection with SAR-CoV, MERS-CoV, and SARS-CoV-2, or variants thereof.

[0666] In embodiments, the infectious disease is or comprises a disease comprising a viral infection, a parasitic infection, or a bacterial infection. In embodiments, the viral infection is caused by a virus of family Flaviviridae, a virus of family Picornaviridae, a virus of family Orthomyxoviridae, a virus of family Coronaviridae, a virus of family Retroviridae, a virus of family Paramyxoviridae, a virus of family Bunyaviridae, or a virus of family Reoviridae.

[0667] In embodiments, the virus of family Coronaviridae comprises a betacoronavirus or an alphacoronavirus, optionally wherein the betacoronavirus is selected from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, and HCoV-OC43, or the alphacoronavirus is selected from a HCoV-NL63 and HCoV-229E. In embodiments, the infectious disease comprises a coronavirus infection 2019 (COVID-19).

[0668] In embodiments, the method requires a single administration. In embodiments, the method requires a plurality of administrations.

[0669] Isolated Cell

[0670] In some aspects of the present disclosure, an isolated cell is provided that comprises the transfected cell in accordance with embodiments of the present disclosure.

[0671] In some aspects, the present disclosure provides an ex vivo gene therapy approach. Accordingly, in embodiments, the method that is used to treat an inherited or acquired disease in a patient in need thereof comprises (a) contacting a cell obtained from a patient (autologous) or another individual (allogeneic) with a transfected cell in accordance with embodiments of the present disclosure; and (b) administering the cell to a patient in need thereof. One of the advantages of ex vivo gene therapy is the ability to “sample” the transduced cells before patient administration. This facilitates efficacy and allows performing safety checks before introducing the cell (s) to the patient. For example, the transduction efficiency and / or the clonality of integration can be assessed before infusion of the product. The present disclosure provides transfected cells and methods that can be effectively used for ex vivo gene modification.

[0672] In embodiments, a composition comprising transfected cells in accordance with the present disclosure comprises a pharmaceutically acceptable carrier, excipient, or diluent.

[0673] Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, N.Y.). For example, pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile, and the fluid should be easy to draw up by a syringe. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0674] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0675] Therapeutic compounds can be prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as collagen, ethylene vinyl acetate, polyanhydrides (e.g., poly[1,3-bis(carboxyphenoxy)propane-co-sebacic-acid] (PCPP-SA) matrix, fatty acid dimer- sebacic acid (FAD-SA) copolymer, poly(lactide-co-glycolide)), polyglycolic acid, collagen, polyorthoesters, polyethyleneglycol-coated liposomes, and polylactic acid. Such formulations can be prepared using standard techniques, or obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811. Semisolid, gelling, soft-gel, or other formulations (including controlled release) can be used, e.g., when administration to a surgical site is desired. Methods of making such formulations are known in the art and can include the use of biodegradable, biocompatible polymers. See, e.g., Sawyer et al., Yale J Biol Med. 2006; 79(3-4): 141-152.

[0676] In embodiments, there is provided a method of transforming a cell using the construct comprising the helper enzyme and / or transgene described herein in the presence of a helper (e.g., without limitation, the helper enzyme) to produce a stably transfected cell which results from the stable integration of a gene of interest into the cell. In embodiments, the stable integration comprises an introduction of a polynucleotide into a chromosome or mini-chromosome of the cell and, therefore, becomes a relatively permanent part of the cellular genome.

[0677] In embodiments, there is provided a transgenic organism that may comprise cells which have been transformed by the methods of the present disclosure. In embodiments, the organism may be a mammal or an insect. When the organism is a mammal, the organism may include, but is not limited to, a mouse, a rat, a chimpanzee, an elephant, a dog, a rabbit, a raccoon, and the like. When the organism is an insect, the organism may include, but is not limited to, a fruit fly, an ant, a mosquito, a bollworm, and the like.

[0678] Methods For Identifying Site-Specific Targeting to a Nucleic Acid

[0679] In aspects, there is provided a method for identifying site-specific targeting to a nucleic acid by a helper enzyme and a targeting element, comprising: (a) transfecting a cell with a donor plasmid, the helper enzyme and a targeting element, and a reporter plasmid, wherein: the donor plasmid comprises a first fragment of a reporter gene under the control of a promoter and a splice-donor site (SD); the reporter plasmid comprises a landing pad for the targeting element comprising site specific DNA binding recognition sites flanking a TTAA followed by a splice acceptor site (SA) and a second fragment of a reporter gene; and (b) splicing and integrating into the landing pad, to permit the reconstitution of the reporter gene from the fragments thereof and thereby causing a reporter readout. In embodiments, the method further comprises (c) amplifying the donor plasmid to identify targeting. In embodiments, the method further comprises (d) sequencing the amplified product to analyze integration in specific sequence regions. In embodiments, the SA and SD are spliced out of the donor plasmid in step (b). In embodiments, the amplifying is via PCR. In embodiments, the sequencing is amplicon sequencing in embodiments, the fluorescent protein is or comprises a monomeric red fluorescent protein (mRFP). In embodiments, the mRFP is selected from mCherry, DsRed, mRFP1, mStrawberry, mOrange, and dTomato. In embodiments, the fluorescent protein is or comprises a green fluorescent protein (GFP). In embodiments, the reporter readout is fluorescence. In embodiments, the promoter is selected from cytomegalovirus (CMV), CIW enhancer fused to the chicken p-actin (GAG), chicken p-actin (CBA), simian vacuolating virus 40 (SV40), p glucuronidase (GUSB), polyubiquitin C gene (UBC), elongation-factor 1a subunit (EF-1 a), and phosphoglycerate kinase (PGK).

[0680] In embodiments, the helper enzyme is a recombinase, integrase or a transposase. In embodiments, the helper enzyme is a mammal-derived transposase. In embodiments, the helper enzyme is derived from Bombyx mori, Xenopus tropicalis, Trichoplusia ni, Myotis lucifugus, Rhinolophus ferrumequinum, Rousetius aegyptiacus, Phyllostomus discolor, Myotis myotis, Pteropus vampyrus, Pipistrellus kuhlii, troglodytes, Molossus molossus, or Homo sapiens.

[0681] In aspects, there is provided a composition, and method of use of a composition, comprising a helper enzyme or a nucleic acid encoding the helper enzyme, an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1 and a non-polar aliphatic amino acid at position 2 of SEQ ID NO: 1, or a position corresponding thereto, one or more amino acid substitutions selected from S8X, C13X, Y281X, N335X, and D416X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, and one or more amino acid substitutions selected from T331X, I332X, R333X, K334X, R336X, G337X, and I338X of SEQ ID NO: 1 or a substitution at a position corresponding thereto.

[0682] In aspects, there is provided a composition, and method of use of a composition, comprising a helper enzyme or a nucleic acid encoding the helper enzyme, an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1 and a non-polar aliphatic amino acid at position 2 of SEQ ID NO: 1, or a position corresponding thereto, and S8X, C13X, N335X, I338X, and D416X of SEQ ID NO: 1 or a substitution at a position corresponding thereto.

[0683] In aspects, there is provided a composition, and method of use of a composition, comprising a helper enzyme or a nucleic acid encoding the helper enzyme, an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1 and a non-polar aliphatic amino acid at position 2 of SEQ ID NO: 1, or a position corresponding thereto, S8X, 013X, R333X, N335X, and D416X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, and / or one or more amino acid substitutions selected from K16X, N19X, N28X, L48X, S56X, N128X, K137X, D208X, N226X, N241X, K286X, N317X, T331X, I332X, K334X, R336X, G337X, I338X, K349X, K369X, E381X, I388X, D480X, M481X, L486X, K525X, K541 R, F552X, Y572X, of SEQ ID NO: 1 or a substitution at a position corresponding thereto.

[0684] In embodiments, targeting element is or comprises one or more of a Cas enzyme, which is optionally catalytically inactive and which is optionally associated with a guide RNA (gRNA), a transcription activator-like effector (TALE) DNA binding domain (DBD), a Zinc finger (ZnF), a catalytically inactive transcription factor, catalytically inactive nickase, a transcriptional activator, a transcriptional repressor, a recombinase, a DNA methyltransferase, a histone methyltransferase, a paternally expressed gene 10 (PEG10), and a transposon-encoded polypeptide D (TnsD) or a variant thereof.

[0685] Definitions

[0686] The following definitions are used in connection with the disclosure disclosed herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of skill in the art to which this disclosure belongs.

[0687] As used herein, “a,” “an,” or “the” can mean one or more than one.

[0688] Further, the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, the language “about 50” covers the range of 45 to 55.

[0689] An “effective amount,” when used in connection with medical uses is an amount that is effective for providing a measurable treatment, prevention, or reduction in the rate of pathogenesis of a disease of interest.

[0690] The term “in vivo" refers to an event that takes place in a subject's body.

[0691] The term “ex vivo" refers to an event which involves treating or performing a procedure on a cell, tissue and / or organ which has been removed from a subject's body. Aptly, the cell, tissue and / or organ may be returned to the subject's body in a method of treatment or surgery.

[0692] As used herein, the term “variant” encompasses but is not limited to nucleic acids or proteins which comprise a nucleic acid or amino acid sequence which differs from the nucleic acid or amino acid sequence of a reference by way of one or more substitutions, deletions and / or additions at certain positions. The variant may comprise one or more conservative substitutions. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids.

[0693] “Carrier” or “vehicle” as used herein refer to carrier materials suitable for drug administration. Carriers and vehicles useful herein include any such materials known in the art, e.g., any liquid, gel, solvent, liquid diluent, solubilizer, surfactant, lipid, or the like, which is nontoxic, and which does not interact with other components of the composition in a deleterious manner.

[0694] The phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio. The terms "pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the disclosure is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the described compositions and methods.

[0695] As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified. As used herein, the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology. Similarly, the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.

[0696] Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the disclosure, the present disclosure, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of' or “consisting essentially of.”

[0697] As used herein, the words “preferred” and “preferably” refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the technology.

[0698] The amount of compositions described herein needed for achieving a therapeutic effect may be determined empirically in accordance with conventional procedures for the particular purpose. Generally, for administering therapeutic agents for therapeutic purposes, the therapeutic agents are given at a pharmacologically effective dose. A “pharmacologically effective amount,” “pharmacologically effective dose,” “therapeutically effective amount,” or “effective amount" refers to an amount sufficient to produce the desired physiological effect or amount capable of achieving the desired result, particularly for treating the disorder or disease. An effective amount as used herein would include an amount sufficient to, for example, delay the development of a symptom of the disorder or disease, alter the course of a symptom of the disorder or disease (e.g., slow the progression of a symptom of the disease), reduce or eliminate one or more symptoms or manifestations of the disorder or disease, and reverse a symptom of a disorder or disease. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized. Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to about 50% of the population) and the ED50 (the dose therapeutically effective in about 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50 / ED50. In embodiments, compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from in vitro assays, including, for example, cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the I C50 as determined in cell culture, or in an appropriate animal model. Levels of the described compositions in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

[0699] As used herein, “methods of treatment” are equally applicable to use of a composition for treating the diseases or disorders described herein and / or compositions for use and / or uses in the manufacture of a medicaments for treating the diseases or disorders described herein.

[0700] SELECTED SEQUENCES

[0701] In embodiments, the present disclosure provides for any of the sequence provided herein, including the below, and a variant sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.

[0702] SEQ ID NO: 1 : amino acid sequence helper transposases.

[0703] SEQ ID NO: 2: amino acid sequence of a variant of a hyperactive helper with P at position 8, R at position 13, and N at position 416 (S8P_C13R_D416N) (572 amino acids)

[0704] EXAMPLES

[0705] Hereinafter, the present disclosure will be described in further detail with reference to examples. These examples are illustrative purposes only and are not to be construed to limit the scope of the present disclosure In addition, various modifications and variations can be made without departing from the technical scope of the present disclosure.

[0706] Example 1 - Design of Transposon System

[0707] Following a screen of numerous DNA-contact mutants for phenotypes that demonstrated an ability to excise a DNA donor element but are deficient in or display a greatly reduced ability to integrate that same DNA donor (Ex+lnt-), a plasmid-based targeting assay was used to discover Ex+lnt- mutants that display strong on-target and reduced off- target integration. Ex+lnt- mut1 (D416N_Y281A_K334A_N335A) was identified as the mutant with the greatest reduction of non-specific integration, with minimal loss of on-target integration. Mut 1 was then used as a template for further mutagenesis. Additional mutations (1-2) were layered onto mut 1 by changing the amino acid at the following DNA-contact positions (331-338). Each position was changed to 1-3 different amino acids to change the amino acid to an amino acid with similar characteristics or to one with vastly different characteristics (alanine). A similar set of mutants was also created using another Ex+lnt- mutant (mut5; D416N_R333A_N335A) as a starting backbone. This mutant also displayed a small reduction of non-specific integration, with minimal loss of on-target integration, but contained mutations at different DNA-contact amino acids, specifically it lacked a mutation at position 281 (FIG. 1). FIG. 3 shows ddPCR analysis of Targeted and Total Integration of helper enzyme mutants. Further mutations to helper enzyme (brackets) reduced total integration 3-4-fold (bars on the right) without affecting targeted integration (bars on the left) compared to their parent mutants.

[0708] Table E: Mutants used in FIG. 3

[0709] One hundred and seventy-four Ex+lnt- mutants based on mut 1 or mut 5 were synthesized (see TABLE A) and screened for targeted genomic integration utilizing an engineered HEK293 cell line containing a landing pad that had been genomically integrated in multiple copies with Sleeping beauty transposase (FIG. 2). This landing pad contained coding sequence of a half of GFP protein after the specific TTAA sequence, and a DNA donor element contained the other half. Upon integration of the DNA donor at a specific targeted site, the two halves of GFP are reunited and can be spliced into a functional GFP protein. Flow cytometry was then used to measure what percentage of cells contained a functional GFP protein. Targeting efficiency of the Ex+lnt- mutants in SB cell line are shown in Table C and Table D.

[0710] While most of these mutants did not improve on-target integration, it is reasoned that these mutants reduced off-target or non-specific integration. Initially, three mut 1 engineered mutants and six mut 5 engineered mutants were screened for both on- and off-target integration via a ddPCR assay. All nine mutants displayed lower levels of off-target integration while also displaying no or minimal reduction to on-target integration (FIG. 3).

[0711] Table C: Targeting efficiency of mutant constructs

[0712] Table D: Targeting efficiency of mutant constructs

[0713] EQUIVALENTS

[0714] While the disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure as come within known or customary practice within the art to which the disclosure pertains and as may be applied to the essential features herein set forth and as follows in the scope of the appended claims.

[0715] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

[0716] INCORPORATION BY REFERENCE All patents and publications referenced herein are hereby incorporated by reference in their entireties.

[0717] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure.

[0718] As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.

Claims

1. CLAIMSWhat is claimed is:1 . A composition comprising a helper enzyme or a nucleic acid encoding the helper enzyme comprising: an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1 and a non-polar aliphatic amino acid at position 2 of SEQ ID NO: 1, or a position corresponding thereto,S8X, C13X, Y281X, N335X, and D416X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, and / or one or more additional amino acid substitutions selected from T331X, I332X, R333X, K334X, R336X, G337X, and I338X of SEQ ID NO: 1 or a substitution at a position corresponding thereto.

2. The composition of claim 1 , wherein the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), and proline (P).

3. The composition of claims 1 or 2, wherein X is selected from a non-polar amino acid, optionally selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), methionine (M), tryptophan (W), and phenylalanine (F), or a polar amino acid, optionally selected from serine (S), cysteine (C), asparagine (N), glutamine (Q), threonine (T), and tyrosine (Y), or a basic amino acid, optionally selected from arginine (R), histidine (H), or an acidic amino acid, optionally selected from aspartic acid (D), and glutamic acid (E), or a positively charged hydrophilic amino acid, optionally selected from lysine (K), arginine (R), and histidine (H).

4. The composition of any one of claims 1-3, wherein the helper enzyme comprises a substitution at S8X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, optionally wherein X is a non-polar residue, optionally selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), methionine (M), tryptophan (W), and phenylalanine (F), or a polar and neutral hydrophilic residue, optionally proline (P).

5. The composition of any one of claims 1-4, wherein the helper enzyme comprises a substitution at C13X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, optionally wherein X is a positively charged hydrophilic amino acid, optionally selected from arginine (R), lysine (K), and histidine (H).

6. The composition of any one of claims 1-5, wherein the helper enzyme comprises a substitution at Y281X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, wherein X is a non-polar amino acid, optionally selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), methionine (M), tryptophan (W), and phenylalanine (F).

7. The composition of any one of claims 1-6, wherein the helper enzyme comprises a substitution at N335X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, wherein X is a non-polar amino acid, optionallyselected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), methionine (M), tryptophan (W), and phenylalanine (F).

8. The composition of any one of claims 1-7, wherein the helper enzyme comprises a substitution at D416X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, wherein X is a polar and positively charged hydrophilic residue, optionally selected from arginine (R) and lysine (K), or a polar and neutral of charge hydrophilic residue, optionally selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C).

9. The composition of any one of claims 1-8, wherein the helper enzyme comprises one or two, or three, or four, or five substitutions selected from: S8P, C13R, Y281A, N335A, and D416N or substitutions corresponding thereto relative to SEQ ID NO: 1.

10. The composition of any one of claims 1-9, wherein the helper enzyme comprises one, or two, or three, or four, substitutions selected from T331A, I332A, I332S, I332M, R333A, R333S, R333K, K334A, R336A, R336S, R336K, G337A, I338A, I338M, and I338S or substitutions corresponding thereto relative to SEQ ID NO: 1.

11. The composition of any one of claims 1-10, wherein the helper enzyme comprises one or two, or three, or four, or five substitutions selected from: S8P, C13R, Y281A, N335A, and D416N or substitutions corresponding thereto relative to SEQ ID NO: 1 , and one, or two, or three, or four substitutions selected from T331A, I332A, I332S, I332M, R333A, R333S, R333K, K334A, R336A, R336S, R336K, G337A, I338A, I338M, and I338S or substitutions corresponding thereto relative to SEQ ID NO: 1.

12. The composition of any one of claims 1-11 , wherein the helper enzyme comprises one, or two, or three, or four, or five, or six, or seven, or eight substitutions selected from: S8P, C13R, Y281A, T331A, I332A, I332S, I332M, R333A, R333S, R333K, K334A, N335A, R336A, R336S, R336K, G337A, I338A, I338M, I338S and D416N or substitutions corresponding thereto relative to SEQ ID NO: 1.

13. The composition of any one of claims 1-12, wherein the helper enzyme comprises or consists of 6 amino acid substitutions.

14. The composition of any one of claims 1-12, wherein the helper enzyme comprises or consists of 7 amino acid substitutions.

15. The composition of any one of claims 1-12, wherein the helper enzyme comprises or consists of 8 amino acid substitutions.

16. The composition of any one of claims 1-15, wherein the helper enzyme comprises a substitution selected from TABLE 1 or a substitution at positions corresponding thereto relative to SEQ ID NO: 1.

17. The composition of any one of claims 1-16, wherein the helper enzyme comprises:S8P_C13R_Y281 A_I332A_K334A_N335A_D416N,S8P_C13R_Y281A_I332A_K334A_N335A_G337A_D416N,S8P_C 13R_Y281 A_l 332A_K334A_N335A_I 338A_D 416 N ,S8P_C 13R_Y281 A_l 332A_K334A_N335A J 338S_D 416 N ,S8P_C13R_Y281A_I332A_K334A_N335A_R336A_D416N,S8P_C13R_Y281A_I332A_K334A_N335A_R336K_D416N,S8P_C13R_Y281A_I332A_K334A_N335A_R336S_D416N,S8P_O13R_Y281A_I332A_R333A_K334A_N335A_D416N,S8P_C13R_Y281A_I332A_R333K_K334A_N335A_D416N,S8P_C13R_Y281A_I332A_R333S_K334A_N335A_D416N,S8P_C13R_Y281A_I332M_K334A_N335A_D416N,S8P_C13R_Y281 AJ332M_K334A_N335A_G337A_D416N,S8P_C 13R_Y281 A J 332 M_K334A_N335A J 338M_D416N ,S8P_C13R_Y281AJ332M_K334A_N335A_R336A_D416N,S8P_C13R_Y281A_I332M_K334A_N335A_R336K_D416N,S8P_C13R_Y281AJ332M_K334A_N335A_R336S_D416N,S8P_C13R_Y281A_I332M_R333A_K334A_N335A_D416N,S8P_C13R_Y281A_I332M_R333K_K334A_N335A_D416N,S8P_C13R_Y281A_I332M_R333S_K334A_N335A_D416N,S8P_C13R_Y281 A_I332S_K334A_N335A_D416N ,S8P_C13R_Y281A_I332S_K334A_N335A_G337A_D416N,S8P_C 13R_Y281 A_l 332S_K334A_N335A J 338A_D 416 N ,S8P_C 13R_Y281 A_l 332S_K334A_N335AJ 338S_D 416 N ,S8P_C13R_Y281A_I332S_K334A_N335A_R336A_D416N,S8P_C13R_Y281A_I332S_K334A_N335A_R336K_D416N,S8P_C13R_Y281A_I332S_K334A_N335A_R336S_D416N,S8P_C13R_Y281A_I332S_R333A_K334A_N335A_D416N,S8P_C13R_Y281A_I332S_R333K_K334A_N335A_D416N,S8P_C13R_Y281A_I332S_R333S_K334A_N335A_D416N,S8P_C13R_Y281 A_K334A_N335A_D416 N ,S8P_C13R_Y281 A_K334A_N335A_G337A_D416 N ,S8P_C13R_Y281A_K334A_N335A_G337A_I338M_D416N,S8P_C13R_Y281 A_K334A_N335A_I338A_D416N ,S8P_C13R_Y281 A_K334A_N335A_I338M_D416N,S8P_C13R_Y281 A_K334A_N335A_I338S_D416N ,S8P_C13R_Y281 A_K334A_N335A_R336A_D416 N ,S8P_C13R_Y281A_K334A_N335A_R336A_G337A_D416N,S8P_C13R_Y281A_K334A_N335A_R336A_I338A_D416N,S8P_C13R_Y281A_K334A_N335A_R336A_I338M_D416N,S8P_C13R_Y281A_K334A_N335A_R336A_I338S_D416N,S8P_C13R_Y281 A_K334A_N335A_R336K_D416 N ,S8P_C13R_Y281A_K334A_N335A_R336K_G337A_D416N,S8P_C13R_Y281A_K334A_N335A_R336K_I338A_D416N,S8P_C13R_Y281A_K334A_N335A_R336K_I338M_D416N,S8P_C13R_Y281A_K334A_N335A_R336K_I338S_D416N,S8P_C13R_Y281 A_K334A_N335A_R336S_D416 N ,S8P_C13R_Y281A_K334A_N335A_R336S_G337A_D416N,S8P_C13R_Y281A_K334A_N335A_R336S_I338A_D416N,S8P_C13R_Y281A_K334A_N335A_R336S_I338M_D416N,S8P_C13R_Y281A_K334A_N335A_R336S_I338S_D416N,S8P_C13R_Y281 A_R333A_K334A_N335A_D416 N ,S8P_C13R_Y281A_R333A_K334A_N335A_G337A_D416N,S8P_C13R_Y281A_R333A_K334A_N335A_I338A_D416N, S8P_C13R_Y281A_R333A_K334A_N335A_I338M_D416N, S8P_C13R_Y281A_R333A_K334A_N335A_I338S_D416N, S8P_C13R_Y281A_R333A_K334A_N335A_R336A_D416N,S8P_O13R_Y281A_R333A_K334A_N335A_R336K_D416N, S8P_O13R_Y281A_R333A_K334A_N335A_R336S_D416N, S8P_C13R_Y281 A_R333K_K334A_N335A_D416N,S8P_C 13R_Y281 A_R333K_K334A_N335A_G337A_D416N , S8P_C13R_Y281A_R333K_K334A_N335A_I338A_D416N, S8P_C13R_Y281A_R333K_K334A_N335A_I338M_D416N, S8P_C13R_Y281A_R333K_K334A_N335A_I338S_D416N, S8P_O13R_Y281A_R333K_K334A_N335A_R336A_D416N, S8P_O13R_Y281A_R333K_K334A_N335A_R336K_D416N,S8P_O13R_Y281A_R333K_K334A_N335A_R336S_D416N, S8P C13R Y281 A_R333S_K334A_N335A_D416 N , S8P_C13R_Y281A_R333S_K334A_N335A_G337A_D416N, S8P_O13R_Y281A_R333S_K334A_N335A_I338A_D416N, S8P_C13R_Y281A_R333S_K334A_N335A_I338M_D416N, S8P_C13R_Y281A_R333S_K334A_N335A_I338S_D416N,S8P_C13R_Y281A_R333S_K334A_N335A_R336A_D416N, S8P_C13R_Y281A_R333S_K334A_N335A_R336K_D416N, S8P_C 13R_Y281 A_R333S_K334A_N335A_R336S_D416N , S8P_C13R_Y281 A_T331 AJ332A_K334A_N335A_D416N, S8P_C13R_Y281 A_T331 AJ332M_K334A_N335A_D416N, S8P_C13R_Y281 A_T331 AJ332S_K334A_N335A_D416N,S8P_C13R_Y281A_T331A_K334A_N335A_D416N,S8P_C13R_Y281A_T331A_K334A_N335A_G337A_D416N,S8P_C 13R_Y281 A_T331 A_K334A_N335A_I 338A_D416N,S8P_C13R_Y281A_T331A_K334A_N335A_I338M_D416N,S8P_C13R_Y281A_T331A_K334A_N335AJ338S_D416N,S8P_C 13R_Y281 A_T331 A_K334A_N335A_R336A_D416N ,S8P_C13R_Y281 A_T331 A_K334A_N335A_R336K_D416N,S8P_C 13R_Y281 A_T331 A_K334A_N335A_R336S_D416N ,S8P_C13R_Y281 A_T331 A_R333A_K334A_N335A_D416N ,S8P_C13R_Y281 A_T331 A_R333K_K334A_N335A_D416N ,S8P_C13R_Y281A_T331A_R333S_K334A_N335A_D416N, of SEQ ID NO: 1 or a substitution at a position corresponding thereto.

18. The composition of any one of claims 1-17, wherein the helper enzyme comprises a deletion of about 30, or about 40, or about 50, or about 60, or about 70, or about 80, or about 90, or about 100 amino acids from an N- terminus or a C-terminus.

19. The composition of any one of claims 1-18, wherein the helper enzyme comprises a deletion at positions about 1-35, or about 1-45, or about 1-55, or about 1-65, or about 1-75, or about 1-85, or about 1-95, or about 1-105, or about 2-35, or about 2-45, or about 2-68 or positions corresponding thereto, wherein the positions are relative to SEQ ID NO: 1.20 The composition of claims 18 or 19, wherein the N terminal deletion yields reduced or ablated off-target effects of the enzyme compared to the enzyme without the N terminal deletion.

21. The composition of any one of claims 18-20, wherein the helper enzyme comprising an N terminal deletion is fused to a DNA binding domain, optionally wherein the DNA binding domain comprises transcription activatorlike effectors (TALEs), and / or zinc finger (ZnF).

22. The composition of any one of claims 1-21, wherein the helper enzyme further comprises a targeting element.

23. The composition of any one of claims 1-22, wherein the helper enzyme is suitable of inserting a donor nucleic acid comprising a transgene in a genomic safe harbor site (GSHS).

24. The composition of claim 23, wherein the GSHS is in an open chromatin location in a chromosome.25 The composition of claims 23 or 24, wherein the GSHS comprises a heterodimeric site located in chromosome 6.

26. The composition of any one of claims 23-25, wherein the GSHS is selected from adeno-associated virus site 1 (AAVS1), chemokine (C-C motif) receptor 5 (CCR5) gene, HIV-1 coreceptor, and human Rosa26 locus.

27. The composition of any one of claims 22-26, wherein the GSHS comprises one or more TTAA integration sites, optionally wherein the TTAA integration sites comprise ttTTAAaa (SEQ ID NO: 880) or taTTAAta (SEQ ID NO: 881) sites.28 The composition of claim 27, wherein the targeting element directs the helper enzyme to either one or more nucleic acid binding sites that are upstream and / or downstream of the TTAA integration sites or to the TTAA integration sites.

29. The composition of claim 27, wherein the targeting element directs the helper enzyme to one or more nucleic acid binding sites that are upstream and / or downstream of the TTAA integration sites and within about 5 to about 30 base pairs of the TTAA integration sites or within about 15 to about 19 base pairs of the TTAA integration sites.

30. The composition of claim 27, wherein the targeting element directs the helper enzyme to two nucleic acid binding sites of the TTAA integration sites, wherein a first site is upstream of TTAA and within about 5 to about 30 base pairs or about 15 to about 19 base pairs of the TTAA and a second site is downstream of TTAA and within about 5 to about 30 base pairs or about 15 to about 19 base pairs of the TTAA.31 . The composition of claim 27, wherein the targeting element directs the helper enzyme to identical targeting regions both upstream and downstream of the TTAA integration sites.

32. The composition of any one of claims 22-31 , wherein the targeting element is or comprises one or more of a transcription activator-like effector (TALE) DNA binding domain (DBD), a Cas enzyme, which is optionally catalytically inactive and which is optionally associated with a guide RNA (gRNA), a Zinc finger (ZnF), a catalytically inactive transcription factor, catalytically inactive nickase, a transcriptional activator, a transcriptional repressor, a recombinase, a DNA methyltransferase, a histone methyltransferase, a paternally expressed gene 10 (PEG10), and a transposon-encoded polypeptide D (TnsD) or a variant thereof.

33. The composition of any one of claims 22-32, wherein the targeting element is inserted after residue S60, or after residue E70, or after residue L80, or after residue N90, or after residue D100, relative to SEQ ID NO: 1 , optionally by way of a linker.

34. The composition of claim 33, wherein the targeting element is inserted immediately after residue G66 relative to SEQ ID NO: 1 , optionally by way of a linker.

35. The composition of claim 22, wherein the targeting element is or comprises a TALE DBD.

36. The composition of claim 35, wherein the TALE DBD comprises one or more repeat sequences.

37. The composition of claim 36, wherein the TALE DBD comprises about 14, or about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences.

38. The composition of claim 36 or claim 37, wherein the repeat sequences each independently comprises about 33 or 34 amino acids.

39. The composition of claim 38, wherein the repeat sequences each independently comprises a repeat variable diresidue (RVD) at residue 12 or 13 of the 33 or 34 amino acids, respectively.

40. The composition of claim 39, wherein the RVD recognizes one base pair in a target nucleic acid sequence.

41. The composition of claim 39 or claim 40, wherein the RVD recognizes a C residue in the target nucleic acid sequence and is selected from HD, N(gap), HA, ND, and HI.

42. The composition of claim 39 or claim 40, wherein the RVD recognizes a G residue in the target nucleic acid sequence and is selected from NN, NH, NK, HN, and NA.

43. The composition of claim 39 or claim 40, wherein the RVD recognizes an A residue in the target nucleic acid sequence and is selected from Nl and NS.

44. The composition of claim 39 or claim 40, wherein the RVD recognizes a T residue in the target nucleic acid sequence and is selected from NG, HG, H(gap), and IG.

45. The composition of claim 22, wherein the targeting element is or comprises a Cas9 enzyme associated with a gRNA or a CasX enzyme associated with a gRNA.

46. The composition of claim 45, wherein the Cas9 enzyme associated with a gRNA comprises a catalytically inactive dCas9 associated with a gRNA or an inactive dCasX associated with a gRNA.

47. The composition of claim 46, wherein catalytically inactive dCas9 comprises at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity to an amino acid sequence of SEQ ID NO: 6 or a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 5 or a codon-optimized form thereof.

48. The composition of claim 45, wherein the target element is or comprises a CasX enzyme associated with a gRNA, optionally wherein the catalytic inactive dCasX.

49. The composition of claim 22, wherein the targeting element is or comprises a Cas12 enzyme associated with a gRNA.

50. The composition of claim 49, wherein the targeting element comprises a catalytically inactive Cas12 associated with a gRNA, optionally wherein the catalytically inactive Cas12 is dCas12j or dCas12a.

51. The composition of claim 22, the targeting element is or comprises a TnsC, TnsB, TnsA, TniQ, Cas6, Cas7, Cas8 enzyme associated with a gRNA.

52. The composition of claim 51, wherein the targeting element comprises a TniQ subdomain of TnsD.

53. The composition of any one of claims 1-52, wherein the composition comprises a linker connecting the helper enzyme and the targeting element.

54. The composition of claim 53, wherein the linker is a covalent linker or a non-covalent linker.

55. The composition of claim 53, wherein the linker comprises less than about 25 amino acids or 75 nucleotides.

56. The composition of claim 53, wherein the linker comprises about 10 amino acids to about 20 amino acids or about 12 amino acids to about 15 amino acids, or about 30 nucleotides to about 60 nucleotides or about 36 nucleotides to about 45 nucleotides.

57. The composition of any one of claims 53-56, wherein the linker is substantially comprised of glycine (G) and serine (S) residues.

58. The composition of any one of claims 53-57, wherein the linker is or comprises (GSS)4 or the linker is GS flanked on either side of a DNA binding domain, optionally TALE and ZnF.

59. The composition of claim 58, wherein the linker comprises an amino acid sequence of AKLAGGAPAVGGGPKAADKFAATGGS (SEQ ID NO: 8) or a variant thereof having a substitution or deletion.

60. The composition of any one of claims 53-59, wherein the linker connects the targeting element to the N-terminus of the helper enzyme or connects the targeting element within the helper enzyme.61 . The composition of any one of claims 53-60, wherein the linker is a non-covalent linker.

62. The composition of claim 61 , wherein the non-covalent linker comprises an epitope tag, optionally wherein the epitope tag comprises an ALFA tag.

63. The composition of claim 62, wherein the ALFA tag further comprises recombinant variable domains of heavy- chain-only antibodies (VHH) (NANOBODY).

64. The composition of any one of claims 53-63, wherein the linker is inserted after residue S60, or after residue E70, or after residue L80, or after residue N90, or after residue D100 relative to SEQ ID NO: 1 .

65. The composition of claim 64, wherein the linker is inserted immediately after residue G66 relative to SEQ ID NO: 1.

66. The composition of any one of claims 1-65, wherein the composition is a nucleic acid, optionally an RNA.

67. The composition of claim 1-66, wherein the RNA is or comprises messenger RNA (mRNA).

68. The composition of claim 1-67, wherein the mRNA is or comprises modified mRNA (mmRNA).

69. The composition of claim 1 -68, wherein the mmRNA comprises one or more of a 5’-m7G cap (capO, cap1 , or cap2), a pseudouridine or n-methyl-pseudouridine substitution, and a poly-A tail of about 30, or of about 50, or of about 100, or of about 150 nucleotides in length.

70. The composition of any one of claims 1-69, wherein the composition further comprises a donor nucleic acid or is suitable for insertion of a donor nucleic acid, optionally wherein the donor nucleic acid is a transposon.71 . The composition of any one of claims 1 -70, wherein the helper enzyme is suitable of inserting a donor nucleic acid comprising a transgene in a genomic safe harbor site (GSHS) and / or wherein the targeting element is suitable for directing the helper enzyme to a GSHS.

72. The composition of any one of claims 1-71, wherein a donor DNA and a helper RNA are suitable for transfection at a donor DNA to helper RNA ratio of about 1 to about 4, or about 1 to about 2, or about 1 to about 1.

73. The composition of any one of claims 1-72, further comprising a nucleic acid encoding a donor comprising a transgene to be integrated, optionally wherein the transgene is defective or substantially absent in a disease state.

74. The composition of claim 73, wherein the transgene comprises a cargo nucleic acid sequence and a first and a second donor end sequences.

75. The composition of claim 74, wherein the cargo nucleic acid sequence is flanked by the first and the second donor end sequences.

76. The composition of any one of claims 1-75, wherein the enzyme or variant thereof is incorporated into a vector or a vector-like particle.

77. The composition of any one of claims 1-76, wherein the vector or a vector-like particle comprises one or more expression cassettes.

78. The composition of claim 77, wherein the vector or a vector-like particle comprises one expression cassette.79 The composition of claim 77 or 78, wherein the expression cassette further comprises the enzyme or variant thereof, the transgene, the donor end sequences, or a combination thereof.80 The composition of claim 77, wherein the enzyme or variant thereof, the transgene, the donor end sequences, or a combination thereof are incorporated into one or more vectors or vector-like particles.81 . The composition of claim 77, wherein the enzyme or variant thereof, the transgene, the donor end sequences, or combination thereof are incorporated into a same vector or vector-like particle.

82. The composition of claim 77, wherein the enzyme or variant thereof, the transgene, the donor end sequences, or combination thereof is incorporated into different vectors or vector-like particles.

83. The composition of any one of claims 76-82, wherein the vector or vector-like particle is nonviral.

84. The composition of any one of claims 70-83, wherein the donor is under the control of at least one tissue-specific promoter.

85. The composition of claim 84, wherein at least one tissue-specific promoter is a single promoter.

86. The composition of claim 84, wherein at least one tissue-specific promoter is under the control of a dual promoter or a tandem promoter.

87. The composition of any one of claims 71-86, wherein the transgene to be integrated comprises at least one gene of interest.

88. The composition of any one of claims 71-86, wherein the transgene to be integrated comprises one gene of interest.

89. The composition of any one of claims 71-86, wherein the transgene to be integrated comprises two or more genes of interest.

90. A host cell comprising the composition any one of claims 1-89.

91. The composition of any one of claims 1-89, wherein the composition is encapsulated in a lipid nanoparticle (LNP).

92. The composition of any one of claims 1-89, wherein the polynucleotide encoding the enzyme or variant thereof and the polynucleotide encoding the donor are in the form of the same LNP, optionally in a co-formulation.

93. The composition of claim 91 or claim 92, wherein the LNP comprises one or more lipids selected from 1 ,2-dioleoyl-3-trimethylammonium propane (DOTAP), a cationic cholesterol derivative mixed with dimethylaminoethanecarbamoyl (DC-Chol), phosphatidylcholine (PC), triolein (glyceryl trioleate), and 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG), 1 , 2-d I my ri stoy l-rac-g ly cero-3- methoxypolyethyleneglycol - 2000 (DMG-PEG 2K), and 1 ,2 distearol -sn-glycerol-3phosphocholine (DSPC) and / or comprising of one or more molecules selected from polyethylenimine (PEI) and poly (I actic-co-glycolic acid) (PLGA), and N-Acetylgalactosamine (GalNAc).

94. A method for inserting a gene into the genome of a cell, comprising contacting a cell with the composition of any one of claims 1-89, 91-93 or host cell of claim 90.

95. A method for treating a disease or disorder ex vivo, comprising contacting a cell with the composition of any one of claims 1-89, 91-93 or host cell of claim 90 and administering the cell to a subject in need thereof.

96. A method for treating a disease or disorder in vivo, comprising administering the composition of any one of claims 1-89, 91-93 or host cell of claim 90 to a subject in need thereof.

97. A composition comprising a helper enzyme or a nucleic acid encoding the helper enzyme comprising:an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1 and a non-polar aliphatic amino acid at position 2 of SEQ ID NO: 1, or a position corresponding thereto, andS8X, C13X, N335X, I338X, and D416X of SEQ ID NO: 1 or a substitution at a position corresponding thereto.

98. The composition of claim 97, wherein the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), and proline (P).

99. The composition of claims 97 or 98, wherein X is selected from a non-polar amino acid, optionally selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), methionine (M), tryptophan (W), and phenylalanine (F), or a polar amino acid, optionally selected from serine (S), cysteine (C), asparagine (N), glutamine (Q), threonine (T), and tyrosine (Y), or a basic amino acid, optionally selected from arginine (R), histidine (H), or an acidic amino acid, optionally selected from aspartic acid (D), and glutamic acid (E), or a positively charged hydrophilic amino acid, optionally selected from lysine (K), arginine (R), and histidine (H).

100. The composition of any one of claims 97-99, wherein the helper enzyme comprises a substitution at S8X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, optionally wherein X is a non-polar residue, optionally selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), methionine (M), tryptophan (W), and phenylalanine (F), or a polar and neutral hydrophilic residue, optionally proline (P).

101. The composition of any one of claims 97-100, wherein the helper enzyme comprises a substitution at C13X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, optionally wherein X is a positively charged hydrophilic amino acid, optionally selected from arginine (R), lysine (K), and histidine (H).

102. The composition of any one of claims 97-101, wherein the helper enzyme comprises a substitution at N335X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, wherein X is a non-polar amino acid, optionally selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), methionine (M), tryptophan (W), and phenylalanine (F).103.The composition of any one of claims 97-102, wherein the helper enzyme comprises a substitution at I338X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, wherein X is a non-polar amino acid, optionally selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), methionine (M), tryptophan (W), and phenylalanine (F), or a polar amino acid, optionally selected from serine (S), cysteine (C), asparagine (N), glutamine (Q), threonine (T), and tyrosine (Y).104.The composition of any one of claims 97-103, wherein the helper enzyme comprises a substitution at D416X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, wherein X is a polar and positively charged hydrophilic residue, optionally selected from arginine (R) and lysine (K), or a polar and neutral of charge hydrophilic residue, optionally selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C).

105. The composition of any one of claims 97-104, wherein the helper enzyme comprises one or two, or three, or four, or five substitutions selected from: S8P, C13R, N335A, I338S, and D416N or substitutions corresponding thereto relative to SEQ ID NO: 1.

106. The composition of claim 97-105, wherein the helper enzyme comprises S8P_C13R_N335A_I338S_D416N of SEQ ID NO: 1 or a substitution at a position corresponding thereto.

107. The composition of any one of claims 97-106, wherein the helper enzyme comprises a deletion of about 30, or about 40, or about 50, or about 60, or about 70, or about 80, or about 90, or about 100 amino acids from an N- terminus or a C-terminus.108.The composition of any one of claims 97-107, wherein the helper enzyme comprises a deletion at positions about 1-35, or about 1-45, or about 1-55, or about 1-65, or about 1-75, or about 1-85, or about 1-95, or about 1- 105, or about 2-35, or about 2-45, or about 2-68 or positions corresponding thereto, wherein the positions are relative to SEQ ID NO: 1.

109. The composition of claims 107 or 108, wherein the N terminal deletion yields reduced or ablated off-target effects of the enzyme compared to the enzyme without the N terminal deletion.

110. The composition of any one of claims 107-109, wherein the helper enzyme comprising an N terminal deletion is fused to a DNA binding domain, optionally wherein the DNA binding domain comprises transcription activatorlike effectors (TALEs), and / or zinc finger (ZnF).111 .The composition of any one of claims 97-110, wherein the helper enzyme further comprises a targeting element.

112. The composition of any one of claims 97-111 , wherein the helper enzyme is suitable of inserting a donor nucleic acid comprising a transgene in a genomic safe harbor site (GSHS).113.The composition of claim 112, wherein the GSHS is in an open chromatin location in a chromosome.114.The composition of claims 112 or 113, wherein the GSHS comprises a heterodimeric site located in chromosome 6.115.The composition of any one of claims 112-114, wherein the GSHS is selected from adeno-associated virus site 1 (AAVS1), chemokine (C-C motif) receptor 5 (CCR5) gene, HIV-1 coreceptor, and human Rosa26 locus.

116. The composition of any one of claims 112-115, wherein the GSHS comprises one or more TTAA integration sites, optionally wherein the TTAA integration sites comprise ttTTAAaa (SEQ ID NO: 880) or taTTAAta (SEQ ID NO: 881) sites.

117. The composition of claim 116, wherein the targeting element directs the helper enzyme to either one or more nucleic acid binding sites that are upstream and / or downstream of the TTAA integration sites or to the TTAA integration sites.

118. The composition of claim 116, wherein the targeting element directs the helper enzyme to one or more nucleic acid binding sites that are upstream and / or downstream of the TTAA integration sites and within about 5 to about 30 base pairs of the TTAA integration sites or within about 15 to about 19 base pairs of the TTAA integration sites.

119. The composition of claim 116, wherein the targeting element directs the helper enzyme to two nucleic acid binding sites of the TTAA integration sites, wherein a first site is upstream of TTAA and within about 5 to about 30 base pairs or about 15 to about 19 base pairs of the TTAA and a second site is downstream of TTAA and within about 5 to about 30 base pairs or about 15 to about 19 base pairs of the TTAA.

120. The composition of claim 116, wherein the targeting element directs the helper enzyme to identical targeting regions both upstream and downstream of the TTAA integration sites.

121. The composition of any one of claims 112-120, wherein the targeting element is or comprises one or more of a transcription activator-like effector (TALE) DNA binding domain (DBD), a Cas enzyme, which is optionally catalytically inactive and which is optionally associated with a guide RNA (gRNA), a Zinc finger (ZnF), a catalytically inactive transcription factor, catalytically inactive nickase, a transcriptional activator, a transcriptional repressor, a recombinase, a DNA methyltransferase, a histone methyltransferase, a paternally expressed gene 10 (PEG10), and a transposon-encoded polypeptide D (TnsD) or a variant thereof.

122. The composition of any one of claims 112-121 , wherein the targeting element is inserted after residue S60, or after residue E70, or after residue L80, or after residue N90, or after residue D100, relative to SEQ ID NO: 1 , optionally by way of a linker.123.The composition of claim 122, wherein the targeting element is inserted immediately after residue G66 relative to SEQ ID NO: 1 , optionally by way of a linker.124.The composition of claim 111, wherein the targeting element is or comprises a TALE DBD.125.The composition of claim 124, wherein the TALE DBD comprises one or more repeat sequences.

126. The composition of claim 125, wherein the TALE DBD comprises about 14, or about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences.

127. The composition of claim 125 or claim 126, wherein the repeat sequences each independently comprises about 33 or 34 amino acids.

128. The composition of claim 127, wherein the repeat sequences each independently comprises a repeat variable diresidue (RVD) at residue 12 or 13 of the 33 or 34 amino acids, respectively.

129. The composition of claim 128, wherein the RVD recognizes one base pair in a target nucleic acid sequence.

130. The composition of claim 128 or claim 129, wherein the RVD recognizes a C residue in the target nucleic acid sequence and is selected from HD, N(gap), HA, ND, and HI.

131. The composition of claim 128 or claim 129, wherein the RVD recognizes a G residue in the target nucleic acid sequence and is selected from NN, NH, NK, HN, and NA.

132. The composition of claim 128 or claim 129, wherein the RVD recognizes an A residue in the target nucleic acid sequence and is selected from Nl and NS.

133. The composition of claim 128 or claim 129, wherein the RVD recognizes a T residue in the target nucleic acid sequence and is selected from NG, HG, H(gap), and IG.134.The composition of claim 111 , wherein the targeting element is or comprises a Cas9 enzyme associated with a gRNA or a CasX enzyme associated with a gRNA.

135. The composition of claim 134, wherein the Cas9 enzyme associated with a gRNA comprises a catalytically inactive dCas9 associated with a gRNA or an inactive dCasX associated with a gRNA.

136. The composition of claim 135, wherein catalytically inactive dCas9 comprises at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity to an amino acid sequence of SEQ ID NO: 6 or a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 5 or a codon-optimized form thereof.

137. The composition of claim 134, wherein the target element is or comprises a CasX enzyme associated with a gRNA, optionally wherein the catalytic inactive dCasX.

138. The composition of claim 111 , wherein the targeting element is or comprises a Cas12 enzyme associated with a gRNA.

139. The composition of claim 138, wherein the targeting element comprises a catalytically inactive Cas12 associated with a gRNA, optionally wherein the catalytically inactive Cas12 is dCas12j or dCas12a.

140. The composition of claim 111, the targeting element is or comprises a TnsC, TnsB, TnsA, TniQ, Cas6, Cas7, Cas8 enzyme associated with a gRNA.

141. The composition of claim 140, wherein the targeting element comprises a TniQ subdomain of TnsD.

142. The composition of any one of claims 1-141, wherein the composition comprises a linker connecting the helper enzyme and the targeting element.143.The composition of claim 142, wherein the linker is a covalent linker or a non-covalent linker.

144. The composition of claim 142, wherein the linker comprises less than about 25 amino acids or 75 nucleotides.

145. The composition of claim 142, wherein the linker comprises about 10 amino acids to about 20 amino acids or about 12 amino acids to about 15 amino acids, or about 30 nucleotides to about 60 nucleotides or about 36 nucleotides to about 45 nucleotides.

146. The composition of any one of claims 142-145, wherein the linker is substantially comprised of glycine (G) and serine (S) residues.

147. The composition of any one of claims 142-146, wherein the linker is or comprises (GSS)4 or the linker is GS flanked on either side of a DNA binding domain, optionally TALE and ZnF.

148. The composition of claim 142, wherein the linker comprises an amino acid sequence of AKLAGGAPAVGGGPKAADKFAATGGS (SEQ ID NO: 8) or a variant thereof having a substitution or deletion.

149. The composition of any one of claims 142-148, wherein the linker connects the targeting element to the N-terminus of the helper enzyme or connects the targeting element within the helper enzyme.

150. The composition of any one of claims 142-149, wherein the linker is a non-covalent linker.

151. The composition of claim 150, wherein the non-covalent linker comprises an epitope tag, optionally wherein the epitope tag comprises an ALFA tag152. The composition of claim 151 , wherein the ALFA tag further comprises recombinant variable domains of heavychain-only antibodies (VHH) (NANOBODY).153.The composition of any one of claims 142-152, wherein the linker is inserted after residue S60, or after residue E70, or after residue L80, or after residue N90, or after residue D100 relative to SEQ ID NO: 1.154.The composition of claim 153, wherein the linker is inserted immediately after residue G66 relative to SEQ ID NO: 1.

155. The composition of any one of claims 97-152, wherein the composition is a nucleic acid, optionally an RNA.

156. The composition of claim 97-155, wherein the RNA is or comprises messenger RNA (mRNA).

157. The composition of claim 97-156, wherein the mRNA is or comprises modified mRNA (mmRNA).158.The composition of claim 97-157, wherein the mmRNA comprises one or more of a 5'-m7G cap (capO, cap1 , or cap2), a pseudouridine or n-methyl-pseudouridine substitution, and a poly-A tail of about 30, or of about 50, or of about 100, or of about 150 nucleotides in length.

159. The composition of any one of claims 97-158, wherein the composition further comprises a donor nucleic acid or is suitable for insertion of a donor nucleic acid, optionally wherein the donor nucleic acid is a transposon.

160. The composition of any one of claims 97-159, wherein the helper enzyme is suitable of inserting a donor nucleic acid comprising a transgene in a genomic safe harbor site (GSHS) and / or wherein the targeting element is suitable for directing the helper enzyme to a GSHS.

161. The composition of any one of claims 97-160, wherein a donor DNA and a helper RNA are suitable for transfection at a donor DNA to helper RNA ratio of about 1 to about 4, or about 1 to about 2, or about 1 to about 1.

162. The composition of any one of claims 97-161 , further comprising a nucleic acid encoding a donor comprising a transgene to be integrated, optionally wherein the transgene is defective or substantially absent in a disease state.163.The composition of claim 162, wherein the transgene comprises a cargo nucleic acid sequence and a first and a second donor end sequences.164.The composition of claim 163, wherein the cargo nucleic acid sequence is flanked by the first and the second donor end sequences.165.The composition of any one of claims 97-164, wherein the enzyme or variant thereof is incorporated into a vector or a vector-like particle.

166. The composition of any one of claims 97-165, wherein the vector or a vector-like particle comprises one or more expression cassettes.

167. The composition of claim 166, wherein the vector or a vector-like particle comprises one expression cassette.168.The composition of claim 166 or 167, wherein the expression cassette further comprises the enzyme or variant thereof, the transgene, the donor end sequences, or a combination thereof.

169. The composition of claim 166, wherein the enzyme or variant thereof, the transgene, the donor end sequences, or a combination thereof are incorporated into one or more vectors or vector-like particles.

170. The composition of claim 166, wherein the enzyme or variant thereof, the transgene, the donor end sequences, or combination thereof are incorporated into a same vector or vector-like particle.

171. The composition of claim 166, wherein the enzyme or variant thereof, the transgene, the donor end sequences, or combination thereof is incorporated into different vectors or vector-like particles.

172. The composition of any one of claims 165-171 , wherein the vector or vector-like particle is nonviral.

173. The composition of any one of claims 159-172, wherein the donor is under the control of at least one tissue-specific promoter.

174. The composition of claim 173, wherein at least one tissue-specific promoter is a single promoter.

175. The composition of claim 174, wherein at least one tissue-specific promoter is under the control of a dual promoter or a tandem promoter.

176. The composition of any one of claims 160-175, wherein the transgene to be integrated comprises at least one gene of interest177. The composition of any one of claims 160-175, wherein the transgene to be integrated comprises one gene of interest.178.The composition of any one of claims 160-175, wherein the transgene to be integrated comprises two or more genes of interest.

179. A host cell comprising the composition any one of claims 97-178.

180. The composition of any one of claims 97-178, wherein the composition is encapsulated in a lipid nanoparticle (LNP).181 .The composition of any one of claims 97-178, wherein the polynucleotide encoding the enzyme or variant thereof and the polynucleotide encoding the donor are in the form of the same LNP, optionally in a co-formulation182. The composition of claim 180 or claim 181 , wherein the LNP comprises one or more lipids selected from 1 ,2- dioleoyl-3-trimethylammonium propane (DOTAP), a cationic cholesterol derivative mixed with dimethylaminoethane-carbamoyl (DC-Chol), phosphatidylcholine (PC), triolein (glyceryl trioleate), and 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG), 1,2- dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol - 2000 (DMG-PEG 2K), and 1,2 distearol -sn-glycerol- 3phosphocholine (DSPC) and / or comprising of one or more molecules selected from polyethylenimine (PEI) and poly (lactic-co-glycolic acid) (PLGA), and N-Acetylgalactosamine (GalNAc).

183. A method for inserting a gene into the genome of a cell, comprising contacting a cell with the composition of any one of claims 97-178, 180-182 or host cell of claim 179.

184. A method for treating a disease or disorder ex vivo, comprising contacting a cell with the composition of any one of claims 97-178, 180-182 or host cell of claim 179 and administering the cell to a subject in need thereof.

185. A method for treating a disease or disorder in vivo, comprising administering the composition of any one of claims 97-178, 180-182 or host cell of claim 179 to a subject in need thereof.

186. A composition comprising a helper enzyme or a nucleic acid encoding the helper enzyme comprising: an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1 and a non-polar aliphatic amino acid at position 2 of SEQ ID NO: 1, or a position corresponding thereto,S8X, C13X, R333X, N335X, and D416X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, and / or one or more additional amino acid substitutions selected from K16X, N19X, N28X, L48X, S56X, N128X, K137X, D208X, N226X, N241X, K286X, N317X, T331X, I332X, K334X, R336X, G337X, I338X, K349X, K369X, E381X, I388X, D480X, M481X, L486X, K525X, K541R, F552X, Y572X, of SEQ ID NO: 1 or a substitution at a position corresponding thereto.

187. The composition of claim 186, further comprising an additional amino acid substitution of Y281X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, wherein X is a non-polar amino acid, optionally selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), methionine (M), tryptophan (W), and phenylalanine (F).

188. The composition of claims 186 or 187, wherein the non-polar aliphatic amino acid is selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), and proline (P).

189. The composition any one of claims 186-188, wherein X is selected from a non-polar amino acid, optionally selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), methionine (M), tryptophan (W), and phenylalanine (F), or a polar amino acid, optionally selected from serine (S), cysteine (C), asparagine (N), glutamine (Q), threonine (T), and tyrosine (Y), or a basic amino acid, optionally selected from arginine (R), histidine (H), or an acidic amino acid, optionally selected from aspartic acid (D), and glutamic acid (E), or a positively charged hydrophilic amino acid, optionally selected from lysine (K), arginine (R), and histidine (H).

190. The composition of any one of claims 186-189, wherein the helper enzyme comprises a substitution at S8X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, optionally wherein X is a non-polar residue, optionally selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), methionine (M), tryptophan (W), and phenylalanine (F) or a polar and neutral hydrophilic residue, optionally proline (P).

191. The composition of any one of claims 186-190, wherein the helper enzyme comprises a substitution at C13X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, optionally wherein X is a positively charged hydrophilic amino acid, optionally selected from arginine (R), lysine (K), and histidine (H).

192. The composition of any one of claims 186-191 , wherein the helper enzyme comprises a substitution at R333X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, optionally wherein X is a non-polar residue, optionally selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I).193.The composition of any one of claims 186-192, wherein the helper enzyme comprises a substitution at N335X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, optionally wherein X is a non-polar residue, optionally selected from alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I).

194. The composition of any one of claims 186-193, wherein the helper enzyme comprises a substitution at D416X of SEQ ID NO: 1 or a substitution at a position corresponding thereto, wherein X is a polar and positively charged hydrophilic residue, optionally selected from arginine (R) and lysine (K), or a polar and neutral of charge hydrophilic residue, optionally selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C).

195. The composition of any one of claims 186-194, wherein the helper enzyme comprises one or two, or three, or four, or five, or six substitutions selected from: S8P, C13R, C13H, R333A, R333S, R333K, N335A, and D416N or substitutions corresponding thereto relative to SEQ ID NO: 1.

196. The composition of any one of claims 186-195, wherein the composition comprises one or two, or three, or four, or five substitutions selected from: K16E, N19D, N28T, L48W, S56A, N128D, K137R, D208G, N226S, N241 I, K286R, N317T, T331A, I332A, I332M, I332S, K334A, K334R, K334S, R336A, R336S, R336K, G337A, I338A, I338M, I338S, K349R, K369R, E381 G, I388T, D480G, M481 R, L486V, K525I, K541R, F552I, Y572C, or substitutions corresponding thereto relative to SEQ ID NO: 1.

197. The composition of any one of claims 186-196, wherein the composition comprises one or two, or three, or four, or five substitutions selected from: S8P, C13R, C13H, R333A, R333S, R333K, N335A, and D416N or substitutions corresponding thereto relative to SEQ ID NO: 1 , and one, or two, or three, or four substitutions selected from K16E, N19D, N28T, L48W, S56A, N128D, K137R, D208G, N226S, N241 I, K286R, N317T, T331A, I332A, I332M, I332S, K334A, K334R, K334S, R336A, R336S, R336K, G337A, I338A, I338M, I338S, K349R, K369R, E381 G, I388T, D480G, M481 R, L486V, K525I, K541 R, F552I, Y572C, or substitutions corresponding thereto relative to SEQ ID NO: 1.198.The composition of any one of claims 186-197, wherein the helper enzyme comprises one, or two, or three, or four, or five, or six, or seven, or eight substitutions selected from: S8P, C13R, C13H, K16E, N19D, N28T, L48W, S56A, N128D, K137R, D208G, N226S, N241I, K286R, N317T, T331A, I332A, I332M, I332S, R333A, R333S, R333K, K334A, K334R, K334S, N335A, R336A, R336S, R336K, G337A, I338A, I338M, I338S, K349R, K369R, E381 G, I388T, D416N, D480G, M481R, L486V, K525I, K541 R, F552I, and Y572C or substitutions corresponding thereto relative to SEQ ID NO: 1.

199. The composition of claim any one of claims 186-198, wherein the helper enzyme comprises or consists of 6 amino acid substitutions.

200. The composition of claim any one of claims 186-198, wherein the helper enzyme comprises or consists of 7 amino acid substitutions.201 .The composition of claim any one of claims 186-198, wherein the helper enzyme comprises or consists of 8 amino acid substitutions.

202. The composition of any one of claims 186-201 , wherein the helper enzyme comprises a substitution selected from TABLE 2 or a substitution at positions corresponding thereto relative to SEQ ID NO: 1.

203. The composition of any one of claims 186-202, wherein the helper enzyme comprises:S8P_C13R_I332A_R333A_K334A_N335A_D416N,S8P_C13R_I332A_R333A_K334A_N335A_D416N,S8P_C13R_I332A_R333A_K334S_N335A_D416N,S8P_C13R_I332A_R333A_N335A_D416N,S8P_C13R_I332A_R333A_N335A_G337A_D416N,S8P_C13R_I332A_R333A_N335A_I338A_D416N,S8P_C13R_I332A_R333A_N335A_I338S_D416N,S8P_C13R_I332A_R333A_N335A_R336A_D416N ,S8P_O13R_I332A_R333A_N335A_R336K_D416N,S8P_C13R_I332A_R333A_N335A_R336S_D416N,S8P_C13R_I332M_R333A_K334A_N335A_D416N,S8P_C13R_I332M_R333A_K334R_N335A_D416N,S8P_C13R_I332M_R333A_K334S_N335A_D416N,S8P_C13R_I332M_R333A_N335A_D416N,S8P_O13R_I332M_R333A_N335A_G337A_D416N,S8P_C13R_I332M_R333A_N335AJ338M_D416N,S8P_C13R_I332M_R333A_N335A_R336A_D416N,S8P_C13R_I332M_R333A_N335A_R336K_D416N,S8P_C13R_I332M_R333A_N335A_R336S_D416N,S8P_C13R_I332S_R333A_K334A_N335A_D416N,S8P_C13R_I332S_R333A_K334R_N335A_D416N,S8P_C13R_I332S_R333A_K334S_N335A_D416N,S8P_C13R_I332S_R333A_N335A_D416N,S8P_C13R_I332S_R333A_N335A_G337A_D416N,S8P_C13R_I332S_R333A_N335A_I338A_D416N,S8P_C13R_I332S_R333A_N335A_I338S_D416N,S8P_C13R_I332S_R333A_N335A_R336K_D416N,S8P_C13R_I332S_R333A_N335A_R336K_D416N,S8P_C13R_I332S_R333A_N335A_R336S_D416N,S8P_C13R_R333A_K334A_N335A_D416N,S8P_C 13R_R333A_K334A_N335A_G337A_D416N, S8P_C13R_R333A_K334A_N335A_I338A_D416N , S8P_C13R_R333A_K334A_N335A_I338M_D416 N , S8P_C13R_R333A_K334A_N335A_I338S_D416N, S8P_C13R_R333A_K334A_N335A_R336A_D416 N, S8P_C13R_R333A_K334A_N335A_R336K_D416 N , S8P_C13R_R333A_K334A_N335A_R336S_D416 N, S8P_C13R_R333A_K334A_N335A_R336S_D416 N, S8P_C13R_R333A_K334R_N335A_D416N, S8P_C13R_R333A_K334R_N335A_G337A_D416N, S8P_C13R_R333A_K334R_N335A_I338A_D416N, S8P_C13R_R333A_K334R_N335A_I338M_D416N, S8P_C13R_R333A_K334R_N335A_I338S_D416N, S8P_C13R_R333A_K334R_N335A_R336A_D416N, S8P_C13R_R333A_K334R_N335A_R336S_D416N, S8P_C13R_R333A_K334S_N335A_D416N, S8P_C13R_R333A_K334S_N335A_G337A_D416N, S8P_C13R_R333A_K334S_N335A_I338A_D416N , S8P_C13R_R333A_K334S_N335A_I338M_D416N, S8P_C13R_R333A_K334S_N335A_I338S_D416N , S8P_C13R_R333A_K334S_N335A_R336A_D416 N, S8P_C13R_R333A_K334S_N335A_R336K_D416 N, S8P_C13R_R333A_K334S_N335A_R336S_D416 N, S8P_C13R_R333A_N335A_D416N, S8P C13R R333A N335A G337A D416N, S8P_C13R_R333A_N335A_G337A_I338M_D416N, S8P_C13R_R333A_N335A_I338A_D416N, S8P_C13R_R333A_N335A_I338M_D416N, S8P_C13R_R333A_N335A_R336A_D416N, S8P_C13R_R333A_N335A_R336A_G337A_D416N, S8P_C13R_R333A_N335A_R336A_I338A_D416N, S8P_C13R_R333A_N335A_R336A_I338S_D416N, S8P_C13R_R333A_N335A_R336K_D416N, S8P_C13R_R333A_N335A_R336K_G337A_D416N,S8P_C 13R_R333A_N335A_R336K_I338A_D416N, S8P_C13R_R333A_N335A_R336K_I338M_D416N, S8P_C13R_R333A_N335A_R336K_I338M_D416N, S8P_C13R_R333A_N335A_R336K_I338S_D416N, S8P_C13R_R333A_N335A_R336S_D416N,S8P_C13R_R333A_N335A_R336S_G337A_D416N,S8P_C13R_R333A_N335A_R336S_I338A_D416N, S8P_C13R_R333A_N335A_R336S_I338M_D416N, S8P_C13R_R333A_N335A_R336S_I338S_D416N, S8P_C13R_T331 A_I332A_R333A_N335A_D416N, S8P_C13R_T331A_I332M_R333A_N335A_D416N, S8P_C13R_T331 AJ332S_R333A_N335A_D416N , S8P_C13R_T331 A_R333A_K334A_N335A_D416 N , S8P_C13R_T331 A_R333A_K334R_N335A_D416 N , S8P_C13R_T331 A_R333A_K334S_N335A_D416 N ,S8P_C13R_T331 A_R333A_N335A_D416 N ,S8P_C13R_T331 A_R333A_N335A_G337A_D416 N , S8P_C 13R_T331 A_R333A_N335A_1338A_D416 N , S8P_C13R_T331A_R333A_N335A_I338M_D416N, S8P_C13R_T331 A_R333A_N335A_I338S_D416N , S8P_C13R_T331 A_R333A_N335A_R336A_D416 N , S8P_C13R_T331 A_R333A_N335A_R336K_D416 N , S8P_C13R_T331 A_R333A_N335A_R336S_D416 N ,S8P_C13R_Y281 A_I332A_K334A_N335A_D416N,S8P_C13R_Y281A_I332A_K334A_N335A_G337A_D416N, S8P_C 13R_Y281 A_l 332A_K334A_N335A J 338A_D 416 N , S8P_C 13R_ Y281 A_l 332A_K334A_N335A J 338S_D 416 N , S8P_C13R_Y281A_I332A_K334A_N335A_R336A_D416N, S8P_C13R_Y281A_I332A_K334A_N335A_R336K_D416N, S8P_C13R_Y281A_I332A_K334A_N335A_R336S_D416N, S8P_C13R_Y281A_I332A_R333A_K334A_N335A_D416N, S8P_C13R_Y281A_I332A_R333K_K334A_N335A_D416N, S8P_O13R_Y281A_I332A_R333S_K334A_N335A_D416N, S8P_C13R_Y281A_I332M_K334A_N335A_D416N,S8P_C13R_Y281 A_I332M_K334A_N335A_G337A_D416N, S8P_C 13R_Y281 A J 332 M_K334A_N335A J 338M_D416N , S8P_C13R_Y281AJ332M_K334A_N335A_R336A_D416N, S8P_C13R_Y281AJ332M_K334A_N335A_R336K_D416N, S8P_C13R_Y281AJ332M_K334A_N335A_R336S_D416N, S8P_C13R_Y281AJ332M_R333A_K334A_N335A_D416N, S8P_C13R_Y281AJ332M_R333K_K334A_N335A_D416N, S8P_C13R_Y281AJ332M_R333S_K334A_N335A_D416N, S8P_C13R_Y281 A_I332S_K334A_N335A_D416N , S8P_C13R_Y281A_I332S_K334A_N335A_G337A_D416N, S8P_C 13R_Y281 A_l 332S_K334A_N335A J 338A_D 416 N , S8P_C 13R_Y281 A_l 332S_K334A_N335A_I 338S_D 416 N , S8P_C13R_Y281A_I332S_K334A_N335A_R336A_D416N, S8P_C13R_Y281A_I332S_K334A_N335A_R336K_D416N, S8P_C13R_Y281A_I332S_K334A_N335A_R336S_D416N, S8P_C13R_Y281A_I332S_R333A_K334A_N335A_D416N, S8P_O13R_Y281A_I332S_R333K_K334A_N335A_D416N, S8P_C13R_Y281A_I332S_R333S_K334A_N335A_D416N, S8P_C13R_Y281 A_K334A_N335A_D416 N ,S8P_C13R_Y281 A_K334A_N335A_G337A_D416 N , S8P_C13R_Y281A_K334A_N335A_G337A_I338M_D416N, S8P_C13R_Y281 A_K334A_N335A_I338A_D416 N , S8P_C13R_Y281 A_K334A_N335A_I338M_D416 N , S8P_C13R_Y281 A_K334A_N335A_I338S_D416N , S8P 013R Y281 A_K334A_N335A_R336A_D416 N , S8P_C13R_Y281A_K334A_N335A_R336A_G337A_D416N, S8P_C13R_Y281A_K334A_N335A_R336A_I338A_D416N, S8P_O13R_Y281A_K334A_N335A_R336A_I338M_D416N, S8P_O13R_Y281A_K334A_N335A_R336A_I338S_D416N, S8P_C13R_Y281 A_K334A_N335A_R336K_D416 N , S8P_C13R_Y281A_K334A_N335A_R336K_G337A_D416N, S8P_O13R_Y281A_K334A_N335A_R336K_I338A_D416N, S8P_C13R_Y281A_K334A_N335A_R336K_I338M_D416N, S8P_O13R_Y281A_K334A_N335A_R336K_I338S_D416N,S8P_C13R.Y281 A_K334A_N335A_R336S_D416 N ,S8P_C13R_Y281A_K334A_N335A_R336S_G337A_D416N,S8P_C13R_Y281A_K334A_N335A_R336S_I338A_D416N,S8P_C13R_Y281A_K334A_N335A_R336S_I338M_D416N,S8P_C13R_Y281A_K334A_N335A_R336S_I338S_D416N,S8P_C13R_Y281 A_R333A_K334A_N335A_D416 N ,S8P_C13R_Y281A_R333A_K334A_N335A_G337A_D416N,S8P_C13R_Y281A_R333A_K334A_N335A_I338A_D416N,S8P_C13R_Y281A_R333A_K334A_N335A_I338M_D416N,S8P_C13R_Y281A_R333A_K334A_N335A_I338S_D416N,S8P_C13R_Y281A_R333A_K334A_N335A_R336A_D416N,S8P_C13R_Y281A_R333A_K334A_N335A_R336K_D416N,S8P_C13R_Y281A_R333A_K334A_N335A_R336S_D416N,S8P_C13R_Y281 A_R333K_K334A_N335A_D416 N ,S8P_C13R_Y281A_R333K_K334A_N335A_G337A_D416N,S8P_C13R_Y281A_R333K_K334A_N335A_I338A_D416N,S8P_C13R_Y281A_R333K_K334A_N335A_I338M_D416N,S8P_C13R_Y281A_R333K_K334A_N335A_I338S_D416N,S8P_C 13R_Y281 A_R333K_K334A_N335A_R336A_D416N ,S8P_C13R_Y281A_R333K_K334A_N335A_R336K_D416N,S8P_C13R_Y281A_R333K_K334A_N335A_R336S_D416N,S8P_C13R_Y281 A_R333S_K334A_N335A_D416 N ,S8P_C13R_Y281A_R333S_K334A_N335A_G337A_D416N,S8P_C13R_Y281A_R333S_K334A_N335A_I338A_D416N,S8P_C13R_Y281A_R333S_K334A_N335A_I338M_D416N,S8P_C13R_Y281A_R333S_K334A_N335A_I338S_D416N,S8P_C13R_Y281A_R333S_K334A_N335A_R336A_D416N,S8P_C13R_Y281A_R333S_K334A_N335A_R336K_D416N,S8P_C13R_Y281A_R333S_K334A_N335A_R336S_D416N, S8P_C13R_Y281 A_T331 AJ332A_K334A_N335A_D416N, S8P_C13R_Y281A_T331A_I332M_K334A_N335A_D416N,S8P_C13R_Y281A_T331A_I332S_K334A_N335A_D416N,S8P_C13R_Y281A_T331A_K334A_N335A_D416N,S8P_C13R_Y281 A_T331 A_K334A_N335A_G337A_D416 N ,S8P_C13R_Y281A_T331A_K334A_N335A_I338A_D416N, S8P_C13R_Y281A_T331A_K334A_N335A_I338M_D416N, S8P_C13R_Y281A_T331A_K334A_N335A_I338S_D416N, S8P_C 13R_Y281 A_T331 A_K334A_N335A_R336A_D416N , S8P_C13R_Y281 A_T331 A_K334A_N335A_R336K_D416 N , S8P_C13R_Y281 A_T331 A_K334A_N335A_R336S_D416N , S8P_C13R_Y281 A_T331 A_R333A_K334A_N335A_D416N , S8P_C 13R_Y281 A_T331 A_R333K_K334A_N335A_D416N , S8P_C 13R_Y281 A_T331 A_R333S_K334A_N335A_D416N , S8P_C13R_R333A_N335A_D416N_K369R, S8P_C13R_R333A_N335A_D416N_M481 R, S8P_C13R_R333A_N335A_D416N_S56A, S8P_C13R_R333A_N335A_D416N_F552I , S8P_C13R_R333A_N335A_D416N_K16E,S8P_C13R_R333A_N335A_D416N_N317T_K525I , S8P_C13R_R333A_N335A_D416N_N317T_K525I_K369R, S8P_C13R_R333A_N335A_D416N_N 128D, S8P_C13R_R333A_N335A_D416N_1388T, S8P_C13R_R333A_N335A_D416N_K349R, S8P_C13R_R333A_N335A_D416 N_K 16E_N 19D, S8P_C13R_R333A_N335A_D416N_L48W,S8P_C13R_R333A_N335A_D416N_N 19D_K369R, S8P_C13R_R333A_N335A_D416N_K 16E_K369R, S8P_C13R_R333A_N335A_D416 N_K 16E_N 19D_K369R, S8P C13R R333A N335A D416N_D208G_K286R, S8P_C13R_R333A_N335A_D416N_K541 R, S8P_C13R_R333A_N335A_D416N_N28T, S8P_C13R_R333A_N335A_D416N_L486V, S8P_C13H_R333A_N335A_D416N_D480G,S8P_C 13R_R333A_N335A_D416N_N 19D_K 137R_N226S, S8P_C13R_R333A_N335A_D416N_K16E_N241 I, S8P_C13R_R333A_N335A_D416N_N19D_E381 G, S8P_C13R_R333A_N335A_D416N_Y572C,S8P_C13R_R333A_N335A_D416N_N19D, of SEQ ID NO: 1 or a substitution at a position corresponding thereto.

204. The composition of any one of claims 186-203, wherein the helper enzyme comprises a deletion of about 30, or about 40, or about 50, or about 60, or about 70, or about 80, or about 90, or about 100 amino acids from an N- terminus or a C-terminus.

205. The composition of any one of claims 186-204, wherein the helper enzyme comprises a deletion at positions about 1-35, or about 1-45, or about 1-55, or about 1-65, or about 1-75, or about 1-85, or about 1-95, or about 1- 105, or about 2-35, or about 2-45, or about 2-68 or positions corresponding thereto, wherein the positions are relative to SEQ ID NO: 1.

206. The composition of claims 204 or 205, wherein the N terminal deletion yields reduced or ablated off-target effects of the enzyme compared to the enzyme without the N terminal deletion.

207. The composition of any one of claims 204-206, wherein the helper enzyme comprising an N terminal deletion is fused to a DNA binding domain, optionally wherein the DNA binding domain comprises transcription activatorlike effectors (TALEs), and / or zinc finger (ZnF).208.The composition of any one of claims 186-207, wherein the helper enzyme further comprises a targeting element.

209. The composition of any one of claims 186-208, wherein the helper enzyme is suitable of inserting a donor nucleic acid comprising a transgene in a genomic safe harbor site (GSHS).

210. The composition of claim 209, wherein the GSHS is in an open chromatin location in a chromosome.

211. The composition of claims 209 or 210, wherein the GSHS comprises a heterodimeric site located in chromosome 6.

212. The composition of any one of claims 209-211 , wherein the GSHS is selected from adeno-associated virus site 1 (AAVS1), chemokine (C-C motif) receptor 5 (CCR5) gene, HIV-1 coreceptor, and human Rosa26 locus.213.The composition of any one of claims 208-212, wherein the GSHS comprises one or more TTAA integration sites, optionally wherein the TTAA integration sites comprise ttTTAAaa (SEQ ID NO: 880) or taTTAAta (SEQ ID NO: 881) sites.214.The composition of claim 213, wherein the targeting element directs the helper enzyme to either one or more nucleic acid binding sites that are upstream and / or downstream of the TTAA integration sites or to the TTAA integration sites.

215. The composition of claim 213, wherein the targeting element directs the helper enzyme to one or more nucleic acid binding sites that are upstream and / or downstream of the TTAA integration sites and within about 5 to about 30 base pairs of the TTAA integration sites or within about 15 to about 19 base pairs of the TTAA integration sites.

216. The composition of claim 213, wherein the targeting element directs the helper enzyme to two nucleic acid binding sites of the TTAA integration sites, wherein a first site is upstream of TTAA and within about 5 to about 30 base pairs or about 15 to about 19 base pairs of the TTAA and a second site is downstream of TTAA and within about 5 to about 30 base pairs or about 15 to about 19 base pairs of the TTAA.

217. The composition of claim 213, wherein the targeting element directs the helper enzyme to identical targeting regions both upstream and downstream of the TTAA integration sites.218.The composition of any one of claims 208-217, wherein the targeting element is or comprises one or more of a transcription activator-like effector (TALE) DNA binding domain (DBD), a Cas enzyme, which is optionally catalytically inactive and which is optionally associated with a guide RNA (gRNA), a Zinc finger (ZnF), a catalytically inactive transcription factor, catalytically inactive nickase, a transcriptional activator, a transcriptional repressor, a recombinase, a DNA methyltransferase, a histone methyltransferase, a paternally expressed gene 10 (PEG10), and a transposon-encoded polypeptide D (TnsD) or a variant thereof.

219. The composition of any one of claims 208-218, wherein the targeting element is inserted after residue S60, or after residue E70, or after residue L80, or after residue N90, or after residue D100, relative to SEQ ID NO: 1 , optionally by way of a linker.

220. The composition of claim 219, wherein the targeting element is inserted immediately after residue G66 relative to SEQ ID NO: 1 , optionally by way of a linker.221 .The composition of claim 208, wherein the targeting element is or comprises a TALE DBD.

222. The composition of claim 221, wherein the TALE DBD comprises one or more repeat sequences.223.The composition of claim 222, wherein the TALE DBD comprises about 14, or about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences.224.The composition of claim 222 or claim 223, wherein the repeat sequences each independently comprises about 33 or 34 amino acids.

225. The composition of claim 224, wherein the repeat sequences each independently comprises a repeat variable diresidue (RVD) at residue 12 or 13 of the 33 or 34 amino acids, respectively.

226. The composition of claim 225, wherein the RVD recognizes one base pair in a target nucleic acid sequence.

227. The composition of claim 225 or claim 226, wherein the RVD recognizes a C residue in the target nucleic acid sequence and is selected from HD, N(gap), HA, ND, and HI.

228. The composition of claim 225 or claim 226, wherein the RVD recognizes a G residue in the target nucleic acid sequence and is selected from NN, NH, NK, HN, and NA.

229. The composition of claim 225 or claim 226, wherein the RVD recognizes an A residue in the target nucleic acid sequence and is selected from Nl and NS.

230. The composition of claim 225 or claim 226, wherein the RVD recognizes a T residue in the target nucleic acid sequence and is selected from NG, HG, H(gap), and IG.

231. The composition of claim 208, wherein the targeting element is or comprises a Cas9 enzyme associated with a gRNA or a CasX enzyme associated with a gRNA.

232. The composition of claim 231, wherein the Cas9 enzyme associated with a gRNA comprises a catalytically inactive dCas9 associated with a gRNA or an inactive dCasX associated with a gRNA.233.The composition of claim 232, wherein catalytically inactive dCas9 comprises at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity to an amino acid sequence of SEQ ID NO: 6 or a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 5 or a codon-optimized form thereof.

234. The composition of claim 231 , wherein the target element is or comprises a CasX enzyme associated with a gRNA, optionally wherein the catalytic inactive dCasX.235.The composition of claim 208, wherein the targeting element is or comprises a Cas12 enzyme associated with a gRNA.

236. The composition of claim 235, wherein the targeting element comprises a catalytically inactive Cas12 associated with a gRNA, optionally wherein the catalytically inactive Cas12 is dCas12j or dCas12a.

237. The composition of claim 208, the targeting element is or comprises a TnsC, TnsB, TnsA, TniQ, Cas6, Cas7, Cas8 enzyme associated with a gRNA.238.The composition of claim 237, wherein the targeting element comprises a TniQ subdomain of TnsD.

239. The composition of any one of claims 186-238, wherein the composition comprises a linker connecting the helper enzyme and the targeting element.

240. The composition of claim 239, wherein the linker is a covalent linker or a non-covalent linker.241 .The composition of claim 239, wherein the linker comprises less than about 25 amino acids or 75 nucleotides.

242. The composition of claim 239, wherein the linker comprises about 10 amino acids to about 20 amino acids or about 12 amino acids to about 15 amino acids, or about 30 nucleotides to about 60 nucleotides or about 36 nucleotides to about 45 nucleotides.

243. The composition of any one of claims 239-242, wherein the linker is substantially comprised of glycine (G) and serine (S) residues.

244. The composition of any one of claims 239-243, wherein the linker is or comprises (GSS)4 or the linker is GS flanked on either side of a DNA binding domain, optionally TALE and ZnF.

245. The composition of claim 239, wherein the linker comprises an amino acid sequence of AKLAGGAPAVGGGPKAADKFAATGGS (SEQ ID NO: 8) or a variant thereof having a substitution or deletion.

246. The composition of any one of claims 239-245, wherein the linker connects the targeting element to the N-terminus of the helper enzyme or connects the targeting element within the helper enzyme.

247. The composition of any one of claims 239-246, wherein the linker is a non-covalent linker.248.The composition of claim 247, wherein the non-covalent linker comprises an epitope tag, optionally wherein the epitope tag comprises an ALFA tag.

249. The composition of claim 248, wherein the ALFA tag further comprises recombinant variable domains of heavy- chain-only antibodies (VHH) (NANOBODY).

250. The composition of any one of claims 239-249, wherein the linker is inserted after residue S60, or after residue E70, or after residue L80, or after residue N90, or after residue D100 relative to SEQ ID NO: 1.251 .The composition of claim 250, wherein the linker is inserted immediately after residue G66 relative to SEQ ID NO: 1.

252. The composition of any one of claims 186-251 , wherein the composition is a nucleic acid, optionally an RNA.253.The composition of claim 186-252, wherein the RNA is or comprises messenger RNA (mRNA).254.The composition of claim 186-253, wherein the mRNA is or comprises modified mRNA (mmRNA).255.The composition of claim 186-254, wherein the mmRNA comprises one or more of a 5'-m7G cap (capO, cap1 , or cap2), a pseudouridine or n-methyl-pseudouridine substitution, and a poly-A tail of about 30, or of about 50, or of about 100, or of about 150 nucleotides in length.

256. The composition of any one of claims 186-255, wherein the composition further comprises a donor nucleic acid or is suitable for insertion of a donor nucleic acid, optionally wherein the donor nucleic acid is a transposon.

257. The composition of any one of claims 186-256, wherein the helper enzyme is suitable of inserting a donor nucleic acid comprising a transgene in a genomic safe harbor site (GSHS) and / or wherein the targeting element is suitable for directing the helper enzyme to a GSHS.

258. The composition of any one of claims 186-257, wherein a donor DNA and a helper RNA are suitable for transfection at a donor DNA to helper RNA ratio of about 1 to about 4, or about 1 to about 2, or about 1 to about 1.

259. The composition of any one of claims 186-258, further comprising a nucleic acid encoding a donor comprising a transgene to be integrated, optionally wherein the transgene is defective or substantially absent in a disease state.

260. The composition of claim 259, wherein the transgene comprises a cargo nucleic acid sequence and a first and a second donor end sequences.

261. The composition of claim 260, wherein the cargo nucleic acid sequence is flanked by the first and the second donor end sequences.

262. The composition of any one of claims 186-261 , wherein the enzyme or variant thereof is incorporated into a vector or a vector-like particle.263.The composition of any one of claims 186-262, wherein the vector or a vector-like particle comprises one or more expression cassettes.264.The composition of claim 263, wherein the vector or a vector-like particle comprises one expression cassette.265.The composition of claim 263 or 264, wherein the expression cassette further comprises the enzyme or variant thereof, the transgene, the donor end sequences, or a combination thereof.

266. The composition of claim 263, wherein the enzyme or variant thereof, the transgene, the donor end sequences, or a combination thereof are incorporated into one or more vectors or vector-like particles.

267. The composition of claim 263, wherein the enzyme or variant thereof, the transgene, the donor end sequences, or combination thereof are incorporated into a same vector or vector-like particle.268.The composition of claim 263, wherein the enzyme or variant thereof, the transgene, the donor end sequences, or combination thereof is incorporated into different vectors or vector-like particles.

269. The composition of any one of claims 262-268, wherein the vector or vector-like particle is nonviral.

270. The composition of any one of claims 259-269, wherein the donor is under the control of at least one tissue-specific promoter.271 .The composition of claim 270, wherein at least one tissue-specific promoter is a single promoter.

272. The composition of claim 270, wherein at least one tissue-specific promoter is under the control of a dual promoter or a tandem promoter.

273. The composition of any one of claims 259-272, wherein the transgene to be integrated comprises at least one gene of interest274. The composition of any one of claims 259-272, wherein the transgene to be integrated comprises one gene of interest.275.The composition of any one of claims 259-272, wherein the transgene to be integrated comprises two or more genes of interest.

276. A host cell comprising the composition any one of claims 186-275.

277. The composition of any one of claims 186-275, wherein the composition is encapsulated in a lipid nanoparticle (LNP).278.The composition of any one of claims 186-275, wherein the polynucleotide encoding the enzyme or variant thereof and the polynucleotide encoding the donor are in the form of the same LNP, optionally in a co-formulation279. The composition of claim 277 or claim 278, wherein the LNP comprises one or more lipids selected from 1 ,2- dioleoyl-3-trimethylammonium propane (DOTAP), a cationic cholesterol derivative mixed with dimethylaminoethane-carbamoyl (DC-Chol), phosphatidylcholine (PC), triolein (glyceryl trioleate), and 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG), 1,2- dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol - 2000 (DMG-PEG 2K), and 1,2 distearol -sn-glycerol- 3phosphocholine (DSPC) and / or comprising of one or more molecules selected from polyethylenimine (PEI) and poly (lactic-co-glycolic acid) (PLGA), and N-Acetylgalactosamine (GalNAc).

280. A method for inserting a gene into the genome of a cell, comprising contacting a cell with the composition of any one of claims 186-275, 277-279 or host cell of claim 276.

281. A method for treating a disease or disorder ex vivo, comprising contacting a cell with the composition of any one of claims 186-275, 277-279 or host cell of claim 276 and administering the cell to a subject in need thereof.

282. A method for treating a disease or disorder in vivo, comprising administering the composition of any one of claims 186-275, 277-279 or host cell of claim 276 to a subject in need thereof.

283. A composition comprising a helper enzyme or a nucleic acid encoding the helper enzyme comprising: an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1 and a non-polar aliphatic amino acid at position 2 of SEQ ID NO: 1, or a position corresponding thereto, and a plurality of amino acid substitutions of any row of TABLE A.

84. A composition comprising a helper enzyme or a nucleic acid encoding the helper enzyme comprising: an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1 and a non-polar aliphatic amino acid at position 2 of SEQ ID NO: 1, or a position corresponding thereto, and a plurality of amino acid substitutions of any row of TABLE B.

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