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WO2024246338A2 - Nouvelles transposases et leurs utilisations - Google Patents

Nouvelles transposases et leurs utilisations Download PDF

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Publication number
WO2024246338A2
WO2024246338A2 PCT/EP2024/065108 EP2024065108W WO2024246338A2 WO 2024246338 A2 WO2024246338 A2 WO 2024246338A2 EP 2024065108 W EP2024065108 W EP 2024065108W WO 2024246338 A2 WO2024246338 A2 WO 2024246338A2
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transposase
seq
variant
amino acid
sequence
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WO2024246338A3 (fr
Inventor
Dimitrie IVANCIC DJERMANOVIC
Avencia SÁNCHEZ-MEJÍAS GARCIA
Marc GÜELL CARGOL
Ravi Das KARN SUDAN
Alejandro AGUDELO FRANCO
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Integra Therapeutics
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Integra Therapeutics
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Publication of WO2024246338A2 publication Critical patent/WO2024246338A2/fr
Publication of WO2024246338A3 publication Critical patent/WO2024246338A3/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/90—Vectors containing a transposable element
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)

Definitions

  • the present invention relates to novel transposases and their use for gene editing.
  • PiggyBac (PB) derived transposase can integrate large cargos across a variety of cellular backgrounds enabling re-writing genetic information for therapeutic use, biomedical research, etc. Re-factoring self-mobilizing genomic elements lead to development of genome engineering tools. Further identification of PB transposases in several species suggested widespread distribution of this transposon system.
  • the present invention provides novel transposases maximizing transposition activity across a variety of cell types.
  • This invention thus relates to a composition comprising at least one transposase ortholog.
  • the present invention relates to a composition
  • a composition comprising at least one transposase selected from the group consisting of: a Poeciliopsis turrubarensis transposase, or a variant thereof; an Anthonomus grandis DR1754440 transposase, or a variant thereof; an Anthonomus grandis DR1754053 transposase, or a variant thereof; a Anthonomus grandis DR1756049 transposase, or a variant thereof; a Xenopus tropicalis transposase, or a variant thereof; a Japanese Medaka transposase, or a variant thereof; a Leptobrachium leishanense transposase, or a variant thereof; a Scalopus aquaticus ScaAqu-5.3491 transposase, or a variant thereof; an Atlantic Salmon transposase, or a variant thereof; a Heliconius butterfly transposase, or a
  • Poeciliopsis turrubarensis transposase or a variant thereof
  • Anthonomus grandis DR1754440 transposase or a variant thereof;
  • Anthonomus grandis DR1754053 transposase or a variant thereof.
  • the at least one transposase is selected from the group consisting of:
  • Poeciliopsis turrubarensis transposase or a variant thereof.
  • Anthonomus grandis DR1754440 transposase or a variant thereof.
  • the at least one transposase is a Poeciliopsis turrubarensis transposase, or a variant thereof.
  • the Poeciliopsis turrubarensis transposase has an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 1; for example having at least 75%, 80%, 85%, 90% 95% or 100% sequence identity with said sequence.
  • the Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 1.
  • the Poeciliopsis turrubarensis transposase variant has an amino acid sequence as set forth in any one of SEQ ID NO: 2 to SEQ ID NO: 13 or SEQ ID NO: 112.
  • the Poeciliopsis turrubarensis transposase variant comprises at least one amino acid substitution on one or more of the amino acids at positions 353, 356, and 432 corresponding to the amino acid numbering of SEQ ID NO: 1.
  • the Poeciliopsis turrubarensis transposase variant has an amino acid sequence as set forth in SEQ ID NO: 112.
  • the at least one transposase is an Anthonomus grandis DR1754440 transposase, or a variant thereof.
  • the Anthonomus grandis DR1754440 transposase has an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 30; for example having at least 75%, 80%, 85%, 90% 95% or 100% sequence identity with said sequence.
  • the Anthonomus grandis DR1754440 transposase has an amino acid sequence as set forth in SEQ ID NO: 30.
  • the Anthonomus grandis DR1754440 transposase variant comprises at least one amino acid substitution on one or more of the amino acids at positions 388, 389, and 393 corresponding to the amino acid numbering of SEQ ID NO: 30.
  • the Anthonomus grandis DR1754440 transposase variant has an amino acid sequence as set forth in SEQ ID NO: 113.
  • the at least one transposase is an Anthonomus grandis DR1756049 transposase, or a variant thereof.
  • the Anthonomus grandis DR1756049 transposase has an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 35 or SEQ ID NO: 108.
  • the Anthonomus grandis DR1756049 transposase has an amino acid sequence as set forth in SEQ ID NO: 35 or SEQ ID NO: 108. [0025] In some embodiments, the Anthonomus grandis DR1756049 transposase variant comprises at least one amino acid substitution on one or more of the amino acids at positions 348, 352, and 429 corresponding to the amino acid numbering of SEQ ID NO: 35.
  • the Anthonomus grandis DR1756049 transposase variant has an amino acid sequence as set forth in SEQ ID NO: 111.
  • the at least one transposase is an Anthonomus grandis DR1754053 transposase, or a variant thereof.
  • the Anthonomus grandis DR1754053 transposase has an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 31; for example having at least 75%, 80%, 85%, 90% 95% or 100% sequence identity with said sequence.
  • the Anthonomus grandis DR1754053 transposase has an amino acid sequence as set forth in SEQ ID NO: 31.
  • the transposase recognizes at least one ITR sequence, preferably at least two ITR sequences, selected from the group consisting of SEQ ID NO: 37 to SEQ ID NO: 84.
  • the Poeciliopsis turrubarensis transposase recognizes a left ITR sequence having at least 75% sequence identity with SEQ ID NO: 37, and a right ITR sequence having at least 75% sequence identity with SEQ ID NO: 38.
  • the Anthonomus grandis DR1754440 transposase recognizes a left ITR sequence having at least 75% sequence identity with SEQ ID NO: 71, and a right ITR sequence having at least 75% sequence identity with SEQ ID NO: 72.
  • the Anthonomus grandis DR1756049 transposase recognizes a left ITR sequence having at least 75% sequence identity with SEQ ID NO: 81, and a right ITR sequence having at least 75% sequence identity with SEQ ID NO: 82.
  • the Anthonomus grandis DR1754053 transposase recognizes a left ITR sequence having at least 75% sequence identity with SEQ ID NO: 73, and a right ITR sequence having at least 75% sequence identity with SEQ ID NO: 74.
  • the composition further comprises a RNA-guided nuclease or nickase.
  • the transposase and the RNA-guided nuclease or nickase are fused together by a covalent linkage or a non-covalent linkage.
  • the covalent linkage comprises a linker
  • the transposase and the RNA-guided nuclease or nickase are not fused together.
  • the composition further comprises at least one nucleic acid molecule comprising at least one transgene of interest to integrate into the genome of one or more cell.
  • the present invention further relates to an in vitro method for the integration of at least one transgene of interest into the genome of one or more cell, comprising contacting said one or more cell with the composition according to the invention, and at least one nucleic acid molecule comprising said at least one transgene of interest.
  • the method is for a targeted integration of the at least one transgene of interest into the genome of one or more cell, wherein the composition further comprises a RNA-guided nuclease or nickase enabling said targeted integration.
  • the present invention further relates to a pharmaceutical composition comprising the composition according to the invention.
  • the present invention further relates to the composition according to the invention, or the pharmaceutical composition according to the invention, for use for treating a genetic disease in a subject in need thereof.
  • transposase refers to an enzyme that binds to the end of a transposon and catalyzes its movement to another part of the genome by a cut-and-paste mechanism or a replicative transposition mechanism.
  • ortholog and “orthologs” refer to genes that evolved in different species from a common ancestral gene by speciation. In general, orthologs retain the same function during evolution. By extension, “ortholog” and “orthologs” also refer to proteins, particularly to transposases, encoded by these genes, that evolved in different species from a common ancestral transposase. Therefore, transposase orthologs retain slightly the same function during evolution, of binding to the end of a transposon and catalyzing its movement to another part of the genome by a cut-and-paste mechanism or by a replicative transposition mechanism.
  • nucleic acid sequence and “nucleotide sequence” may be used interchangeably to refer to any molecule composed of, or comprising, monomeric nucleotides.
  • a nucleic acid may be an oligonucleotide or a polynucleotide.
  • a nucleotide sequence may be a DNA, RNA, or a mix thereof.
  • a nucleotide sequence may be chemically-modified or artificial.
  • transgene refers to an exogenous nucleic acid sequence, in particular an exogenous DNA or cDNA encoding a gene product.
  • the gene product may be an RNA, peptide or protein.
  • the transgene may include or be associated with one or more operational sequences to facilitate or enhance expression, such as a promoter, enhancer(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s) and/or other functional elements.
  • Embodiments of the disclosure may utilize any known suitable promoter, enhancer(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s) and/or other functional elements, unless specified otherwise. Suitable elements and sequences will be well known to those skilled in the art.
  • amino acid sequence refers to a polymer of amino acid residues. Unless specified, a polymer of amino acid residues can be of any length. The terms also apply to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of corresponding naturally-occurring amino acids.
  • binding protein refers to a protein that is able to bind non-covalently to another molecule.
  • a binding protein can bind to, for example, a DNA molecule (a DNA-binding protein), an RNA molecule (an RNA-binding protein) and/or a protein molecule (a protein-binding protein).
  • a protein-binding protein it can bind to one or more molecules of the same protein to form homodimers, homotrimers, etc.; and/or it can bind to one or more molecules of a different protein or proteins.
  • a binding protein can have more than one type of binding activity. For example, zinc finger proteins have DNA-binding, RNA-binding and protein-binding activity.
  • Cas9 or “Cas9 nuclease” refer to an RNA-guided nuclease comprising a Cas9 protein, or a fragment thereof (e.g., a protein comprising an active or inactive DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9).
  • a Cas9 nuclease is also referred to sometimes as a casnl nuclease or a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-associated nuclease.
  • CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids).
  • CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, correct processing of pre-crRNA requires a transencoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer.
  • tracrRNA transencoded small RNA
  • rnc endogenous ribonuclease 3
  • Cas9 protein serves as a guide for ribonuclease 3-aided processing of pre-crRNA.
  • the target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3 ‘-5’ exonucleolytically.
  • DNA-binding and cleavage typically requires protein and both RNAs.
  • single guide RNAs (“sgRNA” or simply “gNRA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species.
  • Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self vs. non-self.
  • Cas9 nuclease sequences and structures are well known to those of skill in the art. Cas9 orthologs have been described in various species, including, but not limited to, S.
  • a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain.
  • a nuclease-inactivated Cas9 protein can interchangeably be referred to as a “dCas9” protein (for nuclease-“dead” Cas9).
  • fusion refers to a molecule in which two or more subunit molecules are linked.
  • the link between the two is covalent; alternatively, the link between the two can be non-covalent and rely, e.g., on intermolecular interactions.
  • the subunit molecules can be the same chemical type of molecule, or can be different chemical types of molecules.
  • fusion protein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins.
  • one protein domain may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) protein, thus forming an “amino-terminal fusion protein” or a “carboxy-terminal fusion protein”, respectively.
  • a fusion protein is a single chain polypeptide which may be fully encoded by a nucleic acid sequence, and includes at least two protein domains directly covalently linked by peptidic bound or optionally covalently linked via a peptidic linker.
  • gene or “genome” as used herein, includes a DNA region encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions.
  • eukaryotic cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells and human cells (e.g., T-cells).
  • linked refers to the juxtaposition of two or more components (such as sequence elements), in which the components are arranged such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components.
  • the term “specificity” refers to the ability to selectively bind a sequence which shares a degree of sequence identity to a selected sequence.
  • sequence identity with a reference sequence, in particular a polypeptide sequence, is meant to encompass having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity; for example, any sub-range comprising or consisting of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, with the reference sequence.
  • insertion and “integration” refer to the addition of a nucleic acid sequence into a second nucleic acid sequence or into a genome or part thereof.
  • specific site-specific
  • targeted and “on-targeted” in relation to insertion or integration, are used herein interchangeably to refer to the insertion of a nucleic acid into a specific site of a second nucleic acid or into a specific site of a genome or part thereof.
  • random “non-targeted” and “off-targeted” refer to non-specific and unintended insertion of a nucleic acid into an unwanted site.
  • total or “overall” refer to the total number of insertions.
  • linker refers to a chemical group or a molecule linking two adjacent molecules or moieties.
  • vector refers to any polynucleotide that can carry, e.g., a second polynucleotide of interest, and e.g., which can transfer gene sequences to target cells.
  • the term includes cloning, and expression vehicles, as well as integrating vectors.
  • expression vector refers to any polynucleotide capable of directing the expression of a nucleic acid.
  • vector and “plasmid” are used interchangeably with the term “nucleic acid construct.”
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below.
  • the percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol, Biol. 48:444-453, 1970) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the term “subject” as used herein, refers to an individual organism, for example, an individual mammal.
  • the subject is a human.
  • the subject is a non-human mammal.
  • the subject is a non-human primate.
  • the subject is a rodent.
  • the subject is a sheep, a goat, a cattle, a cat, or a dog.
  • the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode.
  • the subject is a research animal.
  • treatment refers to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein.
  • treatment refers to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein.
  • treatment may be administered after one or more symptoms have developed and/or after a disease has been diagnosed.
  • treatment may be administered in the absence of symptoms, e.g., to prevent, reduce the likelihood of developing, or delay onset of a symptom or inhibit onset or progression of a disease.
  • treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.
  • transposase activity Novel enzymes having transposase activity are presented herein, and their subsequent use for gene editing, in particular targeted gene insertion.
  • This invention relates to a composition comprising at least one protein having a transposase activity, or a nucleic acid encoding thereof.
  • a protein having transposase activity will be referred to as “transposase”; the term “transposase” thus encompasses transposases orthologs.
  • the present invention relates to a composition comprising at least one transposase, or a nucleic acid encoding thereof.
  • the transposase is selected from the group comprising or consisting of: a Poeciliopsis turrubarensis transposase, or a variant thereof; a Xenopus tropicalis transposase herein interchangeably referred to as “tropical clawed frog” transposase, or a variant thereof; a Japanese Medaka transposase, or a variant thereof; a Leptobrachium leishanense transposase, herein interchangeably referred to as “Leishan spiny toad” transposase, or a variant thereof; a Scalopus aquaticus transposase, herein interchangeably referred to as “ScaAqu- 5.3491 transposase”, or a variant thereof; an Atlantic Salmon transposase, or a variant thereof; a Heliconius butterfly transposase, or a variant thereof; a Takifugu flavidus transposase herein interchangeably
  • the transposase is selected from the group consisting of: a Poeciliopsis turrubarensis transposase, or a variant thereof; a Xenopus tropicalis transposase herein interchangeably referred to as “tropical clawed frog” transposase, or a variant thereof; a Japanese Medaka transposase, or a variant thereof; a Leptobrachium leishanense transposase, herein interchangeably referred to as “Leishan spiny toad” transposase, or a variant thereof; a Scalopus aquaticus transposase, herein interchangeably referred to as “ScaAqu- 5.3491 transposase”, or a variant thereof; an Atlantic Salmon transposase, or a variant thereof; a Heliconius butterfly transposase, or a variant thereof; a Takifugu flavidus transposase herein interchangeably referred to as
  • the transposase is selected from the group consisting of: a Poeciliopsis turrubarensis transposase, or a variant thereof having at least 75% sequence identity with SEQ ID NO: 1, preferably a variant thereof having the amino acid sequence of any one of SEQ ID NO: 2 to SEQ ID NO: 13 or SEQ ID NO: 112, more preferably a variant thereof having the amino acid sequence of SEQ ID NO: 112; an Anthonomus grandis DR1754440 transposase, or a variant thereof having at least 75% sequence identity with SEQ ID NO: 30, preferably a variant thereof having the amino acid sequence of SEQ ID NO: 113; an Anthonomus grandis DR1756049 transposase, or a variant thereof having at least 75% sequence identity with SEQ ID NO: 35 or SEQ ID NO: 108, preferably a variant thereof having the amino acid sequence of SEQ ID NO: 111; an Anthonomus grandis trans
  • the transposase, or a variant thereof is selected from the group comprising or consisting of: a Poeciliopsis turrubarensis transposase, or a variant thereof; a Xenopus tropicalis transposase herein interchangeably referred to as “tropical clawed frog” transposase, or a variant thereof; a Japanese Medaka transposase, or a variant thereof; a Leptobrachium leishanense transposase, herein interchangeably referred to as “Leishan spiny toad” transposase, or a variant thereof; a Scalopus aquaticus transposase, herein interchangeably referred to as “ScaAqu- 5.3491 transposase”, or a variant thereof; an Atlantic Salmon transposase, or a variant thereof; a Heliconius butterfly transposase, or a variant thereof; a Takifugu flavidus transposas
  • the transposase is selected from the group consisting of: a Poeciliopsis turrubarensis transposase, or a variant thereof having at least 75% sequence identity with SEQ ID NO: 1, preferably a variant thereof having the amino acid sequence of any one of SEQ ID NO: 2 to SEQ ID NO: 13 or SEQ ID NO: 112, more preferably a variant thereof having the amino acid sequence of SEQ ID NO: 112; an Anthonomus grandis DR1754440 transposase, or a variant thereof having at least 75% sequence identity with SEQ ID NO: 30, preferably a variant thereof having the amino acid sequence of SEQ ID NO: 113; an Anthonomus grandis DR1756049 transposase, or a variant thereof having at least 75% sequence identity with SEQ ID NO: 35 or SEQ ID NO: 108, preferably a variant thereof having the amino acid sequence of SEQ ID NO: 111; an Anthonomus grandis trans
  • the transposase, or a variant thereof is selected from the group consisting of: a Poeciliopsis turrubarensis transposase, or a variant thereof; a Xenopus tropicalis transposase herein interchangeably referred to as “tropical clawed frog” transposase, or a variant thereof; a Japanese Medaka transposase, or a variant thereof; a Leptobrachium leishanense transposase, herein interchangeably referred to as “Leishan spiny toad” transposase, or a variant thereof; a Scalopus aquaticus transposase, herein interchangeably referred to as “ScaAqu- 5.3491 transposase”, or a variant thereof; an Atlantic Salmon transposase, or a variant thereof; a Heliconius butterfly transposase, or a variant thereof; a Takifugu flavidus transposase herein
  • the transposase, or a variant thereof is selected from the group comprising or consisting of: a Poeciliopsis turrubarensis transposase, or a variant thereof; a Xenopus tropicalis transposase herein interchangeably referred to as “tropical clawed frog” transposase, or a variant thereof; a Japanese Medaka transposase, or a variant thereof; a Leptobrachium leishanense transposase, herein interchangeably referred to as “Leishan spiny toad” transposase, or a variant thereof; a Scalopus aquations transposase, herein interchangeably referred to as “ScaAqu- 5.3491 transposase”, or a variant thereof; an Anthonomus grandis transposase, herein interchangeably referred to as “DR1754440” transposase, or a variant thereof.
  • the transposase, or a variant thereof is selected from the group consisting of: a Poeciliopsis turrubarensis transposase, or a variant thereof; and an Anthonomus grandis transposase, or a variant thereof.
  • Poeciliopsis turrubarensis transposase or a variant thereof
  • Anthonomus grandis DR1754440 transposase or a variant thereof;
  • Anthonomus grandis DR1754053 transposase or a variant thereof.
  • Anthonomus grandis DR1756049 transposase or a variant thereof;
  • the transposase, or a variant thereof is selected from the group consisting of: a Poeciliopsis turrubarensis transposase, or a variant thereof; an Anthonomus grandis transposase, herein interchangeably referred to as “DR1754440” transposase, or a variant thereof.
  • the transposase is selected from the group consisting of: a Poeciliopsis turrubarensis transposase, or a variant thereof having at least 75% sequence identity with SEQ ID NO: 1, preferably a variant thereof having the amino acid sequence of any one of SEQ ID NO: 2 to SEQ ID NO: 13 or SEQ ID NO: 112, more preferably a variant thereof having the amino acid sequence of SEQ ID NO: 112; and an Anthonomus grandis DR1754440 transposase, or a variant thereof having at least 75% sequence identity with SEQ ID NO: 30, preferably a variant thereof having the amino acid sequence of SEQ ID NO: 113.
  • the transposase is a recombinant transposase. In certain embodiments, the transposase is a non-naturally occurring transposase.
  • the transposase is a Poeciliopsis turrubarensis transposase, or a variant thereof.
  • the Poeciliopsis turrubarensis transposase has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 1.
  • the Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 1.
  • the Poeciliopsis turrubarensis transposase is a variant of Poeciliopsis turrubarensis transposase.
  • the variant of Poeciliopsis turrubarensis transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 1.
  • amino acid mutation means substitution, deletion, insertion, and translocation, preferably substitution.
  • the variant of Poeciliopsis turrubarensis transposase comprises at least one amino acid mutation, preferably at least one amino acid substitution, on one or more of the amino acids at positions 18, 22, 200, 238, 336, 342, 353, 356, 388, 413, 432, 547 and 549, corresponding to the amino acid numbering of SEQ ID NO: 1.
  • the variant of Poeciliopsis turrubarensis transposase comprises at least one amino acid mutation, preferably at least one amino acid substitution, on one or more of the amino acids at positions 18, 22, 200, 238, 336, 342, 388, 413, 547 and 549, corresponding to the amino acid numbering of SEQ ID NO: 1.
  • the variant of Poeciliopsis turrubarensis transposase comprises at least one amino acid substitution selected from the group comprising or consisting of W18S, V22S, T200R, I238A, I238R, R336A, Q342L, C388I, C388V, M413K, D547K, S549R, corresponding to the amino acid numbering of SEQ ID NO: 1.
  • the variant of Poeciliopsis turrubarensis transposase comprises at least one amino acid substitution selected from the group comprising or consisting of R353A, K356A, and D432N, corresponding to the amino acid numbering of SEQ ID NO: 1.
  • the variant of Poeciliopsis turrubarensis transposase comprises at least one amino acid substitution selected from the group consisting of W18S, V22S, T200R, I238A, I238R, R336A, Q342L, C388I, C388V, M413K, R353A, K356A, D432N, D547K, S549R, corresponding to the amino acid numbering of SEQ ID NO: 1.
  • the variant of Poeciliopsis turrubarensis transposase comprises at least one amino acid substitution selected from the group consisting of W18S, V22S, T200R, I238A, I238R, R336A, Q342L, C388I, C388V, M413K, D547K, S549R, corresponding to the amino acid numbering of SEQ ID NO: 1.
  • the variant of Poeciliopsis turrubarensis transposase comprises at least one amino acid substitution selected from the group consisting of R353A, K356A, and D432N, corresponding to the amino acid numbering of SEQ ID NO: 1.
  • the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in any one of SEQ ID NO: 2 to SEQ ID NO: 13 or SEQ ID NO: 112. In some embodiments, the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in any one of SEQ ID NO: 2 to SEQ ID NO: 13. In some embodiments, the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 2. In some embodiments, the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 3.
  • the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 6. In some embodiments, the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 7.
  • the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 8. In some embodiments, the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 9. In some embodiments, the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 10. In some embodiments, the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 11.
  • the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 12. In some embodiments, the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 13. In some embodiments, the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 112.
  • the Poeciliopsis turrubarensis transposase is a recombinant transposase.
  • the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 1.
  • the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 1.
  • the transposase is a Xenopus tropicalis transposase, herein interchangeably referred to as “tropical clawed frog” transposase, or a variant thereof.
  • said tropical clawed frog transposase from Xenopus tropicalis has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 14.
  • the Xenopus tropicalis transposase herein interchangeably referred to as “tropical clawed frog” transposase has an amino acid sequence as set forth in SEQ ID NO: 14.
  • the Xenopus tropicalis transposase is a recombinant transposase.
  • the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 14.
  • the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 14.
  • the transposase is a Japanese Medaka transposase, herein interchangeably referred to as ' Sini erca chualsi" transposase or “DR0651552” transposase, or a variant thereof.
  • the Japanese Medaka transposase has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 15.
  • the Japanese Medaka transposase has an amino acid sequence as set forth in SEQ ID NO: 15.
  • the Japanese Medaka transposase is a recombinant transposase.
  • the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 15.
  • the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 15.
  • the transposase is a Leptobrachium leishanense transposase, herein interchangeably referred to as “Leishan spiny toad” transposase, or a variant thereof.
  • said Leishan spiny toad transposase has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 16.
  • said Leishan spiny toad transposase has an amino acid sequence as set forth in SEQ ID NO: 16.
  • the Leishan spiny toad transposase is a recombinant transposase.
  • the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 16.
  • the transposase is a Scalopus aquaticus transposase (or ScaAqu-5.3491 transposase), or a variant thereof.
  • the Scalopus aquaticus transposase has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 17.
  • the Scalopus aquaticus transposase has an amino acid sequence as set forth in SEQ ID NO: 17.
  • the Scalopus aquaticus transposase is a recombinant transposase.
  • the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 17.
  • the transposase is an Atlantic Salmon transposase, or a variant thereof.
  • the Atlantic Salmon transposase has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 18.
  • the Atlantic Salmon transposase has an amino acid sequence as set forth in SEQ ID NO: 18.
  • the Atlantic Salmon transposase is a recombinant transposase.
  • the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 18.
  • the transposase is a Heliconius butterfly transposase, or a variant thereof.
  • the Heliconius butterfly transposase has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 19.
  • the Heliconius butterfly transposase has an amino acid sequence as set forth in SEQ ID NO: 19.
  • the Heliconius butterfly transposase is a recombinant transposase.
  • the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 19. In some embodiments, the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 19.
  • the transposase is a Takifugu flavidus transposase herein interchangeably referred to as “Yellowbelly pufferfish” transposase, or a variant thereof
  • said Yellowbelly pufferfish transposase has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 20.
  • said Y ellowbelly pufferfish transposase has an amino acid sequence as set forth in SEQ ID NO: 20.
  • the Yellowbelly pufferfish transposase is a recombinant transposase.
  • the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 20.
  • the transposase is a Vaquita transposase, or a variant thereof.
  • the Vaquita transposase has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 21.
  • the Vaquita transposase has an amino acid sequence as set forth in SEQ ID NO: 21.
  • the Vaquita transposase transposase is a recombinant transposase.
  • the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 21.
  • the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 21.
  • the transposase is a Oryzias latipes transposase, (also referred to as “Japanese Rice Fish” transposase), or a variant thereof.
  • said Japanese Rice Fish transposase has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 22. [0126] In some embodiments, said Japanese Rice Fish transposase has an amino acid sequence as set forth in SEQ ID NO: 22.
  • the Japanese Rice Fish transposase is a recombinant transposase.
  • the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 22.
  • the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 22.
  • the transposase is a Salvelinus profundus transposase, or a variant thereof.
  • the Salvelinus profundus transposase has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 23.
  • the Salvelinus profundus transposase has an amino acid sequence as set forth in SEQ ID NO: 23.
  • the Salvelinus profundus transposase is a recombinant transposase.
  • the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 23.
  • the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 23.
  • the transposase is a Cuniculus paca transposase, or a variant thereof.
  • the Cuniculus paca transposase has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 24.
  • the Cuniculus paca transposase has an amino acid sequence as set forth in SEQ ID NO: 24.
  • the Cuniculus paca transposase is a recombinant transposase.
  • the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 24. In some embodiments, the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 24.
  • the transposase is a Noctilio leporinus transposase, or a variant thereof.
  • the Noctilio leporinus transposase has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 25.
  • the Noctilio leporinus transposase has an amino acid sequence as set forth in SEQ ID NO: 25.
  • the Noctilio leporinus transposase is a recombinant transposase.
  • the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 25.
  • the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 25.
  • the transposase is a Pipistrellus pipistrellus transposase (or PipPip-6.1914 transposase), or a variant thereof.
  • the Pipistrellus pipistrellus transposase has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 26.
  • the Pipistrellus pipistrellus transposase has an amino acid sequence as set forth in SEQ ID NO: 26.
  • the Pipistrellus pipistrellus transposase is a recombinant transposase.
  • the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 26.
  • the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 26.
  • the transposase is a Philippine tarsier transposase, or a variant thereof.
  • the Philippine tarsier transposase has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 27.
  • the Philippine tarsier transposase has an amino acid sequence as set forth in SEQ ID NO: 27.
  • the Philippine tarsier transposase is a recombinant transposase.
  • the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 27.
  • the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 27.
  • the transposase is a Myotis lucifugus transposase, herein interchangeably referred to as “PiggyBat” transposase, or a variant thereof.
  • said “PiggyBat” transposase from Myotis lucifugus has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 28.
  • said “PiggyBat” transposase from Myotis lucifugus has an amino acid sequence as set forth in SEQ ID NO: 28.
  • the PiggyBat transposase is a recombinant transposase.
  • the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 28.
  • the transposase is a PiggyBac transposase, or a variant thereof.
  • the PiggyBac transposase has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 29.
  • said PiggyBac transposase has an amino acid sequence as set forth in SEQ ID NO: 29.
  • the PiggyBac transposase is a recombinant transposase.
  • the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 29.
  • the transposase is a Anthonomus grandis transposase, herein interchangeably referred to as “DR1754440” transposase or “Antgra4440”, or a variant thereof.
  • the transposase is Anthonomus grandis DR1754440 transposase.
  • said DR1754440 transposase from Anthonomus grandis has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 30.
  • said DR1754440 transposase from Anthonomus grandis has an amino acid sequence as set forth in SEQ ID NO: 30.
  • the Anthonomus grandis DR1754440 transposase is a recombinant transposase.
  • the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 30.
  • the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 30.
  • the Anthonomus grandis DR1754440 transposase is a variant of Anthonomus grandis DR1754440 transposase.
  • the variant of Anthonomus grandis DR1754440 transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 30.
  • the variant of Anthonomus grandis DR1754440 transposase comprises at least one amino acid mutation, preferably at least one amino acid substitution, on one or more of the amino acids at positions 388, 389, and 393, corresponding to the amino acid numbering of SEQ ID NO: 30.
  • the variant of Anthonomus grandis DR1754440 transposase comprises at least one amino acid substitution selected from the group comprising or consisting of R388A, K389A, and K393A, corresponding to the amino acid numbering of SEQ ID NO: 30. In some embodiments, the variant of Anthonomus grandis DR1754440 transposase comprises at least one amino acid substitution selected from the group consisting of R388A, K389A, and K393A, corresponding to the amino acid numbering of SEQ ID NO: 30.
  • the variant of Anthonomus grandis DR1754440 transposase has an amino acid sequence as set forth in SEQ ID NO: 113.
  • the transposase is a Anthonomus grandis transposase, herein interchangeably referred to as “DR1754053” transposase or “Antgra4053”, or a variant thereof.
  • the transposase is Anthonomus grandis DR1754053 transposase.
  • said DR1754053 transposase from Anthonomus grandis has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 31.
  • said DR1754053 transposase from Anthonomus grandis has an amino acid sequence as set forth in SEQ ID NO: 31.
  • the Anthonomus grandis DR1754053 transposase is a recombinant transposase.
  • the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 31.
  • the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 31.
  • the transposase is a Solenopsis invicta transposase, herein interchangeably referred to as “DR3053925” transposase, or a variant thereof.
  • the Solenopsis invicta transposase is a recombinant transposase.
  • the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 32.
  • the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 32.
  • the transposase is a Simochromis diagramma transposase, herein interchangeably referred to as “Simochromis diagramma genomic” transposase, or a variant thereof.
  • the Simochromis diagramma transposase is a recombinant transposase.
  • the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO:
  • the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 33.
  • the transposase is a Nematolebias whitei transposase, herein interchangeably referred to as “Nematolebias whitei chromosome 17” transposase, or a variant thereof.
  • said “Nematolebias whitei chromosome 17” transposase from Nematolebias whitei has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO:
  • said “Nematolebias whitei chromosome 17” transposase from Nematolebias whitei has an amino acid sequence as set forth in SEQ ID NO: 34.
  • the Nematolebias whitei transposase is a recombinant transposase.
  • the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 34. In some embodiments, the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 34.
  • said “DR1756049” transposase from Anthonomus grandis has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 108.
  • said “DR1756049” transposase from Anthonomus grandis has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 35.
  • said “DR1756049” transposase from Anthonomus grandis has an amino acid sequence as set forth in SEQ ID NO: 35 or SEQ ID NO: 108. In some embodiments, said “DR1756049” transposase from Anthonomus grandis has an amino acid sequence as set forth in SEQ ID NO: 108. In some embodiments, said “DR1756049” transposase from Anthonomus grandis has an amino acid sequence as set forth in SEQ ID NO: 35.
  • the Anthonomus grandis DR1756049 transposase is a recombinant transposase.
  • the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 35 or 108.
  • the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 35 or 108.
  • the Anthonomus grandis DR1756049 transposase is a variant of Anthonomus grandis DR1756049 transposase.
  • the variant of Anthonomus grandis DR1756049 transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 35.
  • the variant of Anthonomus grandis DR1756049 transposase comprises at least one amino acid mutation, preferably at least one amino acid substitution, on one or more of the amino acids at positions 348, 352, and 429, corresponding to the amino acid numbering of SEQ ID NO: 35.
  • the variant of Anthonomus grandis DR1756049 transposase comprises at least one amino acid substitution selected from the group consisting of R348A, K352A, and D429N, corresponding to the amino acid numbering of SEQ ID NO: 35.
  • the variant of Anthonomus grandis DR1756049 transposase has an amino acid sequence as set forth in SEQ ID NO: 111.
  • the Anthonomus grandis transposase has an amino acid sequence selected from the group comprising or consisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 108 and SEQ ID NO: 35.
  • the Anthonomus grandis transposase has an amino acid sequence selected from the group comprising or consisting of SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 108.
  • the Anthonomus grandis transposase has an amino acid sequence selected from the group comprising or consisting of SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 35. In some embodiments, the Anthonomus grandis transposase has an amino acid sequence selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 108 and SEQ ID NO: 35. In some embodiments, the Anthonomus grandis transposase has an amino acid sequence selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 108. In some embodiments, the Anthonomus grandis transposase has an amino acid sequence selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 35.
  • the Anthonomus grandis transposase variant has the amino acid sequence of SEQ ID NO: 111 or SEQ ID NO: 113.
  • the transposase is a Coremacera marginata transposase, herein interchangeably referred to as “DR1481656”, or a variant thereof.
  • the transposase is a Coremacera marginata transposase, herein interchangeably referred to as “DR1481656” transposase, or a variant thereof.
  • said “DR1481656” transposase from Coremacera marginata has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 36.
  • said “DR1481656” transposase from Coremacera marginata has an amino acid sequence as set forth in SEQ ID NO: 36.
  • the Coremacera marginata transposase is a recombinant transposase.
  • the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 36. In some embodiments, the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 36.
  • the transposase is a mutant transposase.
  • the mutant transposase comprises at least one amino acid mutation compared to the amino acid sequence of any one of SEQ ID NO: 1 or SEQ ID NO: 14 to SEQ ID NO: 36.
  • the mutant transposase comprises at least one amino acid mutation compared to the amino acid sequence of any one of SEQ ID NO: 14 to SEQ ID NO: 36.
  • the at least one mutation is selected from the group comprising or consisting of substitution, deletion, insertion, and translocation.
  • the mutant transposase comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutation(s) compared to the amino acid sequence of any one of SEQ ID NO: 1 or SEQ ID NO: 14 to SEQ ID NO: 36. In some embodiments, the mutant transposase comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutation(s) compared to the amino acid sequence of any one of SEQ ID NO: 14 to SEQ ID NO: 36. [0200] In some embodiments, the mutant transposase comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitution(s) compared to the amino acid sequence of any one of SEQ ID NO: 1 or SEQ ID NO: 14 to SEQ ID NO: 36. In some embodiments, the mutant transposase comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more substitution(s) compared to the amino acid sequence of any one of SEQ ID NO: 14 to SEQ ID NO: 36.
  • the mutant transposase comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid deletion(s) compared to the amino acid sequence of any one of SEQ ID NO: 1 or SEQ ID NO: 14 to SEQ ID NO: 36. In some embodiments, the mutant transposase comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more deletion(s) compared to the amino acid sequence of any one of SEQ ID NO: 14 to SEQ ID NO: 36.
  • the mutant transposase comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid insertion(s) compared to the amino acid sequence of any one of SEQ ID NO: 1 or SEQ ID NO: 14 to SEQ ID NO: 36. In some embodiments, the mutant transposase comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more insertion(s) compared to the amino acid sequence of any one of SEQ ID NO: 14 to SEQ ID NO: 36.
  • the mutant transposase comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid translocation(s) compared to the amino acid sequence of any one of SEQ ID NO: 1 or SEQ ID NO: 14 to SEQ ID NO: 36. In some embodiments, the mutant transposase comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more translocation(s) compared to the amino acid sequence of any one of SEQ ID NO: 14 to SEQ ID NO: 36.
  • mutant transposase comprises at least one, preferably at least 2, more preferably 3 amino acid substitutions at positions corresponding to positions 372, 375, and/or 450 of hyPB.
  • such mutant may be referred to as “triple mutants” or “X3 mutants”.
  • the transposase is a Poeciliopsis turrubarensis transposase of SEQ ID NO: 1 or Anthonomus grandis transposase of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 35 or SEQ ID NO: 108.
  • the transposase is a Poeciliopsis turrubarensis transposase variant of SEQ ID NO: 112 or a Anthonomus grandis transposase variant of SEQ ID NO: 111 or SEQ ID NO: 113.
  • the transposase is a Poeciliopsis turrubarensis transposase of SEQ ID NO: 1 or a Poeciliopsis turrubarensis transposase variant of SEQ ID NO: 112.
  • the transposase is a Anthonomus grandis transposase of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 35 or SEQ ID NO: 108, or a Anthonomus grandis transposase variant of SEQ ID NO: 111 or SEQ ID NO: 113.
  • Transposases have the ability to recognize and bind specific nucleic acid sequences named Inverted Terminal Repeats (ITRs).
  • ITRs Inverted Terminal Repeats
  • the ITRs typically flank both sites of a nucleic acid sequence that is “cut-and-paste” by the transposase.
  • the transposase recognizes and/or binds to at least one ITR sequence, preferably at least two ITR sequences. In some embodiments, the transposase recognizes and/or binds to a left ITR and a right ITR.
  • left ITR refers to the ITR sequence flanking the 5'-P extremity of the nucleic acid sequence flanked by the ITRs; and “right ITR” refers to the ITR sequence flanking the 3'-OH extremity of the nucleic acid sequence flanked by the ITRs.
  • the transposase recognizes at least one ITR sequence, preferably at least two ITR sequences, selected from the group comprising or consisting of SEQ ID NO: 37 to SEQ ID NO: 84. In some embodiments, the transposase recognizes at least one ITR sequence, preferably at least two ITR sequences, selected from the group consisting of SEQ ID NO: 37 to SEQ ID NO: 84.
  • the Poeciliopsis turrubarensis transposase recognizes a left ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 37, and a right ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 38.
  • the Xenopus tropicalis transposase (also referred to as “tropical clawed frog” transposase) recognizes a left ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 39, and a right ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 40.
  • the Japanese Medaka transposase (also referred to as Siniperca chuatsi transposase, or DR0651552 transposase) recognizes a left ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 41, and a right ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 42.
  • the Leptobrachium leishanense transposase (also referred to as “Leishan spiny toad” transposase) recognizes a left ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 43, and a right ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 44.
  • the Scalopus aquaticus transposase recognizes a left ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 45, and a right ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 46.
  • the Atlantic Salmon transposase recognizes a left ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 47, and a right ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 48.
  • the Heliconius butterfly transposase recognizes a left ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 49, and a right ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 50.
  • the Yellowbelly pufferfish transposase recognizes a left UR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 51, and a right ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 52.
  • the Vaquita transposase recognizes a left ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 53, and a right ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with
  • the Oryzias latipes transposase (also referred to as “Japanese Rice Fish” transposase) recognizes a left ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 55, and a right ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 56.
  • the Salvenius profundus transposase recognizes a left ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 57, and a right ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 58.
  • the Cuniculus paca transposase recognizes a left ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 59, and a right ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 60.
  • the Noctilio leporinus transposase recognizes a left ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 61, and a right ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 62.
  • the Pipistrellus pipistrellus transposase recognizes a left ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 63, and a right ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 64.
  • the Carlito syrichta transposase (also referred to as “Philippine tarsier” transposase) recognizes a left ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 65, and a right ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 66.
  • the Myotis lucifugus transposase (also referred to as “Piggybat” transposase) recognizes a left ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 67, and a right ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 68.
  • the transposase referred to as “PiggyBac” transposase recognizes a left ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 69, and a right ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 70.
  • the Anthonomus grandis transposase (referred to as “DR1754440” transposase) recognizes a left ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 71, and aright ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 72.
  • the Anthonomus grandis transposase (referred to as “DR1754053” transposase) recognizes a left ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 73, and aright ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 74.
  • the Solenopsis invicta transposase (referred to as “DR3053925” transposase) recognizes a left ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 75, and aright ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 76.
  • the Simochromis diagramma transposase recognizes a left ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 77, and a right ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO:
  • the Nematolebias whitei transposase (referred to as “Nematolebias whitei chromosome 17” transposase) recognizes a left ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 79, and a right ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 80.
  • the Anthonomus grandis transposase (referred to as “DR1756049” transposase) recognizes a left ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 81, and aright ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 82.
  • the Coremacera marginata transposase (referred to as “DR1481656” transposase) recognizes a left ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 83, and aright ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 84.
  • the transposase is an Anthonomus grandis transposase, or a Poeciliopsis turrubarensis transposase. In some embodiments, the transposase is an Anthonomus grandis transposase, a Poeciliopsis turrubarensis transposase, or a variant thereof.
  • the transposase is selected from the group comprising or consisting of Anthonomus grandis transposase DR1756049, interchangeably referred to as Antgra6049, Anthonomus grandis transposase DR1754440, interchangeably referred to as Antgra4440, Anthonomus grandis transposase DR1754053, interchangeably referred to as Antgra4053, a Poeciliopsis turrubarensis transposase, or variants thereof.
  • the transposase is selected from the group consisting of Anthonomus grandis DR1756049 transposase, Anthonomus grandis DR1754440 transposase, Anthonomus grandis DR1754053 transposase, and Poeciliopsis turrubarensis transposase, or variants thereof.
  • the transposase is selected from the group comprising or consisting of DR1756049 transposase (Antgra6049) having at least 75% sequence identity with SEQ ID NO: 108, DR1754440 transposase (Antgra4440) having at least 75% sequence identity with SEQ ID NO: 30, DR1754053 transposase (Antgra4053) having at least 75% sequence identity with SEQ ID NO: 31, or a Poeciliopsis turrubarensis transposase having at least 75% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 13.
  • the transposase is selected from the group comprising or consisting of DR1756049 transposase (Antgra6049) having at least 80% sequence identity with SEQ ID NO: 108, DR1754440 transposase (Antgra4440) having at least 80% sequence identity with SEQ ID NO: 30, DR1754053 transposase (Antgra4053) having at least 80% sequence identity with SEQ ID NO: 31, or a Poeciliopsis turrubarensis transposase having at least 80% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 13.
  • the transposase is selected from the group comprising or consisting of DR1756049 transposase (Antgra6049) having at least 85% sequence identity with SEQ ID NO: 108, DR1754440 transposase (Antgra4440) having at least 85% sequence identity with SEQ ID NO: 30, DR1754053 transposase (Antgra4053) having at least 85% sequence identity with SEQ ID NO: 31, or a Poeciliopsis turrubarensis transposase having at least 85% sequence identity with any one of SEQ ID NO: I to SEQ ID NO: 13.
  • the transposase is selected from the group comprising or consisting of DR1756049 transposase (Antgra6049) having at least 90% sequence identity with SEQ ID NO: 108, DR1754440 transposase (Antgra4440) having at least 90% sequence identity with SEQ ID NO: 30, DR1754053 transposase (Antgra4053) having at least 90% sequence identity with SEQ ID NO: 31, or a Poeciliopsis turrubarensis transposase having at least 90% sequence identity with any one of SEQ ID NO: I to SEQ ID NO: 13.
  • the transposase is selected from the group comprising or consisting of DR1756049 transposase (Antgra6049) having at least 95% sequence identity with SEQ ID NO: 108, DR1754440 transposase (Antgra4440) having at least 95% sequence identity with SEQ ID NO: 30, DR1754053 transposase (Antgra4053) having at least 95% sequence identity with SEQ ID NO: 31, or a Poeciliopsis turrubarensis transposase having at least 95% sequence identity with any one of SEQ ID NO: I to SEQ ID NO: 13.
  • the transposase is selected from the group comprising or consisting of DR1756049 transposase (Antgra6049) having 100% sequence identity with SEQ ID NO: 108, DR1754440 transposase (Antgra4440) having 100% sequence identity with SEQ ID NO: 30, DR1754053 transposase (Antgra4053) having 100% sequence identity with SEQ ID NO: 31, or a Poeciliopsis turrubarensis transposase having 100% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 13.
  • composition of the invention comprises:
  • Anthonomus grandis DR1756049 transposase (Antgra6049) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 108, and
  • At least one nucleic acid molecule comprising said at least one transgene of interest, wherein said at least one transgene of interest is flanked with a left ITR (z.e., 5’-P ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 81 and a right ITR (z.e., 3’-OH ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 82.
  • a left ITR z.e., 5’-P ITR
  • a right ITR z.e., 3’-OH ITR
  • composition of the invention consists of:
  • an Anthonomus grandis DR1756049 transposase (Antgra6049) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 108 or SEQ ID NO: 35, preferably an Anthonomus grandis DR1756049 transposase variant of amino acid sequence SEQ ID NO: 111, and
  • At least one nucleic acid molecule comprising at least one transgene of interest, wherein the at least one transgene of interest is flanked with a left ITR (z.e., 5’-P ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 81 and a right ITR (z.e., 3’-OH ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 82.
  • a left ITR z.e., 5’-P ITR
  • a right ITR z.e., 3’-OH ITR
  • composition of the invention comprises:
  • an Anthonomus grandis DR1754440 transposase (Antgra4440) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 30, and
  • At least one nucleic acid molecule comprising said at least one transgene of interest, wherein said at least one transgene of interest is flanked with a left ITR (z.e., 5’-P ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 71 and a right ITR (z.e., 3’-OH ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 72.
  • a left ITR z.e., 5’-P ITR
  • a right ITR z.e., 3’-OH ITR
  • composition of the invention consists of:
  • an Anthonomus grandis DR1754440 transposase (Antgra4440) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 30, preferably an Anthonomus grandis DR1754440 transposase variant of amino acid sequence SEQ ID NO: 113, and - at least one nucleic acid molecule comprising at least one transgene of interest, wherein the at least one transgene of interest is flanked with a left ITR (z.e., 5’-P ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 71 and a right ITR (i.e., 3’-OH ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 72.
  • a left ITR z.e., 5’-P ITR
  • a right ITR i.e., 3
  • Anthonomus grandis DR1754053 transposase (Antgra4053) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 31, and
  • At least one nucleic acid molecule comprising said at least one transgene of interest, wherein said at least one transgene of interest is flanked with a left ITR (i.e., 5’-P ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 73 and a right ITR i.e., 3’-OH ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 74.
  • a left ITR i.e., 5’-P ITR
  • a right ITR i.e., 3’-OH ITR
  • composition of the invention consists of:
  • Anthonomus grandis DR1754053 transposase (Antgra4053) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 31, and
  • At least one nucleic acid molecule comprising at least one transgene of interest, wherein the at least one transgene of interest is flanked with a left ITR (i.e., 5’-P ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 73 and a right ITR (i.e., 3’-OH ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 74.
  • a left ITR i.e., 5’-P ITR
  • a right ITR i.e., 3’-OH ITR
  • composition of the invention comprises:
  • Poeciliopsis turrubarensis transposase having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 13, and
  • At least one nucleic acid molecule comprising said at least one transgene of interest, wherein said at least one transgene of interest is flanked with a left ITR (i.e., 5’-P ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 37 and a right ITR (z.e., 3’-OH ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 38.
  • a left ITR i.e., 5’-P ITR
  • a right ITR z.e., 3’-OH ITR
  • Poeciliopsis turrubarensis transposase having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 13 or SEQ ID NO: 112, preferably SEQ ID NO: 112, and
  • At least one nucleic acid molecule comprising at least one transgene of interest, wherein the at least one transgene of interest is flanked with a left ITR (z.e., 5’-P ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 37 and a right ITR (i.e., 3’-OH ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 38.
  • a left ITR z.e., 5’-P ITR
  • a right ITR i.e., 3’-OH ITR
  • composition of the invention comprises:
  • Poeciliopsis turrubarensis transposase having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with any one of SEQ ID NO: 2 to SEQ ID NO: 13, and
  • At least one nucleic acid molecule comprising said at least one transgene of interest, wherein said at least one transgene of interest is flanked with a left ITR (i.e., 5’-P ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 37 and a right ITR i.e., 3’-OH ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 38.
  • a left ITR i.e., 5’-P ITR
  • a right ITR i.e., 3’-OH ITR
  • composition of the invention consists of:
  • Poeciliopsis turrubarensis transposase having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with any one of SEQ ID NO: 2 to SEQ ID NO: 13 or SEQ ID NO: 112, preferably SEQ ID NO: 112, and
  • At least one nucleic acid molecule comprising at least one transgene of interest, wherein the at least one transgene of interest is flanked with a left ITR (i.e., 5’-P ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 37 and a right ITR (i.e., 3’-OH ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 38.
  • a left ITR i.e., 5’-P ITR
  • a right ITR i.e., 3’-OH ITR
  • the composition of the invention further comprises a sitespecific DNA binding protein.
  • the site-specific DNA binding protein is a RNA-guided nuclease or nickase.
  • composition of the invention further comprises a RNA-guided nuclease or nickase.
  • the RNA-guided nuclease or nickase comprises an active DNA cleavage domain and a guide RNA binding domain.
  • the RNA-guided nuclease or nickase is a Cas protein.
  • the Cas protein is selected from the group comprising or consisting of Cas9, Cas 12a (Cpfl), Cas 12b, Casl2f, and CasX.
  • the Cas protein is selected from the group consisting of Cas9, Casl2a (Cpfl), Casl2b, Casl2f, and CasX. It shall be understood that variants and functional fragments thereof are also encompassed, such as nickase Cas (nCas) or dead Cas (dCas) variants.
  • composition of the invention further comprises at least one RNA-guided nuclease or nickase selected from the group comprising or consisting of:
  • Cas9 protein from Streptococcus pyogenes (SpCas9)
  • Cas9 protein from Staphylococcus aureus (SaCas9)
  • Cas9 protein from Campylobacter jejuni CjCas9
  • Cas9 protein from Spiroplasma syrphidicola; Cas9 protein from Prevotella intermedia;
  • “Ancestral” Cas refered to as LBCA.
  • Cas9 nucleases include, without limitation, Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus haemolyticus Cas9 (ShCas9), and Campylobacter jejuni Cas9 (CjCas9).
  • the RNA-guided nuclease or nickase is a Cas9 protein.
  • the Cas9 protein may be a “Cas9 variant”.
  • a “Cas9 variant”, as used herein, is a protein sharing homology to a Cas9 protein as described herein, and includes fragments thereof.
  • the RNA-guided nuclease or nickase is a Cas9 protein from Streptococcus pyogenes (SpCas9), or a variant thereof.
  • the Cas9 protein from Streptococcus pyogenes (SpCas9), or the variant thereof has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 85.
  • the Cas9 protein from Streptococcus pyogenes has an amino acid sequence as set forth in SEQ ID NO: 85.
  • the RNA-guided nuclease or nickase is a Cas9 protein from Staphylococcus aureus (SaCas9), or a variant thereof.
  • the Cas9 protein from Staphylococcus aureus (SaCas9), or the variant thereof has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 86.
  • the Cas9 protein from Staphylococcus aureus has an amino acid sequence as set forth in SEQ ID NO: 86.
  • the RNA-guided nuclease or nickase is a Cas9 protein from Campylobacter jejuni (CjCas9), or a variant thereof.
  • the Cas9 protein from Campylobacter jejuni (CjCas9), or the variant thereof has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 87.
  • the Cas9 protein from Campylobacter jejuni has an amino acid sequence as set forth in SEQ ID NO: 87.
  • the RNA-guided nuclease or nickase is a Cas9 protein from Corynebacterium ulcerans, or a variant thereof.
  • the Cas9 protein from Corynebacterium ulcerans has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 88.
  • the Cas9 protein from Corynebacterium ulcerans has an amino acid sequence as set forth in SEQ ID NO: 88.
  • the RNA-guided nuclease or nickase is a Cas9 protein from Corynebacterium diphtheria, or a variant thereof.
  • the Cas9 protein from Corynebacterium diphtheria has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 89.
  • the Cas9 protein from Corynebacterium diphtheria has an amino acid sequence as set forth in SEQ ID NO: 89.
  • the RNA-guided nuclease or nickase is a Cas9 protein from Spiroplasma syrphidicola, or a variant thereof.
  • the Cas9 protein from Spiroplasma syrphidicola or the variant thereof has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 90.
  • the Cas9 protein from Spiroplasma syrphidicola has an amino acid sequence as set forth in SEQ ID NO: 90.
  • the RNA-guided nuclease or nickase is a Cas9 protein from Prevotella intermedia, or a variant thereof.
  • the Cas9 protein from Prevotella intermedia, or the variant thereof has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 91.
  • the Cas9 protein from Prevotella intermedia has an amino acid sequence as set forth in SEQ ID NO: 91.
  • the RNA-guided nuclease or nickase is a Cas9 protein from Spiroplasma taiwanense, or a variant thereof.
  • the Cas9 protein from Spiroplasma taiwanense or the variant thereof has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 92.
  • the Cas9 protein from Spiroplasma taiwanense has an amino acid sequence as set forth in SEQ ID NO: 92.
  • the RNA-guided nuclease or nickase is a Cas9 protein from Streptococcus iniae, or a variant thereof.
  • the Cas9 protein from Streptococcus iniae or the variant thereof has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 93.
  • the Cas9 protein from Streptococcus iniae has an amino acid sequence as set forth in SEQ ID NO: 93.
  • the RNA-guided nuclease or nickase is a Cas9 protein from Belliella baltica, or a variant thereof.
  • the Cas9 protein from Belliella baltica, or the variant thereof has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 94.
  • the Cas9 protein from Belliella baltica has an amino acid sequence as set forth in SEQ ID NO: 94.
  • the RNA-guided nuclease or nickase is a Cas9 protein from Psychroflexus lorquis or a variant thereof.
  • the Cas9 protein from Psychroflexus lorquisL or the variant thereof has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 95.
  • the Cas9 protein from Psychroflexus torquisi has an amino acid sequence as set forth in SEQ ID NO: 95.
  • the RNA-guided nuclease or nickase is a Cas9 protein from Streptococcus thermophilus, or a variant thereof.
  • the Cas9 protein from Streptococcus thermophilus has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 96.
  • the Cas9 protein from Streptococcus thermophilus has an amino acid sequence as set forth in SEQ ID NO: 96.
  • the RNA-guided nuclease or nickase is a Cas9 protein from Listeria innocua, or a variant thereof.
  • the Cas9 protein from Listeria innocua has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 97.
  • the Cas9 protein from Listeria innocua has an amino acid sequence as set forth in SEQ ID NO: 97.
  • the RNA-guided nuclease or nickase is a Cas9 protein from Neisseria meningitidis, or a variant thereof.
  • the Cas9 protein from Neisseria meningitidis or the variant thereof has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 98.
  • the Cas9 protein from Neisseria meningitidis has an amino acid sequence as set forth in SEQ ID NO: 98.
  • the RNA-guided nuclease or nickase is a Cas9 nickase from Streptococcus pyogenes (nCas9), or a variant thereof.
  • the Cas9 nickase from Streptococcus pyogenes (nCas9), or the variant thereof has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 99.
  • the Cas9 nickase from Streptococcus pyogenes has an amino acid sequence as set forth in SEQ ID NO: 99.
  • the RNA-guided nuclease or nickase is a dead Cas9 (dCas9), or a variant thereof.
  • the dead Cas9 or the variant thereof has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 100.
  • the dead Cas9 has an amino acid sequence as set forth in SEQ ID NO: 100.
  • the RNA-guided nuclease or nickase is a Casl2a (Cpfl), or a variant thereof.
  • the Casl2a (Cpfl), or the variant thereof has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 101.
  • the Cas 12a (Cpf 1) has an amino acid sequence as set forth in SEQ ID NO: 101.
  • the RNA-guided nuclease or nickase is a UnlCasl2fl, or a variant thereof.
  • the UnlCasl2fl or the variant thereof has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 102.
  • the UnlCasl2fl has an amino acid sequence as set forth in SEQ ID NO: 102.
  • the RNA-guided nuclease or nickase is a CasX, or a variant thereof.
  • the CasX, or the variant thereof has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 103.
  • the CasX has an amino acid sequence as set forth in SEQ ID NO: 103.
  • the RNA-guided nuclease or nickase is Dra2_TnpB, or a variant thereof.
  • the Dra2_TnpB, or the variant thereof has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 104.
  • the Dra2_TnpB has an amino acid sequence as set forth in SEQ ID NO: 104.
  • the RNA-guided nuclease or nickase is an “Ancestral” Cas, refered to as LFCA, or a variant thereof.
  • the LFCA or the variant thereof has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 105.
  • the LFCA has an amino acid sequence as set forth in SEQ ID NO: 105.
  • the RNA-guided nuclease or nickase is an “Ancestral” Cas, referred to as LBCA, or a variant thereof.
  • the LBCA or the variant thereof has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 106.
  • the LBCA has an amino acid sequence as set forth in SEQ ID NO: 106.
  • the RNA-guided nuclease or nickase is a Cas9 nickase from Staphylococcus aureus (SanCas9) or a variant thereof.
  • the Cas9 nickase from Staphylococcus aureus (SanCas9), or the variant thereof has an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 107.
  • the Cas9 nickase from Staphylococcus aureus has an amino acid sequence as set forth in SEQ ID NO: 107.
  • the RNA-guided nuclease or nickase has an amino acid sequence selected from the group comprising or consisting of any one of SEQ ID NO: 85 to SEQ ID NO: 107. In some embodiments, the RNA-guided nuclease or nickase has an amino acid sequence selected from the group consisting of any one of SEQ ID NO: 85 to SEQ ID NO: 107. [0332] The transposase and the RNA-guided nuclease or nickase may be administered separately (z.e., decoupled), or, alternatively, fused or otherwise linked together.
  • the transposase and the RNA-guided nuclease are not fused nor linked together. In some embodiments, the transposase and the RNA-guided nuclease are decoupled or split. In some embodiments, the transposase and the RNA-guided nuclease are to be administered separately (e.g., when contacting cells with the composition of the invention). Illustratively, the transposase and the RNA-guided nuclease may be encoded by distinct vectors.
  • transposase and the RNA-guided nuclease are associated together, fused, or otherwise linked.
  • Methods to associate two proteins, in particular methods to design fusion proteins, are well known in the art.
  • the transposase and the RNA-guided nuclease or nickase are fused together in a fusion protein.
  • the RNA-guided nuclease or nickase is fused in C-terminus or N-terminus of the transposase.
  • the transposase and the RNA-guided nuclease or nickase are covalently or non-covalently linked.
  • the transposase and the RNA-guided nuclease or nickase are covalently linked.
  • the transposase and the RNA- guided nuclease or nickase are non-covalently linked.
  • the fusion protein further comprises at least one linker.
  • the composition further comprises a gRNA.
  • the gRNA is recognized by the RNA-guided nuclease or nickase.
  • the gRNA is complementary and/or specific of at least one locus in the genome of a cell, thereby forcing the localization of the RNA-guided nuclease or nickase to this specific locus.
  • the transposase is fused to an aptamer binding protein
  • the gRNA of the RNA-guided nuclease or nickase comprises at least one aptamer sequence.
  • the aptamer binding protein may be fused in C-terminus or N-terminus of the transposase, optionally through a linker.
  • the at least one aptamer sequence may be DNA or RNA, preferably RNA.
  • the gRNA may comprise more than one aptamer sequence, i.e., 2, 3, 4, 5, 6, 7, 8, 9, or more.
  • the aptamer binding protein is MS2 bacteriophage coat protein (MCP) and the at least one aptamer is a MS2 RNA tetraloop binding sequence.
  • MCP has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 109 (encoded, e.g., by the nucleic acid sequence with SEQ ID NO: 110).
  • the MCP in the transposase MCP-fusion protein binds non-covalently to the at least one MS2 RNA tetraloop binding sequence comprised in the gRNA itself non-covalently bound to a RNA-guided nuclease or nickase; in particular, the binding of the fusion protein to the RNA-guided nuclease or nickase / gRNA complex directs the activity of the transposase of the invention towards the site specifically recognized by the RNA-guided nuclease or nickase / gRNA complex.
  • the present invention further relates to the use of the composition according to the invention for gene editing.
  • gene editing includes gene disruption and gene integration, preferably gene integration.
  • Gene integration may be referred to as gene insertion.
  • the present invention further relates to an in vitro method for the modifying the genome of one or more cell, comprising contacting said one or more cell with the composition according to the invention.
  • one or more cell may refer to a population of cells.
  • the present invention further relates to an in vitro method for the integration of at least one transgene of interest into the genome of one or more cell, comprising contacting said one or more cell with the composition according to the invention, and at least one nucleic acid molecule comprising said at least one transgene of interest.
  • one or more cell may refer to a population of cells.
  • the integration is targeted integration, z. e. , integration of the at least one transgene of interest into a specific locus of the genome.
  • the composition according to the invention preferably comprises a RNA- guided nuclease or nickase and a gRNA.
  • the integration is nontargeted or non-specific integration.
  • the at least one nucleic acid molecule comprising the at least one transgene of interest is a DNA or RNA molecule. In a preferred embodiment, the at least one nucleic acid molecule comprising the at least one transgene of interest is a DNA molecule.
  • the method is for the integration of large nucleic acid sequence, preferably the integration of large transgenes, more preferably the targeted integration of large transgenes.
  • the at least one transgene of interest has a size of at least 5 kb, at least 10 kb, at least 15 kb, at least 20 kb, at least 25 kb, or more.
  • the at least one nucleic acid molecule comprising the at least one transgene of interest has a size of at least 5 kb, at least 10 kb, at least 15 kb, at least 20 kb, at least 25 kb, or more.
  • the at least one nucleic acid molecule comprising the at least one transgene of interest is a transposon (or transposable element), i.e., in some embodiment, the at least one nucleic acid molecule comprising the at least one transgene of interest comprises at least one ITR sequence, preferably at least two ITR sequences, more preferably two ITR sequences.
  • the at least one ITR sequence preferably at least two ITR sequences, is selected from the group of sequences having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with the group consisting of SEQ ID NO: 37 to SEQ ID NO: 84. In some embodiments, the at least one ITR sequence, preferably at least two ITR sequences, is selected from the group consisting of SEQ ID NO: 37 to SEQ ID NO: 84.
  • the at least one ITR sequence is adjacent to the nucleic acid sequence of the at least one gene of interest.
  • the at least two ITR sequences flank the nucleic acid sequence of the at least one gene of interest.
  • the two ITR sequences flank the nucleic acid sequence of the at least one gene of interest.
  • the at least one nucleic acid molecule comprising the at least one transgene of interest comprises a left ITR and a right ITR.
  • left ITR refers to the ITR sequence flanking the 5'-P extremity of the nucleic acid sequence of the at least one gene of interest; and “right ITR” refers to the ITR sequence flanking the 3'-OH extremity of the nucleic acid sequence of the at least one gene of interest.
  • the left ITR is selected from the group comprising or consisting of SEQ ID NO: 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, and 83.
  • the right ITR is selected from the group comprising or consisting of SEQ ID NO: 38, 40, 42, 44, 46, 48, 50, 52, 54, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, and 84.
  • the left ITR is selected from the group consisting of SEQ ID NO: 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, and 83.
  • the right ITR is selected from the group consisting of SEQ ID NO: 38, 40, 42, 44, 46, 48, 50, 52, 54, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, and 84.
  • the nucleic acid sequence of the at least one gene of interest is flanked by the left ITR (or 5’-P ITR) of sequence SEQ ID NO: 37 and the right ITR (3’-OH ITR) of SEQ ID NO: 38. In some embodiments, the nucleic acid sequence of the at least one gene of interest is flanked by the left ITR (or 5’-P ITR) of sequence SEQ ID NO: 39 and the right ITR (3’-OH ITR) of SEQ ID NO: 40.
  • the nucleic acid sequence of the at least one gene of interest is flanked by the left ITR (or 5’-P ITR) of sequence SEQ ID NO: 41 and the right ITR (3’-OH ITR) of SEQ ID NO: 42. In some embodiments, the nucleic acid sequence of the at least one gene of interest is flanked by the left ITR (or 5’-P ITR) of sequence SEQ ID NO: 43 and the right ITR (3’- OH ITR) of SEQ ID NO: 44.
  • the nucleic acid sequence of the at least one gene of interest is flanked by the left ITR (or 5’-P ITR) of sequence SEQ ID NO: 45 and the right ITR (3’-OH ITR) of SEQ ID NO: 46. In some embodiments, the nucleic acid sequence of the at least one gene of interest is flanked by the left ITR (or 5’- P ITR) of sequence SEQ ID NO: 47 and the right ITR (3’-OH ITR) of SEQ ID NO: 48.
  • the nucleic acid sequence of the at least one gene of interest is flanked by the left ITR (or 5’- P ITR) of sequence SEQ ID NO: 53 and the right ITR (3’-OH ITR) of SEQ ID NO: 54. In some embodiments, the nucleic acid sequence of the at least one gene of interest is flanked by the left ITR (or 5’-P ITR) of sequence SEQ ID NO: 55 and the right ITR (3’- OH ITR) of SEQ ID NO: 56.
  • the nucleic acid sequence of the at least one gene of interest is flanked by the left ITR (or 5’-P ITR) of sequence SEQ ID NO: 57 and the right ITR (3’-OH ITR) of SEQ ID NO: 58. In some embodiments, the nucleic acid sequence of the at least one gene of interest is flanked by the left ITR (or 5’- P ITR) of sequence SEQ ID NO: 59 and the right ITR (3’-OH ITR) of SEQ ID NO: 60.
  • the nucleic acid sequence of the at least one gene of interest is flanked by the left ITR (or 5’- P ITR) of sequence SEQ ID NO: 65 and the right ITR (3’-OH ITR) of SEQ ID NO: 66. In some embodiments, the nucleic acid sequence of the at least one gene of interest is flanked by the left ITR (or 5’-P ITR) of sequence SEQ ID NO: 67 and the right ITR (3’- OH ITR) of SEQ ID NO: 68.
  • the nucleic acid sequence of the at least one gene of interest is flanked by the left ITR (or 5’-P ITR) of sequence SEQ ID NO: 69 and the right ITR (3’-OH ITR) of SEQ ID NO: 70. In some embodiments, the nucleic acid sequence of the at least one gene of interest is flanked by the left ITR (or 5’- P ITR) of sequence SEQ ID NO: 71 and the right ITR (3’-OH ITR) of SEQ ID NO: 72.
  • the nucleic acid sequence of the at least one gene of interest is flanked by the left ITR (or 5’-P ITR) of sequence SEQ ID NO: 73 and the right ITR (3’- OH ITR) of SEQ ID NO: 74. In some embodiments, the nucleic acid sequence of the at least one gene of interest is flanked by the left ITR (or 5’-P ITR) of sequence SEQ ID NO: 75 and the right ITR (3’-OH ITR) of SEQ ID NO: 76.
  • the nucleic acid sequence of the at least one gene of interest is flanked by the left ITR (or 5’- P ITR) of sequence SEQ ID NO: 77 and the right ITR (3’-OH ITR) of SEQ ID NO: 78. In some embodiments, the nucleic acid sequence of the at least one gene of interest is flanked by the left ITR (or 5’-P ITR) of sequence SEQ ID NO: 79 and the right ITR (3’- OH ITR) of SEQ ID NO: 80.
  • the nucleic acid sequence of the at least one gene of interest is flanked by the left ITR (or 5’-P ITR) of sequence SEQ ID NO: 81 and the right ITR (3’-OH ITR) of SEQ ID NO: 82. In some embodiments, the nucleic acid sequence of the at least one gene of interest is flanked by the left ITR (or 5’- P ITR) of sequence SEQ ID NO: 83 and the right ITR (3’-OH ITR) of SEQ ID NO: 84.
  • the present invention further relates to a method, preferably an in vitro method, for the integration, preferably the targeted integration, of at least one transgene of interest into the genome of one or more cell, or a population of cells, comprising contacting said one or more cell, or population of cells, with a composition comprising a transposase as described herein, and at least one nucleic acid molecule comprising the at least one transgene of interest, wherein said at least one transgene of interest is flanked with ITR recognized by the transposase.
  • composition comprising an Anthonomus grandis DR1756049 transposase (Antgra6049) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 108, and
  • At least one nucleic acid molecule comprising said at least one transgene of interest, wherein said at least one transgene of interest is flanked with a left ITR (z.e., 5’-P ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 81 and a right ITR (z.e., 3’-OH ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 82.
  • a left ITR z.e., 5’-P ITR
  • a right ITR z.e., 3’-OH ITR
  • the present invention further relates to a method, preferably an in vitro method, for the integration, preferably the targeted integration, of at least one transgene of interest into the genome of one or more cell, or a population of cells, comprising contacting said one or more cell, or population of cells, with:
  • composition comprising an Anthonomus grandis DR1756049 transposase (Antgra6049) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 108 or SEQ ID NO: 35, or having an amino acid sequence as set forth in SEQ ID NO: 111, and
  • the present invention further relates to a method, preferably an in vitro method, for the integration, preferably the targeted integration, of at least one transgene of interest into the genome of one or more cell, or a population of cells, comprising contacting said one or more cell, or population of cells, with:
  • composition comprising an Anthonomus grandis DR1754440 transposase (Antgra4440) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 30, and
  • At least one nucleic acid molecule comprising said at least one transgene of interest, wherein said at least one transgene of interest is flanked with a left ITR (z.e., 5’-P ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 71 and a right ITR (z.e., 3’-OH ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 72.
  • a left ITR z.e., 5’-P ITR
  • a right ITR z.e., 3’-OH ITR
  • the present invention further relates to a method, preferably an in vitro method, for the integration, preferably the targeted integration, of at least one transgene of interest into the genome of one or more cell, or a population of cells, comprising contacting said one or more cell, or population of cells, with: - a composition comprising an Anthonomus grandis DR1754440 transposase (Antgra4440) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 30, or having an amino acid sequence as set forth in SEQ ID NO: 113, and
  • At least one nucleic acid molecule comprising said at least one transgene of interest, wherein said at least one transgene of interest is flanked with a left ITR (z.e., 5’-P ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 71 and a right ITR (z.e., 3’-OH ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 72.
  • a left ITR z.e., 5’-P ITR
  • a right ITR z.e., 3’-OH ITR
  • the present invention further relates to a method, preferably an in vitro method, for the integration, preferably the targeted integration, of at least one transgene of interest into the genome of one or more cell, or a population of cells, comprising contacting said one or more cell, or population of cells, with:
  • composition comprising an Anthonomus grandis DR1754053 transposase (Antgra4053) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 108, and
  • At least one nucleic acid molecule comprising said at least one transgene of interest, wherein said at least one transgene of interest is flanked with a left ITR (z.e., 5’-P ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 73 and a right ITR (z.e., 3’-OH ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 74.
  • a left ITR z.e., 5’-P ITR
  • a right ITR z.e., 3’-OH ITR
  • the present invention further relates to a method, preferably an in vitro method, for the integration, preferably the targeted integration, of at least one transgene of interest into the genome of one or more cell, or a population of cells, comprising contacting said one or more cell, or population of cells, with:
  • composition comprising a Poeciliopsis turrubarensis transposase having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 13, and - at least one nucleic acid molecule comprising said at least one transgene of interest, wherein said at least one transgene of interest is flanked with a left ITR (z.e., 5’-P ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 37 and a right ITR (z.e., 3’-OH ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 38.
  • a left ITR z.e., 5’-P ITR
  • right ITR z.e., 3’-OH ITR
  • the present invention further relates to a method, preferably an in vitro method, for the integration, preferably the targeted integration, of at least one transgene of interest into the genome of one or more cell, or a population of cells, comprising contacting said one or more cell, or population of cells, with:
  • composition comprising a Poeciliopsis turrubarensis transposase having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 13 or SEQ ID NO: 112, preferably having an amino acid sequence as set forth in SEQ ID NO: 112, and
  • At least one nucleic acid molecule comprising at least one transgene of interest, wherein the at least one transgene of interest is flanked with a left ITR (z.e., 5’-P ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 37 and a right ITR (i.e., 3’-OH ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 38.
  • a left ITR z.e., 5’-P ITR
  • a right ITR i.e., 3’-OH ITR
  • the present invention further relates to a method, preferably an in vitro method, for the integration, preferably the targeted integration, of at least one transgene of interest into the genome of one or more cell, or a population of cells, comprising contacting said one or more cell, or population of cells, with:
  • composition comprising a Poeciliopsis turrubarensis transposase having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with any one of SEQ ID NO: 2 to SEQ ID NO: 13, and
  • At least one nucleic acid molecule comprising said at least one transgene of interest, wherein said at least one transgene of interest is flanked with a left ITR i.e., 5’-P ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 37 and a right ITR (z.e., 3’-OH ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 38.
  • a left ITR i.e., 5’-P ITR
  • a right ITR z.e., 3’-OH ITR
  • the present invention further relates to a method, preferably an in vitro method, for the integration, preferably the targeted integration, of at least one transgene of interest into the genome of one or more cell, or a population of cells, comprising contacting said one or more cell, or population of cells, with:
  • composition comprising a Poeciliopsis turrubarensis transposase having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with any one of SEQ ID NO: 2 to SEQ ID NO: 13 or SEQ ID NO: 112, preferably having an amino acid sequence as set forth in SEQ ID NO: 112, and
  • At least one nucleic acid molecule comprising at least one transgene of interest, wherein the at least one transgene of interest is flanked with a left ITR (z.e., 5’-P ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 37 and a right ITR (i.e., 3’-OH ITR) having at least 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 38.
  • a left ITR z.e., 5’-P ITR
  • a right ITR i.e., 3’-OH ITR
  • the present invention further relates to a cell comprising at least one transposase, or nucleic acid encoding thereof, as defined herein.
  • the cell further comprises a RNA-guided nuclease or nickase or a nucleic acid encoding thereof.
  • the cell further comprises at least one nucleic acid molecule comprising the at least one transgene of interest, as defined herein.
  • the present invention further relates to a vector encoding at least one transposase, or nucleic acid encoding thereof, as defined herein.
  • Suitable vector are known in the art.
  • the present invention further relates to an in vivo method for the integration of at least one transgene of interest into the genome of one or more cell, comprising contacting said one or more cell with the composition according to the invention, and at least one nucleic acid molecule comprising said at least one transgene of interest. Any feature of the in vitro method as described herein may be applied to the in vivo method.
  • the present invention further relates to a pharmaceutical composition comprising the transposase according to the invention.
  • the present invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the transposase according to the invention, and a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient is selected in a group comprising or consisting of a solvent, a diluent, a carrier, a vehicle, a dispersion medium, a coating, an antibacterial agent, an antifungal agent, an isotonic agent, an absorption delaying agent and any combinations thereof.
  • the carrier, diluent, solvent or vehicle must be “acceptable” in the sense of being compatible with the transposase, and not be deleterious upon being administered to an individual.
  • the vehicle does not produce an adverse, allergic or other untoward reaction when administered to an individual, preferably a human individual.
  • the pharmaceutical compositions should meet sterility, pyrogenicity, general safety and purity standards as required by regulatory offices, such as, for example, the Food and Drugs Administration (FDA) Office or the European Medicines Agency (EMA).
  • Suitable excipients include, without limitation, mannitol, dextrose, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • Acceptable carriers, solvents, diluents and vehicles for therapeutic use are well known in the pharmaceutical art. The choice of a suitable pharmaceutical carrier, solvent, excipient or vehicle can be made with regard to the intended route of administration and standard pharmaceutical practice.
  • compositions may comprise as, or in addition to, the carrier, vehicle, solvent or diluent any suitable binder, lubricant, suspending agent, coating agent, or solubilizing agent. Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition.
  • the present invention further relates to a medicament comprising the composition or pharmaceutical composition according to the invention.
  • the present invention further relates to the composition according to the invention, or the pharmaceutical composition according to the invention, for use as a medicament.
  • the present invention further relates to the composition, pharmaceutical composition or medicament according to the invention, for use for treating a genetic disease in a subject in need thereof.
  • the composition, pharmaceutical composition, or medicament for use for treating a genetic disease further comprises at least one nucleic acid molecule comprising at least one transgene, wherein the expression of the transgene compensates the genetic defect of the genetic disease.
  • the at least one transgene may encode a protein and thereby compensate the lack or absence of expression of said protein in the genetic disease; or, alternatively, the at least one transgene may encode a siRNA or shRNA or miRNA inhibiting the expression of an overexpressed gene.
  • the present invention further relates to a method for treating a subject in need thereof, comprising administering to said subject the composition, pharmaceutical composition or medicament according to the invention.
  • the present invention further relates to a method for treating a genetic disease in a subject in need thereof, comprising administering to said subject a therapeutically effective dose of the composition, pharmaceutical composition or medicament according to the invention.
  • the method further comprises co-administering to the subject at least one nucleic acid molecule comprising at least one transgene, wherein the expression of the transgene compensates the genetic defect of the genetic disease.
  • composition, pharmaceutical composition, or medicament according to the invention may be administered once, twice, or any number of times.
  • the present invention further relates to the composition or pharmaceutical composition according to the invention, for use for the manufacture of a medicament for treating a genetic disease in a subject in need thereof.
  • Figure 1 is a scheme showing the phylogenetic analysis of PiggyBac derived transposases across eukaryotic genomes.
  • Figure 2 is a histogram showing the transposition activity by PiggyBac derived transposases measured as Stable RFP Cargo integration after 3 weeks.
  • Figure 3 is a histogram representation showing the transposition activity by additional PiggyBac derived transposases measured as Stable RFP Cargo integration after 3 weeks.
  • Figure 4 is a schematic representation of the programable insertion reporter cell line.
  • Figure 5 is a histogram representation showing the programable transposition activity by PiggyBac derived transposases variants measured as GFP reporter reconstitution upon cargo insertion.
  • Figure 6 is a scheme representing the phylogenetic analysis of piggyBac derived transposases across eukaryotic genomes.
  • Figure 7A-7B is a set of histograms assessment of selected transposase orthologs for random integration of an RFP cargo in HEK293T cells, 22 days post plasmid transfection. (A) activity and (B) activity normalized to day 22.
  • Figure 8A-8C is a set of histograms showing random integration of an RFP cargo in HEK293T cells, 22 days post plasmid transfection (Fig. 8A) or 2 weeks post plasmid transfection (Fig. 8B), with other transposase orthologs.
  • Fig. 8C is a histogram showing random integration of an RFP cargo in HEK293T cells, 16 days post plasmid transfection, with transposase orthologs and hyperactive PiggyBac transposase (hyPB).
  • Figure 9 is a histogram showing targeted integration of an RFP cargo in HEK293T cells with the transposase orthologs (3X, coupled to Cas9) and hyperactive PiggyBac transposase (hyPB).
  • Figure 10 is a photograph showing targeted integration compatibility across PiggyBac orthologs.
  • Figure 11A-11B is a set of graphs and schemes showing on-target insertion obtained with the transposase PoeTurrub (from Poeciliopsis Turrubarensis).
  • Fig. 11A NGS reads:PoeTurrub AAVS1::::3’ITR.
  • Fig. 11B Profiles of the top alleles for PoeTurrub 3X NGS reads: The red line delineates the boundary between the AAVS1 genomic region on the left and the 3’ ITR on the right.
  • Figure 12A-12B is a set of graphs and schemes showing on-target insertion obtained with the transposase Antgra6049 (from Anthonomus grandis).
  • Fig. 12A NGS reads: Antgra6049 AAVS1::::3TTR
  • Fig. 12B Profiles of the top alleles for Antgra6049 3X NGS reads: The red line delineates the boundary between the AAVS 1 genomic region on the left and the 3’ ITR on the right.
  • Figure 13A-13B is a set of graphs and schemes showing on-target insertion obtained with the transposase Antgra4440 (from Anthonomus grandis).
  • Fig. 13A NGS reads: Antgra4440 AAVS1::::3TTR.
  • Fig. 13B Profiles of the top alleles for Antgra4440 3X NGS reads: The red line delineates the boundary between the AAVS 1 genomic region on the left and the 3’ ITR on the right.
  • Figure 14A-14B is a set of graphs and schemes showing on-target insertion obtained with the transposase Antgra4053 (from Anthonomus grandis).
  • Fig. 14A NGS reads: Antgra4053 AAVS1::::3TTR.
  • Fig. 14B Profiles of the top alleles for Antgra4053 3X NGS reads: The red line delineates the boundary between the AAVS 1 genomic region on the left and the 3’ ITR on the right.
  • Figure 15 is a scheme showing the percentage of RFP positive cells, i.e., the overall integration obtained with various transposase orthologs tested.
  • Figure 16A-16B is a set of histograms showing ortholog activity in primary cells from a first donor (Fig. 16A) and a second donor (Fig. 16B).
  • Figure 17A-17B is a set of histograms showing targeted integration with ortholog transposases. The best transposases identified in the bioprospecting screen were evaluated for programmable integration by co-transfection of Cas9, AAVS1 targeting gRNA and transposase-transposon plasmid pairs.
  • Fig. 17A shows the percentage of RFP positive cells;
  • Fig. 17B shows junction qPCR A.U.
  • Figure 18 a scheme showing the sequence alignment of the original PiggyBac transposase and the hyperactive PiggyBac transposase with the transposase orthologs.
  • Transposase ORF aminoacidic sequences were codon optimized for Homo Sapiens and ordered as synthesized as gene fragments to TWIST biosciences. Gene fragments were cloned in to CMV based expression vector by Golden Gate assembly using Esp3I. Transposon (cargo vector) plasmid sequences were defined as the first 150bp from the transposon ends from both 5’ and 3’ ITR sequences and synthesized as gene fragments by TWIST biosciences with added overhangs for golden gate assembly. EFla RFP polyA expression cassette was included between ITRs.
  • Triple mutant residue selection was performed by alignment of the functional piggyBacs to the Trichoplusia Ni PiggyBac sequence.
  • Triple mutant encoding plasmids (PBx3) were co-transfected with Cas9 and gRNA, transposon plasmids in to 0.5M HEK293T cells seeded in a p6 plate. Cells were analyzed for RFP expression two days after transfection. Two rounds of enrichment via RFP sorting were performed, one week after transfection and again two weeks after transfection. Genomic DNA was extracted using quiagen columns 4 days after second sorting. 3’ Junction PCR was purified in the cases where the PCR product was sanger sequenced.
  • Genomic DNA was extracted from enriched cellular samples. Subsequently, junction PCR was conducted employing primers containing P5 and P7 adapters. The selected primers for the junction PCR were designed to anneal at the 3’ end of the transposon (forward) and the genomic locus of AAVS1. Illumina reads were processed utilizing CRISPR-A to acquire both the quantity and profile of indels.
  • PiggyBac transposases orthologs sequences from Dfam database are filtered to exclude non-functional sequences. Remaining sequences were prioritized by visual inspection of alignment and ITRs, inclusion of diverse sequences, and synthesis, which led to final selection of 13 transposase sequences (see Figure 6).
  • the on-target insertion was characterized by a junction PCR followed by next generation sequencing to precisely capture and profile the payload-genome junctions at AAVS1. While precise on-target insertion of the pay load in all four examined PiggyBac orthologs can be detected ( Figures 11A, 11B, 12A, 12B, 13A, 13B, 14A, and 14B), no insertion at nearby TTAA site was detected, demonstrating integration on DSB sites generated by Cas9. Furthermore, the lack of plasmid element in the insertion site further demonstrates the clean excision of the payload flanking TTAA by the PiggyBac orthologs.
  • Stem Cell Research Stem cell therapy and regenerative medicine depend on the accurate integration of specific genes into stem cells. The observed efficiency in targeted integration opens new avenues for the genetic modification of stem cells, enabling their use in tissue repair, organ transplantation, and disease modeling.
  • Bioproduction and Biopharmaceuticals In bioproduction processes, such as the production of therapeutic proteins, optimizing host cells for efficient protein expression is crucial. The precise integration of genes, as demonstrated by these orthologs, can improve the development of high-yield cell lines for biopharmaceutical production. Synthetic Biology: The precise and efficient integration of transposons is a cornerstone of synthetic biology. It enables the creation of custom genetic circuits and metabolic pathways for various applications, including biofuel production, environmental remediation, and bioplastic synthesis.
  • the ITRs were annotated using EMBOSS palindrome, by searching for palindromes formed from the DNA sequences flanking the transposase which had a TTAA or TAA motif. From the 83 transposons, 16 top transposons were selected through a manual curation based on them having a transposase with an open reading frame (ORF) between 400-700 amino acids, ITR’s 20- 500 bp away from the beginning or end of the transposase, ITR’s composed of at least 2 different palindromes, and the species they came from (higher priority for mammals).
  • ORF open reading frame
  • Transposase ORF amino acid sequences were codon optimized for Homo Sapiens and ordered and synthesized as gene fragments to TWIST biosciences. Gene fragments were cloned into a CMV based expression vector by Golden Gate assembly using Esp3I restriction enzyme. Transposon (cargo vector) plasmid sequences were defined as the first 150bp from the transposon ends from both 5’ and 3’ ITR sequences and synthesized as gene fragments by TWIST biosciences with added overhangs for golden gate assembly. EFla RFP polyA expression cassette was included between the ITRs.
  • HEK293T cells 120k HEK293T cells were seeded in p24 wells the day prior to transfection.
  • Transposase and transposon DNA was transfected into HEK293T using.
  • ITR vector RFP expression was measured two days after transfection and 20 days after transfection. RFP signal at day 20 was taken as the integration efficiency for each of the tested systems, as it indicated stable transgene integration.
  • the expression of the ITR vector RFP was assessed two days and twenty days post-transfection using cell cytometry with the Cytek AuroraTM CS System.
  • the RFP signal at day twenty was considered indicative of stable transgene integration and was utilized to determine the integration efficiency for each of the tested systems.
  • Triple mutant residue selection was performed by alignment of the functional piggyBacs to the Trichoplusia Ni PiggyBac sequence. Plasmids encoding the triple mutant variants (PBx3) were co-transfected with Cas9, gRNA and transposon plasmids in a 1: 1:3:5 molar ratio into 0.5M Hek23T cells seeded in a p6 plate the day before transfection. Cells were analyzed for RFP expression two days after transfection using cell cytometry with the Cytek AuroraTM CS System. Subsequently, two rounds of enrichment via RFP sorting were conducted with BD FACSAria (Biosciences), one week after transfection and again two weeks after transfection.
  • BD FACSAria Biosciences
  • Genomic DNA was extracted using Quiagen DNeasy Blood & Tissue Kit column’s four days after the second sorting. A 3’ Junction PCR was performed and gel purification using the QIAquick Gel Extraction Kit was performed when necessary before Sanger sequencing the amplified bands.

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Abstract

La présente invention concerne des transposases PiggyBac hautement efficaces identifiées par correction guidée par modèle de langage de transposons extraits du génome.
PCT/EP2024/065108 2023-06-01 2024-05-31 Nouvelles transposases et leurs utilisations Pending WO2024246338A2 (fr)

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