Gene editing tools and compositions for cell engineering

Abstract

The present invention relates to a composition comprising a transposase, optionally a RNA-guided nuclease, and a supercoiled circular dsDNA molecule comprising a transgene flanked by ITRs, in particular for its use as a gene editing tool.

Description

GENE EDITING TOOLS AND COMPOSITIONS FOR CELL ENGINEERING FIELD OF INVENTION

[0001] The present invention relates to gene editing tools and compositions for cell engineering, in particular toward eukaryotic cells generation, more particularly immune cells generation, and preferably CAR-T cells generation.BACKGROUND OF INVENTION

[0002] Traditionally, gene editing is based on the design of artificial endonucleases that induce a double-strand break (DSB) into the sequence of interest in the genome, followed by repair. These First-generation technologies utilizing engineered endonucleases are used mostly for gene inactivation. Recently, technologies that allow editing independent of DSB have been developed. However, these technologies have important limitations due to the sequence PAM restriction presented by endonucleases.

[0003] Base editing (BE; Gaudelli, N. M. et al., (2017) 'Programmable base editing of A»T to G»C in genomic DNA without DNA cleavage', Nature, 551(7681), pp. 464-471) and prime editing (PE; Anzalone, A. V. et al., (2019) 'Search-and-replace genome editing without double-strand breaks or donor DNA', Nature, 576(7785), pp. 149-157) are very promising but have some limitations. BE is limited to transitions A->G or C->T within an editing window, and PE presents some design constraints as edits must be programmed downstream of the gRNA cleavage site and near a PAM sequence. On the other hand, pathogenic genetic defects can range from a few bases to large deletions. BE and PE only target a small number of bases, and HDR-based editing does not scale up well with size and leads to non-efficient results in post-mitotic cells. Methodologies based on NHEJ have been developed such as Homology Independent Targeted Integration (HITI; Suzuki, K. et al., (2016) Tn vivo genome editing via CRISPR / Cas9 mediated homologyindependent targeted integration', Nature, 540(7631), pp. 144-149). This methodologyhas been demonstrated for insertions of several kilobases, but it has been demonstrated to be inefficient for larger cargos. While HITI might work to deliver exons, it may not be efficient enough to robustly deliver cDNAs of genes greater than 4kb such as dystrophin (~14kb) or laminin-a2 (LAMA2, ~9kb).

[0004] Alternatively, viral vector integrases, as well as transposases, are used for gene delivery. However, this technology promotes uncontrolled insertion of the therapeutic gene, which can trigger cell transformation in patients. Non-integrative viral vectors such as AAVs can randomly insert in the DNA of the host cells causing uncontrolled growth. Novel safer techniques for large allele editing are needed.

[0005] One of the main limitations of the CRISPR-Cas systems is its dependency on a PAM motive adjacent to the cut site. Considerable efforts have been made in reducing this restriction, however completely unrestricted PAM Cas systems have not been achieved up to date. In recent years, Cas systems have emerged presenting new features for gene editing, such as staggered cut in the genome for Cas 12 or reduced size in Cas 14 that allowed for easier delivery into cells and tissues of interest. However, these nucleases present greater PAM sequence restrictions compared to Cas9 which greatly limit the number of genetic conditions and mutations that can be addressed using this technology. Delivering a completely sequence-unrestricted Cas nuclease will truly unlock the full potential of the new wave of safer genome editors to treat genetic conditions. More flexible technologies would be required to complement the BE and PE toolbox in small alleles.

[0006] The Inventors have previously developed “FiCAT”, a molecular machinery for precise cut-and-transfer for large alleles editing that solves the issues outlined above, that relies on the coupling of a RNA-guided nuclease (such as a Cas protein) with a transposase (see W02020250181, WO2022129438, WO2023209227). The coupling of the RNA-guided nuclease (e.g. Cas9) module and the transposase combined with adequate engineering of the transposase module have been key in the prototyping of this programmable transposase. The results obtained with this technology validates the suitability of this method to search for safer and more versatile gene editing tools. This technology can facilitate therapeutic developments for genetic diseases and certaincancers. Thanks to its greater safety and precision, FiCAT can allow the development of therapies able to overcome the limitations that other gene therapies are encountering due to concerns about adverse effects.

[0007] Transposons are already very efficient at inserting large genes into the genome, but they do so in a mostly random manner (targeting TTAA motifs in the genome), making it impossible to predict where the desired cargo will be inserted. By incorporating the Cas9 module, FiCAT has achieved complete control on the insertion site and has maintained the same efficiency as current hyperactive transposons. FiCAT has demonstrated to be 500 times more precise than any other approach on creating precise transposases. As stated before, FiCAT can overcome the current AAV-linked hard 4,7kb limitation on cargo size and have demonstrated good efficiencies inserting cargos more than twice as big, up to 1 Ikb.

[0008] Chimeric antigen receptor (CAR)-T cell therapy and in general adoptive cell transfer (ACT) therapies has been revolutionary as it has produced remarkably effective and durable clinical responses in cancer treatment. CARs are engineered synthetic receptors that function to redirect effector cells, most commonly T cells, with macrophages and NK cells that are also been used for this purpose (Klichinsky, M. et al., (2020) 'Human chimeric antigen receptor macrophages for cancer immunotherapy', Nat Biotechnol, 38(8), pp. 947-953; Xie, G. et al., (2020) 'CAR-NK cells: A promising cellular immunotherapy for cancer', EBioMedicine, 59, pp. 102975), to recognize and eliminate cells expressing a specific target antigen. CAR binding to target antigens expressed on the cell surface is independent from the major histocompatibility complex (MHC) receptor resulting in vigorous T cell activation and powerful anti-tumor responses (June, C. H., et al., (2018) 'CAR T cell immunotherapy for human cancer', Science, 359(6382), pp. 1361-1365). CAR-T treatment involves patients’ apheresis, collection of T cells, introduction of the CAR construct, and the autologous administration of the modified CARTs back to the patient. The unprecedented success of anti-CD19 CAR-T cell therapy against B cell malignancies resulted in its approval by the US Food and Drug Administration (FDA) in 2017. This recognition set the stage for the ubiquitous use of CAR-T cell therapies in broader cancer types.

[0009] However, there are major limitations to CAR-T cell therapy that still must be addressed. Currently, one of the major limitations that restrict the widespread adoption of this cell-based therapy is that approved CAR-T treatments only target a single antigen. This can be sufficient in the treatment of hematological malignancies, but it poses a huge hurdle when dealing with solid tumors (Guedan, S., et al., (2022) 'Time 2EVOLVE: predicting efficacy of engineered T-cells - how far is the bench from the bedside?', J Immunother Cancer, 10(5)). Targeting single antigens cannot accurately discriminate most solid tumors to normal tissue, so effective CAR-T therapies should integrate information from multiple antigens using combinations of AND or NOT gates, complex genetic circuits that can expand multi kilobases, to precisely identify target tumoral cells and avoid normal tissue. Naturally, these issues will not be solved without a parallel evolution of safer and more efficient delivery methods that allow bigger payloads and more targeted genomic integrations.

[0010] Other limitations for the implementation of modified T cells as broad-spectrum cancer therapies are life-threatening CAR-T cell-associated toxicities, and among these, the one carrying the major impact in the clinical setting is the cytokine-release syndrome (CRS), which is associated with supraphysiologic cytokine production and massive in vivo T cell expansion. The incidence of CRS in patients receiving the first FDA-approved CAR-T cell therapy was higher than 50% and the overall mortality rate was 5.4% of all patients mostly during the first 30 days after treatment. Critical factors that likely determine the incidence and severity of CRS are mainly the CAR T-cell design and phenotype, CAR T-cell dose infused, leukemia burden and patients own immune system. In order to achieve efficacious therapeutic responses, a CAR-T cell antigen-binding domain must bind its target epitope and reach a minimum threshold level to induce CAR-T cell activation and cytokine secretion. At the same time, however, there is also some threshold level of activation that when surpassed produces toxic levels of cytokines and immune system activation. In other words, the CAR-T cell must remain within its therapeutic window to be clinically effective as overshooting the therapeutic window will lead to toxicity. From an engineering perspective, the degree of CAR-T cell activation and activation kinetics are influenced by several factors including but not limited to thelevel of tumor antigen expressed on malignant cells, tumor burden, antigen binding domain’s affinity to its target epitope, and the CAR’s costimulatory elements.

[0011] Therefore, careful consideration of several components of the CAR’s modular structure is necessary to optimize therapeutic efficacy and limit toxicity. One way to reduce the toxicity of these treatments is to implement novel approaches on the cell engineering process. Current editing techniques are limited in the cargo size they can insert, thus restricting the features that can be inserted. There is thus a need for improved tools and methods for improving the transposon size scalability, in particular in CAR T-Cells.

[0012] It is also worth mentioning that successful T cell-based therapeutic products must also deal with such an intrinsic property as long-term persistence of T cells, a major player that greatly affects the efficiency of these treatments. Limited persistence remains a significant hurdle to the development of effective CAR-T therapies, thus engineering CAR T-cells to improve their survival and prevent the exhausted phenotype represents a logical therapeutic approach. Synthetic biology and genome-editing technology represent a fitting opportunity to control exhaustion-specific pathways through the knock-out or knock-in of specific genes.

[0013] Further, there is also room for improvement in the gene editing tool itself. The state of the art for payload delivery is the use of doggybone vectors (dbDNA™), that can be in vitro enzymatically produced DNA vectors. However, they may lack stability in vivo, trigger immune responses, and / or have insufficient expression efficiency. There is thus an unmet need to improve the gene editing tool, in particular for the generation of CAR-T cells.

[0014] The present invention provides new gene editing solutions for overcoming these limitations, including improving size scalability.SUMMARY

[0015] The present invention relates to a composition comprising:a) a polypeptide comprising (i) at least one transposase, and (ii) optionally at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) optionally, a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease; andc) a nucleic acid molecule comprising a transgene encoding a protein of interest;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0016] The present invention relates to a composition comprising:a) at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) a polypeptide comprising (i) at least one transposase, and (ii) at least one aptamer binding protein (ABP); or a nucleic acid encoding the same;c) a nucleic acid molecule comprising (i) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, and (ii) at least one aptamer sequence; wherein the gRNA is capable of interacting with the RNA-guided nuclease; andd) a nucleic acid molecule comprising a transgene encoding a protein of interest;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0017] In some embodiments, the composition comprises:a) a polypeptide comprising (i) at least one transposase, and (ii) at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease; andc) a nucleic acid molecule comprising a transgene encoding a protein of interest;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0018] In some embodiments, the at least one RNA-guided nuclease and the transposase are fused together through a linker, preferably the at least one RNA-guided nuclease and the transposase are fused together through a linker wherein the at least one RNA-guided nuclease is fused to the C terminal end of the transposase.

[0019] In some embodiments, the aptamer binding protein (ABP) is a MS2 bacteriophage coat protein (MCP) sharing at least 75% identity with SEQ ID NO: 95; and wherein the aptamer sequence is a MS2 RNA tetraloop binding sequence sharing at least 75% identity with SEQ ID NO: 96.

[0020] In some embodiments, the transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase, a Poeciliopsis turrubarensis transposase, an Anthonomus grandis DR1754440 transposase, or variants thereof, preferably a modified hyperactive PiggyBac transposase, comprising one or more amino acid mutations as compared to an unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1.

[0021] In some embodiments, modified hyperactive PiggyBac transposase comprises at least one amino acid mutation selected among the amino acid substitutions Ml 94V, R275A, R277, R347S, R372A, K375A, R376A, E377A, E380A, and D450N, said position number corresponding to the amino acid number of unmodified hyperactive PiggyBac of SEQ ID NO: 1.

[0022] In some embodiments, the modified hyperactive PiggyBac transposase comprises at least one amino acid mutation selected among the amino acid substitutions R372A, K375A and D450N, said position number corresponding to the amino acid number of unmodified hyperactive PiggyBac of SEQ ID NO: 1.

[0023] In some embodiments, the modified hyperactive PiggyBac transposase has an amino acid sequence selected from the group consisting of SEQ ID NO: 2-24.

[0024] In some embodiments, the RNA-guided nuclease is a Cas protein.

[0025] In some embodiments, the Cas protein has at least 80%, 90%, 95%, 99% or at least 100% identity to a Cas protein selected from the group consisting of a Streptococcus pyogenes Cas9 (SpCas9) of SEQ ID NO: 61, Staphylococcus aureus Cas9 (SaCas9) of SEQ ID NO: 62, Cpfl of SEQ ID NO: 63, Campylobacter jejuni Cas9 (CjCas9) of SEQ ID NO: 64, Streptococcus pyogenes Cas9 nickase (nCas9) of SEQ ID NO: 65, CasX of SEQ ID NO: 68, or Staphylococcus aureus Cas9 nickase of SEQ ID NO: 66; preferably wherein said Cas protein is a Cas9 protein selected from the group consisting of a Staphylococcus aureus Cas9 (SaCas9) of SEQ ID NO: 62 and Streptococcus pyogenes Cas9 (SpCas9) of SEQ ID NO: 61.

[0026] In some embodiments, the transgene is a large DNA fragment has a size comprised between 5 kb and 25 kb, preferably between 8 kb and 20 kb.

[0027] In some embodiments, the composition is comprised in a nanoparticle.

[0028] In some embodiments, the transgene encodes a chimeric antigen receptor.

[0029] In some embodiments, the composition further comprises an inhibitor of the protein STING, preferably the inhibitor of the protein STING is a pharmaceutical inhibitor of STING, more preferably the pharmaceutical inhibitor of STING is H-151.

[0030] In some embodiments, the composition further comprises:a) a messenger RNA (mRNA molecule) encoding a polypeptide comprising at least one transposase;b) a nucleic acid molecule comprising a transgene encoding a protein of interest;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0031] In some embodiments, the composition further comprises:a) a messenger RNA (mRNA molecule) encoding a polypeptide comprising (i) at least one transposase, and (ii) at least one RNA-guided nuclease;b) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease; andc) a nucleic acid molecule comprising a transgene encoding a protein of interest;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0032] In some embodiments, the composition further comprises:a) a messenger RNA (mRNA molecule) encoding at least one RNA-guided nuclease;b) a messenger RNA (mRNA molecule) encoding a polypeptide comprising (i) at least one transposase, and (ii) at least one aptamer binding protein (ABP); or a nucleic acid encoding the same;c) a nucleic acid molecule comprising (i) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, and (ii) at least one aptamer sequence; wherein the gRNA is capable of interacting with the RNA-guided nuclease; andd) a nucleic acid molecule comprising a transgene encoding a transgene of interest;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0033] The present invention further relates to a pharmaceutical composition comprising the composition according to the invention, and at least one pharmaceutically acceptable excipient.

[0034] The present invention further relates to the composition of the invention, or the composition of the invention, or the pharmaceutical composition of the invention, for use for treating a genetic disease in a subject in need thereof.

[0035] The present invention further relates to a kit comprising:a) a polypeptide comprising (i) at least one transposase, and (ii) optionally at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease;c) a nucleic acid molecule comprising a transgene encoding a protein of interest; andd) one or more eukaryotic cells comprising the target nucleic acid sequence;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0036] The present invention further relates to a kit comprising:a) at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) a polypeptide comprising (i) at least one transposase, and (ii) at least one aptamer binding protein (ABP); or a nucleic acid encoding the same;c) a nucleic acid molecule comprising (i) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, and (ii) at least one aptamer sequence; wherein the gRNA is capable of interacting with the RNA-guided nuclease;d) a nucleic acid molecule comprising a transgene encoding a protein of interest; ande) one or more eukaryotic cells comprising the target nucleic acid sequence;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0037] In some embodiments, the one or more eukaryotic cells are primary cells.

[0038] In some embodiments, the one or more eukaryotic cells are induced pluripotent stem cells (iPSCs).

[0039] In some embodiments, the one or more eukaryotic cells are hematopoietic stem cells.

[0040] In some embodiments, the one or more eukaryotic cells are immune cells, preferably T cells.

[0041] The present invention further relates to an in vitro method for site specific integration of a transgene into the genome of one or more eukaryotic cells, the method comprising delivering to the one or more eukaryotic cells the composition according to the invention.

[0042] In some embodiments, the one or more eukaryotic cells are primary cells.

[0043] In some embodiments, the one or more eukaryotic cells are induced pluripotent stem cells (iPSCs).

[0044] In some embodiments, the one or more eukaryotic cells are hematopoietic stem cells.

[0045] In some embodiments, the one or more eukaryotic cells are immune cells; preferably T cells.

[0046] In some embodiments, the method comprises the steps of:(i) providing one or more immune cells, preferably activated T cells; and(ii) contacting said immune cell(s), preferably said activated T cells, provided at step (i) with:a) a polypeptide comprising (i) at least one RNA-guided nuclease, and (ii) at least one transposase; or a nucleic acid encoding the same;b) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease; andc) a nucleic acid molecule comprising a transgene encoding a protein of interest, preferably a chimeric antigen receptor;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences;thereby obtaining a population of immune cells comprising the transgene encoding the protein of interest, preferably a population of T cells comprising the transgene encoding the protein of interest.

[0047] In some embodiments, the method comprises the steps of:(i) providing peripheral blood mononuclear cell (PBMC);(ii) isolating immune cell(s), preferably said T cells, from the PBMC provided at step (i);(iii) bringing into contact said immune cell(s), preferably said T cells, with an activating agent; thereby obtaining a population of activated immune cells, preferably activated T cells;(iv) expanding ex vivo said immune cell(s), preferably said activated T cells, obtained at step (iii), in particular for 1 to 7 days, preferably for about 3 days; thereby obtaining an expanded population of activated immune cells, preferably activated T cells;(v) contacting said expanded population of activated immune cells, preferably activated T cells, obtained at step (iv) with a composition comprising:a) a polypeptide comprising (i) at least one RNA-guided nuclease, and (ii) at least one transposase; or a nucleic acid encoding the same;b) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease; andc) a nucleic acid molecule comprising a transgene encoding a protein of interest, preferably a chimeric antigen receptor;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences;thereby obtaining immune cell(s) comprising the transgene encoding the protein of interest, preferably T cells comprising the transgene encoding the protein of interest.

[0048] In some embodiments, the immune cells are T cells, and the transgene encodes a chimeric antigen receptor; thereby obtaining CAR T cells.

[0049] The present invention further relates to a population of eukaryotic cells obtained by the method according to the invention, or a pharmaceutical composition thereof.

[0050] The present invention further relates to a population of CAR T cells obtained by the method according to the invention.

[0051] The present invention further relates to the population of CAR T cells obtained by the method according to the invention, or a pharmaceutical composition comprising thereof, for use for treating cancer.

[0052] The present invention further relates to a method of manufacturing the composition according to the invention, comprising contacting together:a) a polypeptide comprising (i) at least one transposase, and (ii) optionally at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease; andc) a nucleic acid molecule comprising a transgene encoding a protein of interest;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0053] The present invention further relates to a method of manufacturing the composition according to the invention, comprising contacting together:a) at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) a polypeptide comprising (i) at least one transposase, and (ii) at least one aptamer binding protein (ABP); or a nucleic acid encoding the same;c) a nucleic acid molecule comprising (i) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, and (ii) at least one aptamer sequence; wherein the gRNA is capable of interacting with the RNA-guided nuclease; andd) a nucleic acid molecule comprising a transgene encoding a protein of interest;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.DEFINITIONS

[0054] In the present invention, the following terms have the following meanings:

[0055] “About”, when preceding a figure, means plus or less 10% of the value of said figure.

[0056] “And / Or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0057] “At least one” includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, 100, 250, 500, 750, 103,104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, 1015or more.

[0058] “At least 75% of 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.

[0059] “Comprising", "comprises" and "comprised of" are used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. These terms also encompass “consisting of’.

[0060] “Eukaryotic” refers to a cell or to an organism composed of such cells, which cells are not bacterial cells (prokaryotic cells). Eukaryotic cells include, withoutlimitation, fungal cells, plant cells, and animal cells (e.g., mammalian cells and human cells).

[0061] “Flanked” or “flanking” means that a first nucleic acid such as an ITR is adjacent to a second nucleic acid sequence such as a transgene encoding a protein of interest. The first nucleic acid sequence may be separated from the second nucleic acid sequence by any number of nucleotides or not be separated, for example separated by 1000 nucleotides, 500 nucleotides, 100 nucleotides, 50 nucleotides, 20 nucleotides, 19 nucleotides, 18 nucleotides, 17 nucleotides, 16 nucleotides, 15 nucleotides, 14 nucleotides, 13 nucleotides, 12 nucleotides, 11 nucleotides, 10 nucleotides, 9 nucleotides, 8 nucleotides, 7 nucleotides, 6 nucleotides, 5 nucleotides, 4 nucleotides, 3 nucleotides, 2 nucleotides, 1 nucleotide, or 0 nucleotide.

[0062] “Fusion protein” refers to a single-chain hybrid polypeptide which comprises two or more amino acid sequences fused together (i.e., from two or more different proteins and / or peptides). The two or more amino acid sequences can be fused together via a direct peptidic bond or indirectly through a peptidic linker. A fusion protein may be in particular fully encoded by a single nucleic acid sequence.

[0063] “Gene” typically refers to a DNA region encoding a protein (i.e., a coding region). The term may also include DNA regions which do not per se encode a protein (i.e., a non-coding region). The latter include, e.g., regions transcribed into functional non-coding RNA molecules (e.g., transfer RNA, ribosomal RNA, regulatory RNA, etc.). Other non-coding regions regulate the transcription and translation of coding regions (i. e. , regulatory elements), or serve as architectural elements (e.g., scaffold / matrix attachment region), as origins of DNA replication, as centromeres or telomeres, etc. Regulatory elements include, without limitations, promoter sequences, terminators, translational regulatory sequences (e.g., ribosome binding sites [RBS] and internal ribosome entry sites [IRES]), enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions.

[0064] “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. The terms “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. Conversely, the terms “random”, “non-targeted” and “off-targeted” refer to non-specific and unintended insertion of a nucleic acid into an unwanted site. The terms “total” or “overall” refer to the total number of insertions.

[0065] “Linker” refers to a chemical group or a molecule linking two adjacent molecules or moieties.

[0066] “Mutation” refers to a substitution of a residue within a sequence, e.g., a nucleic acid or amino acid sequence, with another residue; and / or to a deletion or insertion of one or more residues within a nucleic acid or amino acid sequence. Mutations are typically described herein by identifying the original residue followed by the position of the residue within the sequence, then the identity of the newly substituted residue. Various methods for making amino acid substitutions (mutations) provided herein are well known in the art.

[0067] “Nuclease” refers to an enzyme catalyzing the hydrolysis of nucleic acids within a nucleic acid sequence. Nuclease activity can result in single-stranded or double-stranded nucleic acid molecules break, wherein the nucleic molecule can be DNA or RNA. Nucleases include Cas proteins.

[0068] “Nucleic acid sequence”, “nucleic acid molecule” 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.

[0069] “Percent identity” between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the twosequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

[0070] “Polypeptide”, “peptide”, “protein” and “amino acid sequence” are used interchangeably to refer to a polymer of amino acid residues. Unless specified, a polymer of amino acid residues can be any length. The term also applies to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of corresponding naturally-occurring amino acids.

[0071] “Subject” refers to a mammal, preferably a human. A subject may be a “patient”, ie., a warm-blooded animal, more preferably a human, who / which is awaiting the receipt of, or is receiving medical care or was / is / will be the object of a medical procedure, or is monitored for the development of a disease. The term “mammal” refers here to any mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is a primate, more preferably a human.

[0072] “Supercoiled circular double-stranded DNA molecule” refers to a closed-loop DNA structure composed of two complementary strands of deoxyribonucleic acid (DNA) that are coiled upon themselves to adopt a supercoiled conformation. This structural configuration results from the introduction of torsional strain, causing the DNA helix to twist and compact beyond its relaxed circular state. The supercoiled form enhances the molecule's stability, reduces its physical size, and alters its accessibility to biological processes such as replication and transcription. This configuration is particularly advantageous for applications requiring efficient cellular uptake, genetic manipulation, and high-level gene expression in host systems.

[0073] “Therapeutically effective amount” is intended to refer to the level or amount of agent that is aimed at, without causing significant negative or adverse side effects to the target, (1) delaying or preventing the onset of a disease; (2) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of a disease; (3) bringing about ameliorations of the symptoms of a disease; (4) reducing the severity or incidence of a disease; or (5) preventing a disease formation. In one embodiment, atherapeutically effective amount is administered prior to the onset of a disease formation, for a prophylactic or preventive action.

[0074] “Transfection” and any declension thereof refers to the introduction of one or several nucleic acid molecules (DNA and / or RNA) into one or more cells by non-viral means, whether in vitro or in vivo. Methods for transfection are well known in the art and include, e.g., lipofection and electroporation.

[0075] “Transgene” refers to an exogenous nucleic acid sequence, in particular an exogenous DNA, encoding a gene product being a protein of interest. In addition to the coding region for the gene product (CDS), 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. Preferred proteins of interest are chimeric antigen receptors (CARs).

[0076] “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.

[0077] “Treatment,” “treat,” and “treating,” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. As used herein, the terms “treatment,” “treat,” and “treating” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed and / or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent, reduce the likelihood of developing, or delay onset of a symptom or inhibit onsetor progression of a disease. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.DETAILED DESCRIPTION

[0078] The present invention relates to a composition comprising:a) a polypeptide comprising (i) at least one transposase, and (ii) optionally at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) optionally, a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease; andc) a nucleic acid molecule comprising a transgene encoding a protein of interest;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0079] The present invention also relates to a composition comprising:a) at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) a polypeptide comprising (i) at least one transposase, and (ii) at least one aptamer binding protein (ABP); or a nucleic acid encoding the same;c) a nucleic acid molecule comprising (i) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, and (ii) at least one aptamer sequence; wherein the gRNA is capable of interacting with the RNA-guided nuclease; andd) a nucleic acid molecule comprising a transgene encoding a protein of interest;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0080] In some embodiments, the composition comprises:a) a polypeptide comprising (i) at least one transposase, and (ii) at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease; andc) a nucleic acid molecule comprising a transgene encoding a protein of interest;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0081] Therefore, the present invention also relates to a composition comprising:a) a polypeptide comprising (i) at least one transposase, and (ii) at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease; andc) a nucleic acid molecule comprising a transgene encoding a protein of interest;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0082] Transposons are chromosomal segments that can undergo transposition, e.g., DNA that can be translocated as a whole in the absence of a complementary sequence inthe host DNA. Transposons can be used to perform long-range DNA engineering in human cells. Common transposon systems used in mammalian cells include, without limitation, Sleeping Beauty (SB), and PiggyBac (PB), isolated from the moth Trichoplusia. PiggyBac has higher transposition activity than SB and it can be excised scarlessly.

[0083] Native DNA transposons typically contain a single gene coding for a transposase protein, which is flanked by Inverted Terminal Repeats (ITRs) that carry transposase binding sites. During their transposition, the transposase protein recognizes these ITRs to catalyze excision and subsequent reintegration of the element elsewhere in a random manner. Moreover, some of these transposons can be adapted for use in gene therapy protocols, employing them as bi-component systems, in which a plasmid contains an expression cassette where a nucleic acid sequence of interest, flanked by the transposon ITRs, can be introduced into a host genome directed by a co-transfected plasmid containing the sequence encoding the transposase enzyme or its mRNA synthesized in vitro. According to the disclosure, a transposon-based system is used to efficiently mediate stable integration and persistent expression of transgenes in a cell, such as therapeutic genes, or chimeric antigen receptors (CAR) for the generation of e.g., CAR-T cells.

[0084] The compositions of the invention comprise a polypeptide comprising a transposase, or a fragment thereof. The compositions of the invention comprise a polypeptide that may consist of a transposase, or a fragment thereof.

[0085] In some embodiments, the transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase (hyPB), a Poeciliopsis turrubarensis transposase or a variant thereof, and an Anthonomus grandis transposase or a variant thereof.

[0086] In some embodiments, the transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase (hyPB), comprising one or more amino acid mutations as compared to an unmodified hyperactive PiggyBac transposase of SEQ IDNO: 1, a Poeciliopsis turrubarensis transposase or a variant thereof, and an Anthonomus grandis transposase or a variant thereof.

[0087] In some embodiments, the transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase (hyPB), comprising one or more amino acid mutations as compared to an unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1, a Poeciliopsis turrubarensis transposase or a variant thereof, an Anthonomus grandis DR1754440 transposase or a variant thereof, an Anthonomus grandis DR1756049 transposase or a variant thereof, and an Anthonomus grandis DR1754053 transposase or a variant thereof.

[0088] In some embodiments, the transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase (hyPB), comprising one or more amino acid mutations as compared to an unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1, a Poeciliopsis turrubarensis transposase or a variant thereof, an Anthonomus grandis DR1754440 transposase or a variant thereof, and an Anthonomus grandis DR1756049 transposase or a variant thereof.

[0089] In some embodiments, the transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase (hyPB), comprising one or more amino acid mutations as compared to an unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1, a Poeciliopsis turrubarensis transposase or a variant thereof, and an Anthonomus grandis DR1754440 transposase or a variant thereof.

[0090] In some embodiments, the transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase (hyPB), comprising one or more amino acid mutations as compared to an unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1, and a Poeciliopsis turrubarensis transposase or a variant thereof.

[0091] In some embodiments, the transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase (hyPB), comprising one or more amino acid mutations as compared to an unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1, an Anthonomus grandis DR1754440 transposase or a variant thereof, anAnthonomus grandis DR1756049 transposase or a variant thereof, and an Anthonomus grandis DR1754053 transposase or a variant thereof.

[0092] In some embodiments, the transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase (hyPB), comprising one or more amino acid mutations as compared to an unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1, an Anthonomus grandis DR1754440 transposase or a variant thereof, and an Anthonomus grandis DR1756049 transposase or a variant thereof.

[0093] In some embodiments, the transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase (hyPB), comprising one or more amino acid mutations as compared to an unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1, and an Anthonomus grandis DR1754440 transposase or a variant thereof.

[0094] In some embodiments, the transposase is 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 DR1756049 transposase or a variant thereof, and an Anthonomus grandis DR1754053 transposase or a variant thereof.

[0095] In some embodiments, the transposase is selected from the group consisting of a Poeciliopsis turrubarensis transposase or a variant thereof, an Anthonomus grandis DR1754440 transposase or a variant thereof, and an Anthonomus grandis DR1756049 transposase or a variant thereof.

[0096] In some embodiments, the transposase is selected from the group consisting of a Poeciliopsis turrubarensis transposase or a variant thereof, and an Anthonomus grandis DR1754440 transposase or a variant thereof.

[0097] In some embodiments, the transposase is selected from the group consisting of an Anthonomus grandis DR1754440 transposase or a variant thereof, an Anthonomus grandis DR1756049 transposase or a variant thereof, and an Anthonomus grandis DR1754053 transposase or a variant thereof.

[0098] In some embodiments, the transposase is selected from the group consisting of an Anthonomus grandis DR1754440 transposase or a variant thereof, and an Anthonomus grandis DR1756049 transposase or a variant thereof.

[0099] In some embodiments, the transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase (hyPB) having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with any of SEQ ID NO: 2-24, a Poeciliopsis turrubarensis transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 25 or a variant thereof, an Anthonomus grandis DR1754440 transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 40 or a variant thereof, an Anthonomus grandis DR1756049 transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 42 or 43 or a variant thereof, and an Anthonomus grandis DR1754053 transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 39 or a variant thereof.

[0100] In some embodiments, the transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase (hyPB) having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with any of SEQ ID NO: 2-24, a Poeciliopsis turrubarensis transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 25 or a variant thereof, an Anthonomus grandisDR1754440 transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 40 or a variant thereof, and an Anlhonomus grandis DR1756049 transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 42 or 43 or a variant thereof.

[0101] In some embodiments, the transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase (hyPB) having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with any of SEQ ID NO: 2-24, Poeciliopsis turrubarensis transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 25 or a variant thereof, and an Anthonomus grandis DR1754440 transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 40 or a variant thereof.

[0102] In some embodiments, the transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase (hyPB) having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with any of SEQ ID NO: 2-24, and a Poeciliopsis turrubarensis transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 25 or a variant thereof.

[0103] In some embodiments, the transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase (hyPB) having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with any of SEQ ID NO: 2-24, an Anthonomus grandis DR1754440 transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 40 or a variant thereof, an Anthonomus grandis DR1756049 transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 42 or 43 or a variant thereof, and an Anthonomus grandis DR1754053 transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 39 or a variant thereof.

[0104] In some embodiments, the transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase (hyPB) having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with any of SEQ ID NO: 2-24, an Anthonomus grandis DR1754440 transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 40 or a variant thereof, and an Anthonomus grandis DR1756049 transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 42 or 43 or a variant thereof.

[0105] In some embodiments, the transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase (hyPB) having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with any of SEQ ID NO: 2-24, and an Anthonomus grandis DR1754440 transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 40 or a variant thereof.

[0106] In some embodiments, the transposase is selected from the group consisting of a Poeciliopsis turrubarensis transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 25 or a variant thereof, an Anthonomus grandis DR1754440 transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 40 or a variant thereof, an Anthonomus grandis DR1756049 transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 42 or 43 or a variant thereof, and an Anthonomus grandis DR1754053 transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 39 or a variant thereof.

[0107] In some embodiments, the transposase is selected from the group consisting of a Poeciliopsis turrubarensis transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 25 or a variant thereof, an Anthonomus grandis DR1754440 transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 40 or a variant thereof, and an Anthonomus grandis DR1756049 transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 42 or 43 or a variant thereof.

[0108] In some embodiments, the transposase is selected from the group consisting of a Poeciliopsis turrubarensis transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 25 or a variant thereof, and an Anthonomus grandis DR1754440 transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 40 or a variant thereof.

[0109] In some embodiments, the transposase is selected from the group consisting of an Anthonomus grandis DR1754440 transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 40 or a variant thereof, an Anthonomus grandis DR1756049 transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 42 or 43 or a variant thereof, and an Anthonomus grandis DR1754053 transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 39 or a variant thereof.

[0110] In some embodiments, the transposase is selected from the group consisting of an Anthonomus grandis DR1754440 transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 40 or a variant thereof, and an Anthonomus grandis DR1756049 transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with SEQ ID NO: 42 or 43 or a variant thereof.

[0111] In some embodiments, the transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase (hyPB) having the amino acid sequence as set forth in any of SEQ ID NO: 2-24, a Poeciliopsis turrubarensis transposase having the amino acid sequence as set forth in SEQ ID NO: 25 or a variant thereof, an Anthonomus grandis DR1754440 transposase having the amino acid sequence as set forth in SEQ ID NO: 40 or a variant thereof, an Anthonomus grandis DR1756049 transposase having the amino acid sequence as set forth in SEQ ID NO: 42 or 43 or a variant thereof, and an Anthonomus grandis DR1754053 transposase having the amino acid sequence as set forth in SEQ ID NO: 39 or a variant thereof.

[0112] In some embodiments, the transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase (hyPB) having the amino acid sequence as set forth in any of SEQ ID NO: 2-24, a Poeciliopsis turrubarensis transposase having the amino acid sequence as set forth in SEQ ID NO: 25 or a variant thereof, an Anthonomus grandis DR1754440 transposase having the amino acid sequence as set forth in SEQ ID NO: 40 or a variant thereof, and an Anthonomus grandis DR1756049 transposase having the amino acid sequence as set forth in SEQ ID NO: 42 or 43 or a variant thereof.

[0113] In some embodiments, the transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase (hyPB) having the amino acid sequence as set forth in any of SEQ ID NO: 2-24, a Poeciliopsis turrubarensis transposase having the amino acid sequence as set forth in SEQ ID NO: 25 or a variant thereof, and an Anthonomus grandis DR1754440 transposase having the amino acid sequence as set forth in SEQ ID NO: 40 or a variant thereof.

[0114] In some embodiments, the transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase (hyPB) having the amino acid sequence as set forth in any of SEQ ID NO: 2-24, and Poeciliopsis turrubarensis transposase having the amino acid sequence as set forth in SEQ ID NO: 25 or a variant thereof.

[0115] In some embodiments, the transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase (hyPB) having the amino acid sequence asset forth in any of SEQ ID NO: 2-24, an Anthonomus grandis DR1754440 transposase having the amino acid sequence as set forth in SEQ ID NO: 40 or a variant thereof, an Anthonomus grandis DR1756049 transposase having the amino acid sequence as set forth in SEQ ID NO: 42 or 43 or a variant thereof, and an Anthonomus grandis DR1754053 transposase having the amino acid sequence as set forth in SEQ ID NO: 39 or a variant thereof.

[0116] In some embodiments, the transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase (hyPB) having the amino acid sequence as set forth in any of SEQ ID NO: 2-24, an Anthonomus grandis DR1754440 transposase having the amino acid sequence as set forth in SEQ ID NO: 40 or a variant thereof, and an Anthonomus grandis DR1756049 transposase having the amino acid sequence as set forth in SEQ ID NO: 42 or 43 or a variant thereof.

[0117] In some embodiments, the transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase (hyPB) having the amino acid sequence as set forth in any of SEQ ID NO: 2-24, and an Anthonomus grandis DR1754440 transposase having the amino acid sequence as set forth in SEQ ID NO: 40 or a variant thereof.

[0118] In some embodiments, the transposase is selected from the group consisting of a Poeciliopsis turrubarensis transposase having the amino acid sequence as set forth in SEQ ID NO: 25 or a variant thereof, an Anthonomus grandis DR1754440 transposase having the amino acid sequence as set forth in SEQ ID NO: 40 or a variant thereof, an Anthonomus grandis DR1756049 transposase having the amino acid sequence as set forth in SEQ ID NO: 42 or 43 or a variant thereof, and an Anthonomus grandis DR1754053 transposase having the amino acid sequence as set forth in SEQ ID NO: 39 or a variant thereof.

[0119] In some embodiments, the transposase is selected from the group consisting of a Poeciliopsis turrubarensis transposase having the amino acid sequence as set forth in SEQ ID NO: 25 or a variant thereof, an Anthonomus grandis DR1754440 transposase having the amino acid sequence as set forth in SEQ ID NO: 40 or a variant thereof, andan Anthonomus grandis DR1756049 transposase having the amino acid sequence as set forth in SEQ ID NO: 42 or 43 or a variant thereof.

[0120] In some embodiments, the transposase is selected from the group consisting of a Poeciliopsis turrubarensis transposase having the amino acid sequence as set forth in SEQ ID NO: 25 or a variant thereof, and an Anthonomus grandis DR1754440 transposase having the amino acid sequence as set forth in SEQ ID NO: 40 or a variant thereof.

[0121] In some embodiments, the transposase is selected from the group consisting of an Anthonomus grandis DR1754440 transposase having the amino acid sequence as set forth in SEQ ID NO: 40 or a variant thereof, an Anthonomus grandis DR1756049 transposase having the amino acid sequence as set forth in SEQ ID NO: 42 or 43 or a variant thereof, and an Anthonomus grandis DR1754053 transposase having the amino acid sequence as set forth in SEQ ID NO: 39 or a variant thereof.

[0122] In some embodiments, the transposase is selected from the group consisting of an Anthonomus grandis DR1754440 transposase having the amino acid sequence as set forth in SEQ ID NO: 40 or a variant thereof, and an Anthonomus grandis DR1756049 transposase having the amino acid sequence as set forth in SEQ ID NO: 42 or 43 or a variant thereof.

[0123] In some embodiments, the transposase has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with any of SEQ ID NO: 2-24, 26-38, 41, and 44. In some embodiments, the transposase has an amino acid sequence selected from the group consisting of SEQ ID NO: 2-24, 26-38, 41, and 44.

[0124] In some embodiments, the transposase has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with any of SEQ ID NO: 26-38, 41, and 44. In some embodiments, the transposase has an amino acid sequence selected from the group consisting of SEQ ID NO: 26-38, 41, and 44.

[0125] In some embodiments, the transposase has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with any of SEQ ID NO: 2, SEQ ID NO: 38, SEQ ID NO: 41, and SEQ ID NO: 44 In some embodiments, the transposase has an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 38, SEQ ID NO: 41, and SEQ ID NO: 44

[0126] In some embodiments, the transposase has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of amino acid sequence identity with any of SEQ ID NO: 38, SEQ ID NO: 41, and SEQ ID NO: 44 In some embodiments, the transposase has an amino acid sequence selected from the group consisting of SEQ ID NO: 38, SEQ ID NO: 41, and SEQ ID NO: 44

[0127] In some embodiments, the transposase is a modified hyperactive PiggyBac transposase (hyPB), comprising one or more amino acid mutations as compared to an unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1. In some embodiments, the transposase is a modified hyperactive PiggyBac transposase (hyPB), comprising one or more amino acid mutations as compared to an unmodified hyperactive PiggyBac transposase of SEQ ID NO: 103.

[0128] In some embodiments, the transposase is a modified hyperactive PiggyBac transposase (hyPB) having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%, of amino acid sequence identity with SEQ ID NO: 1, comprising one or more amino acid mutations as compared to an unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1 (z.e., the amino acid sequence of the modified hyperactive PiggyBac transposase is not 100% identical to SEQ ID NO: 1). In some embodiments, the transposase is a modified hyperactive PiggyBac transposase (hyPB) having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%, of amino acid sequence identity with SEQ ID NO: 103, comprising one or more amino acid mutations as compared to an unmodified hyperactive PiggyBac transposase of SEQ ID NO: 103(z.e., the amino acid sequence of the modified hyperactive PiggyBac transposase is not 100% identical to SEQ ID NO: 103).SEQ ID NO: 1MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEV QPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKPTRRSRVS ALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSA TFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDF LIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGF RGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEY YVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVL KNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMV MYYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNV SSKGEKVQSRKKFMRNLYMGLTSSFMRKRLEAPTLKRYLRDNISNILPKEVPGT SDDSTEEPVMKKRTYCTYCPSKIRRKASASCKKCKKVICREHNIDMCQSCFSEQ ID NO: 103MHHHHHHGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFI DEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKPT RRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRR ESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMS RDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTID EQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRQTNGVPL GEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVRSNKREIP EVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKP QMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIY SHNVSSKGEKVQSRKKFMRNLYMGLTSSFMRKRLEAPTLKRYLRDNISNILPKE VPGTSDDSTEEPVMKKRTYCTYCPSKIRRKASASCKKCKKVICREHNIDMCQSCF

[0129] In some embodiments, the transposase is a modified hyperactive PiggyBac transposase (hyPB) having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%, of amino acid sequence identity with SEQ ID NO: 1, comprising one or more amino acid substitutions, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, as compared to the hyPB of amino acid sequence SEQ ID NO: 1; similarly, the amino acid sequence of the modified hyperactive PiggyBac transposase is not 100% identical to SEQ ID NO: 1.

[0130] In some embodiments, the transposase is a modified hyperactive PiggyBac transposase (hyPB) having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%, of amino acid sequence identity with SEQ ID NO: 103, comprising one or more amino acid substitutions, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, as compared to the hyPB of amino acid sequence SEQ ID NO: 103; similarly, the amino acid sequence of the modified hyperactive PiggyBac transposase is not 100% identical to SEQ ID NO: 103.

[0131] In some embodiments, the modified hyPB comprises (i) one or more amino acid substitutions to increase excision activity as compared to the unmodified hyPB of amino acid sequence SEQ ID NO: 1, and / or (ii) one or more amino acid substitutions to decrease DNA binding activity as compared to the unmodified hyPB of amino acid sequence SEQ ID NO: 1

[0132] In some embodiments, the modified hyPB comprises (i) one or more amino acid substitutions to increase excision activity as compared to the unmodified hyPB of amino acid sequence SEQ ID NO: 103, and / or (ii) one or more amino acid substitutions to decrease DNA binding activity as compared to the unmodified hyPB of amino acid sequence SEQ ID NO: 103.

[0133] In some embodiment, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations to increase excision activity.

[0134] In some embodiment, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations to increase excision activity selected among the amino acid mutations within the region defined by the amino acid position numbers [194-200],[214-222], [434-442] or [446-456], for example amino acid substitution at the position D198, D201, R202, M212 and / or S213; the position number corresponding to the amino acid number of unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1.

[0135] In some embodiment, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations to increase excision activity selected among the amino acid mutations at positions 450, 560, 564, 573, 589, 592, and / or 594; the position number corresponding to the amino acid number of unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1

[0136] In some embodiment, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations to increase excision activity selected among the amino acid mutations at position of M194 and / or D450, the position number corresponding to the amino acid number of unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1, preferably the amino acid substitution selected among M194V and / or D450N.

[0137] In some embodiment, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations to decrease DNA binding activity.

[0138] In some embodiment, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations to decrease DNA binding activity selected among the amino acid mutations at positions 254, 275, 277, 347, 372, 375, and / or 465; the position number corresponding to the amino acid number of unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1.

[0139] In some embodiment, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations to decrease DNA binding activity selected among R275, N347, R372, K375, R376, E377, and E380, the position number corresponding to the amino acid number of unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1

[0140] In some embodiment, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations to decrease DNA binding activity selected amongR372, K375, R376, E377, and E380, the position number corresponding to the amino acid number of unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1, preferably selected among the amino acid substitutions R372A, K375A, R376A, E377A, and / or E380A.

[0141] In some embodiment, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations to decrease DNA binding activity selected among N347, R372, and K375, the position number corresponding to the amino acid number of unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1, preferably selected among the amino acid substitutions N347S, N347A, R372A, K375A, more preferably selected among the amino acid substitutions N347S, N347A.

[0142] In some embodiment, the modified hyperactive PiggyBac transposase comprises one or more amino acid mutations to increase excision activity, as defined above; and one or more amino acid mutations to decrease DNA binding activity, as defined above; compared to the unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1.

[0143] In some embodiment, the modified hyperactive PiggyBac transposase includes at least one amino acid substitution to increase excision activity at position D450, and at least two amino acid substitutions to decrease DNA binding activity at positions N347, R372 and K375, preferably the modified hyperactive PiggyBac transposase of hyperactive PiggyBac includes the double mutations N347S and D450N or triple mutations D450N, R372A and K375A, the position number corresponding to the amino acid number of unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1. In a more preferred embodiment, the modified hyperactive PiggyBac transposase of hyperactive PiggyBac includes the double mutations N347S and D450N, the position number corresponding to the amino acid number of unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1.

[0144] In some embodiment, the modified hyperactive PiggyBac transposase as disclosed in the previous embodiments further comprises at least one mutation in the region defined by the amino acid position numbers [158-169], for example A166S; and / or at least one mutation at position Y527, R518, K525, N463.

[0145] In some embodiments, the modified hyperactive PiggyBac transposase comprises an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%, or 100% identity to modified hyperactive PiggyBac transposase of SEQ ID NO: 2, wherein the modified hyperactive PiggyBac transposase does not comprise SEQ ID NO: 1. In some embodiments, the modified hyperactive PiggyBac transposase has the amino acid sequence as set forth in SEQ ID NO: 2.

[0146] In some embodiments, the modified hyperactive PiggyBac transposase comprises an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%, or 100% identity to modified hyperactive PiggyBac transposase of SEQ ID NO: 104, wherein the modified hyperactive PiggyBac transposase does not comprise SEQ ID NO: 1. In some embodiments, the modified hyperactive PiggyBac transposase has the amino acid sequence as set forth in SEQ ID NO: 104.

[0147] In some embodiments, the modified hyperactive PiggyBac transposase further comprises one or more of the following amino acid mutations at positions 34, 43, 117, 202, 230, 245, 268, 275, 277, 287, 290, 315, 325, 341, 346, 347, 350, 351, 356, 357, 388, 409, 411, 412, 432, 447, 460, 461, 465, 517, 560, 564, 571, 573, 576, 586, 587, 589, 592, and / or 594, the position number corresponding to the amino acid number of the unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1.

[0148] In some embodiments, the modified hyperactive PiggyBac transposase comprises the following mutations or combination of mutations: V34M, T43I, Y177H, R202K, S230N, R245A, D268N, K287A, K290A, K287A / K290A, R315A, G325A, R341A, D346N, N347A, N347S, T350A, S351E, S351P, S351A, K356E, N357A, R388A, K409A, A411T, K412A, K432A, D447A, D447N, D450N, R460A, K461A, W465A, S517A, T560A, S564P, S571N, S573A, K576A, H586A, I587A, M589V, S592G, or F594L, D450N / R372A / K375A, R275A / R277A, K409A / K412A, R460A / K461A, R275A / R277A / N347S / K375A / T560A / S573A / M589V / S592G and R245A / R275A / R277A / R372A / W465A, the position number corresponding to the amino acid number of the unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1.

[0149] In some embodiments, the modified hyperactive PiggyBac transposase comprises the following amino acid substitution or combination of amino acid substitutions:R372A / K375A / D450N,R372A / K375 A / R376A / D450N,K375 A / R376A / E377A / E380A / D450N,R372A / K375 A / R376 A / E377A / E380A / D450N,Ml 94 V,M194V / R372A / K375A, S351A / R372A / K375A / R388A / D450N / W465A / S573A / M589V / S592G / F594L, R245 A / R275 A / R277A / R372A / W465 A / M589 V, R275A / 325A / R372A / T560A,N347A / D450N,N347S / D450N / T560A / S573A / F594L, R202K / R275A / N347S / R372A / D450N / T560A / F594L,R275A / N347S / K375A / D45 0N / S592G,R275A / N347S / R372A / D450N / T560A / F594L, R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L, R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G, R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L, V34M / R275A / G325A / N347S / S351A / R372A / K375A / D450N / T560A / S564P, G325A / N347S / K375A / D450N / S573A / M589V / S592G, S230N / R277A / N347S / K375A / D450N,T43I / R372A / K375A / A411T / D450N, G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, and Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G,the position number corresponding to the amino acid number of the unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1.

[0150] In some embodiments, the modified hyperactive PiggyBac transposases comprises one or more of the following combinations of amino acid substitutions:R372A / K375A / D450N, S351A / R372A / K375A / R388A / D450N / W465A / S573A / M589V / S592G / F594L, R245 A / R275 A / R277A / R372A / W465 A / M589 V,N347A / D450N,N347S / D450N / T560A / S573A / F594L, R202K / R275A / N347S / R372A / D450N / T560A / F594L, R275A / N347S / K375A / D450N / S592G, R275A / N347S / R372A / D450N / T560A / F594L, R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L, R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G, R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L, V34M / R275A / G325A / N347S / S351A / R372A / K375A / D450N / T560A / S564P, G325A / N347S / K375A / D450N / S573A / M589V / S592G, S230N / R277A / N347S / K375A / D450N,T43I / R372A / K375A / A411T / D450N, G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G, and R275A / 325A / R372A / T560A,the position number corresponding to the amino acid number of the unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1.

[0151] In some embodiments, the modified hyperactive PiggyBac transposase comprises the following amino acid substitution or combination of amino acid substitutions:R245 A / R275 A / R277A / R372A / W465 A / M589 V, R275A / 325A / R372A / T560A,N347A / D450N,N347S / D450N / T560A / S573A / F594L, R202K / R275A / N347S / R372A / D450N / T560A / F594L, R275A / N347S / K375A / D450N / S592G,R275A / N347S / R372A / D450N / T560A / F594L,R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L, R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G, R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L, G325A / N347S / K375A / D450N / S573A / M589V / S592G, S230N / R277A / N347S / K375A / D450N, G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, and Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G,the position number corresponding to the amino acid number of the unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1.

[0152] In some embodiments, the modified hyperactive PiggyBac transposase comprises one or more of the following combinations of amino acid substitutions: N347A / D450N,N347S / D450N / T560A / S573A / F594L, R202K / R275A / N347S / R372A / D450N / T560A / F594L, R275A / N347S / K375A / D450N / S592G, R275A / N347S / R372A / D450N / T560A / F594L, R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L, R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G, R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L, G325A / N347S / K375A / D450N / S573A / M589V / S592G, S230N / R277A / N347S / K375A / D450N, G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G,the position number corresponding to the amino acid number of the unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1.

[0153] In some embodiments, the modified hyperactive PiggyBac transposase comprises the following combination of amino acid substitutions:N347A / D450N,N347S / D450N / T560A / S573A / F594L,R202K / R275A / N347S / R372A / D450N / T560A / F594L, R275A / N347S / K375A / D450N / S592G, R275A / N347S / R372A / D450N / T560A / F594L, R275A / R277A / N347S / R372A / D450N / T560A / S564P / F594L, R245A / N347S / R372A / D450N / T560A / S564P / S573A / S592G, R277A / G325A / N347A / K375A / D450N / T560A / S564P / S573A / S592G / F594L, G325A / N347S / K375A / D450N / S573A / M589V / S592G, S230N / R277A / N347S / K375A / D450N, G325A / N347S / S351A / K375A / D450N / S573A / M589V / S592G, Y177H / R275A / G325A / K375A / D450N / T560A / S564P / S592G,the position number corresponding to the amino acid number of the unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1.

[0154] In some embodiments, the modified hyperactive PiggyBac transposase comprises the following combination of amino acid substitutions: R372A / K375A / D450N, the position numbers corresponding to the amino acid numbers of unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1. In some embodiment, the modified hyperactive PiggyBac transposase has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%, or 100% identity with the amino acid sequence of SEQ ID NO: 2. In some embodiment, the modified hyperactive PiggyBac transposase has an amino acid sequence of SEQ ID NO: 2.

[0155] In some embodiment, the modified hyperactive PiggyBac transposase has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%, or 100% identity with the amino acid sequence of any of SEQ ID NO: 2-24. In some embodiments, the modified hyperactive PiggyBac transposase has an amino acid sequence selected among any of SEQ ID NO: 2-24.

[0156] In some embodiment, the modified hyperactive PiggyBac transposase has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%, or 100% identity with the amino acid sequence of any of SEQ ID NO: 3 to SEQ ID NO: 24. In some embodiments, the modified hyperactive PiggyBac transposase has an amino acid sequence selected among any of SEQ ID NO: 3 to SEQ ID NO: 24.

[0157] In some embodiments, the modified hyperactive PiggyBac transposase can comprise one or more mutations relative to hyPB that are involved in the conserved catalytic triad, e.g., at amino acid 268 and / or 346 (e.g., D268N and / or D346N) corresponding to the amino acid numbering of SEQ ID NO: 1.

[0158] In some embodiments, the modified hyperactive PiggyBac transposase can comprise one or more mutations relative to hyPB that are critical for excision, e.g., at amino acid 287, 287 / 290 and / or 460 / 461 (e.g., K287A, K287A / K290A, and / or R460A / K461 A) corresponding to the amino acid numbering of SEQ ID NO: 1.

[0159] In some embodiments, the modified hyperactive PiggyBac transposase can comprise one or more mutations relative to hyPB that are involved in target joining, e.g., at amino acid 351, 356, and / or 379 (e.g., S351E, S351P, S351A, and / or K356E) corresponding to the amino acid numbering of SEQ ID NO: 1.

[0160] In some embodiments, the modified hyperactive PiggyBac transposase can comprise one or more mutations relative to hyPB that are critical for integration, e.g., at amino acid 560, 564, 571, 573, 589, 592, and / or 594 (e.g., T560A, S564P, S571N, S573A, M589V, S592G, and / or F594L) corresponding to the amino acid numbering of SEQ ID NO: 1

[0161] In some embodiments, the modified hyperactive PiggyBac transposase can comprise one or more mutations relative to hyPB that are involved in alignment, e.g., at amino acid 325, 347, 350, 357 and / or 465 (e.g., G325A, N347A, N347S, T350A and / or W465A) corresponding to the amino acid numbering of SEQ ID NO: 1.

[0162] In some embodiments, the modified hyperactive PiggyBac transposase can comprise one or more mutations relative to hyPB that are well conserved, e.g., at aminoacid 576 and / or 587 (e.g., K576A and / or I587A) corresponding to the amino acid numbering of SEQ ID NO: 1.

[0163] In some embodiments, the modified hyperactive PiggyBac transposase can comprise one or more mutations relative to hyPB that are involved in Zn2+binding, e.g., 586 (e.g., H586A) corresponding to the amino acid numbering of SEQ ID NO: 1.

[0164] In some embodiments, the programmable transposase can comprise one or more mutations relative to hyPB that are involved in integration, e.g., 315, 341, 372, and / or 375 (e.g., R315A, R341A, R372A, and / or K375A) corresponding to the amino acid numbering of SEQ ID NO: 1.

[0165] In some embodiments, the modified hyperactive PiggyBac transposase comprises an amino acid sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 1 In some embodiments, the modified hyperactive PiggyBac transposase is selected for its high specificity of DNA integration into a genome compared to hyperactive PiggyBac. In some embodiments, the modified hyperactive PiggyBac transposase comprises an amino acid sequence having one or more of the modifications disclosed herein relative to SEQ ID NO: 1, and retains at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence set forth in SEQ ID NO: 2 to SEQ ID NO: 24.

[0166] In some embodiments, the modified hyperactive PiggyBac transposase can comprise a mutation of one or more of amino acids selected from amino acid: 245, 275, 277, 325, 347, 351, 372, 375, 388, 450, 465, 560, 564, 573, 589, 592, 594 corresponding to the amino acid numbering of SEQ ID NO: 1.

[0167] In some embodiments, the modified hyperactive PiggyBac transposase mutation can comprise one or more of the amino acid modifications selected from: R245 A, R275 A, R277A, R275A / R277A, G325A, N347A, N347S, S351E, S351P, S351A, R372A, K375A, R388A, D450N, W465A, T560A, S564P, S573A, M589V, S592G, or F594L corresponding to the amino acid numbering of SEQ ID NO: 1.

[0168] In some embodiments, the modified hyperactive PiggyBac transposase mutation can comprise one or more of the amino acid modifications selected from: R245 A, G325 A, N347S, N347A, D450N, and S573P, corresponding to the amino acid numbering of SEQ ID NO: 1

[0169] In an embodiment, the modified hyperactive PiggyBac transposase comprises the amino acid modification D450N corresponding to the amino acid numbering of SEQ ID NO: 1

[0170] In an embodiment, the modified hyperactive PiggyBac transposase comprises the amino acid modifications R245A and D450, corresponding to the amino acid numbering of SEQ ID NO: 1

[0171] In an embodiment, the modified hyperactive PiggyBac transposase comprises the amino acid modifications R245A, G325A, and S573P, corresponding to the amino acid numbering of SEQ ID NO: 1.

[0172] In an embodiment, the modified hyperactive PiggyBac transposase comprises the amino acid modifications R245A, G325A, D450 and S573P, corresponding to the amino acid numbering of SEQ ID NO: 1.

[0173] In an embodiment, the modified hyperactive PiggyBac transposase comprises the amino acid modification N347S or N347A, corresponding to the amino acid numbering of SEQ ID NO: 1

[0174] In an embodiment, the modified hyperactive PiggyBac transposase comprises the amino acid modifications N347S and D450N, corresponding to the amino acid numbering of SEQ ID NO: 1

[0175] In another, the modified hyperactive PiggyBac transposase comprises the amino acid modifications N347A and D450N, corresponding to the amino acid numbering of SEQ ID NO: 1 In some embodiments, this modified hyperactive PiggyBac transposase has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%, or 100% identity with the amino acid sequence of SEQ ID NO: 12. In someembodiments, this modified hyperactive PiggyBac transposase comprises the amino acid sequence of SEQ ID NO: 12.

[0176] In some embodiments, the modified hyperactive PiggyBac transposase comprises the amino acid sequence SEQ ID NO: 1, wherein:amino acid residue at position 34 is V or M,amino acid residue at position 43 is T or I,amino acid residue at position 177 is Y or H,amino acid residue at position 202 is R or K,amino acid residue at position 230 is S or N,amino acid residue at position 245 is A,amino acid residue at position 268 is D or N,amino acid residue at position 277 is R or A,amino acid residue at position 275 is R or A,amino acid residue at position 277 is R or A,amino acid residue at position 325 is A or G,amino acid residue at position 347 is S, or A,amino acid residue at position 351 is E, P or A,amino acid residue at position 372 is R or A,amino acid residue at position 375 is K or A,amino acid residue at position 388 is R or A,amino acid residue at position 409 is K or A,amino acid residue at position 411 is A or T,amino acid residue at position 412 is K or A,amino acid residue at position 450 is D or N,amino acid residue at position 460 is R or A,amino acid residue at position 465 is W or A,amino acid residue at position 517 is S or A,amino acid residue at position 560 is T or A,amino acid residue at position 564 is P or S,amino acid residue at position 571 is S or N,amino acid residue at position 573 is S or A,amino acid residue at position 576 is K or A,amino acid residue at position 586 is H or A,amino acid residue at position 587 is I or A,amino acid residue at position 589 is M or V,amino acid residue at position 592 is G or S, and / or,amino acid residue at position 594 is L or F.

[0177] In some embodiments, the modified hyperactive PiggyBac transposase comprises or consists of an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%, or 100% sequence identity with the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24

[0178] In some embodiments, the modified hyperactive PiggyBac transposase is not a HimarlC9 mutant.

[0179] In some embodiments, the transposase is a Poeciliopsis turrubarensis transposase, or a variant thereof.

[0180] In some embodiments, the Poeciliopsis turrubarensis transposase has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 25.SEQ ID NO: 25MSSRRFTAEEASLQILNWDSDVDEEISETEDPSEPEDNAADDPDCHFSQDEEDSE DDSSSDENQETQQSSSTKGTWTSKDGKIRWSSSPHQSQGRLSSSNVIKMTPGPT RFAVTRVDDIESAFQLFISPPIEKIILEMTNLEGRRVFQEKWKPLDPTNLHAYIGILVLAGVYRSKGEATASLWNEENGRPIFRATMSLETFHMISRVIRFDNRDTRAGRR ERDKLAAIRDVWDKWVEILPLLYNPGPHVTVDERLIPFRGRCPFRQYMPKKPA KYGIKIWAACDAKSSYAWNLQVYTGKPAGGAPEKNQGMRVVLEMTEGLQGH NITCDNFFTSYRLGDELQKRKLTMLGTVRKNKPELPSELLMMKGRPLHSSKFAF TEKTTLVSYCPKRNKNVLVMSTMHKDASLSTREDMKPQMILDYNSTKGGVDN LDKVTATYSCQRKTARWPLVIFYNIVDVSAYNAYVLWIEINQQWNASKLHRRR LFLEELGKALVTPHIQNRVRPARSLAAAAVIAKVQGRASDQPAMEPFDTGAKK RKRCQVCSSRDDSKTSTSCVRCRKYICRKHTVTFCPSCGER

[0181] In some embodiments, the Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 25.

[0182] In some embodiments, the Poeciliopsis turrubarensis transposase is a variant of Poeciliopsis turrubarensis transposase. In some embodiments, the variant of Poeciliopsis turrubarensis transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 25. It will be understood that variant of Poeciliopsis turrubarensis transposase has less than 100% sequence identity with SEQ ID NO: 25.

[0183] As used herein, “amino acid mutation” means substitution, deletion, insertion, and translocation, preferably substitution.

[0184] In some embodiments, 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: 25 In some embodiments, 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: 25. In some embodiments, 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 353, 356, and 432, corresponding to the amino acid numbering of SEQ ID NO: 25.

[0185] In some embodiments, 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, R353A, K356A, D432N, D547K, S549R, corresponding to the amino acid numbering of SEQ ID NO: 25. In some embodiments, 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: 25. In some embodiments, 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: 25. In some embodiments, 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: 25. In some embodiments, 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: 25. In some embodiments, 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: 25.

[0186] In some embodiments, the variant Poeciliopsis turrubarensis transposase has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with any one of SEQ ID NO: 26 to SEQ ID NO: 38. In some embodiments, the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in any one of SEQ ID NO: 26 to SEQ ID NO: 38.In some embodiments, the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 26. In some embodiments, the variant ofPoeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 27. In some embodiments, the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 28. In some embodiments, the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 29 In some embodiments, the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 30. In some embodiments, the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 31. In some embodiments, the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 32. In some embodiments, the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 33. In some embodiments, the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 34 In some embodiments, the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 35. In some embodiments, the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 36. In some embodiments, the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 37. In some embodiments, the variant of Poeciliopsis turrubarensis transposase has an amino acid sequence as set forth in SEQ ID NO: 38.SEQ ID NO: 26MSSRRFTAEEASLQILNSDSDVDEEISETEDPSEPEDNAADDPDCHFSQDEEDSE DDSSSDENQETQQSSSTKGTWTSKDGKIRWSSSPHQSQGRLSSSNVIKMTPGPT RFAVTRVDDIESAFQLFISPPIEKIILEMTNLEGRRVFQEKWKPLDPTNLHAYIGIL VLAGVYRSKGEATASLWNEENGRPIFRATMSLETFHMISRVIRFDNRDTRAGRR ERDKLAAIRDVWDKWVEILPLLYNPGPHVTVDERLIPFRGRCPFRQYMPKKPA KYGIKIWAACDAKSSYAWNLQVYTGKPAGGAPEKNQGMRVVLEMTEGLQGH NITCDNFFTSYRLGDELQKRKLTMLGTVRKNKPELPSELLMMKGRPLHSSKFAF TEKTTLVSYCPKRNKNVLVMSTMHKDASLSTREDMKPQMILDYNSTKGGVDN LDKVTATYSCQRKTARWPLVIFYNIVDVSAYNAYVLWIEINQQWNASKLHRRRLFLEELGKALVTPHIQNRVRPARSLAAAAVIAKVQGRASDQPAMEPFDTGAKK RKRCQVCSSRDDSKTSTSCVRCRKYICRKHTVTFCPSCGERSEQ ID NO: 27 MSSRRFTAEEASLQILNWDSDSDEEISETEDPSEPEDNAADDPDCHFSQDEEDSE DDSSSDENQETQQSSSTKGTWTSKDGKIRWSSSPHQSQGRLSSSNVIKMTPGPT RFAVTRVDDIESAFQLFISPPIEKIILEMTNLEGRRVFQEKWKPLDPTNLHAYIGIL VLAGVYRSKGEATASLWNEENGRPIFRATMSLETFHMISRVIRFDNRDTRAGRR ERDKLAAIRDVWDKWVEILPLLYNPGPHVTVDERLIPFRGRCPFRQYMPKKPA KYGIKIWAACDAKSSYAWNLQVYTGKPAGGAPEKNQGMRVVLEMTEGLQGH NITCDNFFTSYRLGDELQKRKLTMLGTVRKNKPELPSELLMMKGRPLHSSKFAF TEKTTLVSYCPKRNKNVLVMSTMHKDASLSTREDMKPQMILDYNSTKGGVDN LDKVTATYSCQRKTARWPLVIFYNIVDVSAYNAYVLWIEINQQWNASKLHRRR LFLEELGKALVTPHIQNRVRPARSLAAAAVIAKVQGRASDQPAMEPFDTGAKK RKRCQVCSSRDDSKTSTSCVRCRKYICRKHTVTFCPSCGERSEQ ID NO: 28 MSSRRFTAEEASLQILNWDSDVDEEISETEDPSEPEDNAADDPDCHFSQDEEDSE DDSSSDENQETQQSSSTKGTWTSKDGKIRWSSSPHQSQGRLSSSNVIKMTPGPT RFAVTRVDDIESAFQLFISPPIEKIILEMTNLEGRRVFQEKWKPLDPTNLHAYIGIL VLAGVYRSKGEATASLWNEENGRPIFRATMSLERFHMISRVIRFDNRDTRAGRR ERDKLAAIRDVWDKWVEILPLLYNPGPHVTVDERLIPFRGRCPFRQYMPKKPA KYGIKIWAACDAKSSYAWNLQVYTGKPAGGAPEKNQGMRVVLEMTEGLQGH NITCDNFFTSYRLGDELQKRKLTMLGTVRKNKPELPSELLMMKGRPLHSSKFAF TEKTTLVSYCPKRNKNVLVMSTMHKDASLSTREDMKPQMILDYNSTKGGVDN LDKVTATYSCQRKTARWPLVIFYNIVDVSAYNAYVLWIEINQQWNASKLHRRR LFLEELGKALVTPHIQNRVRPARSLAAAAVIAKVQGRASDQPAMEPFDTGAKK RKRCQVCSSRDDSKTSTSCVRCRKYICRKHTVTFCPSCGERSEQ ID NO: 29 MSSRRFTAEEASLQILNWDSDVDEEISETEDPSEPEDNAADDPDCHFSQDEEDSE DDSSSDENQETQQSSSTKGTWTSKDGKIRWSSSPHQSQGRLSSSNVIKMTPGPT RFAVTRVDDIESAFQLFISPPIEKIILEMTNLEGRRVFQEKWKPLDPTNLHAYIGIL VLAGVYRSKGEATASLWNEENGRPIFRATMSLETFHMISRVIRFDNRDTRAGRR ERDKLAAIRDVWDKWVEALPLLYNPGPHVTVDERLIPFRGRCPFRQYMPKKPA KYGIKIWAACDAKSSYAWNLQVYTGKPAGGAPEKNQGMRVVLEMTEGLQGH NITCDNFFTSYRLGDELQKRKLTMLGTVRKNKPELPSELLMMKGRPLHSSKFAF TEKTTLVSYCPKRNKNVLVMSTMHKDASLSTREDMKPQMILDYNSTKGGVDN LDKVTATYSCQRKTARWPLVIFYNIVDVSAYNAYVLWIEINQQWNASKLHRRR LFLEELGKALVTPHIQNRVRPARSLAAAAVIAKVQGRASDQPAMEPFDTGAKK RKRCQVCSSRDDSKTSTSCVRCRKYICRKHTVTFCPSCGERSEQ ID NO: 30 MSSRRFTAEEASLQILNWDSDVDEEISETEDPSEPEDNAADDPDCHFSQDEEDSE DDSSSDENQETQQSSSTKGTWTSKDGKIRWSSSPHQSQGRLSSSNVIKMTPGPT RFAVTRVDDIESAFQLFISPPIEKIILEMTNLEGRRVFQEKWKPLDPTNLHAYIGIL VLAGVYRSKGEATASLWNEENGRPIFRATMSLETFHMISRVIRFDNRDTRAGRR ERDKLAAIRDVWDKWVERLPLLYNPGPHVTVDERLIPFRGRCPFRQYMPKKPA KYGIKIWAACDAKSSYAWNLQVYTGKPAGGAPEKNQGMRVVLEMTEGLQGH NITCDNFFTSYRLGDELQKRKLTMLGTVRKNKPELPSELLMMKGRPLHSSKFAF TEKTTLVSYCPKRNKNVLVMSTMHKDASLSTREDMKPQMILDYNSTKGGVDN LDKVTATYSCQRKTARWPLVIFYNIVDVSAYNAYVLWIEINQQWNASKLHRRR LFLEELGKALVTPHIQNRVRPARSLAAAAVIAKVQGRASDQPAMEPFDTGAKK RKRCQVCSSRDDSKTSTSCVRCRKYICRKHTVTFCPSCGERSEQ ID NO: 31 MSSRRFTAEEASLQILNWDSDVDEEISETEDPSEPEDNAADDPDCHFSQDEEDSE DDSSSDENQETQQSSSTKGTWTSKDGKIRWSSSPHQSQGRLSSSNVIKMTPGPT RFAVTRVDDIESAFQLFISPPIEKIILEMTNLEGRRVFQEKWKPLDPTNLHAYIGIL VLAGVYRSKGEATASLWNEENGRPIFRATMSLETFHMISRVIRFDNRDTRAGRRERDKLAAIRDVWDKWVEILPLLYNPGPHVTVDERLIPFRGRCPFRQYMPKKPA KYGIKIWAACDAKSSYAWNLQVYTGKPAGGAPEKNQGMRVVLEMTEGLQGH NITCDNFFTSYALGDELQKRKLTMLGTVRKNKPELPSELLMMKGRPLHSSKFAF TEKTTLVSYCPKRNKNVLVMSTMHKDASLSTREDMKPQMILDYNSTKGGVDN LDKVTATYSCQRKTARWPLVIFYNIVDVSAYNAYVLWIEINQQWNASKLHRRR LFLEELGKALVTPHIQNRVRPARSLAAAAVIAKVQGRASDQPAMEPFDTGAKK RKRCQVCSSRDDSKTSTSCVRCRKYICRKHTVTFCPSCGERSEQ ID NO: 32 MSSRRFTAEEASLQILNWDSDVDEEISETEDPSEPEDNAADDPDCHFSQDEEDSE DDSSSDENQETQQSSSTKGTWTSKDGKIRWSSSPHQSQGRLSSSNVIKMTPGPT RFAVTRVDDIESAFQLFISPPIEKIILEMTNLEGRRVFQEKWKPLDPTNLHAYIGIL VLAGVYRSKGEATASLWNEENGRPIFRATMSLETFHMISRVIRFDNRDTRAGRR ERDKLAAIRDVWDKWVEILPLLYNPGPHVTVDERLIPFRGRCPFRQYMPKKPA KYGIKIWAACDAKSSYAWNLQVYTGKPAGGAPEKNQGMRVVLEMTEGLQGH NITCDNFFTSYRLGDELLKRKLTMLGTVRKNKPELPSELLMMKGRPLHSSKFAF TEKTTLVSYCPKRNKNVLVMSTMHKDASLSTREDMKPQMILDYNSTKGGVDN LDKVTATYSCQRKTARWPLVIFYNIVDVSAYNAYVLWIEINQQWNASKLHRRR LFLEELGKALVTPHIQNRVRPARSLAAAAVIAKVQGRASDQPAMEPFDTGAKK RKRCQVCSSRDDSKTSTSCVRCRKYICRKHTVTFCPSCGERSEQ ID NO: 33 MSSRRFTAEEASLQILNWDSDVDEEISETEDPSEPEDNAADDPDCHFSQDEEDSE DDSSSDENQETQQSSSTKGTWTSKDGKIRWSSSPHQSQGRLSSSNVIKMTPGPT RFAVTRVDDIESAFQLFISPPIEKIILEMTNLEGRRVFQEKWKPLDPTNLHAYIGIL VLAGVYRSKGEATASLWNEENGRPIFRATMSLETFHMISRVIRFDNRDTRAGRR ERDKLAAIRDVWDKWVEILPLLYNPGPHVTVDERLIPFRGRCPFRQYMPKKPA KYGIKIWAACDAKSSYAWNLQVYTGKPAGGAPEKNQGMRVVLEMTEGLQGH NITCDNFFTSYRLGDELQKRKLTMLGTVRKNKPELPSELLMMKGRPLHSSKFAF TEKTTLVSYIPKRNKNVLVMSTMHKDASLSTREDMKPQMILDYNSTKGGVDN LDKVTATYSCQRKTARWPLVIFYNIVDVSAYNAYVLWIEINQQWNASKLHRRRLFLEELGKALVTPHIQNRVRPARSLAAAAVIAKVQGRASDQPAMEPFDTGAKK RKRCQVCSSRDDSKTSTSCVRCRKYICRKHTVTFCPSCGERSEQ ID NO: 34 MSSRRFTAEEASLQILNWDSDVDEEISETEDPSEPEDNAADDPDCHFSQDEEDSE DDSSSDENQETQQSSSTKGTWTSKDGKIRWSSSPHQSQGRLSSSNVIKMTPGPT RFAVTRVDDIESAFQLFISPPIEKIILEMTNLEGRRVFQEKWKPLDPTNLHAYIGIL VLAGVYRSKGEATASLWNEENGRPIFRATMSLETFHMISRVIRFDNRDTRAGRR ERDKLAAIRDVWDKWVEILPLLYNPGPHVTVDERLIPFRGRCPFRQYMPKKPA KYGIKIWAACDAKSSYAWNLQVYTGKPAGGAPEKNQGMRVVLEMTEGLQGH NITCDNFFTSYRLGDELQKRKLTMLGTVRKNKPELPSELLMMKGRPLHSSKFAF TEKTTLVSYVPKRNKNVLVMSTMHKDASLSTREDMKPQMILDYNSTKGGVDN LDKVTATYSCQRKTARWPLVIFYNIVDVSAYNAYVLWIEINQQWNASKLHRRR LFLEELGKALVTPHIQNRVRPARSLAAAAVIAKVQGRASDQPAMEPFDTGAKK RKRCQVCSSRDDSKTSTSCVRCRKYICRKHTVTFCPSCGERSEQ ID NO: 35 MSSRRFTAEEASLQILNWDSDVDEEISETEDPSEPEDNAADDPDCHFSQDEEDSE DDSSSDENQETQQSSSTKGTWTSKDGKIRWSSSPHQSQGRLSSSNVIKMTPGPT RFAVTRVDDIESAFQLFISPPIEKIILEMTNLEGRRVFQEKWKPLDPTNLHAYIGIL VLAGVYRSKGEATASLWNEENGRPIFRATMSLETFHMISRVIRFDNRDTRAGRR ERDKLAAIRDVWDKWVEILPLLYNPGPHVTVDERLIPFRGRCPFRQYMPKKPA KYGIKIWAACDAKSSYAWNLQVYTGKPAGGAPEKNQGMRVVLEMTEGLQGH NITCDNFFTSYRLGDELQKRKLTMLGTVRKNKPELPSELLMMKGRPLHSSKFAF TEKTTLVSYCPKRNKNVLVMSTMHKDASLSTREDKKPQMILDYNSTKGGVDN LDKVTATYSCQRKTARWPLVIFYNIVDVSAYNAYVLWIEINQQWNASKLHRRR LFLEELGKALVTPHIQNRVRPARSLAAAAVIAKVQGRASDQPAMEPFDTGAKK RKRCQVCSSRDDSKTSTSCVRCRKYICRKHTVTFCPSCGERSEQ ID NO: 36MSSRRFTAEEASLQILNWDSDVDEEISETEDPSEPEDNAADDPDCHFSQDEEDSE DDSSSDENQETQQSSSTKGTWTSKDGKIRWSSSPHQSQGRLSSSNVIKMTPGPT RFAVTRVDDIESAFQLFISPPIEKIILEMTNLEGRRVFQEKWKPLDPTNLHAYIGIL VLAGVYRSKGEATASLWNEENGRPIFRATMSLETFHMISRVIRFDNRDTRAGRR ERDKLAAIRDVWDKWVEILPLLYNPGPHVTVDERLIPFRGRCPFRQYMPKKPA KYGIKIWAACDAKSSYAWNLQVYTGKPAGGAPEKNQGMRVVLEMTEGLQGH NITCDNFFTSYRLGDELQKRKLTMLGTVRKNKPELPSELLMMKGRPLHSSKFAF TEKTTLVSYCPKRNKNVLVMSTMHKDASLSTREDMKPQMILDYNSTKGGVDN LDKVTATYSCQRKTARWPLVIFYNIVDVSAYNAYVLWIEINQQWNASKLHRRR LFLEELGKALVTPHIQNRVRPARSLAAAAVIAKVQGRASDQPAMEPFDTGAKK RKRCQVCSSRKDSKTSTSCVRCRKYICRKHTVTFCPSCGERSEQ ID NO: 37 MSSRRFTAEEASLQILNWDSDVDEEISETEDPSEPEDNAADDPDCHFSQDEEDSE DDSSSDENQETQQSSSTKGTWTSKDGKIRWSSSPHQSQGRLSSSNVIKMTPGPT RFAVTRVDDIESAFQLFISPPIEKIILEMTNLEGRRVFQEKWKPLDPTNLHAYIGIL VLAGVYRSKGEATASLWNEENGRPIFRATMSLETFHMISRVIRFDNRDTRAGRR ERDKLAAIRDVWDKWVEILPLLYNPGPHVTVDERLIPFRGRCPFRQYMPKKPA KYGIKIWAACDAKSSYAWNLQVYTGKPAGGAPEKNQGMRVVLEMTEGLQGH NITCDNFFTSYRLGDELQKRKLTMLGTVRKNKPELPSELLMMKGRPLHSSKFAF TEKTTLVSYCPKRNKNVLVMSTMHKDASLSTREDMKPQMILDYNSTKGGVDN LDKVTATYSCQRKTARWPLVIFYNIVDVSAYNAYVLWIEINQQWNASKLHRRR LFLEELGKALVTPHIQNRVRPARSLAAAAVIAKVQGRASDQPAMEPFDTGAKK RKRCQVCSSRDDRKTSTSCVRCRKYICRKHTVTFCPSCGERSEQ ID NO: 38 MSSRRFTAEEASLQILNWDSDVDEEISETEDPSEPEDNAADDPDCHFSQDEEDSE DDSSSDENQETQQSSSTKGTWTSKDGKIRWSSSPHQSQGRLSSSNVIKMTPGPT RFAVTRVDDIESAFQLFISPPIEKIILEMTNLEGRRVFQEKWKPLDPTNLHAYIGIL VLAGVYRSKGEATASLWNEENGRPIFRATMSLETFHMISRVIRFDNRDTRAGRR ERDKLAAIRDVWDKWVEILPLLYNPGPHVTVDERLIPFRGRCPFRQYMPKKPAKYGIKIWAACDAKSSYAWNLQVYTGKPAGGAPEKNQGMRVVLEMTEGLQGH NITCDNFFTSYRLGDELQKRKLTMLGTVAKNAPELPSELLMMKGRPLHSSKFAF TEKTTLVSYCPKRNKNVLVMSTMHKDASLSTREDMKPQMILDYNSTKGGVDN LNKVTATYSCQRKTARWPLVIFYNIVDVSAYNAYVLWIEINQQWNASKLHRRR LFLEELGKALVTPHIQNRVRPARSLAAAAVIAKVQGRASDQPAMEPFDTGAKK RKRCQVCSSRDDSKTSTSCVRCRKYICRKHTVTFCPSCGER

[0187] In some embodiments, the transposase is a Poeciliopsis turrubarensis transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 25, or a variant thereof having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with any one of SEQ ID NO: 26 to SEQ ID NO: 38.

[0188] In some embodiments, the transposase is a Poeciliopsis turrubarensis transposase having an amino acid sequence as set forth in SEQ ID NO: 25, or a variant thereof having an amino acid sequence as set forth in any one of SEQ ID NO: 26 to SEQ ID NO: 38.

[0189] In certain embodiments, the Poeciliopsis turrubarensis transposase is a recombinant transposase. In some embodiments, the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 25.

[0190] In some embodiments, the transposase is a Anthonomus grandis transposase, herein interchangeably referred to as “DR1754440” transposase or “Antgra4440”, or a variant thereof. In some embodiments, the transposase is Anthonomus grandis DR1754440 transposase.

[0191] In some embodiments, said DR1754440 transposase from Anthonomus grandis has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 40.SEQ ID NO: 40MGYMCPSGFSDRVIYIPMVFYSMSRKKLTRAELQDLADNLEELSSGEEVQSPYE DSSSEEDPFHNSEESDELYQPSDTSDEDSGDIESDANENLTQPNDGVISAENSDQ EAETPVTTGNIIWSLPTNTYVPRLSIPNESQCIIHPSIAANASPLDIFTKICPRSLFTF IAESTNQRIKIYKENKKKQATFTDAGEIMITIGCTLVMCFNKVPKLRHYWSSHPS LGNKAIKSAISKNRCLFLLSKLYFNNPVKPEGASKIYYVQELLSCFKYTFQKYRS DSTSQSIDESMVGFKGRSSLKQYMPQKPVKRGIKLWSRCDAKTGYTYDANVY CGKDDVERSGTLGETVVKKLCETIQNPNVVLAFDRFFTSVRLMDSLQYPAVGT AIITRKDMPKKFIEKRKMSRGECEFLSNQTNSIAIKWQDTKQVILLSNCHTPDMT CVKRKDKTGQRVDVPCPTAIAYYNEIMGGVDLADQMSGVYDFGRKSCKWWK KVFYRLLMIAVVNSWVIYNDLRRTQKKIPLLEFLFTLSEDLIEEGQQQTSVKLRR NSCGRPSKRSKVMKNVGDHLPIETSKRRRCARCALQKKERRTTTICAMCDVGL CKHCFALYHSS

[0192] In some embodiments, said DR1754440 transposase from Anthonomus grandis has an amino acid sequence as set forth in SEQ ID NO: 40.

[0193] In certain embodiments, the Anthonomus grandis DR1754440 transposase is a recombinant transposase. In some embodiments, the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 40.

[0194] In some embodiments, the Anthonomus grandis DR1754440 transposase is a variant of Anthonomus grandis DR1754440 transposase. In some embodiments, the variant of Anthonomus grandis DR1754440 transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 40.

[0195] In some embodiments, the variant of Anthonomus grandis DR1754440 transposase comprises at least one amino acid mutation, preferably at least one aminoacid 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: 40.

[0196] In some embodiments, 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 K393 A, corresponding to the amino acid numbering of SEQ ID NO: 40. 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: 40.

[0197] In some embodiments, the variant of Anthonomus grandis DR1754440 transposase has an amino acid sequence with at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 41. In some embodiments, the variant of Anthonomus grandis DR1754440 transposase has an amino acid sequence as set forth in SEQ ID NO: 41.SEQ ID NO: 41MGYMCPSGFSDRVIYIPMVFYSMSRKKLTRAELQDLADNLEELSSGEEVQSPYE DSSSEEDPFHNSEESDELYQPSDTSDEDSGDIESDANENLTQPNDGVISAENSDQ EAETPVTTGNIIWSLPTNTYVPRLSIPNESQCIIHPSIAANASPLDIFTKICPRSLFTF IAESTNQRIKIYKENKKKQATFTDAGEIMITIGCTLVMCFNKVPKLRHYWSSHPS LGNKAIKSAISKNRCLFLLSKLYFNNPVKPEGASKIYYVQELLSCFKYTFQKYRS DSTSQSIDESMVGFKGRSSLKQYMPQKPVKRGIKLWSRCDAKTGYTYDANVY CGKDDVERSGTLGETVVKKLCETIQNPNVVLAFDRFFTSVRLMDSLQYPAVGT AIITAADMPAKFIEKRKMSRGECEFLSNQTNSIAIKWQDTKQVILLSNCHTPDMT CVKRKDKTGQRVDVPCPTAIAYYNEIMGGVDLANQMSGVYDFGRKSCKWWK KVFYRLLMIAVVNSWVIYNDLRRTQKKIPLLEFLFTLSEDLIEEGQQQTSVKLRR NSCGRPSKRSKVMKNVGDHLPIETSKRRRCARCALQKKERRTTTICAMCDVGL CKHCFALYHSS

[0198] In some embodiments, the transposase is a Anthonomus grandis DR1754440 transposase having an amino acid sequence with at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 40, or a variant thereof having an amino acid sequence with at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 41. In some embodiments, the transposase is a Anthonomus grandis DR1754440 transposase having the amino acid sequence of SEQ ID NO: 40, or a variant thereof having the amino acid sequence of SEQ ID NO: 41.

[0199] In some embodiments, the transposase is a Anthonomus grandis transposase, herein interchangeably referred to as “DR1754053” transposase or “Antgra4053”, or a variant thereof. In some embodiments, the transposase is Anthonomus grandis DR1754053 transposase.

[0200] In some embodiments, said DR1754053 transposase from Anthonomus grandis has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 39.SEQ ID NO: 39MSARWSYEKEQEKLLRLWEISDNSSEVPEILDEEPIESGSEGEFDDHVSQRSEES DSEQEILEDDIVEEFSPEVKSRYLWGVDKKTKWVANPGNKAVRTRQENIIIHLP GPKGVAKEAKTPFDCWNIFIDELIIDSIVECTNRLIQTKSENYNDKSIVQQTTALE MKALIGLLYMCGRFRASRLNLDELWASDGSGIDMFRATMPLKRFRFLLSCLRF DNKDSREERLKIDKLAPIRAIFDRFVENSQAAYVPSEYLTIDEKLESFRGRCGFR QYIPNI<PAI<YGLI<VFALVDSI<TFYVLNLEAYVGQQPEGPYALSNI<PADIVLRL VEPIRGTRRNITFDNWFTSYDLVIKLLKEYKLTSVGTLRKNKREIPGELLTTKRE ERSSKFAFQSDVTLVSYIPKKNRNVLLLSTLHHDNTIDPDSRDQNKPEIIMFYNM TKGGVDTVDQLSATYSVSRNSRRWPLTLFFSLLNSAAINAFVIFNCNTNENLKRRFFLKDLSLSLVKPFQMSRLQNPRISLAVRRKISDIQGEIVPTTVGEDTKRQRFSR RCYICPRNKDRKSFYTCRACNKFVCLSHAAQTCDNCSSIDST

[0201] In some embodiments, said DR1754053 transposase from Anthonomus grandis has an amino acid sequence as set forth in SEQ ID NO: 39.

[0202] In certain embodiments, the Anthonomus grandis DR1754053 transposase is a recombinant transposase. In some embodiments, the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 39. In some embodiments, the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 39.

[0203] In some embodiments, the transposase is a Anthonomus grandis transposase, herein interchangeably referred to as “DR1756049” transposase or “Antgra6049”, or a variant thereof. In some embodiments, the transposase is Anthonomus grandis DR1756049 transposase.

[0204] In some embodiments, said “DR1756049” transposase from Anthonomus grandis has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%sequence identity with SEQ ID NO: 42 or SEQ ID NO: 43.

[0205] In some embodiments, said “DR1756049” transposase from Anthonomus grandis has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 42.

[0206] In some embodiments, said “DR1756049” transposase from Anthonomus grandis has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 43.SEQ ID NO: 42MCMALVYEFSIVSASAGDTSRFTGRKCTYKHVFFRNRFLLVISSFPDMDEENPG PSRPKRRHTDFKMMRPLTLSEQEELLYLSESEEDPFVDSGSEYEPTDHESSSDED ELPDVAALEDASLTDIENNETISVQNSNSDIPTWTDVPNQNDFLFSGVPGLRVNL TSDNPIDFFQLFITDDFLEHIATETNNYAEQLFLSKETKEKSRITEWKPLTVPTLK TFLGLWLHMGNIQLNRLQDYWKTDELYNLSCFRNNMSRNRFLVILRCLHFSKN PERGDPDYGDRLCRIRWLQNFFNDRIDKIYYPNKELSLDESMVLWRGRLVFRQ YIKNKKHKFGVKLYILSEPDGLVLKARIYTGDEKDMKGELGHAANVVLSLTKN YLDKGHSLYMDNYYNSIALCKELILKKTYATGTLRLNRKGNPPEVSSKKLKKG ESITKYSKGIAVGKWRDKREVLFISTEFSGELIKQPNRRNEEKEKPNAIINYNKN MSGIDRQDQMLAYYASERKTIRWYKKLAIHLISMMLLNSYHLYNKYSTQQKLS YYDYRHNIIKNLLSEAKEKPPEPPKVKQNQPSHLPEKCEMDNKGRQMRKRCRV CYTQKMRKNTPFFCPVCPDKPGLCLGNCFRIYHDEKSEQ ID NO: 43MMRPLTLSEQEELLYLSESEEDPFVDSGSEYEPTDHESSSDEDELPDVAALEDAS LTDIENNETISVQNSNSDIPTWTDVPNQNDFLFSGVPGLRVNLTSDNPIDFFQLFI TDDFLEHIATETNNYAEQLFLSKETKEKSRITEWKPLTVPTLKTFLGLWLHMGN IQLNRLQDYWKTDELYNLSCFRNNMSRNRFLVILRCLHFSKNPERGDPDYGDR LCRIRWLQNFFNDRIDKIYYPNKELSLDESMVLWRGRLVFRQYIKNKKHKFGV KLYILSEPDGLVLKARIYTGDEKDMKGELGHAANVVLSLTKNYLDKGHSLYM DNYYNSIALCKELILKKTYATGTLRLNRKGNPPEVSSKKLKKGESITKYSKGIAV GKWRDKREVLFISTEFSGELIKQPNRRNEEKEKPNAIINYNKNMSGIDRQDQML AYYASERKTIRWYKKLAIHLISMMLLNSYHLYNKYSTQQKLSYYDYRHNIIKN LLSEAKEKPPEPPKVKQNQPSHLPEKCEMDNKGRQMRKRCRVCYTQKMRKNT PFFCPVCPDKPGLCLGNCFRIYHDEK

[0207] In some embodiments, said “DR1756049” transposase from Anthonomus grandis has an amino acid sequence as set forth in SEQ ID NO: 43 or SEQ ID NO: 42. In some embodiments, said “DR1756049” transposase from Anthonomus grandis has an aminoacid sequence as set forth in SEQ ID NO: 42. In some embodiments, said “DR1756049” transposase from Anthonomus grandis has an amino acid sequence as set forth in SEQ ID NO: 43

[0208] In certain embodiments, the Anthonomus grandis DR1756049 transposase is a recombinant transposase. In some embodiments, the recombinant transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 43 or 42. In some embodiments, the recombinant transposase has less than 100% sequence identity with SEQ ID NO: 43 or 42.

[0209] In some embodiments, the Anthonomus grandis DR1756049 transposase is a variant of Anthonomus grandis DR1756049 transposase. In some embodiments, the variant of Anthonomus grandis DR1756049 transposase comprises at least one amino acid mutation compared to the amino acid sequence of SEQ ID NO: 43.

[0210] In some embodiments, 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: 43.

[0211] In some embodiments, 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: 43

[0212] In some embodiments, the variant of Anthonomus grandis DR1756049 transposase has an amino acid sequence with at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 44. In some embodiments, the variant of Anthonomus grandis DR1756049 transposase has an amino acid sequence as set forth in SEQ ID NO: 44.SEQ ID NO: 44MMRPLTLSEQEELLYLSESEEDPFVDSGSEYEPTDHESSSDEDELPDVAALEDAS LTDIENNETISVQNSNSDIPTWTDVPNQNDFLFSGVPGLRVNLTSDNPIDFFQLFI TDDFLEHIATETNNYAEQLFLSKETKEKSRITEWKPLTVPTLKTFLGLWLHMGN IQLNRLQDYWKTDELYNLSCFRNNMSRNRFLVILRCLHFSKNPERGDPDYGDR LCRIRWLQNFFNDRIDKIYYPNKELSLDESMVLWRGRLVFRQYIKNKKHKFGV KLYILSEPDGLVLKARIYTGDEKDMKGELGHAANVVLSLTKNYLDKGHSLYM DNYYNSIALCKELILKKTYATGTLALNRAGNPPEVSSKKLKKGESITKYSKGIA VGKWRDKREVLFISTEFSGELIKQPNRRNEEKEKPNAIINYNKNMSGIDRQNQM LAYYASERKTIRWYKKLAIHLISMMLLNSYHLYNKYSTQQKLSYYDYRHNIIK NLLSEAKEKPPEPPKVKQNQPSHLPEKCEMDNKGRQMRKRCRVCYTQKMRKN TPFFCPVCPDKPGLCLGNCFRIYHDEK

[0213] In some embodiments, the transposase is an Anthonomus grandis transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with any of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42 or SEQ ID NO: 43; or a variant thereof having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 44 or SEQ ID NO: 41

[0214] In some embodiments, the transposase is an Anthonomus grandis transposase having an amino acid sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42 and SEQ ID NO: 43; or a variant thereof having the amino acid sequence of SEQ ID NO: 44 or SEQ ID NO: 41.

[0215] In some embodiments, the Anthonomus grandis transposase has an amino acid sequence selected from the group comprising or consisting of SEQ ID NO: 40, SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 43. In some embodiments, the Anthonomus grandis transposase has an amino acid sequence selected from the group comprising or consisting of SEQ ID NO: 40, SEQ ID NO: 39 and SEQ ID NO: 42 In someembodiments, the Anthonomus grandis transposase has an amino acid sequence selected from the group comprising or consisting of SEQ ID NO: 40, SEQ ID NO: 39 and SEQ ID NO: 43. In some embodiments, the Anthonomus grandis transposase has an amino acid sequence selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 43 In some embodiments, the Anthonomus grandis transposase has an amino acid sequence selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 39 and SEQ ID NO: 42. In some embodiments, the Anthonomus grandis transposase has an amino acid sequence selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 39 and SEQ ID NO: 43

[0216] In some embodiments, the Anthonomus grandis transposase variant has the amino acid sequence of SEQ ID NO: 44 or SEQ ID NO: 41.

[0217] In some embodiments, the transposase is a Poeciliopsis turrubarensis transposase or an Anthonomus grandis transposase. In some embodiments, the transposase is a Poeciliopsis turrubarensis transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 25, or a variant thereof having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with any one of SEQ ID NO: 26 to SEQ ID NO: 38; or an Anthonomus grandis transposase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with any of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42 or SEQ ID NO: 43; or a variant thereof having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 44 or SEQ ID NO: 41. In some embodiments, the transposase is a Poeciliopsis turrubarensis transposase having an amino acid sequence SEQ ID NO: 25, or a variant thereof having an amino acid sequence selected from the group consisting of SEQ ID NO: 26 to SEQ ID NO: 38; or an Anthonomus grandis transposase an amino acid sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NO: 40, SEQ ID NO: 42 or SEQ ID NO: 43; or a variant thereof having an amino acid sequence of SEQ ID NO: 44 or SEQ ID NO: 41.

[0218] Transposases can recognize and bind specific nucleic acid sequences named Inverted Terminal Repeats (ITRs). The ITRs typically flank both sites of a nucleic acid sequence that is “cut-and-paste” by the transposase.

[0219] Thus, in some embodiments, 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. As used herein, “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.

[0220] In some embodiments, 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: 45 to SEQ ID NO: 60 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: 45 to SEQ ID NO: 60

[0221] In some embodiments, 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: 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.SEQ ID NO: 45TTAACCCTTGTGTTATGTTTCGGGTCAATTTGACCCGTTTCGATTTTTGAGTT GACCAAAGTGCAGGTTATTCTTATTTTTTTTCCATGAAATCTTGTGACTTTTC CGCATCTAGGGTCATGAACTTCTGAGTAAAATCTGGAGTCTTTGSEQ ID NO: 46

[0222] TCTTTCATAAGCGTGTATGTAATCTAAAATAAAGTTCTGACTGGTTT GACTTCATTTTTTGACTGTTTTGAGACGGATTACACAATACGGGTCAAAATG ACCCGCAACATCATAAATGTAATTATTTCTCAACATAATACAAGGGTTAA

[0223] In some embodiments, 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: 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.SEQ ID NO: 49TTAACCCTTTCGGCCCCAATGGGACAAATAAATCCCAACATACATTTCATAA CATCTGCTGGGCGCTATCTGTCCCGTTTTTAATGGAAATTGTCATTAGCGGG TATTTTAAGGTTTTAACAATTTTTCGACAGGCAGCGACACGCTTTTSEQ ID NO: 50TCCTTTTAAAAGGACCGCCGTTTTTTCCAAAACTGGAAATTGAAAAAAAAAT CATGATTTTTTCTCGACTTATGGGACCTTCTATTGACCGTTTTTAAGAAAAA AATATACAAATTCGGAAAAATATAGTTTCAGGGTCAAATGGGTTAA

[0224] In some embodiments, 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: 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.SEQ ID NO: 47TTAACCCTCCGTTAGTAAGGTGGGGTATATCATACCCCAACCTACTTAATAT CTCTATAATTTTGTCCGTTGCAAAATACGTAGCGCCATCTCGTAAGTACCTT TAAATATAATGTCAAGTGATAGAAAGCACGTGTTTTGGAGCGCGTCSEQ ID NO: 48CTTTCAGACCAATACAAAAACATTATTTATTGATAATATTTCAAAAATGTTT AGTTTATATTCGAAACATGTTTGTTTTTTTATTGGGGTATGCTATACCCCAGC TTACCAATCTTGTGACAATTACCAACCTTACCAACGGAGGGTTAA

[0225] In some embodiments, 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: 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.SEQ ID NO: 51TTAACACGTTCACTGCCAATGACTCAACATATGAGTCACGGCGGTACCCCTC CTATGGCCAGTGGCTCAACTGTTGAGTCATGTGCATGGCTTTAGTATATGAG TTTAGTATTGTTTCGGCTTCCGCAGGGGATACGTCTCGTTTCACCGSEQ ID NO: 52CAAAGTAGATTTAGGCCCAAAAAGCAAAAAAAAAACACAATATTAATCATA CAAAGTATTAAAAATCCTATTTTCGAACTACTTTTTGCAAATAGTCCCGACG GCTTCTGAACAAGGGCACAAAATACTTATAGGCAGTCAACGTGTTAA

[0226] In some embodiments, the hyPB transposase recognizes ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with any of SEQ ID NO: 53 to SEQ ID NO: 60. In some embodiments, the hyPB transposase recognizes ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with any of SEQ ID NO: 53 to SEQ ID NO: 54 In some embodiments, the hyPB transposase recognizes ITR sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with any of SEQ ID NO: 55 to SEQ ID NO: 60.

[0227] In some embodiments, the transposase comprises the amino acid sequence SEQ ID NO: 100, SEQ ID NO: 101, and / or SEQ ID NO: 102

[0228] In some embodiments, the polypeptide comprising the transposase or fragment thereof further comprises a nuclear localization signal selected from the group consisting of SEQ ID NO: 97, SEQ ID NO: 98, and / or SEQ ID NO: 99

[0229] In some embodiments, the transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase, a Poeciliopsis turrubarensis transposase, an Anthonomus grandis DR1754440 transposase, or variants thereof, preferably a modified hyperactive PiggyBac transposase, comprising one or more amino acid mutations as compared to an unmodified hyperactive PiggyBac transposase of SEQ ID NO: 1.

[0230] In some embodiments, modified hyperactive PiggyBac transposase comprises at least one amino acid mutation selected among the amino acid substitutions Ml 94V, R275A, R277, R347S, R372A, K375A, R376A, E377A, E380A, and D450N, said position number corresponding to the amino acid number of unmodified hyperactive PiggyBac of SEQ ID NO: 1.

[0231] In some embodiments, the modified hyperactive PiggyBac transposase comprises at least one amino acid mutation selected among the amino acid substitutions R372A, K375A and D450N, said position number corresponding to the amino acid number of unmodified hyperactive PiggyBac of SEQ ID NO: 1.

[0232] In some embodiments, the modified hyperactive PiggyBac transposase has an amino acid sequence selected from the group consisting of SEQ ID NO: 2-24.

[0233] In some embodiments, the composition of the invention comprises a RNA-guided nuclease.

[0234] In some embodiments, the RNA-guided nuclease comprises an active DNA cleavage domain and a guide RNA binding domain.

[0235] In some embodiments, the RNA-guided nuclease is a Cas protein.

[0236] Non-limitative exemples of Cas proteins include Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), or Campylobacter jejuni Cas9 (CjCas9), or a variant thereof, nickase Cas9 (nCas9), dead Cas9 (dCas9)), Cas 12a protein,Casl2b protein, Casl2f protein, Cpfl protein, Cas beta (also referred to as Cas-beta-M67), or CasX protein, including variants and functional fragments thereof.

[0237] In some embodiments, the Cas protein may be any Cas protein known from the art.

[0238] In some embodiments, the composition of the invention further comprises at least one RNA-guided nuclease selected from the group consisting of: Cas9 protein from Streptococcus pyogenes (SpCas9); Cas9 protein from Staphylococcus aureus (SaCas9); Cas 12a (Cpfl); Cas9 protein from Campylobacter jejuni (CjCas9); Cas9 nickase from Streptococcus pyogenes Cas9 (nCas9); Cas9 nickase from Staphylococcus aureus Cas9 (SanCas9); Humanized Cas9; CasX; Dead Cas9; TnpB; Casl2f; Cas9 protein from Corynebacterium ulcerans; Cas9 protein from Corynebacterium diphtheria; Cas9 protein from Spiroplasma syrphidicola; Cas9 protein from Prevotella intermedia; Cas9 protein from Spiroplasma taiwanense; Cas9 protein from Streptococcus iniae; Cas9 protein from Belliella baltica; Cas9 protein from Psychroflexus torquisi; Cas9 protein from Streptococcus thermophilus; Cas9 protein from Listeria innocua; Cas9 protein from Neisseria meningitidis; UnlCasl2fl; “Ancestral” Cas, referred to as LBCA; “Ancestral” Cas referred to as LFCA; Cas beta (also referred to as Cas-beta-M67); and variants thereof. In some embodiments, the composition of the invention further comprises at least one RNA-guided nuclease selected from the group consisting of: Cas9 protein from Streptococcus pyogenes (SpCas9); Cas9 protein from Staphylococcus aureus (SaCas9); Cas 12a (Cpfl); Cas9 protein from Campylobacter jejuni (CjCas9); Cas9 nickase from Streptococcus pyogenes Cas9 (nCas9); Cas9 nickase from Staphylococcus aureus Cas9 (SanCas9); Humanized Cas9; CasX; Dead Cas9; TnpB; Casl2f; Cas9 protein from Corynebacterium ulcerans; Cas9 protein from Corynebacterium diphtheria; Cas9 protein from Spiroplasma syrphidicola; Cas9 protein from Prevotella intermedia; Cas9 protein from Spiroplasma taiwanense; Cas9 protein from Streptococcus iniae; Cas9 protein from Belliella baltica; Cas9 protein from Psychroflexus torquisi; Cas9 protein from Streptococcus thermophilus; Cas9 protein from Listeria innocua; Cas9 protein from Neisseria meningitidis; UnlCasl2fl; “Ancestral” Cas, referred to as LBCA; “Ancestral” Cas referred to as LFCA; and variants thereof.

[0239] In some embodiments, the composition of the invention further comprises at least one RNA-guided nuclease selected from the group consisting of: Cas9 protein from Streptococcus pyogenes (SpCas9) having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 61; Cas9 protein from Staphylococcus aureus (SaCas9) having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 62; Casl2a (Cpfl) having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 63; Cas9 protein from Campylobacter jejuni (CjCas9) having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 64; Cas9 nickase from Streptococcus pyogenes Cas9 (nCas9) having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 65; Cas9 nickase from Staphylococcus aureus Cas9 (SanCas9) having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 66; Humanized Cas9 having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 67; CasX having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 68; Dead Cas9 having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 69;TnpB having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 70; Casl2f having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 71; Cas9 protein from Corynebacterium ulcerans having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 72; Cas9 protein from Corynebacterium diphtheria having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 73; Cas9 protein from Spiroplasma syrphidicola having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 74; Cas9 protein from Prevotella intermedia having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 75; Cas9 protein from Spiroplasma taiwanense having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 76; Cas9 protein from Streptococcus iniae having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 77;Cas9 protein from Belliella baltica having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 78; Cas9 protein from Psychroflexus torquisi having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 79; Cas9 protein from Streptococcus thermophilus having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 80;Cas9 protein from Listeria innocua having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 81; Cas9 protein from Neisseria meningitidis having at least 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 82;UnlCasl2fl having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 83; “Ancestral” Cas, referred to as LBCA having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 84; “Ancestral” Cas referred to as LFCA having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 85; Cas beta (also referred to as Cas-beta-M67) having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 119; and variants thereof.

[0240] In some embodiments, the composition of the invention further comprises at least one RNA-guided nuclease selected from the group consisting of: Cas9 protein from Streptococcus pyogenes (SpCas9) having the amino acid sequence as set forth in SEQ ID NO: 61; Cas9 protein from Staphylococcus aureus (SaCas9) having the amino acid sequence as set forth in SEQ ID NO: 62; Casl2a (Cpfl) having the amino acid sequence as set forth in SEQ ID NO: 63; Cas9 protein from Campylobacter jejuni (CjCas9) having the amino acid sequence as set forth in SEQ ID NO: 64; Cas9 nickase from Streptococcus pyogenes Cas9 (nCas9) having the amino acid sequence as set forth in SEQ ID NO: 65;Cas9 nickase from Staphylococcus aureus Cas9 (SanCas9) having the amino acid sequence as set forth in SEQ ID NO: 66; Humanized Cas9 having the amino acid sequence as set forth in SEQ ID NO: 67; CasX having the amino acid sequence as set forth in SEQ ID NO: 68; Dead Cas9 having the amino acid sequence as set forth in SEQ ID NO: 69; TnpB having the amino acid sequence as set forth in SEQ ID NO: 70; Casl2f having the amino acid sequence as set forth in SEQ ID NO: 71; Cas9 protein from Corynebacterium ulcerans having the amino acid sequence as set forth in SEQ ID NO: 72; Cas9 protein from Corynebacterium diphtheria having the amino acid sequence as set forth in SEQ ID NO: 73; Cas9 protein from Spiroplasma syrphidicola having theamino acid sequence as set forth in SEQ ID NO: 74; Cas9 protein from Prevotella intermedia having the amino acid sequence as set forth in SEQ ID NO: 75; Cas9 protein from Spiroplasma taiwanense having the amino acid sequence as set forth in SEQ ID NO: 76; Cas9 protein from Streptococcus iniae having the amino acid sequence as set forth in SEQ ID NO: 77; Cas9 protein from Belliella baltica having the amino acid sequence as set forth in SEQ ID NO: 78; Cas9 protein from Psychroflexus torquisi having the amino acid sequence as set forth in SEQ ID NO: 79; Cas9 protein from Streptococcus thermophilus having the amino acid sequence as set forth in SEQ ID NO: 80; Cas9 protein from Listeria innocua having the amino acid sequence as set forth in SEQ ID NO: 81; Cas9 protein from Neisseria meningitidis having the amino acid sequence as set forth in SEQ ID NO: 82; UnlCasl2fl having the amino acid sequence as set forth in SEQ ID NO: 83; “Ancestral” Cas, referred to as LBCA having the amino acid sequence as set forth in SEQ ID NO: 84; “Ancestral” Cas referred to as LFCA having the amino acid sequence as set forth in SEQ ID NO: 85; Cas beta (also referred to as Cas-beta-M67) having the amino acid sequence as set forth in SEQ ID NO: 119; and variants thereof.

[0241] In some embodiments, the composition of the invention further comprises at least one RNA-guided nuclease selected from the group consisting of: Cas9 protein from Streptococcus pyogenes (SpCas9); Cas9 protein from Staphylococcus aureus (SaCas9); Cas 12a (Cpfl); Cas9 protein from Campylobacter jejuni (CjCas9); Cas9 nickase from Streptococcus pyogenes Cas9 (nCas9); Cas9 nickase from Staphylococcus aureus Cas9 (SanCas9); Humanized Cas9; CasX; TnpB; Casl2f; Cas9 protein from Corynebacterium ulcerans; Cas9 protein from Corynebacterium diphtheria; Cas9 protein from Spiroplasma syrphidicola; Cas9 protein from Prevotella intermedia; Cas9 protein from Spiroplasma taiwanense; Cas9 protein from Streptococcus iniae; Cas9 protein from Belliella baltica; Cas9 protein from Psychroflexus torquisi; Cas9 protein from Streptococcus thermophilus; Cas9 protein from Listeria innocua; Cas9 protein from Neisseria meningitidis; UnlCasl2fl; “Ancestral” Cas, referred to as LBCA; “Ancestral” Cas referred to as LFCA; Cas beta (also referred to as Cas-beta-M67); and variants thereof.

[0242] In some embodiments, the composition of the invention further comprises at least one RNA-guided nuclease selected from the group consisting of: Cas9 protein from Streptococcus pyogenes (SpCas9) having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 61; Cas9 protein from Staphylococcus aureus (SaCas9) having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 62; Casl2a (Cpfl) having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 63; Cas9 protein from Campylobacter jejuni (CjCas9) having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 64; Cas9 nickase from Streptococcus pyogenes Cas9 (nCas9) having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 65; Cas9 nickase from Staphylococcus aureus Cas9 (SanCas9) having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 66; Humanized Cas9 having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 67; CasX having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 68; TnpB having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 70; Casl2f having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 71; Cas9 protein from Corynebacterium ulcerans having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 72; Cas9 protein from Corynebacterium diphtheria having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 73; Cas9 protein from Spiroplasma syrphidicola having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 74; Cas9 protein from Prevotella intermedia having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 75; Cas9 protein from Spiroplasma taiwanense having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 76; Cas9 protein from Streptococcus iniae having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 77; Cas9 protein from Belliella baltica having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 78; Cas9 protein from Psychroflexus torquisi having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 79; Cas9 protein from Streptococcus thermophilus having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 80; Cas9 protein from Listeria innocua having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 81; Cas9 protein from Neisseria meningitidis having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 82; UnlCasl2fl having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 83; “Ancestral” Cas, referred to as LBCA having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 84; “Ancestral” Cas referred to as LFCA having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 85; and variants thereof.

[0243] In some embodiments, the composition of the invention further comprises at least one RNA-guided nuclease having a nucleic acid sequence selected from the group consisting of SEQ ID NO: 61-85 and SEQ ID NO: 119. In some embodiments, the composition of the invention further comprises at least one RNA-guided nuclease having a nucleic acid sequence selected from the group consisting of SEQ ID NO: 61-85.

[0244] In some embodiments, the composition of the invention further comprises at least one RNA-guided nuclease selected from the group consisting of: Cas9 protein from Streptococcus pyogenes (SpCas9) having the amino acid sequence as set forth in SEQ ID NO: 61; Cas9 protein from Staphylococcus aureus (SaCas9) having the amino acid sequence as set forth in SEQ ID NO: 62; Casl2a (Cpfl) having the amino acid sequence as set forth in SEQ ID NO: 63; Cas9 protein from Campylobacter jejuni (CjCas9) having the amino acid sequence as set forth in SEQ ID NO: 64; Cas9 nickase from Streptococcus pyogenes Cas9 (nCas9) having the amino acid sequence as set forth in SEQ ID NO: 65;Cas9 nickase from Staphylococcus aureus Cas9 (SanCas9) having the amino acid sequence as set forth in SEQ ID NO: 66; Humanized Cas9 having the amino acid sequence as set forth in SEQ ID NO: 67; CasX having the amino acid sequence as set forth in SEQ ID NO: 68; TnpB having the amino acid sequence as set forth in SEQ ID NO: 70; Casl2f having the amino acid sequence as set forth in SEQ ID NO: 71; Cas9 protein from Corynebacterium ulcerans having the amino acid sequence as set forth in SEQ ID NO: 72; Cas9 protein from Corynebacterium diphtheria having the amino acid sequence as set forth in SEQ ID NO: 73; Cas9 protein from Spiroplasma syrphidicola having the amino acid sequence as set forth in SEQ ID NO: 74; Cas9 protein fromPrevotella intermedia having the amino acid sequence as set forth in SEQ ID NO: 75;Cas9 protein from Spiroplasma taiwanense having the amino acid sequence as set forth in SEQ ID NO: 76; Cas9 protein from Streptococcus iniae having the amino acid sequence as set forth in SEQ ID NO: 77; Cas9 protein from Belliella baltica having the amino acid sequence as set forth in SEQ ID NO: 78; Cas9 protein from Psychroflexus torquisi having the amino acid sequence as set forth in SEQ ID NO: 79; Cas9 protein from Streptococcus thermophilus having the amino acid sequence as set forth in SEQ ID NO: 80; Cas9 protein from Listeria innocua having the amino acid sequence as set forth in SEQ ID NO: 81; Cas9 protein from Neisseria meningitidis having the amino acid sequence as set forth in SEQ ID NO: 82; UnlCasl2fl having the amino acid sequence as set forth in SEQ ID NO: 83; “Ancestral” Cas, referred to as LBCA having the amino acid sequence as set forth in SEQ ID NO: 84; “Ancestral” Cas referred to as LFCA having the amino acid sequence as set forth in SEQ ID NO: 85; Cas beta (also referred to as Cas-beta-M67) having the amino acid sequence as set forth in SEQ ID NO: 119; and variants thereof. In some embodiments, the composition of the invention further comprises at least one RNA-guided nuclease selected from the group consisting of: Cas9 protein from Streptococcus pyogenes (SpCas9) having the amino acid sequence as set forth in SEQ ID NO: 61; Cas9 protein from Staphylococcus aureus (SaCas9) having the amino acid sequence as set forth in SEQ ID NO: 62; Cas 12a (Cpfl) having the amino acid sequence as set forth in SEQ ID NO: 63; Cas9 protein from Campylobacter jejuni (CjCas9) having the amino acid sequence as set forth in SEQ ID NO: 64; Cas9 nickase from Streptococcus pyogenes Cas9 (nCas9) having the amino acid sequence as set forth in SEQ ID NO: 65; Cas9 nickase from Staphylococcus aureus Cas9 (SanCas9) having the amino acid sequence as set forth in SEQ ID NO: 66; Humanized Cas9 having the amino acid sequence as set forth in SEQ ID NO: 67; CasX having the amino acid sequence as set forth in SEQ ID NO: 68; TnpB having the amino acid sequence as set forth in SEQ ID NO: 70; Casl2f having the amino acid sequence as set forth in SEQ ID NO: 71; Cas9 protein from Corynebacterium ulcerans having the amino acid sequence as set forth in SEQ ID NO: 72; Cas9 protein from Corynebacterium diphtheria having the amino acid sequence as set forth in SEQ ID NO: 73; Cas9 protein from Spiroplasma syrphidicola having the amino acid sequence as set forth in SEQ ID NO: 74; Cas9 protein from Prevotella intermedia having the amino acid sequence as set forth in SEQ ID NO:75; Cas9 protein from Spiroplasma taiwanense having the amino acid sequence as set forth in SEQ ID NO: 76; Cas9 protein from Streptococcus iniae having the amino acid sequence as set forth in SEQ ID NO: 77; Cas9 protein from Belliella baltica having the amino acid sequence as set forth in SEQ ID NO: 78; Cas9 protein from Psychroflexus torquisi having the amino acid sequence as set forth in SEQ ID NO: 79; Cas9 protein from Streptococcus thermophilus having the amino acid sequence as set forth in SEQ ID NO: 80; Cas9 protein from Listeria innocua having the amino acid sequence as set forth in SEQ ID NO: 81; Cas9 protein from Neisseria meningitidis having the amino acid sequence as set forth in SEQ ID NO: 82; UnlCasl2fl having the amino acid sequence as set forth in SEQ ID NO: 83; “Ancestral” Cas, referred to as LBCA having the amino acid sequence as set forth in SEQ ID NO: 84; “Ancestral” Cas referred to as LFCA having the amino acid sequence as set forth in SEQ ID NO: 85; and variants thereof.

[0245] In some embodiments, the Cas protein has at least 80%, 90%, 95%, 99% or at least 100% identity to a Cas protein selected from the group consisting of a Streptococcus pyogenes Cas9 (SpCas9) of SEQ ID NO: 61, Staphylococcus aureus Cas9 (SaCas9) of SEQ ID NO: 62, Cpfl of SEQ ID NO: 63, Campylobacter jejuni Cas9 (CjCas9) of SEQ ID NO: 64, Streptococcus pyogenes Cas9 nickase (nCas9) of SEQ ID NO: 65, CasX of SEQ ID NO: 68, or Staphylococcus aureus Cas9 nickase of SEQ ID NO: 66; preferably wherein said Cas protein is a Cas9 protein selected from the group consisting of a Staphylococcus aureus Cas9 (SaCas9) of SEQ ID NO: 62, Streptococcus pyogenes Cas9 (SpCas9) of SEQ ID NO: 61, and Cas beta (also referred to as Cas-beta-M67) of SEQ ID NO: 119. In some embodiments, the Cas protein has at least 80%, 90%, 95%, 99% or at least 100% identity to a Cas protein selected from the group consisting of a Streptococcus pyogenes Cas9 (SpCas9) of SEQ ID NO: 61, Staphylococcus aureus Cas9 (SaCas9) of SEQ ID NO: 62, Cpfl of SEQ ID NO: 63, Campylobacter jejuni Cas9 (CjCas9) of SEQ ID NO: 64, Streptococcus pyogenes Cas9 nickase (nCas9) of SEQ ID NO: 65, CasX of SEQ ID NO: 68, or Staphylococcus aureus Cas9 nickase of SEQ ID NO: 66; preferably wherein said Cas protein is a Cas9 protein selected from the group consisting of a Staphylococcus aureus Cas9 (SaCas9) of SEQ ID NO: 62 and Streptococcus pyogenes Cas9 (SpCas9) of SEQ ID NO: 61.

[0246] In some embodiments, the Cas protein is selected from the group comprising or consisting of Cas9, Casl2a (Cpfl), Casl2b, Casl2f, and CasX. In some embodiments, 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. In some embodiments, the Cas protein is selected from the group comprising or consisting of Cas9, Cas 12a (Cpfl), Cas 12b, Casl2f, and CasX. In some embodiments, 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.

[0247] In some embodiments, the RNA-guided nuclease is a Cas9 protein, including variants and functional fragments thereof. In some embodiments, the RNA-guided nuclease is a Cas9 protein. In some embodiments, 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.

[0248] In some embodiments, the Cas9 protein has at least 80%, 90%, 95%, 99% or at least 100% amino acid sequence identity with the sequence of the proteins selected from the group consisting of Streptococcus pyogenes Cas9 (SpCas9) of SEQ ID NO: 61, Staphylococcus aureus Cas9 (SaCas9) of SEQ ID NO: 62, Cpfl of SEQ ID NO: 63, Campylobacter jejuni Cas9 (CjCas9) of SEQ ID NO: 64, Streptococcus pyogenes Cas9 nickase (nCas9) of SEQ ID NO: 65, and Staphylococcus aureus Cas9 nickase of SEQ ID NO: 66

[0249] In some embodiments, the Cas9 protein has at least 80%, 90%, 95%, 99% or at least 100% amino acid sequence identity with the sequence of the proteins selected from the group consisting of Staphylococcus aureus Cas9 (SaCas9) of SEQ ID NO: 62 and Streptococcus pyogenes Cas9 (SpCas9) of SEQ ID NO: 61.

[0250] In some embodiments, the Cas9 protein is a Staphylococcus aureus Cas9 (SaCas9) of SEQ ID NO: 62. In some embodiments, the Cas9 protein is a Streptococcus pyogenes Cas9 (SpCas9) of SEQ ID NO: 61.

[0251] In some embodiments, the Cas9 variant comprises the amino acid sequence of a Cas9 protein with one or several amino acid substitutions. In some embodiments, the RNA-guided nuclease is a variant or a functional fragment of a Cas9 protein.

[0252] In some embodiments, the Cas9 variant is humanized Cas9 (hCas9) or a functional fragment thereof. As used herein, the term “humanized Cas9” or “hCas9” refers to a sequence optimized Cas9 protein for human cells. In some embodiments, the hCas9 protein has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 67.

[0253] In some embodiments, the RNA-guided nuclease is a CasX protein. In some embodiments, the CasX has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 68.

[0254] In some embodiments, the RNA-guided nuclease is a deadCas9 protein. In some embodiments, the deadCas9 has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 69.

[0255] In some embodiments, the RNA-guided nuclease is a TnpB (Transposase B from transposon PsiTn554) protein. In some embodiments, the TnpB has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 70.

[0256] In some embodiments, the RNA-guided nuclease is a Casl2f protein. In some embodiments, the Casl2f protein is from the bacterium Acidibacillus sulfuroxidans (AsCasl2f). In some embodiments, the Casl2f has an amino acid sequence having at leastabout 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 71.

[0257] In some embodiments, the RNA-guided nuclease is a Cas beta (Cas-beta-M67) protein. In some embodiments, the Cas beta has an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 119.

[0258] In some embodiments, the RNA-guided nuclease is a Cas9 protein from Streptococcus pyogenes (SpCas9), or a variant thereof.

[0259] In some embodiments, the Cas9 protein from Streptococcus pyogenes (SpCas9), or the variant thereof has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 61SEQ ID NO: 61MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKNLIGALLFG SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVE EDI<I<HERHPIFGNIVDEVAYHEI<YPTIYHLRI<I<LADSTDI<ADLRLIYLALAHMI KFRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENPINASRVDAKAILSARLS KSRRLENLIAQLPGEKRNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTY DDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNSEITKAPLSASMIKRYD EHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILE KMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD NREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQ SFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGE QKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGAYHDL LKIIKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEERLKTYAHLFDDKVMKQLK RRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGHSLHEQIANLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIE MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYY LQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSD NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQL VETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFD SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYE KLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSIT GLYETRIDLSQLGGD

[0260] In some embodiments, the Cas9 protein from Streptococcus pyogenes (SpCas9) has an amino acid sequence as set forth in SEQ ID NO: 61.

[0261] In some embodiments, the RNA-guided nuclease is a Cas9 protein from Staphylococcus aureus (SaCas9), or a variant thereof.

[0262] In some embodiments, the Cas9 protein from Staphylococcus aureus (SaCas9), or the variant thereof has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 62SEQ ID NO: 62MAPKKKRKVGIHGVPAAKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKE ANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEA RVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKAL EEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFI DTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILV NEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSE DIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIF NRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK VIN AIIKKYGLPND III ELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQ EGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGN RTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDF INRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKER NI<GYI<HHAEDALIIANADFIFI<EWI<I<LDI<AI<I<VMENQMFEEI<QAESMPEIETE QEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIV NNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPL YKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKL SLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQA EFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRI IKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG

[0263] In some embodiments, the Cas9 protein from Staphylococcus aureus (SaCas9) has an amino acid sequence as set forth in SEQ ID NO: 62.

[0264] In some embodiments, the RNA-guided nuclease is a Casl2a (Cpfl), or a variant thereof.

[0265] In some embodiments, the Casl2a (Cpfl), or the variant thereof has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 63.SEQ ID NO: 63MAPKKKRKVSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAE DYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEIN LRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFD NRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTK QKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLE KLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAV VTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGS SEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDES FYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKD KETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPN KMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSI SRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEE GKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRA SLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINK CPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEII NNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELV EKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCAT GGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIA DSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRN PKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMAL MSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGA YNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH

[0266] In some embodiments, the Cast 2a (Cpfl) has an amino acid sequence as set forth in SEQ ID NO: 63

[0267] In some embodiments, the RNA-guided nuclease is a Cas9 protein from Campylobacter jejuni (CjCas9), or a variant thereof.

[0268] In some embodiments, the Cas9 protein from Campylobacter jejuni (CjCas9), or the variant thereof has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 64SEQ ID NO: 64MARILAFDIGIS SIGWAF SENDELKDCGVRIFTKVENPKTGESLALPRRLARS AR KRLARRKARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLISPYELRFRAL NELLSKQDFARVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLANYQSVG EYLYKEYFQKFKENSKEFTNVRNKKESYERCIAQSFLKDELKLIFKKQREFGFSF SKKFEEEVLSVAFYKRALKDFSHLVGNCSFFTDEKRAPKNSPLAFMFVALTRIIN LLNNLKNTEGILYTKDDLNALLNEVLKNGTLTYKQTKKLLGLSDDYEFKGEKG TYFIEFKKYKEFIKALGEHNLSQDDLNEIAKDITLIKDEIKLKKALAKYDLNQNQ IDSLSKLEFKDHLNISFKALKLVTPLMLEGKKYDEACNELNLKVAINEDKKDFL PAFNETYYI<DEVTNPVVLRAH<EYRI< VLNALLI<I<YGI<VHI<INIELAREVGI<NH SQRAKIEKEQNENYKAKKDAELECEKLGLKINSKNILKLRLFKEQKEFCAYSGE KIKISDLQDEKMLEIDHIYPYSRSFDDSYMNKVLVFTKQNQEKLNQTPFEAFGN DSAI<WQI<IEVLAI<NLPTI<I<QI<RILDI<NYI<DI<EQI<NFI<DRNLNDTRYIARLVL NYTKDYLDFLPLSDDENTKLNDTQKGSKVHVEAKSGMLTSALRHTWGFSAKD RNNHLHHAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAKKISELDYKNKRK FFEPFSGFRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQSYGGKEGVL KALELGKIRKVNGKIVKNGDMFRVDIFKHKKTNKFYAVPIYTMDFALKVLPNK AVARSKKGEIKDWILMDENYEFCFSLYKDSLILIQTKDMQEPEFVYYNAFTSST VSLIVSKHDNKFETLSKNQKILFKNANEKEVIAKSIGIQNLKVFEKYIVSALGEV TKAEFRQREDFKK

[0269] In some embodiments, the Cas9 protein from Campylobacter jejuni (CjCas9) has an amino acid sequence as set forth in SEQ ID NO: 64.

[0270] In some embodiments, the RNA-guided nuclease is a Cas9 nickase from Streptococcus pyogenes (nCas9), or a variant thereof.

[0271] In some embodiments, the Cas9 nickase from Streptococcus pyogenes (nCas9), or the variant thereof has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 65SEQ ID NO: 65MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVE EDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLS KSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTY DDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYD EHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILE KMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD NREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQ SFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGE QKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDL LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLK RRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKE DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENI VIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGK SDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKR QLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFY KVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKS EQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGG FDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGY KEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYN KHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGD

[0272] In some embodiments, the Cas9 nickase from Streptococcus pyogenes (nCas9) has an amino acid sequence as set forth in SEQ ID NO: 65.

[0273] In some embodiments, the RNA-guided nuclease is a Cas9 nickase from Staphylococcus aureus (SanCas9) or a variant thereof.

[0274] In some embodiments, the Cas9 nickase from Staphylococcus aureus (SanCas9), or the variant thereof has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 66SEQ ID NO: 66MAPKKKRKVGIHGVPAAKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKE ANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEA RVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKAL EEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFI DTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAY NADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILV NEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSE DIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIF NRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIII ELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQ EGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEEASKKGN RTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDF INRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKER NI<GYI<HHAEDALIIANADFIFI<EWI<I<LDI<AI<I<VMENQMFEEI<QAESMPEIETE QEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIV NNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPL YKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKL SLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQA EFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRI IKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG

[0275] In some embodiments, the Cas9 nickase from Staphylococcus aureus (SanCas9) has an amino acid sequence as set forth in SEQ ID NO: 66.

[0276] In some embodiments, the RNA-guided nuclease is a humanized Cas9 (hCas9), or a variant thereof.

[0277] In some embodiments, the humanized Cas9 (hCas9), or the variant thereof has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 67.SEQ ID NO: 67MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVE EDI<I<HERHPIFGNIVDEVAYHEI<YPTIYHLRI<I<LVDSTDI<ADLRLIYLALAHMI KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLS KSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTY DDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYD EHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILE KMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD NREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQ SFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGE QKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDL LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLK RRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKE DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENI VIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGK SDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKR QLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFY KVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKS EQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGG FDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGY KEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYN KHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGD

[0278] In some embodiments, the humanized Cas9 (hCas9) has an amino acid sequence as set forth in SEQ ID NO: 67.

[0279] In some embodiments, the RNA-guided nuclease is a CasX, or a variant thereof.

[0280] In some embodiments, the CasX, or the variant thereof has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 68.SEQ ID NO: 68MAPKKKRKVSMQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLR ERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGL MSRVAQPAPKNIDQRKLIPVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKG KPHTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVTR ESNHPVKPLEQIGGNSCASGPVGKALSDACMGAVASFLTKYQDIILEHQKVIKK NEKRLANLKDIASANGLAFPKITLPPQPHTKEGIEAYNNVVAQIVIWVNLNLWQ KLKIGRDEAKPLQRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGK VFWQNLAGYKRQEALLPYLSSEEDRKKGKKFARYQFGDLLLHLEKKHGEDWG KVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGL KEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKD GVI<I<LNLYLIINYFI<GGI<LRFI<I<II<PEAFEANRFYTVINI<I<SGEIVPMEVNFNFD DPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPA LFVALTFERREVLDSSNIKPMNLIGIDRGENIPAVIALTDPEGCPLSRFKDSLGNP THILRIGESYKEKQRTIQAAKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLP SKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELK VEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKK RFSHRPVQEKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGN TDKRAFVETWQSFYRKKLKEVWKPAVTS

[0281] In some embodiments, the CasX has an amino acid sequence as set forth in SEQ ID NO: 68

[0282] In some embodiments, the RNA-guided nuclease is a dead Cas9 (dCas9), or a variant thereof.

[0283] In some embodiments, the dead Cas9, or the variant thereof has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 69.SEQ ID NO: 69MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVE EDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLS KSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTY DDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYD EHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILE KMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD NREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQ SFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGE QKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDL LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLK RRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKE DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGK SDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKR QLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFY KVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKS EQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGG FDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGY KEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYN KHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGD

[0284] In some embodiments, the dead Cas9 has an amino acid sequence as set forth in SEQ ID NO: 69

[0285] In some embodiments, the RNA-guided nuclease is TnpB (Transposase B from transposon PsiTn554), or a variant thereof.

[0286] In some embodiments, the TnpB (Transposase B from transposon PsiTn554), or the variant thereof has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 70.SEQ ID NO: 70MIRNKAFVVRLYPNAAQTELINRTLGSARFVYNHFLARRIAAYKESGKGLTYG QTSSELTLLKQAEETSWLSEVDKFALQNSLKNLETAYKNFFRTVKQSGKKVGF PRFRKKRTGESYRTQFTNNNIQIGEGRLKLPKLGWVKTKGQQDIQGKILNVTVR RIHEGHYEASVLCEVEIPYLPAAPKFAAGVDVGIKDFAIVTDGVRFKHEQNPKY YRSTLKRLRKAQQTLSRRKKGSARYGKAKTKLARIHKRIVNKRQDFLHKLTTS LVREYEIIGTEHLKPDNMRKNRRLALSISDAGWGEFIRQLEYKAAWYGRLVSKVSPYFPSSQLCHDCGFKNPEVKNLAVRTWTCPNCGETHDRDENAALNIRREAL VAAGISDTLNAHGGYVRPASAGNGLRSENHATLVV

[0287] In some embodiments, the TnpB (Transposase B from transposon PsiTn554) has an amino acid sequence as set forth in SEQ ID NO: 70.

[0288] In some embodiments, the RNA-guided nuclease is a Casl2f, or a variant thereof.

[0289] In some embodiments, the Casl2f, or the variant thereof has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 71.SEQ ID NO: 71MIK VYRYEIVKPLDLDWKEFGTILRQLQQETRF ALNK ATQL AWEWMGF S SD Y KDNHGEYPKSKDILGYTNVHGYAYHTIKTKAYRLNSGNLSQTIKRATDRFKAY QKEILRGDMSIPSYKRDIPLDLIKENISVNRMNHGDYIASLSLLSNPAKQEMNVK RKISVIIIVRGAGKTIMDRILSGEYQVSASQIIHDDRKNKWYLNISYDFEPQTRVL DLNKIMGIDLGVAVAVYMAFQHTPARYKLEGGEIENFRRQVESRRISMLRQGK YAGGARGGHGRDKRIKPIEQLRDKIANFRDTTNHRYSRYIVDMAIKEGCGTIQM EDLTNIRDIGSRFLQNWTYYDLQQKIIYKAEEAGIKVIKIDPQYTSQRCSECGNID SGNRIGQAIFKCRACGYEANADYNAARNIAIPNIDKIIAESIK

[0290] In some embodiments, the Casl2f has an amino acid sequence as set forth in SEQ ID NO: 71

[0291] In some embodiments, the RNA-guided nuclease is a Cas9 protein from Corynebacterium ulcerans. or a variant thereof.

[0292] In some embodiments, the Cas9 protein from Corynebacterium ulcerans, or the variant thereof has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 72.SEQ ID NO: 72MTNAVANHHVLWAKFDNVSEPYPLLAHLLDTATAATCLFNHWLRKGLRDRLS TELGPDAEKILGFVAGIHDLGKANPYFQAQRRNKKEEWITLRDAIQKAGFPLSN GTSALFEETKEKRRHENITLSILGWEITKFLQVKDVWPQLAIIGHHGNFSAPGFL SDEDDLEDIEDIFDDNGWSPTHELLVSSLLQAVGLEKQPEIKHISPASAILISGLV VLADRIASQSEMASDGLQALQKEELFFHQPEKWIANRKAFCREIIENTVGTYHP WESEAAGIRAVLGDYEPRFTQKAALNAGDGLFNVMETTGAGKTEAALLRHVK RKERLLFFLPTQATTNAIMDRIGKIFDGTPNVASLAHGLAVTEDFYAHPILPVQG SSDDANYKDNGGLYPTEFVRSAGTPRLLAPVCVGTIDQALMGALPSKFNHLRL LALANAHVVVDEVHTMDQYQSELMSGLLEWWSATDTPVTLLTATMPAWQRE KFHLSYTGKDPHFKGVFPSLEDWSTPSKNTETSQENIPTEAFTIPINIDKIAHNEIV DSHVQWVIEQRKLFPQARIGIICNTVGRAQSIAEALAHESPIVLHSRMTAGHRKE AATKLEQAIGKKGTANATLVIGTQAIEASLDIDLDLLRTELCPAPSLIQRAGRLW RRLDPQREVRVPGMVGKKLTIAVVDSPSTGQTLPYLRSQLYRVESWLKQRDRI EFPADIQDFIDATTPGLQELFQKVSLPEDCGSAEEREALADDYLNEVASWVTKQ RQAGTSRIDFAKHGKPRQVLASDCVVEDFLQITSANNLEESATRLIDYPSISAILC DPTGTIPGAWTDSVEKLIAISAKDSESLRRALRASISIPHSKKFLPITSREIPLSEAK TLLSGYSAVHIQPDEYDLQSGLKGPQK

[0293] In some embodiments, the Cas9 protein from Corynebacterium ulcerans has an amino acid sequence as set forth in SEQ ID NO: 72.

[0294] In some embodiments, the RNA-guided nuclease is a Cas9 protein from Corynebacterium diphtheria, or a variant thereof.

[0295] In some embodiments, the Cas9 protein from Corynebacterium diphtheria, or the variant thereof has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 73.SEQ ID NO: 73MNPHEELWAKQKGLAKPYPLLAHLLDSAAVAGALWDHWLRQDLRQMFIEEL GSNAREIIQFVVGSHDIGKATPLFQYQKAQKGEVWDSIRYAIDRTGRYQKPLPS SYLVKKTSGGPNRHEQWSSFASKNEYLKPSAAAKENWIGLAIGGHHGRFEPVG YGRHQRKAAEDLAKSGWSAAQQDLLRALEKASGITRASLPSELSPELTLVLSGL TILADRISSTESFVITGARMIDDGTLHLATPIDWLKTRKLDSEKHVAKTVGIYHG WNNHESAIHSILKGYDPRPLQTIALQNQVGLLNLMAPTGNGKTEAAILRHSLKE NDRLIFLLPTQATSNAIMRRVQGIYSDTPNAAALAHSLASVEDFYQTPLSVFDD HYDPSKEQFES SMSGGL YPS SF VC SGAARLLAPICIGTVDQALATALPGKWIHL RILALANAHIVIDEVHTLDHYQTALLENILPILAKLKTKITFLTATMPSWQRTKL LTAYGGEDLQIPPTVFPAAETVLPGQFNRTLIDSDSTTIDFTMEETSYDHLVESH VI<WHQTTRLNAPHARIGLICNTVI<RAQEIAAALEI<TNDRIVLLHSRMTTEHRR RSAELLESLLGPNGNRKTITVVGTQAIEASLDIDLDILRTELCPAPSLVQRAGRV WRRNDPYRSSRITADHI<PISVVFIAEAI<DWQVLPYLRAETSRTQRWLEI<HNQM FLPQMAQEFIDAATVDLDTATSEMDLDALALMGIHLMKADGAKARIQDVLNS DSKVSDFALLTSKNEIDEAQTRLIEEGTHLRIILGDENESIPGGWKHGLSSLLKLK ASDRESLRTALLASIPLLVSEKQKQLLYQHNLVPLSSSKTVLAGFYFLPKAQNFY SKNLGFIWPEEKD

[0296] In some embodiments, the Cas9 protein from Corynebacterium diphtheria has an amino acid sequence as set forth in SEQ ID NO: 73.

[0297] In some embodiments, the RNA-guided nuclease is a Cas9 protein from Spiroplasma syrphidicola. or a variant thereof.

[0298] In some embodiments, the Cas9 protein from Spiroplasma syrphidicola, or the variant thereof has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 74.SEQ ID NO: 74MNYKKLILGLDLGIASCGWAVTGQMEDGNWVLDDFGVRLFQTPENSKDGTTN AAARRLKRGARRLIKRRKNRIKDLKNLFEKINFINKASLDKYINEHSATNLVEDF NRHELYNPYFLRSIGITEKLTREELVWSLIHIANRRGYKNKFAFDIEGDGKKRET KLDEAISNALISSNLTISQEIVRNKKFRDAKNKKALLVRNKGGKEGENNFQFLF ARDDYKKEVDLLLAKQAKFYPELTEEIRAKAADIIFRQRDFEDGPGPKKQELRE IYKKENKQFSKNFTQLEGRCTFLRELSVGYKSSILFDLFHIISEVSKISKYIEENDQ LAQDIISSFLYNEAGKKGKTLLKEILKKHHINDDIFDTNAYKNIDFKTNYLNLLK EVFGNDVLKNLSLNRLEDNIYHQLGFIIHTNITPERKEKAINQWLLENNIILAKEK LNILLKPNSSISTTVKTSFKWMSIAISNFLKGIPYGKFQAQFIKEDNFKLPESYAK QYQKYLTGEKTFEMFAPIIDPDLWRNPIVFRAINQARKVIKKLFEKYTFIDQINIE LTREMGLSFSDRKKVKERQDDSLKENAKAKEFLMANGIIVNDTNVLKYKLWIQ QNKKSLYSGKEITIADLGASNVLQIDHIIPYSKLADDSFNNKVLVFSKENQEKGN QFADQYVKSLGTENYNNYKKRVNYLLFQNQINQKKAEYLLCSNQNEEILNDFV SRNLNDTRYITRYVTNWLKAEFELQSRFGLAKPKIMTLNGAITSRFRRTWLRNS PWGLEKKS

[0299] In some embodiments, the Cas9 protein from Spiroplasma syrphidicola has an amino acid sequence as set forth in SEQ ID NO: 74.

[0300] In some embodiments, the RNA-guided nuclease is a Cas9 protein from Prevotella intermedia, or a variant thereof.

[0301] In some embodiments, the Cas9 protein from Prevotella intermedia, or the variant thereof has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 75.SEQ ID NO: 75MKRILGLDLGTTSIGWALVNEAENNNEASSIVRLGVRVNPLTVDEKSNFEKGK AITTNADRQLRHGARINLQRYKLRRQNLHDCLQKQGWLGTEAMYEEGKASTF ETYKLRAKAAEEEISLHEFARVLFMLNKKRGYKSNRKANNKEDGQLFDGMTIAKKLYEEHLTPAEYSLQLLNKGKKFTQGYYRSDLNAELERIWDEQKKYYPEILT DEFKQQLEGKTKTNTSKIFLAKYGIYSADLKGLDRKFQPLKWRVEALQQQVDK EVLAFVISDLKGQIANTSGLLGAISDRSKELYFNKQTVGQYLWASLEENPHISIK NKPFYRQDYLDEFEKIWETQAAFHKQLTPELKQEIRDIIIFYQRPLKSKKSLISVC ELEQRKVKATIDGKEKEITIGPKVAPKSSPVFQEFRIWQNLNNVLLIDNDTNEKR PLDEVERNLLYKELSIKAKLSKTEALKILNKKGKQWDLNYRELEGNRTQAILFD CYNRIITLTGHEECDFKKIKASEIRHYVSTIFKNLGFSTEILDFDPSLKKHELEKQP MYQLWHLLYSYESDNSRTGNESLLRKLETTFGFPEEYATVLCDVVFEEDYGNL SVKAMREILPYLQAGNDYSQACAYAGYNHSRHSLTKEELDQKVYKERLELLPK NSLRNPVVEKILNQMINVINAIIDEYGKPDEIRIEMARELKSSAADRKKTTHAISQ GNAENQRIREILEKEF SLS YISRNDIIKYKLYEELEPNYYKTL YSDTYITKDKLF S KDFDIEHIIPKARLFDDSFSNKTLEARNINLEKSNKTAFDFIKEKYGEDGAEAYK KKLDMLLENDAISRPKYNNLLRAEADIPSDFINRDLRNTQYIAKKACEILGELVK TVTPTTGKITNRLREDWQLVDVMKELNFEKYEKLGLTEIVEDRDGRKIKRIKD WTKRNDHRHHAMDALAIAFTKPSFIQYLNNLNARSNKGDSIYAIENKELHYEE GKLRFNAPIPVNEFRAEAKRHLSAILVSIKAKNKVMTQNVNKIKTKHGIIKKIQL TPRGPLHNETIYGTKMRPIIKMVKVGAALDEATINKVSSPAIREALLKRLNEYSG NAKKAFTGKNTLEKNPIYLNAGRTKTVPSLVKTVEWESFHPTRKLIDKDLNVD KVVDKGIREILKARLEEFNGDAKKAFSNLEENPIYLDEAKKIALKRVSIEGVLSA IPLHTLKNQAGKPITGKDGKPVLGNYVQTSNNHHIAFYYDEDGNLQDNAVSFF EAAERKSQGIPVIDKDYNRDKGWRFLFTMKQNEYFVFPNEATGFIPSEVDLTDE ANYGIISPNLYRVQKVSRIDKGTSASRDYWFRHHLETILNDDAKLKNLAFKRIR GLLELKDIIKVRINSTGKIVAVGEYD

[0302] In some embodiments, the Cas9 protein from Prevotella intermedia has an amino acid sequence as set forth in SEQ ID NO: 75.

[0303] In some embodiments, the RNA-guided nuclease is a Cas9 protein from Spiroplasma laiwanense. or a variant thereof.

[0304] In some embodiments, the Cas9 protein from Spiroplasma laiw anense. or the variant thereof has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 76.SEQ ID NO: 76MWSRKILKAGSRLFDEANLSDKIASKRREQRGRRRNLRRKITWKQDLINLFVK YNFLQKENDFYELDFNFDLLELRKKAINSKIELEQLLIILFNYIKHRGSFNYREDL SELKNISQEELETSSEFKLPVDIQFELKEENNKFREINNEKSLINHEWYVKEINLIL DAQIENKLINLDFKKDYLKLFNRKREYYDGPGPKDKNLLNPSKYGWKNQEEFF DRFAGKDTYDSKEQRAPKHSLTSYLFNILNDLNNLSINGDRNQLTYENKKDLIN LTLINQKEKAENITLKKIAKYLKINEKNITGYRLKPNSNESIFTVFESANKMRSIL VKNNKSIDFICLENIDKIDKIVDILTKYQSIEDKSLKLEELNFDFFDKETCEKLAVI SLTGTHALSKKTMSKLIEEMFHDNLNHMEALAKLKIKPDYKLKVDLTNFKTIPI LREKINEMYISPVVKRALIESLKIIKELERHFKDFEIKDIVIEMAKKNSAEKKQFIS KIQRQNVDLVKKLSNDYSLDENKLNFKMKEKFLLLSEQ

[0305] In some embodiments, the Cas9 protein from Spiroplasma taiwanense has an amino acid sequence as set forth in SEQ ID NO: 76.

[0306] In some embodiments, the RNA-guided nuclease is a Cas9 protein from Streptococcus iniae. or a variant thereof.

[0307] In some embodiments, the Cas9 protein from Streptococcus iniae, or the variant thereof has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 77.SEQ ID NO: 77MRKPYSIGLDIGTNSVGWAVITDDYKVPSKKMRIQGTTDRTSIKKNLIGALLFD NGETAEATRLKRTTRRRYTRRKYRIKELQKIFSSEMNELDIAFFPRLSESFLVSD DKEFENHPIFGNLKDEITYHNDYPTIYHLRQTLADSDQKADLRLIYLALAHIIKF RGHFLIEGNLDSENTDVHVLFLNLVNIYNNLFEEDIVETASIDAEKILTSKTSKSR RLENLIAEIPNQKRNMLFGNLVSLALGLTPNFKTNFELLEDAKLQISKDSYEEDLDNLLAQIGDQYADLFIAAKKLSDAILLSDIITVKGASTKAPLSASMVQRYEEHQ QDLALLKNLVKKQIPEKYKEIFDNKEKNGYAGYIDGKTSQEEFYKYIKPILLKL DGTEKLISKLEREDFLRKQRTFDNGSIPHQIHLNELKAIIRRQEKFYPFLKENQKK IEKLFTFKIPYYVGPLANGQSSFAWLKRQSNESITPWNFEEVVDQEASARAFIER MTNFDTYLPEEKVLPKHSPLYEMFMVYNELTKVKYQTEGMKRPVFLSSEDKEE IVNLLFKKERKVTVKQLKEEYFSKMKCFHTVTILGVEDRFNASLGTYHDLLKIF KDKAFLDDEANQDILEEIVWTLTLFEDQAMIERRLVKYADVFEKSVLKKLKKR HYTGWGRLSQKLINGIKDKQTGKTILGFLKDDGVANRNFMQLINDSSLDFAKII KNEQEKTIKNESLEETIANLAGSPAIKKGILQSIKIVDEIVKIMGQNPDNIVIEMAR ENQSTMQGIKNSRQRLRKLEEVHKNTGSKILKEYNVSNTQLQSDRLYLYLLQD GKDMYTGKELDYDNLSQYDIDHIIPQSFIKDNSIDNTVLTTQASNRGKSDNVPNI ETVNKMKSFWYKQLKSGAISQRKFDHLTKAERGALSDFDKAGFIKRQLVETRQ ITKHVAQILDSRFNSNLTEDSKSNRNVKIITLKSKMVSDFRKDFGFYKLREVND YHHAQDAYLNAVVGTALLKKYPKLEAEFVYGDYKHYDLAKLMIQPDSSLGKA TTRMFFYSNLMNFFKKEIKLADDTIFTRPQIEVNTETGEIVWDKVKDMQTIRKV MSYPQVNIVMKTEVQTGGFSKESIWPKGDSDKLIARKKSWDPKKYGGFDSPIIA YSVLVVAKIAKGKTQKLKTIKELVGIKIMEQDEFEKDPIAFLEKKGYQDIQTSSII KLPKYSLFELENGRKRLLASAKELQKGNELALPNKYVKFLYLASHYTKFTGKE EDREKKRSYVESHLYYFDVRLSQVFRVTNVEF

[0308] In some embodiments, the Cas9 protein from Streptococcus iniae has an amino acid sequence as set forth in SEQ ID NO: 77.

[0309] In some embodiments, the RNA-guided nuclease is a Cas9 protein from Belliella ballica. or a variant thereof.

[0310] In some embodiments, the Cas9 protein from Belliella ballica. or the variant thereof has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 78.SEQ ID NO: 78MKKILGLDLGTTSIGWAFIKEPEKDVVGSEIVDMGVRIVPLSSDEENDFAKGNTI SINADRTLKRGARRNLQRFKQRRNALLEIFKEKKLISTNFKYAEDGPSSTFSTLN LRAKAAKEKIELQDLVKVLLQINKKRGYKSSRKAKSEEDDGSAIDSMGIAKELY ENDLTPGQWVYEALQKGRKNVPDFYRSDLQEEFKKIVNYQSEFFPDIFNASFVE DWMGKASTPTKQYFNKKGVQLAENKGKREERRLQEYKWRAEAVNFKIDLSEI ALILSQINSQISNSSGYLGAISDRSKELYFKNLTVGQYLYQQIKKNPHTRLKGQV FYRQDYLDEFERIWSVQSSFYPQLNDALKREVRDITIFFQRRLKSQKHLISNCEF EDHHKVVPKSHPVFQEFRIWQNLNNLLLIKKDNLNEKFDLELESKIALANELAF KRELNVKDALKILGLKPNEWEFNFTKIEGNRTNQAFFDAFAKIIELEDGEPIDLG DLKADDILDQFSEAFLRIGIDTELLQVNSDIEGAEYEKQSYIQFWHLLYSSEDDQ KLKLNLIRKFGFKPEHAKILASISLQDDHASLSSRAIKKILPHLQSGLIYDKACTY AGYNHS S SFTKDENEKRELRAELELLKKNSLRNP VVEKILNQMINVVNAILKDP ELGRPDEIRVEMARELKANAEQRKNMTSNIASATRDHDKYREILKSEFGLKRVT KNDLLRYKLWLETDGISLYTGKPIEASKLFSKEYDIEHIIPKARLFDDSFSNKTIC ERQLNIDKANVTAFSFLQNKLSADEFEQYQSRVKSLYGKLSKAKIQKLLMAND KIPEDFIARQLQETRYISKKAKEILFEISRRVSVTTGTITDKLREDWGLVEIMKEL NWEKYDKLGLTYTIEGKHGERLNKIKDWSKRNDHRHHAMDALTVALTKPAYI QYLNNLNAKGLNNKKGTEVFAIEQKYLKRENGKLCFIPPIENIRSEAKKHLSRIL VSYKAKNKVVTINKNKTKSKAGLNEQIALTPRGQLHKETVYGKSFHYSTKFEKI GASFNVQKINTVAKKEEREALLKRLAENGNDPKKAFTGKNTLNKMPIYLDLGK NIKLSEKVKTVVLEQNYTIRKNIDPDLKVDKVIDVGIKRILESRLEEFGGNAKLA FSNLEENPIWLNKEKGISIKRVKISGVSNVESLHVKKDHFGEPILDQEGNEIPVDF VSTGNNHHVAIYEDENGNLQEEVVSFFEAVVRQNQGLPIIKKNHTLGWKFLFTL KQNEYFVFPSDDFVPADVDLMDEQNYHLISPNLFRVQKIARKNYVFNNHLETK AVDNDLLKSKKELSKITYHFYQTPEHLRGIIKIRINHLGKIIQIGEY

[0311] In some embodiments, the Cas9 protein from Belliella baltica has an amino acid sequence as set forth in SEQ ID NO: 78.

[0312] In some embodiments, the RNA-guided nuclease is a Cas9 protein from Psychroflexus lorqiiisi. or a variant thereof.

[0313] In some embodiments, the Cas9 protein from Psychroflexus torquisi, or the variant thereof has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 79.SEQ ID NO: 79MKRILGLDLGTNSIGWSLIEHDFKNKQGQIEGLGVRIIPMSQEILGKFDAGQSISQ TADRTKYRGVRRLYQRDNLRRERLHRVLKILDFLPKHYSESIDFQDKVGQFKP KQEVKLNYRKNEKNKHEFVFMNSFIEMVSEFKNAQPELFYNKGNGEETKIPYD WTLYYLRKKALTQQITKEELAWLILNFNQKRGYYQLRGEDIDEDKNKKYMQL KVNNLIDSGAKVKGKVLYNVIFDNGWKYEKQIVNKDEWEGRTKEFIITTKTLK NGNIKRTYKAVDSEIDWAAIKAKTEQDINKANKTVGEYIYESLLDNPSQKIRGK LVKTIERKFYKEEFEKLLSKQIELQPELFNESLYKACIKELYPRNENHQSNNKKQ GFEYLFTEDIIFYQRPLKSQKSNISGCQFEHKIYKQKNKKTGKLELIKEPIKTISRS HPLFQEFRIWQWLQNLKIYNKEKIENGKLEDVTTQLLPNNEAYVTLFDFLNTKK ELEQKQFIEYFVKKKLIDKKEKEHFRWNFVEDKKYPFSETRAQFLSRLAKVKGI KNTEDFLNKNTQVGSKENSPFIKRIEQLWHIIYSVSDLKEYEKALEKFAEKHNLE KDSFLKNFKKFPPFVSDYASYSKKAISKLLPIMRMGKYWSESAVPTQVKERSLSI MERVKVLPLKEGYSDKDLADLLSRVSDDDIPKQLIKSFISFKDKNPLKGLNTYQ ANYLVYGRHSETGDIQHWKTPEDIDRYLNNFKQHSLRNPIVEQVVMETLRVVR DIWEHYGNNEKDFFKEIHVELGREMKSPAGKREKLSQRNTENENTNHRIREVL KELMNDASVEGGVRDYSPSQQEILKLYEEGIYQNPNTNYLKVDEDEILKIRKKN NPTQKEIQRYKLWLEQGYISPYTGKIIPLTKLFTHEYQIEHIIPQSRYYDNSLGNKI ICESEVNEDI<DNI<TAYEYLI<VEI<GSIVFGHI<LLNLDEYEAHVNI<YFI<I<NI<TI<L KNLLSEDIPEGFINRQLNDSRYISKLVKGLLSNIVRENGEQEATSKNLIPVTGVVT SKLKQDWGLNDKWNEIIAPRFKRLNKLTNSNDFGFWDNDINAFRIQVPDSLIKG FSKKRIDHRHHALDALVVACTSRNHTHYLSALNAENKNYSLRDKLVIKNENGD YTKTFQIPWQGFTIEAKNNLEKTVVSFKKNLRVINKTNNKFWSYKDENGNLNL GKDGKPKKKLRKQTKGYNWAIRKPLHKETVSGIYNINAPKNKIATSVRTLLTEII<NEI<HLAI< ITDLRIRETILPNHLKHYLNNKGEANF SEAF SQGGIEDLNKKITTLN EGKKHQPIYRVKIFEVGSKFSISEDENSAKSKKYVEAAKGTNLFFAIYLDEENKKRNYETIPLNEVITHQKQVAGFPKSERLSVQPDSQKGTFLFTLSPNDLVYVPNNEE LENRDLFNLGNLNVEQISRIYKFTDSSDKTCNFIPFQVSKLIFNLKKKEQKKLDV DFIIQNEFGLGSPQSKNQKSIDDVMIKEKCIKLKIDRLGNISKA

[0314] In some embodiments, the Cas9 protein from Psychroflexus torquisi has an amino acid sequence as set forth in SEQ ID NO: 79.

[0315] In some embodiments, the RNA-guided nuclease is a Cas9 protein from Streptococcus thermophilus, or a variant thereof.

[0316] In some embodiments, the Cas9 protein from Streptococcus thermophilus, or the variant thereof has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 80.SEQ ID NO: 80MTKPYSIGLDIGTNSVGWAVTTDNYKVPSKKMKVLGNTSKKYIKKNLLGVLLF DSGITAEGRRLKRTARRRYTRRRNRILYLQEIFSTEMATLDDAFFQRLDDSFLVP DDKRDSKYPIFGNLVEEKAYHDEFPTIYHLRKYLADSTKKADLRLVYLALAHM IKYRGHFLIEGEFNSKNNDIQKNFQDFLDTYNAIFESDLSLENSKQLEEIVKDKIS KLEKKDRILKLFPGEKNSGIF SEFLKLIVGNQ ADFRKCFNLDEK ASLHF SKES YD EDLETLLGYIGDDYSDVFLKAKKLYDAILLSGFLTVTDNETEAPLSSAMIKRYN EHKEDLALLKEYIRNISLKTYNEVFKDDTKNGYAGYIDGKTNQEDFYVYLKKL LAEFEGADYFLEKIDREDFLRKQRTFDNGSIPYQIHLQEMRAILDKQAKFYPFLA KNKERIEKILTFRIPYYVGPLARGNSDF AW SIRKRNEKITPWNFED VIDKE SSAE AFINRMTSFDLYLPEEKVLPKHSLLYETFNVYNELTKVRFIAESMRDYQFLDSK QKKDIVRLYFKDKRKVTDKDIIEYLHAIYGYDGIELKGIEKQFNSSLSTYHDLLN IINDKEFLDDSSNEAIIEEIIHTLTIFEDREMIKQRLSKFENIFDKSVLKKLSRRHYT GWGKLSAKLINGIRDEKSGNTILDYLIDDGISNRNFMQLIHDDALSFKKKIQKAQ IIGDEDKGNIKEVVKSLPGSPAIKKGILQSIKIVDELVKVMGGRKPESIVVEMARE NQYTNQGKSNSQQRLKRLEKSLKELGSKILKENIPAKLSKIDNNALQNDRLYLY YLQNGKDMYTGDDLDIDRLSNYDIDHIIPQAFLKDNSIDNKVLVSSASNRGKSDDVPSLEVVKKRKTFWYQLLKSKLISQRKFDNLTKAERGGLSPEDKAGFIQRQLV ETRQITKHVARLLDEKFNNKKDENNRAVRTVKIITLKSTLVSQFRKDFELYKVR EINDFHHAHDAYLNAVVASALLKKYPKLEPEFVYGDYPKYNSFRERKSATEKV YFYSNIMNIFKKSISLADGRVIERPLIEVNEETGESVWNKESDLATVRRVLSYPQ VNVVKKVEEQNHGLDRGKPKGLFNANLSSKPKPNSNENLVGAKEYLDPKKYG GYAGISNSFTVLVKGTIEKGAKKKITNVLEFQGISILDRINYRKDKLNFLLEKGY KDIELIIELPKYSLFELSDGSRRMLASILSTNNKRGEIHKGNQIFLSQKFVKLLYH AKRISNTINENHRKYVENHKKEFEELFYYILEFNENYVGAKKNGKLLNSAFQS WQNHSIDELCSSFIGPTGSERKGLFELTSRGSAADFEFLGVKIPRYRDYTPSSLLK DATLIHQSVTGLYETRIDLAKLGEG

[0317] In some embodiments, the Cas9 protein from Streptococcus thermophilus has an amino acid sequence as set forth in SEQ ID NO: 80.

[0318] In some embodiments, the RNA-guided nuclease is a Cas9 protein from Listeria innocua. or a variant thereof.

[0319] In some embodiments, the Cas9 protein from Listeria innocua, or the variant thereof has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 81.SEQ ID NO: 81MKKPYTIGLDIGTNSVGWAVLTDQYDLVKRKMKIAGDSEKKQIKKNFWGVRL FDEGQTAADRRMARTARRRIERRRNRISYLQGIFAEEMSKTDANFFCRLSDSFY VDNEKRNSRHPFFATIEEEVEYHKNYPTIYHLREELVNSSEKADLRLVYLALAHI IKYRGNFLIEGALDTQNTSVDGIYKQFIQTYNQVFASGIEDGSLKKLEDNKDVA KILVEKVTRKEKLERILKLYPGEKSAGMFAQFISLIVGSKGNFQKPFDLIEKSDIE CAKDSYEEDLESLLALIGDEYAELFVAAKNAYSAVVLSSIITVAETETNAKLSAS MIERFDTHEEDLGELKAFIKLHLPKHYEEIFSNTEKHGYAGYIDGKTKQADFYK YMKMTLENIEGADYFIAKIEKENFLRKQRTFDNGAIPHQLHLEELEAILHQQAK YYPFLKENYDKIKSLVTFRIPYFVGPLANGQSEFAWLTRKADGEIRPWNIEEKVDFGKSAVDFIEKMTNKDTYLPKENVLPKHSLCYQKYLVYNELTKVRYINDQGK TSYFSGQEKEQIFNDLFKQKRKVKKKDLELFLRNMSHVESPTIEGLEDSFNSSYS TYHDLLKVGIKQEILDNPVNTEMLENIVKILTVFEDKRMIKEQLQQFSDVLDGV VLI<I<LERRHYTGWGRLSAI<LLMGIRDI<QSHLTILDYLMNDDGLNRNLMQLIN DSNLSFKSIIEKEQVTTADKDIQSIVADLAGSPAIKKGILQSLKIVDELVSVMGYP PQTIVVEMARENQTTGKGKNNSRPRYKSLEKAIKEFGSQILKEHPTDNQELRNN RLYLYYLQNGKDMYTGQDLDIHNLSNYDIDHIVPQSFITDNSIDNLVLTSSAGN REKGDDVPPLEIVRKRKVFWEKLYQGNLMSKRKFDYLTKAERGGLTEADKAR FIHRQLVETRQITKNVANILHQRFNYEKDDHGNTMKQVRIVTLKSALVSQFRKQ FQLYKVRDVNDYHHAHDAYLNGVVANTLLKVYPQLEPEFVYGDYHQFDWFK ANKATAKKQFYTNIMLFFAQKDRIIDENGEILWDKKYLDTVKKVMSYRQMNIV KKTEIQKGEFSKATIKPKGNSSKLIPRKTNWDPMKYGGLDSPNMAYAVVIEYA KGKNKLVFEKKIIRVTIMERKAFEKDEKAFLEEQGYRQPKVLAKLPKYTLYECE EGRRRMLASANEAQKGNQQVLPNHLVTLLHHAANCEVSDGKSLDYIESNREM FAELLAHVSEFAKRYTLAEANLNKINQLFEQNKEGDIKAIAQSFVDLMAFNAM GAPASFKFFETTIERKRYNNLKELLNSTIIYQSITGLYESRKRLDD

[0320] In some embodiments, the Cas9 protein from Listeria innocua has an amino acid sequence as set forth in SEQ ID NO: 81.

[0321] In some embodiments, the RNA-guided nuclease is a Cas9 protein from Neisseria meningitidis, or a variant thereof.

[0322] In some embodiments, the Cas9 protein from Neisseria meningitidis, or the variant thereof has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 82.SEQ ID NO: 82MAAFKPNPINYILGLDIGIASVGWAMVEIDEDENPICLIDLGVRVFERAEVPKTG DSLAMARRLARSVRRLTRRRAHRLLRARRLLKREGVLQAADFDENGLIKSLPN TPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVADNAHALQTGDFRTPAELALNKFEKESGHIRNQRGDYSHTFSRKDLQAELIL LFEKQKEFGNPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKA AKNTYTAERFIWLTKLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQAR KLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAISRALEKEGLKDKKSPLNL SPELQDEIGTAFSLFKTDEDITGRLKDRIQPEILEALLKHISFDKFVQISLKALRRI VPLMEQGKRYDEACAEIYGDHYGKKNTEEKIYLPPIPADEIRNPVVLRALSQAR KVINGVVRRYGSPARIHIETAREVGKSFKDRKEIEKRQEENRKDREKAAAKFRE YFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLGRLNEKGYVEIDHALPFS RTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVETSRFP RSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRVFAS NGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKE MNAFDGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADT PEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDE GVSVLRVPLTQLKLKDLEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFY KYDKAGNRTQQVKAVRVEQVQKTGVWVRNHNGIADNATMVRVDVFEKGDK YYLVPIYSWQVAKGILPDRAVVQGKDEEDWQLIDDSFNFKFSLHPNDLVEVITK KARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILEGIGVKTALSFQKYQIDELG KEIRPCRLKKRPPVR

[0323] In some embodiments, the Cas9 protein from Neisseria meningitidis has an amino acid sequence as set forth in SEQ ID NO: 82.

[0324] In some embodiments, the RNA-guided nuclease is a UnlCasl2fl, or a variant thereof.

[0325] In some embodiments, the UnlCasl2fl, or the variant thereof has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 83.SEQ ID NO: 83MAKNTITKTLKLRIVRPYNSAEVEKIVADEKNNREKIALEKNKDKVKEACSKH LKVAAYCTTQVERNACLFCKARKLDDKFYQKLRGQFPDAVFWQEISEIFRQLQ KQAAEIYNQSLIELYYEIFIKGKGIANASSVEHYLSDVCYTRAAELFKNAAIASG LRSKIKSNFRLKELKNMKSGLPTTKSDNFPIPLVKQKGGQYTGFEISNHNSDFIIK IPFGRWQVKKEIDKYRPWEKFDFEQVQKSPKPISLLLSTQRRKRNKGWSKDEGT EAEIKKVMNGDYQTSYIEVKRGSKIGEKSAWMLNLSIDVPKIDKGVDPSIIGGID VGVKSPLVCAINNAFSRYSISDNDLFHFNKKMFARRRILLKKNRHKRAGHGAK NKLKPITILTEKSERFRKKLIERWACEIADFFIKNKVGTVQMENLESMKRKEDSY FNIRLRGFWPYAEMQNKIEFKLKQYGIEIRKVAPNNTSKTCSKCGHLNNYFNFE YRKKNKFPHFKCEKCNFKENADYNAALNISNPKLKSTKEEP

[0326] In some embodiments, the UnlCasl2fl has an amino acid sequence as set forth in SEQ ID NO: 83

[0327] In some embodiments, the RNA-guided nuclease is an “Ancestral” Cas, referred to as LBCA, or a variant thereof.

[0328] In some embodiments, the LBCA, or the variant thereof has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 84.SEQ ID NO: 84MKKDYSIGLDIGTNSVGWAVTDDNYNLVRKKMKVLGNTDKKSIGKALWGVR LFDAAETAEERRMHRTTRRRYTRRRQRIDLLQEIFQEEISKVDPSFFIRLNESRLH PEDI<TDDRHPLFGDNETDI<DYHI<QYPTIYHLRI<HLMESDEI<HDIRLVYLALHH IIKYRGHFLIEGDLNSENTDVEELFKQLVQVFNDTFEEEHLSEEAIDIEEILTDKKS RSTRAKEVVKLFGSEKKQACISALIKLIVGLKGNLKKVFGDAEDTSIHFSKDNY EEDLEAIRDIIPDEYADLFEAIKALYDAIVLSGILGGSTSNTKAKVSASMIARYEQ HQKDLKQLKQFVKEHLPEKYNEIFSDNTKNGYSAYIEGGTSQEDFYKYLKKILK ELEEAEYLLEEIENENFLPKQRTSDNGVIPYQIHLEELRAILKNQGKYYPFLKENAEKIESILTFRIPYYVGPLARGNGRFAWMIRKKDGKITPWNFDEVVDKEKSAEE FIERMTNNCTYLPGENVLPKNSLLYEKFTVLNELNNVRLTTDKGKTRRFSAEQK QEIFDDLFRKNRKVTKKKLEDYLKREYEEFDSADISGIDGEFKSSLGSYHDFCKI VVKGNSLDEEDYKDIIEEIIKWLTVFEDRKMLRRRLEKYSEILTEEQIKKICKRH YTGWGRLSRKLLTGIRDKETGKSIIDVLRETDSSNRNFMQLLSDEDLSFKEEIEQ ANAEAEGENLHEIVEDLPGSPAIKRGILQALKIVDEIVKVMGHEPKNIFVEMARE NQKTGRGRRSTKKRLKRLQEALKNLGSNLLKELPRDDNELRNDRLFLYYTQM GKCMYTGEPIDLDDLSNYDIDHIIPQSFIKDDSLDNRVLVSREENARKTDNFPSP EIRRKMKSFWQMLLKAGLISKKKFDRLTRADRGDFTDDELAGFIARQLVETRQI TKHVATLLKQRYPTEKDEEDKTIRNAKIVSVKANLVSEFRQDFGLYKCREVND YHHAHDAYLNAVVGNALLI<I<YPQLAAEFII<GDYRI<NNAREENI<ANAI<MHFY SNIMNSFTSDVKIADETGEIVWDKEKDIATVRKVMNYHQVLITRKVEEEKGGFF DQTILSKGNSKKLIPLKKNLDPEKYGGYNSPTVAYSVLVEYDIEKGKKKKLKTV KQLVGIPIRERAKLEKNPIKYLEKKGYQNPKVDLLIKIPKNSLFELDGGRRRILA AAKELKNANQLVLPAEEYTLLDKVAKIIKKNNSESIEYVEEHLSEFDELLESLID YSPKLALQDKNLEKIKEAFEQLNLADKKEVAKEIINLLHCTATTANAALKFLGG SKNRMRYTSIKELLNASLIHQSITGLYETRIDLGKLGED

[0329] In some embodiments, the LBCA has an amino acid sequence as set forth in SEQ ID NO: 84

[0330] In some embodiments, the RNA-guided nuclease is an “Ancestral” Cas, referred to as LFCA, or a variant thereof.

[0331] In some embodiments, the LFCA, or the variant thereof has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 85.SEQ ID NO: 85MKKDYSIGLDIGTNSVGWAVITDDYKVVRKKMKVLGNTDKKSIKKNLWGVRL FDSGETAEDTRLKRTTRRRYTRRRNRICYLQEIFQEEMNKVDDSFFHRLDESFLVPEDI<I<YDRHPIFGNLEEEVAYHEQYPTIYHLRI<HLADSSEI<ADLRLVYLALA HIIKYRGHFLIEGDLNTENTDVEELFKQFVQVYNQTFEEQHLSDETIDVEEILTEK VSKSRRAENVLKLFPNEKKNGLFGQLIKLIVGLQGNFKKVFDLSEDAKLQFSKD TYEEDLENLLAMIGDEYADLFLAAKNLYDAILLSGILTTTDNNTKAKLSASMIK RYEEHQKDLAQLKQFIKEHLPDKYNEIFSDSSKNGYAGYIEGKTSQEDFYKYLK KILSKIDGAEYFLEKIEQENFLRKQRTFDNGVIPHQIHLEELRAILRRQGKYYPFL KENQEKIEQILTFRIPYYVGPLARGNSRFAWLTRKSDEPITPWNFDEVVDKEKSA EAFIERMTNYDTYLPNEI<VLPI<HSLLYEI<FTVFNELTI<VRYVTDRGI<TQNFSAE QKQEIFDDLFKKNRKVTKKKLENYLKKEYEYFDSPDITGIEDEFNASLGTYHDL LKILKSKDFLDDEENEEILEDIVKILTVFEDRKMIRKRLEKYSDILTEEQLKKLER RHYTGWGRLSRKLINGIRDKQSGKTILDYLIDDDSSNRNFMQLINDDNLSFKEEI EKAQVIGETENLHEIVQDLPGSPAIKKGILQSLKIVDELVKVMGHEPKNIVVEM ARENQTTSRGRRNSI<QRLI<RLEEALI<NLGSNLLI<EHPVDNQQLQNDRLYLYY LQNGKDMYTGQELDIDNLSNYDIDHIIPQSFIKDNSIDNRVLVSSEENRGKSDNV PSKEVVRKMKSFWQKLLNAGLISQRKFDNLTKAERGGLTEDDKAGFIKRQLVE TRQITKHVANILDSRFNTEKDEEDNTIRNVKIITLKSNLVSQFRKDFGLYKVREIN DYHHAHDAYLNAVVGTALLKKYPQLEPEFVYGDYRKNNAREENKATAKKHF YSNIMNFFASEVKIADETGEIVWDKEKDIATVRKVLSYHQVNIVKKVEVQKGG FSKETILPKGNSNKLIPRKNNWDPKKYGGFDSPTVAYSVLVTYDIEKGKKKKLK TVKELVGITIMERSAFEKNPIAYLEKKGYQNPQEDVLIKLPKYSLFELENGRRR MLASAKELQKGNQMVLPAHLVTLLYHAKRIDKSNNSESLEYVEEHRNEFDELL DYIIDFSEKYILADKNLEKIKKLYEQNNEADIKELAKSFINLLTFTAMGAPAAFK FFGETIDRKRYTSIKELLNATLIHQSITGLYETRIDLGKLGED

[0332] In some embodiments, the LFCA has an amino acid sequence as set forth in SEQ ID NO: 85

[0333] In some embodiments, the RNA-guided nuclease is a Cas beta (Cas-beta-M67), or a variant thereof.

[0334] In some embodiments, the Cas beta (Cas-beta-M67), or the variant thereof has an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 119.SEQ ID NO: 119MPKKKRKVGKNRSSSSDLSPLERSLRKVGENRLERLRVREEKIRKHIEQHPRGK NDHQALHFLLHQIEVERNDLYRNL1<DPEYVPI<PAI<QRRERRQINVAI<PPTRPI< KEKGPQPESTKYVIRPPVPGKNLPAFASKYEARDTRDDSYQDGRSWTSAPYVE VELPILGADKVIQKLMKFVQKDERSIVRDWATKTYSSIEAAREALLVGAQVSED VSVWRGLLAETKNAQNFAALSDDQIEAAMSKEAKGADLRPRRAALLVAQRH WVDQTVKAIKESAPSGVDKDTLDRRLRAGLRGFHTAANRGKHRNPQFPYLTA EKPVVPMESVVQSVLAFLDDPDDQRYTKDKERDKKRHRVTVLQKELGKARPR KRLELQTPKWAGRPTVKGTISKRRDAALVWDTSKEANGLCLALPIGGMPKIDV EQFIYQDGTSLLSDCQIASKTTKKGAACAVLPLKPKHDFLRWFTKHVENHNPD APLERRCLHNTTQFVIVDPEGPRPRLFVRPVFKFYDPGKTVPNTHETWKKPDCR YLVGIDRGINYVLRAVVVDTEEKKVIADIGLPGRKHEWRMIRDEIAYHQQMRD LARNTGKHASVVAKHVRALALARKKDRALGKFATVEAVARLVKKCEQDYGS GNYCFVLEDLDMGAMNLKRNNRVKHMAVMEEALVNQMRKQGYAYDGRRG RVDGVRHEGAWYTSQVSPFGWWAKRDEVEEAWKRDKTRPIGRKVGNWYEM PEPGQDGDRPDTYRKGYWSKPKNAEGKPYGRNRFSVRPGDRKPDAERRFCWG SELFWDPNVKSFKGKEFPEGVVLDADFVGALNIALRPLVNDGQGKGFKAEDM AREHTILNPQFKIACQIPVYEFVEEDGDKWAALRRIMLLEKRPAATKKAGQAK KKK

[0335] In some embodiments, the Cas beta (Cas-beta-M67) has an amino acid sequence as set forth in SEQ ID NO: 119.

[0336] In some embodiments, the RNA-guided nuclease has an amino acid sequence selected from the group comprising or consisting of any one of SEQ ID NO: 61 to SEQ ID NO: 85 and SEQ ID NO: 119 In some embodiments, the RNA-guided nuclease has an amino acid sequence selected from the group consisting of any one of SEQ ID NO: 61 to SEQ ID NO: 85 and SEQ ID NO: 119 In some embodiments, the RNA-guided nuclease has an amino acid sequence selected from the group comprising or consisting ofany one of SEQ ID NO: 61 to SEQ ID NO: 85. In some embodiments, the RNA-guided nuclease has an amino acid sequence selected from the group consisting of any one of SEQ ID NO: 61 to SEQ ID NO: 85

[0337] In certain embodiments, the RNA-guided nuclease is not dead Cas9. In certain embodiments, the RNA-guided nuclease is not a catalytically dead Cas protein.

[0338] The composition may comprise at least one transposase as described herein, and at least one RNA-guided nuclease as described herein; or a nucleic acid encoding the same.

[0339] The transposase and the RNA-guided nuclease may be separate (z.e., decoupled), or, alternatively, fused or otherwise linked together. When the transposase and the RNA-guided nuclease coupled, associated, fused or otherwise linked together, the resulting construct may be herein referred to as “FiCAT”.

[0340] In one embodiment, 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. Illustratively, the transposase and the RNA-guided nuclease may be encoded by distinct vectors.

[0341] In another embodiment, the 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.

[0342] In some embodiments, the transposase and the RNA-guided nuclease are fused together in a fusion protein, optionally through a linker (z.e., a fusion protein comprising the transposase, the RNA-guided nuclease, and a linker). In some embodiments, the RNA-guided nuclease is fused in C-terminus or N-terminus of the transposase. In certain embodiments, the transposase and the RNA-guided nuclease are covalently or non-covalently linked, preferably through a linker. In one embodiment, the transposase and the RNA-guided nuclease are covalently linked. In another embodiment, the transposase and the RNA-guided nuclease are non-covalently linked.

[0343] In one embodiment, the fusion protein comprises, from N-ter to C-ter, at least one transposase as described herein, at least one linker, and at least one RNA-guided nuclease as described herein (z.e., [transposase]-[linker]-[RNA-guided nuclease]). In another embodiment, the fusion protein comprises, from N-ter to C-ter, at least one RNA-guided nuclease as described herein, at least one linker, and at least one transposase as described herein (z.e., [RNA-guided nuclease]-[linker]-[transposase]).

[0344] In some embodiments, the linker comprises at least 1 amino acid, at least 10 amino acids, at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, or at least 80 amino acids. In some embodiments, the linker comprises from 1 to 100 amino acids. In some embodiments, the linker comprises from 1 to 90 amino acids, from 1 to 80 amino acids, from 1 to 70 amino acids, from 1 to 60 amino acids, from 1 to 50 amino acids, from 1 to 40 amino acids, from 1 to 30 amino acids, from 1 to 20 amino acids, or from 1 to 10 amino acids. In some embodiments, the linker comprises from 10 to 100 amino acids, from 20 to 100 amino acids, from 30 to 100 amino acids, from 40 to 100 amino acids, from 50 to 100 amino acids, from 60 to 100 amino acids, from 70 to 100 amino acids, from 80 to 100 amino acids, or from 90 to 100 amino acids. In some embodiments, the linker comprises from 1 to 40 amino acids, from 1 to 50 amino acids, from 1 to 60 amino acids, from 1 to 70 amino acids, or from 1 to 80 amino acids. In some embodiments, the linker comprises from 1 to 40 amino acids, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 amino acids. In some embodiments, the linker comprises at most 100 amino acids, at most 90 amino acids, at most 80 amino acids, at most 70 amino acids, at most 60 amino acids, at most 50 amino acids, at most 40 amino acids, at most 30 amino acids, at most 20 amino acids, or at most 10 amino acids.

[0345] In some embodiments, the linker is a peptidic linker with an amino acid sequence of any of SEQ ID NO: 87-94 and SEQ ID NO: 120-125

[0346] In some embodiments, the fusion protein further comprises at least one linker. In some embodiments, the linker is a peptidic linker selected from the group consisting of GGS3x linker, GGS4x linker, GGS5x linker, GGS6x linker, GGS7x linker, GGS8xlinker, XTEN linker, and linker B. In some embodiments, the linker is a peptidic linker with an amino acid sequence of any of SEQ ID NO: 87-94.

[0347] In some embodiments, the linker is a peptidic linker is selected from the group consisting of short linker having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 120, P9-rigid linker having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 121, (GGGGS)*10 linker having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 122, (PPPGSS)*7 linker having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 123, (EAAAK)*5-(GGGGS)*5 linker having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 124, and XTEN linker having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 125.

[0348] In some embodiments, the linker comprises from 1 to 20 repeats of SEQ ID NO: 126, from 5 to 15 repeats of SEQ ID NO: 126, or about 10 repeats of SEQ ID NO: 126.In other words, the linker may consist of an amino acid sequence of SEQ ID NO: 126, (SEQ ID NO: 126)2, (SEQ ID NO: 126)3, (SEQ ID NO: 126)4, (SEQ ID NO: 126)5, (SEQ ID NO: 126)6, (SEQ ID NO: 126)7, (SEQ ID NO: 126)8, (SEQ ID NO: 126)9, (SEQ ID NO: 126)io, (SEQ ID NO: 126)i i, (SEQ ID NO: 126)12, (SEQ ID NO: 126)13, (SEQ ID NO: 126)14, (SEQ ID NO: 126)is, (SEQ ID NO: 126)16, (SEQ ID NO: 126)17, (SEQ ID NO: 126)18, (SEQ ID NO: 126)19, or (SEQ ID NO: 126)20.

[0349] In some embodiments, the linker is a (GGGGS)*10 linker having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 122

[0350] In some embodiment, the fusion protein comprising the transposase and the RNA-guided nuclease has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 86.

[0351] In some embodiment, the fusion protein comprising the transposase and the RNA-guided nuclease comprises or consists of:- at least one RNA-guided nuclease, preferably a Cas beta with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 119;- at least one transposase, preferably having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, and SEQ ID NO: 104, more preferably a transposase with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 104; and- at least one linker selected from the group consisting of short linker having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 120, P9-rigid linker having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 121, (GGGGS)*10 linker having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 122, (PPPGSS)*7 linker having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 123, (EAAAK)*5-(GGGGS)*5 linker having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 124, and XTEN linker having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 125, preferably a (GGGGS)* 10 linker having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 122;wherein the at least one linker is localized between the at least one RNA-guided nuclease and the at least one transposase, andpreferably wherein the linker comprises from 1 to 40 amino acids, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 amino acids.

[0352] In one embodiment, the fusion protein comprising the transposase and the RNA-guided nuclease comprises, from N-ter to C-ter: (i) at least one RNA-guided nuclease, preferably a Cas beta with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 119; (ii) at least one linker selected from the group consisting of short linker having at least 80% identity with SEQ ID NO: 120, P9-rigid linker having at least 80% identity with SEQ ID NO: 121, (GGGGS)* 10 linker havingat least 80% identity with SEQ ID NO: 122, (PPPGSS)*7 linker having at least 80% identity with SEQ ID NO: 123, (EAAAK)*5-(GGGGS)*5 linker having at least 80% identity with SEQ ID NO: 124, and XTEN linker having at least 80% identity with SEQ ID NO: 125, preferably a (GGGGS)*10 linker having at least 80% identity with SEQ ID NO: 122; and (iii) at least one transposase, preferably having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, and SEQ ID NO: 104, more preferably a transposase with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 104;preferably wherein the linker comprises from 1 to 40 amino acids, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 amino acids.

[0353] In one embodiment, the fusion protein comprising the transposase and the RNA-guided nuclease comprises, from N-ter to C-ter: (i) at least one transposase, preferably having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, and SEQ ID NO: 104, more preferably a transposase with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 104; (ii) at least one linker selected from the group consisting of short linker having at least 80% identity with SEQ ID NO: 120, P9-rigid linker having at least 80% identity with SEQ ID NO: 121, (GGGGS)*10 linker having at least 80% identity with SEQ ID NO: 122, (PPPGSS)*7 linker having at least 80% identity with SEQ ID NO: 123, (EAAAK)*5-(GGGGS)*5 linker having at least 80% identity with SEQ ID NO: 124, and XTEN linker having at least 80% identity with SEQ ID NO: 125, preferably a (GGGGS)* 10 linker having at least 80% identity with SEQ ID NO: 122; and (iii) at least one RNA-guided nuclease, preferably a Cas beta with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 119;

[0354] preferably wherein the linker comprises from 1 to 40 amino acids, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 amino acids.

[0355] In some embodiment, the fusion protein comprising the transposase and the RNA-guided nuclease comprises or consists of:- at least one RNA-guided nuclease, preferably a Cas beta with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 119;- at least one transposase, preferably having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, and SEQ ID NO: 104, more preferably a transposase with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 104; and- at least one linker comprising from 1 to 20 repeats of SEQ ID NO: 126, from 5 to 15 repeats of SEQ ID NO: 126, or about 10 repeats of SEQ ID NO: 126;wherein the at least one linker is localized between the at least one RNA-guided nuclease and the at least one transposase, andpreferably wherein the linker comprises from 1 to 40 amino acids, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 amino acids.

[0356] In one embodiment, the fusion protein comprising the transposase and the RNA-guided nuclease comprises, from N-ter to C-ter: (i) at least one RNA-guided nuclease, preferably a Cas beta with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 119; (ii) at least one linker comprising from 1 to 20 repeats of SEQ ID NO: 126, from 5 to 15 repeats of SEQ ID NO: 126, or about 10 repeats of SEQ ID NO: 126; and (iii) at least one transposase, preferably having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, and SEQ ID NO: 104, more preferably a transposase with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 104;preferably wherein the linker comprises from 1 to 40 amino acids, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 amino acids.

[0357] In another embodiment, the fusion protein comprising the transposase and the RNA-guided nuclease comprises, from N-ter to C-ter: (i) at least one transposase, preferably having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, and SEQ ID NO: 104, more preferably a transposase with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 104; (ii) at least one linker comprising from 1 to 20 repeats of SEQ ID NO: 126, from 5 to 15 repeats of SEQ ID NO: 126, or about 10 repeats of SEQ ID NO: 126; and (iii) at least one RNA-guided nuclease, preferably a Cas beta with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 119;preferably wherein the linker comprises from 1 to 40 amino acids, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 amino acids.

[0358] The compositions of the invention comprise a gRNA. The gRNA is at least partially complementary to a target nucleic acid sequence; further, it will be apparent to the person skilled in the art that the gRNA is capable of interacting with the RNA-guided nuclease. Hence, the gRNA is used to guide the RNA-guided nuclease to a specific site; when the transposase and the RNA-guided nuclease are associated together, fused, or otherwise linked, the integration activity of the transposase is thus directed to this specific site, enabling targeted (specific) integration. In some embodiments, the gRNA is recognized by the RNA-guided nuclease. Typically, 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 (and the associated, fused, or otherwise linked transposase) to this specific locus.

[0359] In some embodiments, the transposase is fused to an aptamer binding protein (ABP), and the gRNA of the RNA-guided nuclease 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 as described hereinabove. The at least one aptamer sequence may be DNA or RNA, preferably RNA. The gRNA may comprise more than one aptamer sequence, z.e., 2, 3, 4, 5, 6, 7, 8, 9, or more. It will be noted that when the transposase is fused to the RNA-guided nuclease, the transposase is not further fused to an ABP.

[0360] In some embodiments, the aptamer binding protein is MS2 bacteriophage coat protein (MCP) and the at least one aptamer is a MS2 RNA tetraloop binding sequence. In some embodiments, MCP has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 95. In some embodiments, MS2 RNA tetraloop binding sequence has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 96.

[0361] Examples of covalent and non-covalent association of transposases with RNA-guided nuclease and gRNA are disclosed in W02020250181 and WO2022129438, the entire content of which is incorporated herein by reference. It will be understood that 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; in particular, the binding of the fusion protein to the RNA-guided nuclease / gRNA complex directs the activity of the transposase of the invention towards the site specifically recognized by the RNA-guided nuclease / gRNA complex. Non-limitative examples of gRNA include those of SEQ ID NO: 111-113.

[0362] In some embodiments, the at least one RNA-guided nuclease and the transposase are fused together through a linker, preferably the at least one RNA-guided nuclease and the transposase are fused together through a linker wherein the at least one RNA-guided nuclease is fused to the C terminal end of the transposase.

[0363] In some embodiments, the aptamer binding protein (ABP) is a MS2 bacteriophage coat protein (MCP) sharing at least 75% identity with SEQ ID NO: 95;and wherein the aptamer sequence is a MS2 RNA tetraloop binding sequence sharing at least 75% identity with SEQ ID NO: 96.

[0364] The compositions of the invention comprise a nucleic acid molecule comprising a transgene encoding a protein of interest; wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences. In other words, the compositions of the invention comprise a supercoiled circular dsDNA molecule comprising a transgene flanked by ITRs, wherein the transgene encodes a protein of interest.

[0365] It is known in the art that supercoiled DNA refers to a form of DNA that is twisted and coiled beyond its relaxed, double-helix structure. This supercoiling can occur naturally in cellular DNA as a way to compact the DNA and fit it within the limited space of the cell nucleus or in plasmids. Hence, supercoiled circular dsDNA vectors are more compact, and thus advantageous for improved transfection efficiency.

[0366] Non-limitative examples of conditions that influence the supercoiled state of DNA include the activity of topoisomerases and DNA binding agents, mechanical conditions such as conditions used for in vitro supercoiling, and ionic conditions, in particular regarding Mg2+or K+ions levels.

[0367] The person skilled in the art readily knows how to measure the degree of DNA supercoiling in a DNA molecule, namely by measuring the superhelical density (o), which is calculated from the linking number (Lk), wherein Lk is the total number of times one strand wraps around the other, and its difference from the linking number of a relaxed molecule (Lko) gives the specific linking difference (ALk), which is then normalized by the length of the DNA to get the superhelical density. Further methods include using psoralen compounds to detect intercalation levels or employing gel electrophoresis with intercalating agents like chloroquine to separate topoisomers; or by electrophoresis without restriction enzyme; or the use of optical tweezers to measure DNA supercoiling by suspending a DNA molecule between two trapped beads and applying controlled forces and torques; or the use of psoralen / trimethyl-psoralen (TMP) crosslinking andsequencing; or the use of plasmid topoisomer analysis (agarose / chloroquine gels); or the use of supercoiling-sensitive reporter genes combined with fluorescence sensors; or the use of type IB topoisomerase (Topi) inhibitors-based assays such as described in Seol Y, et al. Single-Molecule Supercoil Relaxation Assay as a Screening Tool to Determine the Mechanism and Efficacy of Human Topoisomerase IB Inhibitors. Mol Cancer Ther. 2015 Nov;14(ll):2552-9; or commercially available assays such as ThermoFisher Scientific’s NanoDrop One / OneC Spectrophotometer, that relies on absorbance measurement to assess the hypochromic shift of supercoiled DNA compared to native DNA.

[0368] Hence, in some embodiments, the degree or level of supercoiling is measured by electrophoresis assays, fluorescence assays, absorbance assays, spectrophotometry assays, optical tweezers-based assays, Topi -based assays, agarose / chloroquine electrophoresis assays, optionally associated with psoralen / trimethyl-psoralen (TMP) crosslinking and sequencing, or any combination thereof. In some embodiments, the degree or level of supercoiling is measured by electrophoresis, preferably by electrophoresis without restrictions enzymes.

[0369] In some embodiments, the supercoiled circular dsDNA molecule is positively supercoiled (over-twisted) or negatively supercoiled (under-twisted), preferably positively supercoiled.

[0370] In some embodiments, the supercoiled circular dsDNA molecule is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or at least 100% supercoiled. In some preferred embodiments, the supercoiled circular dsDNA molecule is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or at least 100% supercoiled.

[0371] In some embodiments, the supercoiled circular dsDNA molecule is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% more supercoiled compared to doggybone DNA (dbDNA™). In some embodiments, the supercoiled circular dsDNA molecule is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% more supercoiled compared to plasmid DNA. In some embodiments, the supercoiled circular dsDNA molecule is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% more supercoiled compact compared to relaxed DNA.

[0372] In some embodiments, the supercoiled circular dsDNA molecule yields an expression of the transgene that is about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more, higher compared to a doggybone DNA (dbDNA™) vector comprising the same transgene. In some embodiments, the supercoiled circular dsDNA molecule yields an expression of the transgene that is about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more, higher compared to a plasmid DNA vector comprising the same transgene. In some embodiments, the supercoiled circular dsDNA molecule yields an expression of the transgene that is about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more, higher compared to a relaxed DNA vector comprising the same transgene.

[0373] In some embodiments, the supercoiled circular dsDNA molecule has increased transfection efficiency by about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more, compared to a doggybone DNA (dbDNA™) vector comprising the same transgene. In some embodiments, the supercoiled circular dsDNA molecule has increased transfection efficiency by about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more, compared to a plasmid DNA vector comprising the same transgene. Insome embodiments, the supercoiled circular dsDNA molecule has increased transfection efficiency by about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more, compared to a relaxed DNA vector comprising the same transgene.

[0374] In some embodiments, the supercoiled circular dsDNA molecule is about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more, more compact compared to doggybone DNA (dbDNA™). In some embodiments, the supercoiled circular dsDNA molecule is about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more, more compact compared to plasmid DNA. In some embodiments, the supercoiled circular dsDNA molecule is about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more, more compact compared to relaxed DNA.

[0375] In some embodiments, the supercoiled circular dsDNA molecule is about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more, more stable compared to doggybone DNA (dbDNA™), in particular in vivo. In some embodiments, the supercoiled circular dsDNA molecule is about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more, more stable compared to plasmid DNA, in particular in vivo. In some embodiments, the supercoiled circular dsDNA molecule is about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more, more stable compared to relaxed DNA, in particular in vivo.

[0376] In some embodiments, the supercoiled circular dsDNA molecule is about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more, less immunogenic compared to doggybone DNA (dbDNA™). In some embodiments, the supercoiled circular dsDNA molecule is about 1.1 -fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold,1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more, less immunogenic compared to plasmid DNA. In some embodiments, the supercoiled circular dsDNA molecule is about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more, less immunogenic compared to relaxed DNA.

[0377] In some embodiments, the supercoiled circular dsDNA molecule is about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more, less cytotoxic compared to doggybone DNA (dbDNA™). In some embodiments, the supercoiled circular dsDNA molecule is about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more, less cytotoxic compared to plasmid DNA. In some embodiments, the supercoiled circular dsDNA molecule is about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more, less cytotoxic compared to relaxed DNA.

[0378] In some embodiments, the supercoiled circular dsDNA molecule is about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more, safer compared to doggybone DNA (dbDNA™). In some embodiments, the supercoiled circular dsDNA molecule is about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more, safer compared to plasmid DNA. In some embodiments, the supercoiled circular dsDNA molecule is about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more, safer compared to relaxed DNA.

[0379] Supercoiled circular double-stranded DNA vectors can be generated through methods known from the art. The supercoiled circular DNA molecule may be isolated and purified. The person skilled in the art readily knows how to measure the degree of supercoiling, illustratively by linking number, twist, and / or writhe of the DNA molecule; such means may include topoisomerase assays, DNA relaxation assays,spectrophotometric methods, and / or agarose gel electrophoresis. Means to produce supercoiled circular double-stranded DNA vectors have been described in the art.

[0380] In one embodiment, the supercoiled circular dsDNA molecule further comprises on or more components selected from the group consisting of an origin of replication (ori), selectable marker genes, promoter regions, multiple cloning site (MCS), restriction enzyme sites, a transcription termination signal, enhancers, a polyadenylation signal, introns, and any combinations thereof. In some embodiments, the supercoiled circular dsDNA molecule further comprises an origin of replication. In some embodiments, the supercoiled circular dsDNA molecule further comprises at least one selectable marker gene (illustratively, an antibiotic resistance gene).

[0381] In another embodiment, the supercoiled circular dsDNA molecule does not comprise a selectable marker gene, nor an origin of replication. In some embodiments, the supercoiled circular dsDNA molecule does not comprise a selectable marker gene. In some embodiments, the supercoiled circular dsDNA molecule does not comprise an antibiotic resistance gene. In some embodiments, the supercoiled circular dsDNA molecule does not comprise an origin of replication, in particular the supercoiled circular dsDNA molecule does not comprise a bacterial origin of replication.

[0382] In some embodiments, the supercoiled circular dsDNA molecule comprises:- a transgene encoding a protein of interest,- ITRs flanking the transgene ( / . e. , at least one ITR sequence adj acent to the 5 ’P extremity of the transgene, and at least one ITR sequence adjacent to the 3 ’OH extremity of the transgene),- an origin of replication, and- at least one selectable marker gene.

[0383] In some embodiments, more than 90% of the nucleic acid molecules of the composition that comprise the transgene are supercoiled. In some embodiments, more than 95%, 96%, 97%, 98%, or 99% of the nucleic acid molecules of the composition thatcomprise the transgene are supercoiled. In some embodiments, less than 10% of the nucleic acid molecules of the composition that comprise the transgene are not supercoiled or are relaxed. In some embodiments, less than 5%, 4%, 3%, 2%, or 1% of the nucleic acid molecules of the composition that comprise the transgene are not supercoiled or are relaxed.

[0384] In some embodiments, the supercoiled circular dsDNA molecule that comprises the transgene is a supercoiled circular dsDNA vector. Such molecules can be prepared by methods known in the art, illustratively by means using topoisomerases, DNA binding agents, mechanical manipulation, in vitro supercoiling, or any combination thereof. Alternatively, supercoiled circular dsDNA vectors are available commercially, such as GenCircle™ (GenScript) or Minicircle™ (Plasmid Factory). In some embodiments, the supercoiled circular dsDNA molecule that comprises the transgene is a GenCircle™ molecule or a Minicircle™ molecule.

[0385] In some embodiments, the supercoiled circular dsDNA molecule has a length of at least 102bp, at least 103bp, at least 104bp, at least 105bp, at least 106bp, at least 107bp, or at least 108bp.

[0386] In some embodiments, the supercoiled circular dsDNA molecule has a vector backbone from about 200 bp to about 600 bp.

[0387] In some embodiments, the supercoiled circular dsDNA molecule has less than 5% residual host DNA.

[0388] The transgene is comprised in the supercoiled circular dsDNA molecule; therefore, the transgene is also a DNA sequence and has the same structural features as the molecule comprising thereof (supercoiled, circular).

[0389] In some embodiments, the transgene has a size or length of at least 10 base pairs (bp), such as at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1 000, 2000, 3 000, 4000, 5 000, 6000, 7000, 8 000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000 bp or more. In some embodiments, the transgene has a size or length of at least 2 kilo-base-pair (kb), such asat least 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 50 kb, or more. In some embodiments, the transgene has a size or length from 2 kb to 100 kb. In some embodiments, the transgene has a size or length from 2 kb to 90 kb, from 2 kb to 80 kb, from 2 kb to 70 kb, from 2 kb to 60 kb, from 2 kb to 50 kb, from 2 kb to 40 kb, from 2 kb to 30 kb, from 2 kb to 20 kb, or from 2 kb to 10 kb. In some embodiments, the transgene has a size or length from 2 kb to 100 kb, from 2.5 kb to 100 kb, from 4.7 kb to 100 kb, from 5 kb to 100 kb, from 8 kb to 100 kb, from 10 kb to 100 kb, from 15 kb to 100 kb, from 20 kb to 100 kb, from 30 kb to 100 kb, or from 50 kb to 100 kb. In some embodiments, the transgene has a size or length between 8 kb and 20 kb; for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. In some embodiments, the transgene has a size or length of 2 kb or more, including 2, 3, 4, 5, 6, 7, 8 or more than 8 kb. In some embodiments, the transgene has a size or length of more than 20 kb, for example more than 20, 25, 30, 35, 40, 45 50, or more than 50 kb. In some embodiments, the transgene has a size or length from 1 kb to 20 kb. In some embodiments, the transgene is a large DNA fragment having a size or length from 5 kb to 25 kb, preferably from 8 kb to 20 kb. In some embodiments, the transgene is a large DNA fragment having a size or length comprised between 5 kb and 25 kb, preferably between 8 kb and 20 kb. In some embodiments, the transgene has a size or length from 2.5 kb to 6.3 kb. In some embodiments, the transgene has a size or length from 5 kb to 50 kb, from 8 kb to 50 kb, from 11 kb to 50 kb, or from 15 kb to 50 kb. In some embodiments, the transgene has a size or length of more than 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, or more.

[0390] In some embodiments, the transgene encodes a protein of interest selected from the group consisting of a chimeric antigen receptor (CAR), an enzyme, a transcription factor, a growth factor, a trophic factor, a hormone, a cytokine, an antibody, an antigen, a receptor, an immune regulator, a differentiation factor, a suicide protein, a cell-cycle modifying protein, an anti-proliferative protein, an angiogenic factor, an anti-angiogenic factor, a genome editor, a nuclease, a recombinase, a neurotransmitter, and a reporter, including any precursor thereof, as well as fusion proteins.

[0391] In some embodiments, the transgene encodes a chimeric antigen receptor (CAR). CARs are engineered receptors that are grafted onto T-cells to enable them to recognize and target specific cancer cells. The most common targets for CAR-T cell therapies are surface proteins on cancer cells. In some embodiments, the CAR is a surface protein. In some embodiments, the CAR is a cancer surface protein.

[0392] In some embodiments, the transgene encodes a chimeric antigen receptor (CAR) selected from the group consisting of CD19 (CAR19), BCMA (B-cell maturation antigen), CD22, CD20, Mesothelin, HER2 / ErbB2 (Human epidermal growth factor receptor 2), EGFR (Epidermal growth factor receptor), Glypican-3 (GPC3), R0R1 (Receptor tyrosine kinase-like orphan receptor 1), MUC1 (Mucin 1), NKG2D ligands, PSMA (Prostate-specific membrane antigen), and IL-13Ra2 (Interleukin- 13 receptor alpha 2).

[0393] In some embodiments, the transgene encodes a chimeric antigen receptor (CAR) selected from the group consisting of CD19 (CAR19), BCMA (B-cell maturation antigen), CD22, CD20, Mesothelin, HER2 / ErbB2 (Human epidermal growth factor receptor 2), EGFR (Epidermal growth factor receptor), Glypican-3 (GPC3), R0R1 (Receptor tyrosine kinase-like orphan receptor 1), and MUC1 (Mucin 1).

[0394] In some embodiments, the transgene encodes a chimeric antigen receptor (CAR) selected from the group consisting of CD19 (CAR19), BCMA (B-cell maturation antigen), and CD22.

[0395] In some embodiments, the transgene encodes a chimeric antigen receptor (CAR) CD 19 (CAR19).

[0396] It will be understood that the compositions of the invention comprise a nucleic acid molecule comprising a transgene encoding a protein of interest, wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0397] Hence, it will be apparent for the person skilled in the art to use an ITR suitable for the corresponding transposase. The person skilled in the art readily knows what ITRs are recognized by a given transposase, therefore the person skilled in the art knows what ITR should be flanking the transgene in the supercoiled circular dsDNA molecule of the composition. Certain ITRs and their corresponding transposases are also described hereinabove. For example, if the composition comprises the Poeciliopsis turrubarensis transposase as described herein, the ITR flanking the transgene are SEQ ID NO: 45 and SEQ ID NO: 46

[0398] In some embodiments, on the supercoiled circular dsDNA molecule comprising the transgene, the ITR flanking the transgene are selected depending on the transposase used. In some embodiments, on the supercoiled circular dsDNA molecule comprising the transgene, the ITR flanking the transgene are selected from the ITR sequences described hereinabove, and are suitable for the transposase used as described hereinbefore.

[0399] In some embodiments, at least one ITR sequence is adjacent to the 5’P extremity of the transgene sequence (“left” ITR), and at least one ITR sequence is adjacent to the 3 ’OH extremity of the transgene sequence (“right” ITR), wherein the ITR sequences may be the same or different, preferably different.

[0400] In one embodiment, the ITR sequences flanking the transgene are separated by a spacer sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acids. In another embodiment, the ITR sequences flanking the transgene are in direct contact with the transgene.

[0401] In some embodiments, the supercoiled circular dsDNA molecule comprising the transgene comprises at least one ITR sequence, preferably at least two ITR sequences, having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with one or more sequence selected from the group consisting of SEQ ID NO: 45 to SEQ ID NO: 60 In some embodiments, the supercoiled circular dsDNA molecule comprising the transgene comprises at least one ITR sequence, preferably at least two ITR sequences, having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with one or more sequence selected from the group consisting of SEQ ID NO: 45 to SEQ ID NO: 52. In some embodiments, the supercoiled circular dsDNA molecule comprising the transgene comprises at least one ITR sequence, preferably at least two ITR sequences, having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with one or more sequence selected from the group consisting of SEQ ID NO: 52 to SEQ ID NO: 60. In some embodiments, the supercoiled circular dsDNA molecule comprising the transgene comprises at least one ITR sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO: 45 and at least one ITR sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO: 46 In some embodiments, the supercoiled circular dsDNA molecule comprising the transgene comprises at least one ITR sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO: 47 and at least one ITR sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO: 48. In some embodiments, the supercoiled circular dsDNA molecule comprising the transgene comprises at least one ITR sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO: 49 and at least one ITR sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO: 50. In some embodiments, the supercoiled circular dsDNA molecule comprising the transgene comprises at least one ITR sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO: 51 and at least one ITR sequence having at least 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO: 52.

[0402] In some embodiments, the supercoiled circular dsDNA molecule comprising the transgene comprises at least one ITR sequence, preferably at least two ITR sequences, selected from the group consisting of SEQ ID NO: 45 to SEQ ID NO: 60. In some embodiments, the supercoiled circular dsDNA molecule comprising the transgene comprises at least one ITR sequence, preferably at least two ITR sequences, selected from the group consisting of SEQ ID NO: 45 to SEQ ID NO: 52. In some embodiments, the supercoiled circular dsDNA molecule comprising the transgene comprises at least one ITR sequence, preferably at least two ITR sequences, selected from the group consisting of SEQ ID NO: 52 to SEQ ID NO: 60. In some embodiments, the supercoiled circular dsDNA molecule comprising the transgene comprises at least one ITR sequence of SEQ ID NO: 45 and at least one ITR sequence of SEQ ID NO: 46. In some embodiments, the supercoiled circular dsDNA molecule comprising the transgene comprises at least one ITR sequence of SEQ ID NO: 47 and at least one ITR sequence of SEQ ID NO: 48. In some embodiments, the supercoiled circular dsDNA molecule comprising the transgene comprises at least one ITR sequence of SEQ ID NO: 49 and at least one ITR sequence of SEQ ID NO: 50 In some embodiments, the supercoiled circular dsDNA molecule comprising the transgene comprises at least one ITR sequence of SEQ ID NO: 51 and at least one ITR sequence of SEQ ID NO: 52.

[0403] In some embodiments, the supercoiled circular dsDNA molecule comprising the transgene is not a doggybone DNA (dbDNA™).

[0404] In some embodiments, the composition of the invention further comprises an inhibitor or antagonist of the protein STING, preferably the inhibitor of the protein STING is a pharmaceutical inhibitor of STING. As used herein, the expressions “STING inhibitor” and “STING antagonist” are used interchangeably. In certain embodiments, the inhibitor or antagonist of the protein STING is a selective inhibitor or antagonist of the protein STING, ie., it does not modulate other proteins and / or pathways.

[0405] In a non-exhaustive manner, examples of small molecule inhibitors targeting STING are disclosed in Kaifeng Liu et al. “Development of small molecule inhibitors / agonists targeting STING for disease”; Biomedicine & Pharmacotherapy, Vol.132, 2020; Cui X et al., “STING modulators: Predictive significance in drug discovery.” Eur JMed Chem. 2019Nov 15; 182: 111591 ; orLiuB etal., “A cell-based high throughput screening assay for the discovery of cGAS-STING pathway agonists.” Antiviral Res.2017 Nov; 147:37-46.

[0406] In some embodiments, the inhibitor of STING is selected from the group consisting of H-151, CCCP, C-176, Omaveloxolone, SN-011, STING-IN-2, C-178, PROTAC STING Degrader- 1, STING-IN-3, SN-001, LB244, STING-IN-6, JAK-IN-23, STING-IN-7, STING-IN-4, SN-008, STING-IN-5, C-di-IMP, PPARa agonist 4, STING modulator-3, Eupenici sirenin C, STING-IN-8, BSP16, Anti-inflammatory agent 65, and Cladophorol A.

[0407] In a preferred embodiment, the inhibitor of STING is H-151. It is known in the art that H-151 acts by inhibiting STING and this results in reduced expression of inflammatory cytokines upon dsDNA sensing. H-151 has CAS No. 941987-60-6, and is commercially available at, for example, Merck™ Cat. No.: SML2437, InvivoGen Cat. No.: inh-hl51 or MedChemExpress Cat. No.: HY- 112693.

[0408] In some embodiments, the composition of the invention further comprises an anticancer agent. Anticancer agents are known in the art and comprise, illustratively, chemotherapy agents, targeted therapy agents, immunotherapy agents, hormone therapy agents, monoclonal antibodies, tyrosine kinase inhibitors, antimetabolites, alkylating agents, taxanes, platinum-based drugs, topoisomerase inhibitors, proteasome inhibitors, histone deacetylase inhibitors, checkpoint inhibitors, oncolytic viruses.

[0409] In certain embodiments, the transposase is present in the composition under the form of mRNA. In certain embodiments, the transposase and the RNA-guided nuclease are present in the composition under the form of mRNA.

[0410] In some embodiments, the composition comprises:a) a messenger RNA (mRNA molecule) encoding a polypeptide comprising at least one transposase;b) a nucleic acid molecule comprising a transgene encoding a protein of interest;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0411] In some embodiments, the composition comprises:a) a messenger RNA (mRNA molecule) encoding a polypeptide comprising (i) at least one transposase, and (ii) at least one RNA-guided nuclease;b) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease; andc) a nucleic acid molecule comprising a transgene encoding a protein of interest;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0412] In some embodiments, the composition comprises:a) a messenger RNA (mRNA molecule) encoding at least one RNA-guided nuclease;b) a messenger RNA (mRNA molecule) encoding a polypeptide comprising (i) at least one transposase, and (ii) at least one aptamer binding protein (ABP); or a nucleic acid encoding the same;c) a nucleic acid molecule comprising (i) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, and (ii) at least one aptamer sequence; wherein the gRNA is capable of interacting with the RNA-guided nuclease; andd) a nucleic acid molecule comprising a transgene encoding a transgene of interest;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0413] The transposases and RNA-guided nucleases have been described hereinabove.

[0414] In some embodiments, the mRNA molecule encoding the RNA-guide nuclease has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 105.

[0415] In some embodiments, the mRNA molecule encoding the transposase has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 107, preferably the mRNA molecule encoding the transposase does not have the nucleic acid sequence of SEQ ID NO: 106

[0416] In some embodiments, the composition of the invention is comprised in a nanoparticle. In some embodiments, the composition of the invention is comprised in a nanoparticle selected from the group consisting of liposomes, solid lipid nanoparticles, nanostructured lipid carriers, lipid-core micelles, lipid emulsions, vesicles, exosomes, phospholipid-based nanoparticles, lipid-polymer hybrids, and self-assembled lipid nanoparticles.

[0417] In some embodiments, the composition of the invention is not comprised in, nor associated with, a viral vector, in particular an integrative viral vector.

[0418] The present invention further relates to a pharmaceutical composition comprising the composition according to the invention, and at least one pharmaceutically acceptable excipient.

[0419] The present invention further relates to a kit comprising:a) a polypeptide comprising (i) at least one transposase, and (ii) optionally at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease;c) a nucleic acid molecule comprising a transgene encoding a protein of interest; andd) one or more eukaryotic cells comprising the target nucleic acid sequence;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0420] The present invention further relates to a kit comprising:a) at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) a polypeptide comprising (i) at least one transposase, and (ii) at least one aptamer binding protein (ABP); or a nucleic acid encoding the same;c) a nucleic acid molecule comprising (i) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, and (ii) at least one aptamer sequence; wherein the gRNA is capable of interacting with the RNA-guided nuclease;d) a nucleic acid molecule comprising a transgene encoding a protein of interest; ande) one or more eukaryotic cells comprising the target nucleic acid sequence;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0421] The present invention further relates to a kit comprising:a) a polypeptide comprising (i) at least one transposase, and (ii) at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease;c) a nucleic acid molecule comprising a transgene encoding a protein of interest; andd) one or more eukaryotic cells comprising the target nucleic acid sequence;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0422] The transposases and RNA-guided nucleases have been described hereinabove.

[0423] In one embodiment, the at least one transposase and the at least one RNA-guided nuclease are provided in the form of mRNAs. In another embodiment, the at least one transposase and the at least one RNA-guided nuclease are provided in the form of proteins.

[0424] In some embodiments, the one or more eukaryotic cells are selected from the group consisting of primary cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells, and immune cells. In some embodiments, the one or more eukaryotic cells are selected from the group consisting of primary cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells, and T cells. In some embodiments, the one or more eukaryotic cells are selected from the group consisting of hematopoietic stem cells, and immune cells, preferably T cells. In some embodiments, the one or more eukaryotic cells are selected from the group consisting of primary cells, induced pluripotent stem cells (iPSCs), and hematopoietic stem cells. In some embodiments, the one or more eukaryotic cells are selected from the group consisting of primary cells and induced pluripotent stem cells (iPSCs). In some embodiments, the one or more eukaryotic cells are selected from the group consisting of induced pluripotent stem cells (iPSCs), hematopoietic stem cells, and immune cells. In some embodiments, the one or more eukaryotic cells are selected from the group consisting of induced pluripotent stem cells (iPSCs) and hematopoietic stem cells. In some embodiments, the one or more eukaryotic cells are selected from the group consisting of primary cells and immune cells, preferably T cells.

[0425] In one embodiment, the one or more eukaryotic cells are primary cells.

[0426] In some embodiments, the primary cells are selected from the group consisting of fibroblasts, keratinocytes, endothelial cells, hepatocytes, cardiomyocytes, neurons, chondrocytes, osteoblasts, adipocytes, lymphocytes, macrophages, dendritic cells, smooth muscle cells, skeletal muscle cells, epithelial cells, mesenchymal stem cells, schwann cells, astrocytes, microglia, melanocytes, pancreatic beta cells, retinal cells, glial cells, peripheral blood mononuclear cell (PBMC), and oligodendrocytes.

[0427] In some embodiments, the primary cells are selected from the group consisting of PBMC, lymphocytes, macrophages, dendritic cells. In some embodiments, the primary cells are selected from the group consisting of lymphocytes, macrophages, dendritic cells.

[0428] In some embodiments, the primary cells are lymphocytes. In some embodiments, the primary cells are T cells.

[0429] In some embodiments, the primary cells are PBMC. In certain embodiments, the primary cells are lymphocytes, preferably T cells, isolated from PBMC.

[0430] In another embodiment, the one or more eukaryotic cells are induced pluripotent stem cells (iPSCs). In some embodiments, the iPSCs are blood-derived iPSCs. Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cell that is generated by reprogramming somatic (adult) cells to a pluripotent state, allowing them to differentiate into virtually any cell type in the body. iPSCs have similar characteristics to embryonic stem cells, including the ability to self-renew and differentiate into various cell lineages. iPSCs are typically obtained after contacting a somatic cell with reprograming factor such as, e.g., Oct4, Sox2, Klf4, and / or c-Myc.

[0431] In another embodiment, the one or more eukaryotic cells are hematopoietic stem cells. Hematopoietic stem cells (HSCs) or “Hematopoietic Stem and Progenitor Cells” (HSPCs) are multipotent stem cells found primarily in the bone marrow that have the ability to give rise to all types of blood cells, including red blood cells, white blood cells, and platelets. They are essential for the continuous replenishment of the blood system throughout a person's life. HSCs are capable of both self-renewal (producing more stemcells) and differentiation (producing specialized blood cells), making them critical for maintaining the hematopoietic system and supporting immune function.

[0432] In another embodiment, the one or more eukaryotic cells are immune cells, preferably T cells. In some embodiments, the immune cells are selected from the group consisting of T lymphocytes (T cell), B lymphocytes (B cells), natural killer cells (NK), macrophages, dendritic cells, neutrophils, basophils, eosinophils, monocytes, plasma cells, mast cells, regulatory T cells, helper T cells, cytotoxic T cells, memory T cells, antigen-presenting cells, innate lymphoid cells, gamma delta T cells. In some embodiments, the immune cells are lymphocytes, preferably T cells. In some embodiments, the immune cells are selected from the group consisting of regulatory T cells, helper T cells, cytotoxic T cells, and memory T cells. In some embodiments, the immune cells are selected from the group consisting of helper T cells, cytotoxic T cells, and memory T cells. In some embodiments, the immune cells are helper T cells. In some embodiments, the immune cells are cytotoxic T cells. In some embodiments, the immune cells are memory T cells.

[0433] The present invention further relates to the composition of the invention, or the pharmaceutical composition of the invention, for use for treating a genetic disease in a subject in need thereof.

[0434] The present invention further relates to a method of treating a genetic disease in a subject in need thereof, comprising administering to said subject a therapeutically effective dose of the composition of the invention, or the pharmaceutical composition of the invention.

[0435] The present invention further relates to the use of the composition of the invention, or the pharmaceutical composition comprising thereof, for the manufacture of a medicament for treating a genetic disease in a subject in need thereof.

[0436] The present invention further relates to an in vitro method for site specific integration of a transgene into the genome of one or more eukaryotic cells, the method comprising delivering to the one or more eukaryotic cells the composition according to the invention.

[0437] In some embodiments, the composition of the invention is delivered to the one or more eukaryotic cells by transfection. In some embodiments, the composition is transfected in the one or more eukaryotic cells by at least one technique selected from the group comprising or consisting of lipofection, electroporation, sonication, nanoparticles, microinjection, and PEI.

[0438] In some embodiments, the composition of the invention is delivered to the one or more eukaryotic cells at a concentration from about 104cells / mL to about IO10cells / mL.

[0439] In some embodiments, the one or more eukaryotic cells are selected from the group consisting of primary cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells, and immune cells. In some embodiments, the one or more eukaryotic cells are selected from the group consisting of primary cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells, and T cells. In some embodiments, the one or more eukaryotic cells are selected from the group consisting of hematopoietic stem cells, and immune cells, preferably T cells. In some embodiments, the one or more eukaryotic cells are selected from the group consisting of primary cells, induced pluripotent stem cells (iPSCs), and hematopoietic stem cells. In some embodiments, the one or more eukaryotic cells are selected from the group consisting of primary cells and induced pluripotent stem cells (iPSCs). In some embodiments, the one or more eukaryotic cells are selected from the group consisting of induced pluripotent stem cells (iPSCs), hematopoietic stem cells, and immune cells. In some embodiments, the one or more eukaryotic cells are selected from the group consisting of induced pluripotent stem cells (iPSCs) and hematopoietic stem cells. In some embodiments, the one or more eukaryotic cells are selected from the group consisting of primary cells and immune cells, preferably T cells.

[0440] In one embodiment, the one or more eukaryotic cells are primary cells.

[0441] In some embodiments, the primary cells are selected from the group consisting of fibroblasts, keratinocytes, endothelial cells, hepatocytes, cardiomyocytes, neurons, chondrocytes, osteoblasts, adipocytes, lymphocytes, macrophages, dendritic cells, smooth muscle cells, skeletal muscle cells, epithelial cells, mesenchymal stem cells,schwann cells, astrocytes, microglia, melanocytes, pancreatic beta cells, retinal cells, glial cells, peripheral blood mononuclear cell (PBMC), and oligodendrocytes.

[0442] In some embodiments, the primary cells are selected from the group consisting of PBMC, lymphocytes, macrophages, dendritic cells. In some embodiments, the primary cells are selected from the group consisting of lymphocytes, macrophages, dendritic cells.

[0443] In some embodiments, the primary cells are lymphocytes. In some embodiments, the primary cells are T cells.

[0444] In some embodiments, the primary cells are PBMC. In certain embodiments, the primary cells are lymphocytes, preferably T cells, isolated from PBMC.

[0445] In another embodiment, the one or more eukaryotic cells are induced pluripotent stem cells (iPSCs). In some embodiments, the iPSCs are blood-derived iPSCs. Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cell that is generated by reprogramming somatic (adult) cells to a pluripotent state, allowing them to differentiate into virtually any cell type in the body. iPSCs have similar characteristics to embryonic stem cells, including the ability to self-renew and differentiate into various cell lineages. iPSCs are typically obtained after contacting a somatic cell with reprograming factor such as, e.g., Oct4, Sox2, Klf4, and / or c-Myc.

[0446] In another embodiment, the one or more eukaryotic cells are hematopoietic stem cells. Hematopoietic stem cells (HSCs) are multipotent stem cells found primarily in the bone marrow that have the ability to give rise to all types of blood cells, including red blood cells, white blood cells, and platelets. They are essential for the continuous replenishment of the blood system throughout a person's life. HSCs are capable of both self-renewal (producing more stem cells) and differentiation (producing specialized blood cells), making them critical for maintaining the hematopoietic system and supporting immune function.

[0447] In another embodiment, the one or more eukaryotic cells are immune cells, preferably T cells. In some embodiments, the immune cells are selected from the group consisting of T lymphocytes (T cell), B lymphocytes (B cells), natural killer cells (NK),macrophages, dendritic cells, neutrophils, basophils, eosinophils, monocytes, plasma cells, mast cells, regulatory T cells, helper T cells, cytotoxic T cells, memory T cells, antigen-presenting cells, innate lymphoid cells, gamma delta T cells. In some embodiments, the immune cells are lymphocytes, preferably T cells. In some embodiments, the immune cells are selected from the group consisting of regulatory T cells, helper T cells, cytotoxic T cells, and memory T cells. In some embodiments, the immune cells are selected from the group consisting of helper T cells, cytotoxic T cells, and memory T cells. In some embodiments, the immune cells are helper T cells. In some embodiments, the immune cells are cytotoxic T cells. In some embodiments, the immune cells are memory T cells.

[0448] In some embodiments, the method comprises the steps of:(i) providing one or more immune cells, preferably activated T cells; and(ii) contacting said immune cell(s), preferably activated T cells, provided at step (i) with:a) a polypeptide comprising (i) at least one RNA-guided nuclease, and (ii) at least one transposase; or a nucleic acid encoding the same;b) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease; andc) a nucleic acid molecule comprising a transgene encoding a protein of interest, preferably a chimeric antigen receptor;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences;thereby obtaining a population of immune cells comprising the transgene encoding the protein of interest, preferably a population of T cells comprising the transgene encoding the protein of interest.

[0449] In some embodiments, the method comprises the steps of:(i) providing peripheral blood mononuclear cell (PBMC);(ii) isolating immune cell(s), preferably T cells, from the PBMC provided at step (i);(iii) bringing into contact said immune cell(s), preferably said T cells, with an activating agent; thereby obtaining a population of activated immune cells, preferably activated T cells;(iv) expanding ex vivo said immune cell(s), preferably activated T cells, obtained at step (iii), in particular for 1 to 7 days, preferably for about 3 days; thereby obtaining an expanded population of activated immune cells, preferably activated T cells;(v) contacting said expanded population of activated immune cells, preferably activated T cells, obtained at step (iv) with a composition comprising:a) a polypeptide comprising (i) at least one RNA-guided nuclease, and (ii) at least one transposase; or a nucleic acid encoding the same;b) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease; andc) a nucleic acid molecule comprising a transgene encoding a protein of interest, preferably a chimeric antigen receptor;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences;thereby obtaining immune cell(s) comprising the transgene encoding the protein of interest, preferably T cells comprising the transgene encoding the protein of interest.

[0450] In some embodiments, activation is performed with CD3 and / or CD28. Commercially available solution suitable for this use include TRANSACT (Milteny).

[0451] In some embodiments, the eukaryotic cells, preferably the T cells, are expanded for 1 to 4 days, preferably 2 to 3 days, in the presence of IL-7 and IL-15 at a dose from 1 ng / mL to 100 ng / mL, preferably about 10 ng / mL.

[0452] In some embodiments, the immune cells are T cells, and the transgene encodes a chimeric antigen receptor; thereby obtaining CAR T cells.

[0453] The present invention thus also relates to a method of generating CAR-T cells, comprising performing the steps as described hereinabove, wherein the eukaryotic cells or the immune cells are T cells and wherein the transgene is a CAR.

[0454] In some embodiments, the supercoiled circular double-stranded DNA (dsDNA) molecule comprising the transgene encoding the CAR has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with the nucleic acid sequence of SEQ ID NO: 110.

[0455] Methods for determining transgene insertion in a genome are known in the art and include, non-limitatively, sequencing or quantitative PCR. Non-limitative examples of suitable primers are those of SEQ ID NO: 114-118.

[0456] The present invention further relates to a population of eukaryotic cells obtained by the method according to the invention, or a pharmaceutical composition thereof.

[0457] The present invention further relates to a population of CAR-T cells obtained by the method according to the invention.

[0458] The present invention further relates to the population of eukaryotic cells obtained by the method according to the invention, or a pharmaceutical composition; for use as a medicament.

[0459] The present invention further relates to the population of CAR T cells obtained by the method according to the invention, or a pharmaceutical composition comprising thereof, for use as a medicament.

[0460] The present invention further relates to the population of CAR T cells obtained by the method according to the invention, or a pharmaceutical composition comprising thereof, for use for treating cancer in a subject in need thereof.

[0461] The present invention further relates to a method for treating cancer in a subject in need thereof comprising administering to said subject a therapeutically effective dose of the population of CAR T cells obtained by the method according to the invention, or the pharmaceutical composition comprising thereof.

[0462] The present invention further relates to the use of the population of CAR T cells obtained by the method according to the invention, or a pharmaceutical composition comprising thereof, for the manufacture of a medicament for treating cancer in a subject in need thereof.

[0463] The present invention further relates to a method of manufacturing the composition according to the invention, comprising contacting together:a) a polypeptide comprising (i) at least one transposase, and (ii) optionally at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) optionally, a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease; andc) a nucleic acid molecule comprising a transgene encoding a protein of interest;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0464] The present invention further relates to a method of manufacturing the composition according to the invention, comprising contacting together:a) a polypeptide comprising (i) at least one transposase, and (ii) at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease; andc) a nucleic acid molecule comprising a transgene encoding a protein of interest;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0465] The present invention further relates to a method of manufacturing the composition according to the invention, comprising contacting together:a) at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) a polypeptide comprising (i) at least one transposase, and (ii) at least one aptamer binding protein (ABP); or a nucleic acid encoding the same;c) a nucleic acid molecule comprising (i) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, and (ii) at least one aptamer sequence; wherein the gRNA is capable of interacting with the RNA-guided nuclease; andd) a nucleic acid molecule comprising a transgene encoding a protein of interest;

[0466] wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0467] The transposases and RNA-guided nucleases have been described hereinabove.

[0468] The present invention also relates to a composition comprising:a) a polypeptide comprising (i) at least one transposase, and (ii) optionally at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) optionally, a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease; andc) a nucleic acid molecule comprising a transgene encoding a protein of interest;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0469] The present invention also relates to a composition comprising:a) at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) a polypeptide comprising (i) at least one transposase, and (ii) at least one aptamer binding protein (ABP); or a nucleic acid encoding the same;c) a nucleic acid molecule comprising (i) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, and (ii) at least one aptamer sequence; wherein the gRNA is capable of interacting with the RNA-guided nuclease; andd) a nucleic acid molecule comprising a transgene encoding a protein of interest;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0470] In some embodiments, the transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase, a Poeciliopsis turrubarensis transposase, an Anthonomus gran dis DR1754440 transposase, or variants thereof.

[0471] In some embodiments, the transposase is a modified hyperactive PiggyBac transposase having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 2-24, preferably having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 2.

[0472] In some embodiments, the transposase is a Poeciliopsis turrubarensis transposase having at least 80% sequence identity with SEQ ID NO: 25, or a variant thereof having at least 80% sequence identity with any one of SEQ ID NO: 26 to SEQ ID NO: 38

[0473] In some embodiments, the transposase is an Anthonomus grandis DR1754440 transposase having an amino acid sequence with at least 80% sequence identity with SEQ ID NO: 40, or a variant thereof having an amino acid sequence with at least 80% sequence identity with SEQ ID NO: 41.

[0474] In some embodiments, the transgene encodes a chimeric antigen receptor.

[0475] In some embodiments, the composition further comprises an inhibitor of the protein STING, preferably the inhibitor of the protein STING is a pharmaceutical inhibitor of STING, more preferably the pharmaceutical inhibitor of STING is H-151.

[0476] The present invention also relates to a composition comprising:a) a messenger RNA (mRNA molecule) encoding a polypeptide comprising (i) at least one transposase, and (ii) at least one RNA-guided nuclease;b) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease; andc) a nucleic acid molecule comprising a transgene encoding a protein of interest;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0477] The present invention also relates to a pharmaceutical composition comprising the composition as described herein, and at least one pharmaceutically acceptable excipient.

[0478] The present invention also relates to a kit comprising:a) a polypeptide comprising (i) at least one transposase, and (ii) optionally at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease;c) a nucleic acid molecule comprising a transgene encoding a protein of interest; andd) one or more eukaryotic cells comprising the target nucleic acid sequence;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0479] In some embodiments, the one or more eukaryotic cells are selected from the group consisting of primary cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells, and immune cells, preferably T cells.

[0480] The present invention also relates to an in vitro method for site specific integration of a transgene, preferably a chimeric antigen receptor, into the genome of one or more immune cells, preferably T cells and more preferably activated T cells, comprising the steps of:(i) providing one or more immune cells, preferably T cells and more preferably activated T cells; and(ii) contacting said immune cell(s), preferably T cells and more preferably activated T cells, provided at step (i) with:a) a polypeptide comprising (i) at least one RNA-guided nuclease, and (ii) at least one transposase; or a nucleic acid encoding the same;b) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease; andc) a nucleic acid molecule comprising a transgene encoding a protein of interest, preferably a chimeric antigen receptor;wherein the nucleic acid molecule comprising the transgene encoding a protein of interest, preferably a chimeric antigen receptor, is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene encoding a protein of interest, preferably a chimeric antigen receptor, is flanked by inverted terminal repeat (ITR) sequences;thereby obtaining a population of immune cells comprising the transgene encoding the protein of interest, preferably a population of T cells comprising the transgene encoding the chimeric antigen receptor.

[0481] The present invention also relates to a population of immune cells obtained by the method described hereinabove, preferably a population of CAR T cells obtained by the method described hereinabove.

[0482] The present invention also relates to a method of manufacturing the composition described hereinabove, comprising contacting together:a) a polypeptide comprising (i) at least one transposase, and (ii) optionally at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) optionally, a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease; andc) a nucleic acid molecule comprising a transgene encoding a protein of interest;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

[0483] The present invention also relates to a composition comprising:a) a polypeptide comprising (i) at least one transposase, and (ii) optionally at least one Cas protein, preferably a Cas protein having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO: 119, more preferably a Cas protein having the amino acid sequence SEQ ID NO: 119; or a nucleic acid encoding the same;b) optionally, a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the Cas protein; andc) a nucleic acid molecule comprising a transgene encoding a protein of interest, preferably a chimeric antigen receptor;preferably wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences;optionally wherein said at least one transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase, preferably having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 2-24, more preferably SEQ ID NO: 2; a Poeciliopsis turrubarensis transposase, preferably having at least 80% sequence identity with SEQ ID NO: 25, or a variant thereof, preferably having at least 80% sequence identity with any one of SEQ ID NO: 26 to SEQ ID NO: 38; and an Anthonomus grandis DR1754440 transposase, preferably having an amino acid sequence with at least 80% sequence identity with SEQ ID NO: 40, or a variant thereof, preferably having an amino acid sequence with at least 80% sequence identity with SEQ ID NO: 41.

[0484] The present invention also relates to a composition comprising:a) at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) a polypeptide comprising (i) at least one Cas protein, preferably a Cas protein having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO: 119, morepreferably a Cas protein having the amino acid sequence SEQ ID NO: 119, and (ii) at least one aptamer binding protein (ABP); or a nucleic acid encoding the same;c) a nucleic acid molecule comprising (i) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, and (ii) at least one aptamer sequence; wherein the gRNA is capable of interacting with the Cas protein; andd) a nucleic acid molecule comprising a transgene encoding a protein of interest, preferably a chimeric antigen receptor;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences;optionally wherein said at least one transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase, preferably having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 2-24, more preferably SEQ ID NO: 2; a Poeciliopsis turrubarensis transposase, preferably having at least 80% sequence identity with SEQ ID NO: 25, or a variant thereof, preferably having at least 80% sequence identity with any one of SEQ ID NO: 26 to SEQ ID NO: 38; and an Anthonomus grandis DR1754440 transposase, preferably having an amino acid sequence with at least 80% sequence identity with SEQ ID NO: 40, or a variant thereof, preferably having an amino acid sequence with at least 80% sequence identity with SEQ ID NO: 41.

[0485] The present invention further relates to a kit comprising:a) a polypeptide comprising (i) at least one transposase, and (ii) optionally at least one Cas protein, preferably a Cas protein having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO: 119, more preferably a Cas protein having the amino acid sequence SEQ ID NO: 119; or a nucleic acid encoding the same;b) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the Cas protein;c) a nucleic acid molecule comprising a transgene encoding a protein of interest; andd) optionally one or more eukaryotic cells comprising the target nucleic acid sequence;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences,optionally wherein said at least one transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase, preferably having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 2-24, more preferably SEQ ID NO: 2; a Poeciliopsis turrubarensis transposase, preferably having at least 80% sequence identity with SEQ ID NO: 25, or a variant thereof, preferably having at least 80% sequence identity with any one of SEQ ID NO: 26 to SEQ ID NO: 38; and an Anthonomus grandis DR1754440 transposase, preferably having an amino acid sequence with at least 80% sequence identity with SEQ ID NO: 40, or a variant thereof, preferably having an amino acid sequence with at least 80% sequence identity with SEQ ID NO: 41.

[0486] The present invention further relates to an in vitro method for site specific integration of a transgene, preferably a chimeric antigen receptor, into the genome of one or more immune cells, preferably T cells and more preferably activated T cells, comprising the steps of:(i) providing one or more immune cells, preferably T cells and more preferably activated T cells; and(ii) contacting said immune cell(s), preferably said T cells and more preferably said activated T cells, provided at step (i) with:a) a polypeptide comprising (i) at least one Cas protein, preferably a Cas protein having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO: 119,more preferably a Cas protein having the amino acid sequence SEQ ID NO: 119, and (ii) at least one transposase; or a nucleic acid encoding the same;b) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the Cas protein; andc) a nucleic acid molecule comprising a transgene encoding a protein of interest, preferably a chimeric antigen receptor;wherein the nucleic acid molecule comprising the transgene encoding a protein of interest, preferably a chimeric antigen receptor, is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene encoding a protein of interest, preferably a chimeric antigen receptor, is flanked by inverted terminal repeat (ITR) sequences;optionally wherein said at least one transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase, preferably having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 2-24, more preferably SEQ ID NO: 2; a Poeciliopsis turrubarensis transposase, preferably having at least 80% sequence identity with SEQ ID NO: 25, or a variant thereof, preferably having at least 80% sequence identity with any one of SEQ ID NO: 26 to SEQ ID NO: 38; and an Anthonomus grandis DR1754440 transposase, preferably having an amino acid sequence with at least 80% sequence identity with SEQ ID NO: 40, or a variant thereof, preferably having an amino acid sequence with at least 80% sequence identity with SEQ ID NO: 41,thereby obtaining a population of immune cells comprising the transgene encoding the protein of interest, preferably a population of T cells comprising the transgene encoding the chimeric antigen receptor.

[0487] The present invention further relates to a population of immune cells obtained by the method described herein, preferably a population of CAR T cells.

[0488] The present invention further relates to a composition comprising:a) a polypeptide comprising (i) at least one transposase, and (ii) optionally at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) optionally, a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease; andc) a nucleic acid molecule comprising a transgene encoding a protein of interest.

[0489] The present invention also relates to a composition comprising:a) at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) a polypeptide comprising (i) at least one transposase, and (ii) at least one aptamer binding protein (ABP); or a nucleic acid encoding the same;c) a nucleic acid molecule comprising (i) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, and (ii) at least one aptamer sequence; wherein the gRNA is capable of interacting with the RNA-guided nuclease; andd) a nucleic acid molecule comprising a transgene encoding a protein of interest.

[0490] The present invention further relates to a composition comprising:a) a polypeptide comprising (i) at least one transposase, and (ii) at least one Cas beta (Cas-beta-M67) having the amino acid sequence of SEQ ID NO: 119, or a variant thereof having an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 119; or a nucleic acid encoding the same;b) optionally, a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease; andc) a nucleic acid molecule comprising a transgene encoding a protein of interest;optionally wherein said at least one transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase, preferably having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 2-24, more preferably SEQ ID NO: 2; a Poeciliopsis turrubarensis transposase, preferably having at least 80% sequence identity with SEQ ID NO: 25, or a variant thereof, preferably having at least 80% sequence identity with any one of SEQ ID NO: 26 to SEQ ID NO: 38; and an Anthonomus grandis DR1754440 transposase, preferably having an amino acid sequence with at least 80% sequence identity with SEQ ID NO: 40, or a variant thereof, preferably having an amino acid sequence with at least 80% sequence identity with SEQ ID NO: 41.

[0491] The present invention also relates to a composition comprising:a) at least one Cas beta (Cas-beta-M67) having the amino acid sequence of SEQ ID NO: 119, or a variant thereof having an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with SEQ ID NO: 119; or a nucleic acid encoding the same;b) a polypeptide comprising (i) at least one transposase, and (ii) at least one aptamer binding protein (ABP); or a nucleic acid encoding the same;c) a nucleic acid molecule comprising (i) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, and (ii) at least one aptamer sequence; wherein the gRNA is capable of interacting with the RNA-guided nuclease; andd) a nucleic acid molecule comprising a transgene encoding a protein of interest;optionally wherein said at least one transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase, preferably having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 2-24, more preferably SEQ ID NO: 2; a Poeciliopsis turrubarensis transposase, preferably having at least 80% sequence identity with SEQ ID NO: 25, or a variant thereof, preferably having at least 80% sequence identity with any one of SEQ ID NO:26 to SEQ ID NO: 38; and an Anthonomus grandis DR1754440 transposase, preferably having an amino acid sequence with at least 80% sequence identity with SEQ ID NO: 40, or a variant thereof, preferably having an amino acid sequence with at least 80% sequence identity with SEQ ID NO: 41.BRIEF DESCRIPTION OF THE DRAWINGS

[0492] Figure 1 is a scheme showing the optimization of T cell gene editing / writing platform.

[0493] Figure 2 is a scheme showing the outline of the T cells editing / writing workflow. PBMCs from healthy donors are thawed and T cells are isolated from other cell types. T cells are then cultured in the presence of an activating (CD3 / CD28) reagent with a special medium supplemented with IL7 and IL15. Expansion occurs up to 3 days after thawing. After the nucleofection, T cells were allowed to recover from the electrical pulse and expansion continue for up to 12-15 days. During this last expansion an aliquot of cells is periodically collect to do flow cytometry, and at the end point the cells were pelleted and frozen for later use.

[0494] Figure 3A-3B is a set of histograms showing side by side comparison of recombinant protein and mRNA as editing / writing machinery to generate a KO of B2M and / or integrate a minimal payload encoding GFP (dbDNA). (Fig. 3A) Comparison of editing efficiency between mRNA and recombinant protein for Cas9+gRNA and FiCAT decoupled condition. (Fig. 3B) Comparison of payload integration between mRNA and recombinant protein for hyPB mediated random integration and FiCAT precise integration. EPI condition represents dbDNA-only electroporated cells. Data is taken from flow cytometry analysis at day 11 post-electroporation. n=2 biological replicate for each condition. Error bars represent SEM.

[0495] Figure 4A-4G is a histogram showing improvement of payload integration upon treatment with the STING inhibitor (STINGi) H-151. (Fig. 4A) Payload integration comparison between untreated cells and H-151 -treated T cells for hyPB-mediated randomintegration and FiCAT precise insertion. Data is taken from flow cytometry analysis at day 11 post-electroporation. n=2 biological replicate for each condition. Error bars represent SEM. (Fig. 4B-4C) Comparison at 3 hours (4B) and 24 hours (4C) after nucleofection. (Fig. 4D-4E-4G)) Comparison on different readouts with either untreated T cells (left column) or H-151 treated T cells (right column) for each one of the tested conditions, in (from left to right) EPI condition, hyPB-mediated transduction, Cas9 RNP-mediated transduction, and FICAT RNP-mediated transduction. (4D) improved viability as a function of a percentage (%) of live cells normalized to mock, (4E) efficiency of transduction as a function of a % of GFP+ cells, and (4G) B2M KO as a % of B2M-negative cells. (4F) comparison of the performance of different transductions for B2M KO efficiency as a % of GFP+ cells. From left to right: dnDNA-only electroporated cells (EPI), EPI with H-151 treatment (EPI H151), hyPB-mediated transduction only (hyPB), hyPB-mediated transduction with H-151 treatment (hyPB H151), Cas9 RNP-mediated transduction only (Cas9 RNP), Cas9 RNP-mediated transduction with H-151 treatment (Cas9 RNP Hl 51), FiCAT RNP-mediated transduction only (FiCAT RNP), FiCAT RNP-mediated transduction with H-151 treatment (FiCAT RNP Hl 51). The fraction of GFP+ cells which are B2M-positive is provided in the upper part of each column, and the fraction of GFP+ cells which are B2M-negative is provided in the lower part.

[0496] Figure 5A-5C is set of scheme and histograms showing side by side comparison of linear and circular payload for integration efficiency. (Fig. 5A) Cartoons depicting morphological features of circular and linear double strand DNA. (Fig. 5B) Delivery comparison of doggybone (dbDNA™) and Gencircle™ DNAs at 1 day postelectroporation. (Fig. 5C) Payload integration comparison between Gencircle™ and doggyboneDNA (dbDNA™) electroporated T cells for hyPB-mediated random integration and FiCAT precise insertion. Data is taken from flow cytometry analysis at day 14 post-electroporation. n=2-4 biological replicate for each condition. Error bars represent SEM.

[0497] Figure 6A-6C is a histogram showing generation of CAR-T cells with the gene writing workflow of the invention. (Fig. 6A) Payload integration comparison between minimal GFP and CAR19-GFP DNA donors. For FiCAT conditions, either B2M orTRAC locus were targeted. (Fig. 6B) Junction PCR to detect transposon insertion into TRAC locus. The specificity of this PCR prevents the episomal payload to be amplified.(Fig. 6C) CAR19 detection assay using CD 19 recombinant protein. Data is taken from flow cytometry analysis at day 12 post-electroporation n=2 biological replicate for each condition. Error bars represent SEM.

[0498] Figure 7 is a histogram showing the generation of CAR-T cells with PiggyBac orthologs. The plot compares CAR19 payload integration with PiggyBac orthologs (Poecillopsis turrubarensis and Anthonomous Grandi) to...

Claims

CLAIMS1. A composition comprising:a) a polypeptide comprising (i) at least one transposase, and (ii) optionally at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) optionally, a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease; andc) a nucleic acid molecule comprising a transgene encoding a protein of interest;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

2. A composition comprising:a) at least one RNA-guided nuclease; or a nucleic acid encoding the same; b) a polypeptide comprising (i) at least one transposase, and (ii) at least one aptamer binding protein (ABP); or a nucleic acid encoding the same;c) a nucleic acid molecule comprising (i) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, and (ii) at least one aptamer sequence; wherein the gRNA is capable of interacting with the RNA-guided nuclease; andd) a nucleic acid molecule comprising a transgene encoding a protein of interest;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

3. The composition according to claim 1 or 2, wherein said transposase is selected from the group consisting of a modified hyperactive PiggyBac transposase, a Poeciliopsis turrubarensis transposase, an Anthonomus grandis DR1754440 transposase, or variants thereof.

4. The composition according to claim 1 or 2, wherein said transposase is a modified hyperactive PiggyBac transposase having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 2-24, preferably having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:

25. The composition according to claim 1 or 2, wherein said transposase is a Poeciliopsis turrubarensis transposase having at least 80% sequence identity with SEQ ID NO: 25, or a variant thereof having at least 80% sequence identity with any one of SEQ ID NO: 26 to SEQ ID NO:

386. The composition according to claim 1 or 2, wherein said transposase is an Anthonomus grandis DR1754440 transposase having an amino acid sequence with at least 80% sequence identity with SEQ ID NO: 40, or a variant thereof having an amino acid sequence with at least 80% sequence identity with SEQ ID NO: 41.

7. The composition according to any one of claims 1 to 6, wherein the transgene encodes a chimeric antigen receptor.

8. The composition according to any one of claims 1 to 7, further comprising an inhibitor of the protein STING, preferably the inhibitor of the protein STING is a pharmaceutical inhibitor of STING, more preferably the pharmaceutical inhibitor of STING is H-151.

9. The composition according to claim 1, comprising:a) a messenger RNA (mRNA molecule) encoding a polypeptide comprising (i) at least one transposase, and (ii) at least one RNA-guided nuclease;b) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease; andc) a nucleic acid molecule comprising a transgene encoding a protein of interest;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

10. A pharmaceutical composition comprising the composition according to any one of claims 1 to 9, and at least one pharmaceutically acceptable excipient.

11. A kit compri sing :a) a polypeptide comprising (i) at least one transposase, and (ii) optionally at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease;c) a nucleic acid molecule comprising a transgene encoding a protein of interest;andd) one or more eukaryotic cells comprising the target nucleic acid sequence;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

12. The kit according to claim 11, wherein the one or more eukaryotic cells are selected from the group consisting of primary cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells, and immune cells, preferably T cells.

13. An in vitro method for site specific integration of a transgene, preferably a chimeric antigen receptor, into the genome of one or more immune cells, preferably T cells and more preferably activated T cells, comprising the steps of:(i) providing one or more immune cells, preferably T cells and more preferably activated T cells; and(ii) contacting said immune cell(s), preferably said T cells and more preferably said activated T cells, provided at step (i) with:a) a polypeptide comprising (i) at least one RNA-guided nuclease, and (ii) at least one transposase; or a nucleic acid encoding the same;b) a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease; andc) a nucleic acid molecule comprising a transgene encoding a protein of interest, preferably a chimeric antigen receptor;wherein the nucleic acid molecule comprising the transgene encoding a protein of interest, preferably a chimeric antigen receptor, is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene encoding a protein of interest, preferably a chimeric antigen receptor, is flanked by inverted terminal repeat (ITR) sequences;thereby obtaining a population of immune cells comprising the transgene encoding the protein of interest, preferably a population of T cells comprising the transgene encoding the chimeric antigen receptor.

14. A population of immune cells obtained by the method according to claim 13, preferably a population of CAR T cells obtained by the method according to claim 1315. A method of manufacturing the composition according to claim 1, comprising contacting together:a) a polypeptide comprising (i) at least one transposase, and (ii) optionally at least one RNA-guided nuclease; or a nucleic acid encoding the same;b) optionally, a guide RNA (gRNA) at least partially complementary to a target nucleic acid sequence, wherein the gRNA is capable of interacting with the RNA-guided nuclease; andc) a nucleic acid molecule comprising a transgene encoding a protein of interest;wherein the nucleic acid molecule comprising the transgene is a supercoiled circular double-stranded DNA (dsDNA) molecule, and wherein the transgene is flanked by inverted terminal repeat (ITR) sequences.

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