Engineered transposases with improved excision and integration activities and methods of use thereof

Abstract

Provided herein are novel engineered variants of PiggyBac transposase that maintain essential catalytic activity while having sequence divergence from known variants. The present disclosure also relates to methods for assessing transposase activity using novel reporter systems, and the use of these engineered transposases in applications such as genome modification, gene transfer, and cell engineering.

Description

Attorney Docket No: 230702000640ENGINEERED TRANSPOSASES WITH IMPROVED EXCISION AND INTEGRATION ACTIVITIES AND METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of United States provisional Patent Application Serial No. 63 / 747,853, filed January 21, 2025, the contents of which are incorporated herein by reference in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (230702000640seqlist.xml; Size:21,440 bytes; and Date of Creation: January 20, 2026) is herein incorporated by reference in its entirety.FIELD

[0003] The present disclosure relates to the field of genetic engineering tools and more specifically to modified transposase enzymes.BACKGROUND

[0004] Transposons, mobile genetic elements capable of moving within genomes, have long been harnessed as tools for genetic engineering across various organisms. Among these, the piggyBac transposon, originally isolated from the cabbage looper moth, stands out for its unique properties and versatile applications in genome engineering. The piggyBac transposon system has garnered significant attention due to its ability to transpose without leaving a footprint at the excised site, making it a potentially ideal tool for seamless genetic modifications.

[0005] Research has extensively explored the potential of the piggyBac transposase system in various genome engineering applications. The works by Li, X., et al (Li, Xianghong, et al. "piggyBac transposase tools for genome engineering." Proceedings of the National Academy of Sciences 110.25 (2013): E2279-E2287.) and Chen, Q., et al (Chen, Qiujia, et al."Structural basis of seamless excision and specific targeting by piggyBac transposase." Nature communications 11.1 (2020): 3446.) underscore the significance of piggyBac, particularly in mammalian cells, as an effective tool for genome manipulation. Key attributes such as its precise excision capability, large cargo size, and ability to mediate long-term 1MF-366023694Attorney Docket No: 230702000640 expression make piggyBac a promising alternative to other transposon systems like Sleeping Beauty. Moreover, the development of modified versions of the piggyBac transposase, such as Exc+Int- mutants, has expanded its utility by enabling reversible transgenesis and precise control over transgene integration.

[0006] In the realm of plant genome engineering, the work by Nishizawa-Yokoi, A. and Toki, S. (Nishizawa-Yokoi, Ayako, and Seiichi Toki. "Precise genetic engineering with piggyBac transposon in plants." Plant Biotechnology 40.4 (2023): 255-262.) highlights the successful application of the piggyBac transposon in precise genome editing, focusing specifically on rice. Through a combination of piggyBac transposition and homologous recombination-mediated gene targeting, researchers have achieved efficient and precise modification of target genes, paving the way for tailored genetic modifications in agriculturally important crops. Additionally, the design of a piggyBac-mediated transgenesis system for temporary expression of sequence- specific nucleases offers a promising strategy for eliminating transgenes from host genomes, a crucial step towards generating transgene-free plants with desired traits.

[0007] Expanding on the utility of the piggyBac system, the work (Nishizawa-Yokoi, Ayako, and Seiichi Toki. "Precise genetic engineering with piggyBac transposon in plants." Plant Biotechnology 40.4 (2023): 255-262.) introduces a novel approach for temporary expression of CRISPR / Cas9 in plants, particularly in vegetatively propagated crops like rice. By leveraging piggyBac-mediated transgenesis, researchers have demonstrated successful integration and subsequent excision of CRISPR / Cas9 expression cassettes from the host genome, enabling targeted mutagenesis without leaving unnecessary sequences behind. This strategy holds potential for streamlining genome editing processes in crops, if efficiency of transposon integration and excision are enhanced.

[0008] Thus, the piggyBac transposase system represents a powerful tool in the field of genome engineering, with applications ranging from mammalian cells to plants. Ongoing research aimed at refining and expanding the capabilities of this system promises to unlock new possibilities for precise genetic modifications, ultimately advancing our understanding of fundamental biological processes and facilitating the development of novel therapeutics, improved crops, and biotechnological solutions.2MF-366023694Attorney Docket No: 230702000640

[0009] Document US20100287633 discloses hyperactive PiggyBac transposases with enhanced mobility compared to the wild-type transposase. These modified transposases contain specific amino acid substitutions that improve their activity, including mutations at positions such as M185L, A187G, and M226F. US20100287633 describes proteins comprising at least 80% sequence identity to SEQ ID NO:2 and containing at least one of several specified amino acid substitutions. These hyperactive variants demonstrated increased transposition efficiency in cellular assays and could be used for various applications including gene transfer and genome modification. Meanwhile, document US20180072999 describes PiggyBac transposases with reduced integration activity while maintaining excision capabilities. US20180072999 discloses mutations R372A and K375A that create transposases capable of excising transposons but with significantly diminished or eliminated integration activity. These modified transposases are particularly useful for generating transgene-free induced pluripotent stem cells (iPSCs), as they allow for the removal of integrated transgenes without subsequent reintegration. US20180072999 also describes hyperactive variants of these excision-positive / integration-negative transposases. For its part, document WO2023122716 presents next-generation transpososomes featuring rationally engineered hyperactive PiggyBac transposases capable of transposing elements with LE / LE (Left End / Left End) configuration. WO2023122716 includes modified transposases lacking N-terminal amino acids and / or containing additional C-terminal domains. These modifications overcome inhibition by N-terminal phosphorylation and enable efficient transposition of symmetric transposon ends. WO2023122716 also describes variants that maintain the ability to excise transposons but lack integration activity, making them valuable tools for genome engineering applications.

[0010] There is a desire in the art for transposases with altered activity to facilitate genome modification, gene transfer, and cell engineering.BRIEF SUMMARY

[0011] The present disclosure provides novel engineered variants of the PiggyBac transposase that maintain essential catalytic activity while having sequence divergence from known variants. The present disclosure also relates to methods for assessing transposase activity using novel reporter systems, and the use of these engineered transposases in applications such as genome modification, gene transfer, and cell engineering.3MF-366023694Attorney Docket No: 230702000640

[0012] The present disclosure provides novel engineered PiggyBac transposase variants designed through consensus sequence analysis, ancestral sequence reconstruction, and deep learning methods. The key variant, TnPB_hyC57a, maintains approximately 77.3% sequence identity to the wild-type sequence while preserving the core catalytic domain containing the conserved DDD motif. TnPB_hyC57a exhibits seamless transposon excision activity comparable to reference PiggyBac transposases as shown in reporter systems in yeast Saccharomyces cerevisiae and in model plant Nicotiana benthamiana. While TnPB_hyC57a lacks transposon integration activity, the TnPB_hyC57a E381V / S411A mutant shows transposon integration activity in yeast assays.

[0013] The present disclosure provides a secondary variant, TnPB_hyL43a, which maintains 49.5% sequence identity to the wild-type sequence while preserving the core catalytic domain containing the conserved DDD motif. While TnPB_hyL43a does not exhibit seamless transposon excision activity, it shows transposon integration activity comparable to reference PiggyBac transposases in yeast assays.

[0014] The engineered variants maintain essential functional elements including the catalytic domain, DNA binding domains for recognition of terminal inverted repeats, and the C-terminal cysteine-rich domain involved in DNA binding, while having relatively low sequence similarity to previously reported mutations and preserving or enhancing transposition activity. This development provides new tools for genetic manipulation, as demonstrated through both computational and experimental analyses.

[0015] The present disclosure includes a robust excision reporter system in yeast comprising a URA3 selectable marker interrupted by a transposon carrying an mCherry fluorescent protein gene, flanked by inverted terminal repeat (ITR) sites, allowing quantitative assessment of transposase activity through selective growth conditions and colony counting.

[0016] The present disclosure includes a robust integration reporter system in yeast comprising a transposon donor plasmid unable to replicate in yeast, consisting of a G418 resistance selectable marker co-expressed with a mCherry fluorescent protein gene, flanked by inverted terminal repeat (ITR) sites. This plasmid is transformed into cells which express the various transposase variants from replicative plasmids, allowing the selection of integration events by G418 resistance and confirmation by red fluorescence, allowing4MF-366023694Attorney Docket No: 230702000640 quantitative assessment of transposase activity through selective growth conditions and colony counting.

[0017] The present disclosure includes a robust excision reporter system in model plant Nicotiana benthamiana comprising a Neongreen fluorescent protein gene interrupted by a transposon carrying an mScarlet fluorescent protein gene, flanked by inverted terminal repeat (ITR) sites, and with all fluorescent marker genes carrying nuclear localization signals. This system allows quantitative assessment of transposase activity through fluorescent microscopy analysis and green / red fluorescent nuclei counting.DESCRIPTION OF THE FIGURES

[0018] The present application can be understood by reference to the following description taken in conjunction with the accompanying figures.

[0019] FIG. 1. Excision reporter construct for test of transposase activity in Saccharomyces cerevisiae. Each plasmid contained one transposase variant, whose expression was driven from a strong constitutive promoter. In the same plasmid, an excision reporter cassette contained the URA3 gene interrupted by the transposon, containing the gene encoding for the mCherry red fluorescent protein, flanked by ITR sites. Finally, a G418 antibiotic resistance gene allows for selection of transformed yeast. If the designed transposase has excisionase activity, it excises the transposon in a scarless manner, reestablishing the URA3 gene and allowing selection in uracil deficient plates.

[0020] FIG. 2. Summary of two independent excision experiments in S. cerevisiae. Yeast cells were transformed with the reporter plasmids, grown for 1.5 hours and plated in media for selection of either G418 or G418 URA. The ratio of colonies from URA versus G418 plates allows to estimate the rate of excision of the transposon from the reporter plasmid. Excision rate is calculated as the ratio between the number of colonies in minimal medium+G418 over number of colonies in complete medium+G418.

[0021] FIG. 3. Representative images of plates from URA selection. Only cells that excise transposon are able to grown in this media. Differences between designed TnPB_hyC57a variant and reference hyPB R372A / K375A / D450N transposase are shown.

[0022] FIG. 4. Excision activity of TnPB_hyC57a mutants in S. cerevisiae. E381V, S411A, and E381V / S411A variants of TnPB_hyC57a were compared to TnPB_hyC57a in an5MF-366023694Attorney Docket No: 230702000640 excision experiment. Yeast cells were transformed with the reporter plasmids, grown for 1.5 hours and plated in media for selection of either G418 or G418 URA(-). The ratio of colonies from URA(-) versus G418 plates was used to estimate the rate of excision of the transposon from the reporter plasmid. Excision rate was calculated as the ratio between the number of colonies in minimal medium+G418 over number of colonies in complete medium+G418, and then variant activities calculated relative to the TnPB_hyC57a excision rate.

[0023] FIG. 5. Integration reporter assay in S. cerevisiae. Yeast replicating plasmids were constructed, harboring the transposase variants under the control of a strong constitutive promoter, and containing an URA3 selection marker and CEN / ARS sequences for selection and replication in yeast respectively. Yeast cells transformed with each transposon variant were subsequently transformed with a plasmid unable to replicate in yeast, containing the transposon with G418 selection marker and mCherry red fluorescent protein co-expressed from a constitutive promoter by means of a T2A ribosomal skipping peptide. If the designed transposase has integrase activity, it excises the transposon from the non-replicating plasmid and integrates it into the yeast genome.

[0024] FIG. 6. Integration activity of transposase variants in S. cerevisiae. Yeast cells expressing the transposase variants from replicating plasmids were transformed with the transposon containing non-replicating plasmids, grown for 1.5 hours and plated in media for selection of G418 URA(-). After two days of incubation at 28°C, mCherry fluorescent colonies were counted in plates to calculate the number of integration events. A control replicating plasmid with G418 resistance was also transformed in cells that expressed the transposases to estimate total transformation efficiency for each yeast line. The integration rate was calculated as the number of G418 resistant and mCherry(+) colonies in the nonreplicating plasmid transformation divided by the number of G418 resistant colonies in the transformation efficiency control. Each variant was tested at least twice in independent experiments.

[0025] FIG. 7. Representative plates of a transposon integration experiment in S. cerevisiae. Top row shows the transposition events, due to integration of the transposon to the yeast genome, conferring G418 resistance and mCherry fluorescence. Bottom row shows the transformation efficiency for all yeast lines, by using a replicative plasmid with G418 resistance. The integration rate is calculated dividing the number of integration colonies by the total transformation colonies. The reference hyPB transposase shows notable integration 6MF-366023694Attorney Docket No: 230702000640 activity, while the reference hyPB R372A / K375A / D450N variant is highly impaired for integration activity. For new transposase designs, TnPB_hyC57a shows no integration activity, while TnPB_hyL43a shows integration activity.

[0026] FIG. 8 Excision reporter constructs for determination of transposase activity in Nicotiana benthamiana. One set of plasmids contained each transposase variant, expressed using a strong plant viral promoter. A second set of plasmids contained transposase variants flanked by nuclear localization signals. Finally, the excision reporter plasmid contained an excision reporter cassette consisting of the Neongreen fluorescent gene interrupted by the transposon, containing the gene encoding for the mScarlet fluorescent protein, flanked by ITR sites. Both fluorescent protein encoding genes contained nuclear localization signals. All plasmids have elements to allow replication in Agrobacterium tumefaciens, and genes of interest were flanked by left border (LB) and right border (RB) sequences to allow transformation of plant cells by Agrobacterium T-DNA. Transformed plant cells fluoresce red by expression of the mScarlett gene. If the designed transposase has excisionase activity, it excises the transposon in a scarless manner, reestablishing the Neongreen gene and allowing fluorescent detection by microscopy.

[0027] FIG. 9. Excision activity of transposase variants in N. benthamiana. Agrobacterium cells harboring the transposase and the reporter plasmids were grown, induced byaceto syringone addition, and syringe infiltrated into N. benthamiana leaves. After 5 days, leaf discs were cut and analyzed by epifluorescence microscopy in six different fields. Rate of excision was calculated as the number of Neongreen (+) nuclei divided by the number of mScarlet (+) nuclei. All variants were tested in two independent experiments.

[0028] FIG. 10. Representative images of epifluorescence microscopy analysis for the excisionase activity of the transposase variants in N. benthamiana leaves. Cells transformed by Agrobacterium T-DNA are observed by mScarlet fluorescence. Seamless excision events reconstitute the Neongreen gene and are observed as green fluorescence. When reporter constructed is transformed alone, only mScarlet fluorescence is observed. Co-infiltration of the reporter together with transposase expressing T-DNA constructs trigger seamless excision events. Addition on nuclear localization signals (NLS) to N-terminal and C-terminal ends of reference hyPB variant is highly important for its activity in plants. The designed TnPB_hyC57a variant is highly functional, independent of the use of additional NLS sequences.7MF-366023694Attorney Docket No: 230702000640DETAILED DESCRIPTION

[0029] The following description sets forth exemplary methods, parameters and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.1. General description

[0030] The present invention provides novel engineered PiggyBac transposase variants with modified properties for gene transfer and genome engineering applications. These variants are designed to overcome limitations in currently available transposases while maintaining high efficiency and specificity for TTAA target sites.

[0031] In one embodiment, the invention provides novel PiggyBac transposase sequences with consensus-based mutations. In some embodiments, the novel transposases have one or more improved qualities. The novel transposases described herein have preserved transposition activity. The modified transposases maintain the core catalytic domain containing the conserved DDD motif and include specific mutations selected from phylogenetic analysis and structure-based design.

[0032] In another embodiment, the invention provides hyperactive variants of the modified transposases containing additional mutations that enhance transposition activity. These hyperactive variants are designed by incorporating specific amino acid substitutions that have been shown to improve transposase efficiency while maintaining target site specificity.

[0033] The modified transposases of the invention can be used in various applications including, but not limited to, generation of stable cell lines, development of transgenic organisms, protein production systems, genetic modification of stem cells, cell therapy, and genome engineering. The variants described herein offer improved efficiency and control over transposition events.2. Definitions

[0034] In order to provide greater clarity to the disclosure in this invention, the following are definitions of some relevant terms and concepts that will aid in understanding the content presented herein.8MF-366023694Attorney Docket No: 230702000640

[0035] PiggyBac transposase refers to a protein capable of catalyzing the excision of a transposon from a donor polynucleotide and the subsequent integration of the transposon into a target site. The wild-type PiggyBac transposase originates from Trichoplusia ni (cabbage looper moth).

[0036] PiggyBac transposon or "transposable element" refers to a polynucleotide that is able to excise from a donor polynucleotide and integrate into a target site, containing a nucleic acid sequence flanked by cis-acting nucleotide sequences on the termini.

[0037] LE / LE configuration refers to a transposon design where both ends contain Left End (LE) sequences, creating a symmetric arrangement of terminal inverted repeats.

[0038] Phosphorylation sites refers to specific amino acid residues, particularly serine residues in the N-terminal region of the transposase, that can be modified by phosphate group addition by cellular kinases.

[0039] Modified transposase refers to any PiggyBac transposase variant that contains one or more amino acid substitutions, deletions, or additions compared to the wild-type sequence, while maintaining transposase activity.

[0040] Hyperactive transposase refers to a modified transposase that exhibits enhanced transposition activity compared to the wild-type transposase, as measured by excision and / or integration assays.

[0041] Excision-positive / integration-negative variant or "Exc+ / Int-" refers to a modified transposase that maintains the ability to excise transposons but shows significantly reduced or eliminated integration activity.

[0042] Inverted Terminal Repeats or "ITRs" refer to the DNA sequences at both ends of a transposon that are specifically recognized by the PiggyBac transposase. These may be Left End (LE) or Right End (RE) sequences.

[0043] TTAA target site refers to the specific tetranucleotide sequence where PiggyBac transposons are inserted into and excised from the genome.9MF-366023694Attorney Docket No: 230702000640

[0044] Excision reporter system refers to a genetic construct designed to measure transposase excision activity, comprising a selectable marker interrupted by a transposon, where successful excision restores marker function.

[0045] Consensus sequence refers to a protein or nucleotide sequence derived from the alignment of multiple related sequences, representing the most common amino acid at each position.

[0046] Core catalytic domain refers to the region of the transposase containing the conserved DDD motif essential for catalytic activity.

[0047] C-terminal domain or "CD" refers to the cysteine-rich region at the C-terminus of the PiggyBac transposase involved in DNA binding.

[0048] Colony count assay refers to a method for measuring transposition activity by counting colonies that survive selection after transposition events.

[0049] Excision assay refers to a method for measuring the ability of a transposase to remove a transposon from its original location, typically assessed by PCR analysis.

[0050] Integration assay refers to a method for measuring the ability of a transposase to insert a transposon into a new location in the genome.

[0051] URA3 marker refers to the gene encoding orotidine-5'-phosphate decarboxylase, which can be used as a selectable marker in yeast by allowing growth in media lacking uracil.

[0052] Transpososome refers to the complex formed between the transposase protein and the transposon DNA during the transposition reaction.

[0053] Conservative amino acid substitutions refers to replacements of amino acids with others having similar chemical properties that are less likely to disrupt protein function.

[0054] pLDDT score refers to the predicted Local Distance Difference Test score, a measure of confidence in protein structure prediction.

[0055] Ancestral sequence reconstruction or "ASR" refers to computational methods used to infer the sequence of ancestral proteins from which current proteins evolved.10MF-366023694Attorney Docket No: 230702000640

[0056] Deep learning models refers to computational approaches using artificial neural networks to predict protein properties or design new protein sequences.

[0057] Consensus-based mutations refers to amino acid changes selected based on the most common residues found at specific positions in related sequences.

[0058] G418 refers to geneticin, an aminoglycoside antibiotic used for positive selection of Saccharomyces cerevisiae yeast cells that have been transformed with the G418 and kanamycin resistance gene Tn903.

[0059] mCherry refers to the gene encoding for a red fluorescent protein derived from Discosoma sp. and engineered to function as a monomeric protein.

[0060] Neongreen refers to the gene encoding for a green fluorescent protein derived from Branchiostoma lanceolatum and engineered to function as a monomeric protein.

[0061] mScarlet refers to the gene encoding for a red fluorescent protein derived from a synthetic construct engineered to function as a monomeric protein.

[0062] Nuclear localization signal (NLS) refers to the DNA sequences encoding amino acid sequences that target proteins to localize to the nucleus of the cells, confining the target protein and its activity to this subcellular localization.

[0063] T2A ribosomal skipping peptide refers to an amino acid sequence of viral origin that allows the co-translation of two genes from a single expressed mRNA in eukaryotic hosts, by inducing ribosomal skipping during protein synthesis.3. Detailed description

[0064] The present invention relates to novel PiggyBac transposase variants designed through multiple complementary approaches. The wild-type PiggyBac transposase from Trichoplusia ni comprises the amino acid sequence:

[0065] MGSSLDDEHILSALLQSDDELVGEDSDSEISDHVSEDDVQSDTEEAFIDEVHE VQPTSSGSEILDEQNVIEQPGSSLASNKILTLPQRTIRGKNKHCWSTSKSTRRSRVSAL NHVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTGATFRDT NEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMD DKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRMYIPN11MF-366023694Attorney Docket No: 230702000640 KPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRN ITCDNWFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPL TEVSYKPKPAKMVYEESSCDEDASINESTGKPQMVMYYNQTKGGVDTEDQMCSVM TCSRKTNRWPMAEEYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNEYMSETSSF MRKREEAPTEKRYERDNISNIEPNEVPGTSDDSTEEPVTKKRTYCTYCPSKIRRKANA SCKKCKKVICREHNIDMCQSCF (SEQ ID NO:1)

[0066] The novel variants have been designed through consensus sequence analysis, ancestral sequence reconstruction, and deep learning methods. These approaches yielded several key variants, including TnPB_C57a, which maintains the core catalytic domain containing the conserved DDD motif while exhibiting approximately 77.8% sequence identity to the wild-type sequence. This variant comprises the sequence:

[0067] MATEEDKEEIEPSEEQTDDEEGASSSDSEIEDHEEEDDVQPSTDSTFISGARHD QPNVSASDPEEQDDSEEEPGTSEVSNRIITPPQRTIRGKNKHCWSTSKGSKRTRTSAINI VRSQRGPTRMCRNIYDPEECFKEFITDEIISEIVKWTNAEISEKRRDKATSATFRDTNE DEIRAEIGIETETAAMKDNHESTDEEFDKSFSGRYVSVMSRDRFDFEIRCERFDDKSE RPTIRENDIFTPIRKIWDEFINQCRQNYTPGSYETIDEQEEGFRGRCPFRMYIPNKPNKY GIKIVMMCDSGTKYMINAMPYEGKGTNTNGEPEGEYYVKEETKPVHGTNRNITCDN WFTSVPEAKSEEQEPYKETEVGTIRSNKREIPEEEKNSRSRPVGTSMFCYDGPETEVS YKPKPSKMVYEESSCDEDGSINETTGKPEMIMFYNQTKGGVDTFDQMCSVMSCSRK TNRWPMAVFYGMENIAFINSYIIYCHNVESKGEKPESRRRFMKKESMQETEPWMRK REEAPTEKRYERDNITNIEPNEVPETSDRSTEEPVAKKRKYCTYCPSKKRAKSDSSCK KCKKAICGEHNIDMCQSCF (SEQ ID NO:2, TnPB_C57a, 77.8 %identity with SEQ ID:1);

[0068] MATLLDKEEILPSEEQTDDELGASSSDSEVEDHLEEDDVQPSTDSTFISGARHD QPNVSASDPLEQDDSLEEPGTSLVSNRIITPPQRTIRGKNKHCWSTSKPSKRTRTSAINI VRSQRGPTRMCRNIYDPLLCFKLFITDEIISEIVKWTNAEISLKRRDKATSATFRDTNE DEIRALIGILTLTAAMKDNHLSTDELFDKSFSGRYVSVMSRDRFDFLIRCLRFDDKSL RPTIRENDIFTPIRKIWDLFINQCRQNYTPGSYLTIDEQLLGFRGRCPFRVYIPNKPNKY GIKIVMMCDSGTKYMINAMPYLGKGTNTNGLPLGEYYVKELTKPVHGTNRNITCDN WFTSVPLAKSLLQEPYKLTLVGTIRSNKREIPEELKNSRSRPVGTSMFCYDGPLTLVS YKPKPSKMVYLLSSCDEDGSINETTGKPEMIMFYNQTKGGVDTFDQMCSVMSCSRK TNRWPMAVFYGMLNIAFINSYIIYCHNVLSKGEKPLSRRRFMKKLSMGLTEPWMRK RLEAPTLKRYLRDNITNILPKEVPETSDRSTEEPVAKKRKYCTYCPSKKRAKSSSSCK12MF-366023694Attorney Docket No: 230702000640 KCKKAICGEHNIDMCQSCF (SEQ ID N0:3, TnPB_hyC57a, 77.3 %identity with SEQ ID:1);

[0069] MATLLDKEEILPSEEQTDDELGASSSDSEVEDHLEEDDVQPSTDSTFISGARHD QPNVSASDPLEQDDSLEEPGTSLVSNRIITPPQRTIRGKNKHCWSTSKPSKRTRTSAINI VRSQRGPTRMCRNIYDPLLCFKLFITDEIISEIVKWTNAEISLKRRDKATSATFRDTNE DEIRAEIGIETETAAMKDNHESTDEEFDKSFSGRYVSVMSRDRFDFEIRCERFDDKSE RPTIRENDIFTPIRKIWDEFINQCRQNYTPGSYETIDEQEEGFRGRCPFRVYIPNKPNKY GIKIVMMCDSGTKYMINAMPYEGKGTNTNGEPEGEYYVKEETKPVHGTNRNITCDN WFTSVPEAKSEEQEPYKETEVGTIASNAREIPEEEKNSRSRPVGTSMFCYDGPETEVS YKPKPSKMVYEESSCDEDGSINETTGKPEMIMFYNQTKGGVDTFNQMCSVMSCSRK TNRWPMAVFYGMENIAFINSYIIYCHNVESKGEKPESRRRFMKKESMGETEPWMRK REEAPTEKRYERDNITNIEPKEVPETSDRSTEEPVAKKRKYCTYCPSKKRAKSSSSCK KCKKAICGEHNIDMCQSCF (SEQ ID NO:4, TnPB_hyC57aEx, 76.8 %identity with SEQ ID:1).

[0070] MGSSLDDEHILSALLQSDDELVGEDSDSEISDHVSEDDVQSDTEEAFIDEVHE VQPTSSGSEILDEQNVIEQPGSSLASNKILTLPQRTIRGKNKHCWSTSKSTRRSRVSAL NHVRMGRGPSEKVKDIYNPLDSFMVFWTDEIINNIVKYTNKEMKKKKPKSKKGPNY TSVDELKIKAYLGIEVYRARERLNDVPIELIFDPGITDRFRIVMSKEEYKFLRENIRFYD ESTRPFIEPYDPFYEFSEIFKLFKKNLIDSYIPGEVLGIDSILIPFDGYAPYSIYRPDKKYK NGLLIYVLCDAGTNYLLNFRPYGGSSQKTDGLPLGTDIVLTLTEPVRDRERLVVVDS DFASLPLARLLQQPPYRLGVIGPVSPDDPAIPASLQLTADRPVGSSRAVRLGDVTLVA YRPTPDTVVYLLASRLDGYAVNPATGKPVHYEIYEKYKGAVDKLEETIEKYGSHRK TDKWYLALFDFMLNVALNNGYVVYKWNLSKKGEPVPDLETYLEELYIGLTTPYIKK RLKDPNLSRRLRKAQKNYIPKKDPGAKPKDGKPPGNKKEKYCYYCDKSLRIKATSV CVLCNKPICPKHAVDRCPSCG (SEQ ID NO:5, TnPB_L43a, 50% identity with SEQ ID:1)

[0071] MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHE VQPTSSGSEILDEQNVIEQPGSSLASNKILTLPQRTIRGKNKHCWSTSKPTRRSRVSAL NHVRMGRGPSEKVKDIYNPLDSFMVFWTDEIINNIVKYTNKEMKKKKPKSKKSPNY TSVDELKIKAYLGIEVYRARERLNDVPIELIFDPGITDRFRIVMSKEEYKFLRENIRFYD ESTRPFIEPYDPFYEFSEIFKLFKKNLIDSYIPGEVLGIDSILIPFDGYAPYSVYRPDKKY KNGLLIYVLCDAGTNYLLNFRPYGGSSQKTDGLPLGTDIVLTLTEPVRDRERLVVVD SDFASLPLARLLQQPPYRLGVIGPVSPDDPAIPASLQLTADRPVGSSRAVRLGDVTLV13MF-366023694Attorney Docket No: 230702000640 AYRPTPDTVVYLLASRLDGYAVNPATGKPVHYEIYEKYKGAVDKLEETIEKYGSHR KTDKWYLALFDFMLNVALNNGYVVYKWNLSKKGEPVPDLETYLEELYIGLTTPYIK KRLKDPNLSRRLRKAQKNYIPKKDPGAKPKDGKPPGNKKEKYCYYCDKSLRIKASS VCVLCNKPICPKHAVDRCPSCG (SEQ ID N0:6, TnPB_hyL43a, 49.5% identity with SEQ ID:1)

[0072] The variants maintain essential functional elements including the catalytic domain, DNA binding domains for recognition of terminal inverted repeats, and the C-terminal cysteine-rich domain involved in DNA binding. Through careful design and selection, these variants avoid sequence similarities with previously reported mutations while preserving or enhancing transposition activity.

[0073] To assess the transposon excision functionality of these novel variants, we developed a robust seamless excision reporter system in S. cerevisiae yeast. This system comprises a URA3 selectable marker interrupted by a transposon carrying an mCherry fluorescent protein gene, all flanked by inverted terminal repeat (ITR) sites. Successful transposition restores URA3 function, only if the transposon is removed without leaving a DNA sequence trace or genetic scar, necessary for in-frame restoration of the URA3 gene. This assay allows the quantitative assessment of transposase activity through selective growth conditions and colony counting.

[0074] To assess the transposon genomic integration functionality of these novel variants, we developed a transposon integration assay in S. cerevisiae yeast. This system consists of yeast cells that contain a replicative plasmid that expresses the transposase variant from a strong constitutive promoter. These cells are then transformed with a transposon donor plasmid that only replicates in Escherichia coli, but is unable to replicate in S. cerevisiae. The transposon contains a G418 resistance gene, linked by a T2A ribosomal skipping peptide to the mCherry red fluorescent protein, and both genes are under the control of a strong constitutive promoter. Without the presence of transposases, this transposon donor plasmid is unable to transpose into the yeast genome, and very few to no yeast cells grow under G418 resistance selection, and no cells show red fluorescence. When the transposon donor plasmid is transformed into yeast cells expressing an active transposase, the transposon is excised from the plasmid and integrated into the yeast genome, giving rise to G418 resistant, red fluorescent yeast colonies.14MF-366023694Attorney Docket No: 230702000640

[0075] To assess the transposon excision functionality of these novel variants in plants, we developed a robust seamless excision reporter system in Nicotiana benthamiana. This system comprises a Neongreen fluorescent protein interrupted by a transposon carrying an mScarlet fluorescent protein gene, flanked by inverted terminal repeat (ITR) sites. Successful transposition restores Neongreen fluorescence, only if the transposon is removed without leaving a DNA sequence trace or genetic scar, necessary for in-frame restoration of the Neongreen gene. In order to induce transposition, the reporter plasmid has to be cotransformed into plant cells with a construction expressing the designed transposases, using a strong constitutive plant viral promoter. All plasmids contained replication origins and antibiotic selection markers that allowed for transformation and selection of Agrobacterium tumefaciens bacteria. Additionally, sequences of transposase variants and excision reporter were flanked by left border (LB) and right border (RB) T-DNA sequences, to allow DNA transfer and transformation of plant cells by Agrobacterium. All fluorescent proteins contained a NLS to direct the fluorescence in the plant nuclei. By means of fluorescence microscopy, this assay allows the quantitative assessment of transposase activity through fluorescent nuclei counting.

[0076] In some embodiments, the present invention provides hyperactive transposase proteins, wherein the protein comprises at least 75% sequence identity to SEQ ID NO:1, and comprises a substitution in at least one of the following amino acid positions in SEQ ID NO:1: position 2, position 3, position 4, position 7, position 9, position 12, position 13, position 14, position 15, position 17, position 22, position 23, position 24, position 25, position 30, position 31, position 34, position 35, position 41, position 42, position 44, position 45, position 46, position 49, position 50, position 51, position 52, position 53, position 54, position 57, position 58, position 60, position 62, position 63, position 65, position 66, position 67, position 68, position 69, position 70, position 72, position 75, position 78, position 81, position 83, position 85, position 103, position 105, position 107, position 109, position 112, position 114, position 139, position 161, position 162, position 163, position 165, position 177, position 179, position 180, position 184, position 185, position 188, position 189, position 194, position 198, position 202, position 204, position 206, position 207, position 226, position 231, position 235, position 240, position 244, position 253, position 256, position 263, position 264, position 282, position 289, position 296, position 309, position 315, position 318, position 322, position 333, position 339, position 340, position 352, position 357, position 367, position 371, position 372, position 15MF-366023694Attorney Docket No: 230702000640 375, position 381, position 397, position 411, position 424, position 429, position 434, position 436, position 438, position 449, position 450, position 457, position 469, position 470, position 474, position 478, position 482, position 486, position 490, position 496, position 497, position 500, position 501, position 504, position 505, position 507, position 509, position 512, position 513, position 514, position 533, position 538, position 542, position 546, position 553, position 557, position 566, position 568, position 570, position 571, position 572, position 580, and / or position 583.

[0077] In some embodiments, the protein comprises at least 75% sequence identity to SEQ ID NO: 1, and comprises at least one of the following amino acid substitutions in SEQ ID NO: 1: an alanine for the glycine at position 2; a threonine for the serine at position 3; a leucine for the serine at position 4; a lysine for the aspartate at position 7; a glutamate for the histidine at position 9; a proline for the serine at position 12; a serine for the alanine at position 13; a glutamate for the leucine at position 14; a glutamate for the leucine at position 15; a threonine for the serine at position 17; a glycine for the valine at position 22; an alanine for the glycine at position 23; a serine for the glutamate at position 24; a serine for the aspartate at position 25; a valine for the isoleucine at position 30; a glutamate for the serine at position 31; a leucine for the valine at position 34; a glutamate for the serine at position 35; a proline for the serine at position 41; a serine for the aspartate at position 42; a aspartate for the glutamate at position 44; a serine for the glutamate at position 45; a threonine for the alanine at position 46; a serine for the aspartate at position 49; a glycine for the glutamate at position 50; an alanine for the valine at position 51; an arginine for the histidine at position 52; a histidine for the glutamate at position 53; an aspartate for the valine at position 54; an asparagine for the threonine at position 57; a valine for the serine at position 58; an alanine for the glycine at position 60; an aspartate for the glutamate at position 62; a proline for the isoleucine at position 63; a glutamate for the aspartate at position 65; a glutamine for the glutamate at position 66; an aspartate for the glutamine at position 67; an aspartate for the asparagine at position 68; a serine for the valine at position 69; a leucine for the isoleucine at position 70; a glutamate for the glutamine at position 72; a threonine for the serine at position 75; a valine for the alanine at position 78; an arginine for the lysine at position 81; an isoleucine for the leucine at position 83; a proline for the leucine at position 85; a glycine for the serine and a proline for the glycine at position 103; a lysine for the arginine at position 105; a threonine for the serine at position 107; a threonine for the valine at position 109; an isoleucine for the leucine at position 112; an isoleucine for the histidine at position 114; an 16MF-366023694Attorney Docket No: 230702000640 isoleucine for the phenylalanine at position 139; an aspartate for the glutamate at position 161; a lysine for the serine at position 162; an alanine for the methionine at position 163; a serine for the glycine at position 165; an arginine for the tyrosine at position 177; a leucine for the phenylalanine at position 179; an isoleucine for the phenylalanine at position 180; a threonine for the valine at position 184; a leucine for the methionine at position 185; an alanine for the valine at position 188; a methionine for the arginine at position 189; a leucine for the methionine at position 194; a glutamate for the aspartate at position 198; a lysine for the arginine at position 202; a phenylalanine for the leucine at position 204; a glycine for the methionine at position 206; an arginine for the valine at position 207; a phenylalanine for the methionine at position 226; a leucine for the isoleucine at position 231; an isoleucine for the leucine at position 235; a isoleucine for the valine at position 240; an isoleucine for the valine at position 244; an asparagine for the histidine at position 253; an arginine for the isoleucine at position 256; a serine for the alanine at position 263; a tyrosine for the histidine at position 264; a valine for the methionine at position 282; an asparagine for the serine at position 289; a valine for the leucine at position 296; an alanine for the glycine at position 309; a lysine for the arginine at position 315; an asparagine for the glutamine at position 318; a leucine for the valine at position 322; a threonine for the serine at position 333; a threonine for the serine at position 339; an asparagine for the cysteine at position 340; a valine for the isoleucine at position 352; a serine for the asparagine at position 357; a leucine for the isoleucine at position 367; an isoleucine for the valine at position 371; an alanine for the arginine at position 372; an alanine for the lysine at position 375; a glutamate for the valine at position 381; a tyrosine for the phenylalanine at position 397; a serine for the alanine at position 411; a glycine for the alanine at position 424; a threonine for the serine at position 429; a glutamate for the glutamine at position 434; an isoleucine for the valine at position 436; a phenylalanine for the tyrosine at position 438; a phenylalanine for the leucine at position 449; an asparagine for the aspartate at position 450; a serine for the threonine at position 457; a valine for the leucine at position 469; a phenylalanine for the leucine at position 470; a leucine for the isoleucine at position 474; a phenylalanine for the cysteine at position 478; a tyrosine for the phenylalanine at position 482; a cysteine for the serine at position 486; a leucine for the serine at position 490; a proline for the valine at position 496; a leucine for the glutamine at position 497; an arginine for the lysine at position 500; an arginine for the lysine at position 501; a lysine for the arginine at position 504; a lysine for the asparagine at position 505; a serine for the tyrosine at position 507; a glycine or glutamine for the serine at17MF-366023694Attorney Docket No: 230702000640 position 509; a glutamate for the serine at position 512; a proline for the serine at position 513; a tryptophan for the phenylalanine at position 514; a threonine for the serine at position 533; a lysine for the asparagine at position 538; a glutamate for the glycine at position 542; an arginine for the aspartate at position 546; an alanine for the threonine at position 553; a lysine for the threonine at position 557; a lysine for the isoleucine at position 566; an alanine for the arginine at position 568; a serine for the alanine at position 570; a serine or aspartate for the asparagine at position 571; a serine for the alanine at position 572; an alanine for the valine at position 580; and / or a glycine for the arginine at position 583.

[0078] Provided herein are hyperactive transposase proteins comprising an amino acid sequence with at least about 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO: 3 and / or SEQ ID NO: 6. In some embodiments, the hyperactive transposase proteins comprise an amino acid sequence with at least 99% identity to the amino acid sequence of SEQ ID NO: 3 and / or SEQ ID NO: 6.

[0079] In some embodiments, the hyperactive transposase proteins comprise a core catalytic domain as described herein. In some embodiments, the hyperactive transposase proteins comprise a core catalytic domain comprising a DDD motif.

[0080] In some embodiments, the hyperactive transposase proteins comprise one or more nuclear localization signals (NLS). The one or more NLS may be located at the N and / or the C terminus of the hyperactive transposase proteins. In some embodiments, hyperactive transposase proteins comprising the one or more NLS have an increased excision rate compared to hyperactive transposase proteins without the one or more NLS. In some embodiments, the hyperactive transposase protein is TnPB_hyC57a with or without the one or more NLS. In some embodiments, the excision rate for TnPB_hyC57a without the one or more NLS is not increased compared to TnPB_hyC57a with the one or more NLS.

[0081] In some embodiments, the hyperactive transposase proteins have excision and / or integration activity. Excision and / or integration activity can be measured using a seamless excision and / or seamless integration assay. In some embodiments, the seamless excision assay is performed in yeast. In some embodiments, the seamless excision assay comprises a yeast transposon excision assay. In some embodiments, the seamless excision assay comprises the yeast transposon excision assay shown in FIG. 1. In some embodiments, the yeast transposon excision assay comprises an excision report system.18MF-366023694Attorney Docket No: 230702000640

[0082] In some embodiments, the seamless excision assay is performed in Nicotiana benthamiana. In some embodiments, the seamless excision assay performed in Nicotiana benthamiana comprises transforming plasmids comprising a transposase expression and excision reporter into A. tumefaciens cells and infiltrating the cells into Nicotiana benthamiana.

[0083] In some embodiments, the hyperactive transposase proteins have excision and integration activity. Excision and integration activity can be measured using a seamless excision and a seamless integration assay. In some embodiments, the seamless excision assay comprises a yeast transposon excision assay. In some embodiments, the seamless integration assay is performed in yeast. In some embodiments, the seamless integration assay comprises a yeast transposon integration assay.

[0084] In some embodiments, the seamless excision assay is used to generate an excision rate. In some embodiments, the excision rate is the percent of transformed cells wherein excision occurred in the seamless excision assay, as described herein.

[0085] In some embodiments, the seamless integration assay is used to generate an integration rate. In some embodiments, the integration rate is the percent of transformed cells wherein integration occurred in the seamless integration assay, as described herein.

[0086] In some embodiments, the hyperactive transposase protein comprises an amino acid sequence with at least about 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the hyperactive transposase protein comprises an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the hyperactive transposase protein comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the hyperactive transposase protein is TnPB_hyC57a. In some embodiments, the hyperactive transposase protein is TnPB_hyC57a with one or more NLS at the C and / or N terminus as described herein.

[0087] In some embodiments, the hyperactive transposase protein has improved excision activity compared to a transposase isolated from Trichoplusia ni without one or more amino acid substitutions. In some embodiments, the hyperactive transposase protein has about 2-fold, about 5 fold, or about 10 fold improved excision activity. In some embodiments, the hyperactive transposase protein has between about 2-fold and about 6-fold improved excision19MF-366023694Attorney Docket No: 230702000640 activity. In some embodiments, the hyperactive transposase proteins has about 6-fold improved excision activity. In some embodiments, the hyperactive transposase protein has about 2-fold improved excision activity. In some embodiments, the excision activity is measured using a seamless excision assay as described herein. In some embodiments, the seamless excision assay is performed in yeast. In some embodiments, the transposase isolated from Trichoplusia ni without one or more amino acid substitutions comprises the amino acid of SEQ ID NO: 1. In some embodiments, the hyperactive transposase protein has improved excision activity compared to hyPB (SEQ ID NO: 7). In some embodiments, the hyperactive transposase protein has improved excision activity compared to a transposase with an amino acid sequence of any of SEQ ID NO 1 and SEQ ID NO: 7-8.

[0088] In some embodiments, the hyperactive transposase protein has between about 2-fold and about 6-fold improved excision activity compared to a transposase comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the hyperactive transposase proteins has about 6-fold improved excision activity. In some embodiments, the hyperactive transposase protein has about 2-fold improved excision activity. In some embodiments, the excision activity is measured using a seamless excision assay as described herein. In some embodiments, the seamless excision assay is performed in yeast.

[0089] In some embodiments, the hyperactive transposase proteins described herein have excision and integration activity. In some embodiments, the hyperactive transposase proteins comprise an amino acid sequence with at 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the hyperactive transposase protein comprises an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the hyperactive transposase protein comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the hyperactive transposase protein is TnPB_hyL43A.

[0090] In some embodiments, the hyperactive transposase protein comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the hyperactive transposase protein is TnPB_hyL43A. In some embodiments, the hyperactive transposase protein has similar or increased integration activity compared to a transposase isolated from Trichoplusia ni without one or more amino acid substitutions. In some embodiments, the hyperactive transposase protein has similar or increased integration activity to a transposase isolated from Trichoplusia ni. In some embodiments, the hyperactive transposase protein has about 2-fold,20MF-366023694Attorney Docket No: 230702000640 about 5-fold, or about 10-fold improved integration activity. In some embodiments, the hyperactive transposase protein has about 2-fold improved integration activity. In some embodiments, the integration activity is measured using a seamless integration assay as described herein. In some embodiments, the seamless integration assay is performed in yeast. In some embodiments, the transposase isolated from Trichoplusia ni without one or more amino acid substitutions comprises the amino acid of SEQ ID NO: 1. In some embodiments, the hyperactive transposase protein has improved integration activity compared to hyPB. In some embodiments, the hyperactive transposase protein has improved integration activity compared to a transposase with an amino acid sequence of any of SEQ ID NO 1 and SEQ ID NO: 8-11.

[0091] In some embodiments, the hyperactive transposase protein has similar or increased integration compared to a transposase comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the integration activity is measured using a seamless integration assay as described herein. In some embodiments, the seamless integration assay is performed in yeast.

[0092] In some embodiments, the hyperactive transposase protein has an excision rate of between about 15% and about 60%. In some embodiments, the hyperactive transposase protein has an excision rate of about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, about 55%, or about 60%. In some embodiments, the hyperactive transposase protein has an excision rate of between about 15% and about 55%, about 15% and about 50%, about 15% and about 45%, about 15% and about 40%, about 15% and about 35%, about 15% and about 30%, about 15% and about 25%, or about 15% and about 20%. In some embodiments, the hyperactive transposase protein has an excision rate of between about 15% and about 45%, about 15% and about 40%, about 15% and about 35%, about 15% and about 30%, about 15% and about 25%, or about 15% and about 20%. In some embodiments, the hyperactive transposase protein has an excision rate of between about 20% and about 60%, about 25% and about 60%, about 30% and about 60%, about 35% and about 60%, about 40% and about 60%, about 45% and about 60%, about 50% and about 60%, or about 55% and about 60%.

[0093] In some embodiments, the hyperactive transposase protein has an integration rate of between about 15% and about 25%. In some embodiments, the hyperactive transposase protein has an integration rate of about 15%, about 20%, or about 25%.21MF-366023694Attorney Docket No: 230702000640

[0094] In some embodiments, the hyperactive transposase protein comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the hyperactive transposase protein is TnPB_hyC57a. In some embodiments, the hyperactive transposase protein has an excision rate of between about 40% and about 55% as measured seamless excision assay performed in Nicotiana benthamiana. In some embodiments, has an excision rate of about 51.5%.

[0095] In some embodiments, the hyperactive transposase protein comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the hyperactive transposase protein is TnPB_hyC57a. In some embodiments, the hyperactive transposase protein has an excision rate of between about 15% and about 25% as measured seamless excision assay performed in yeast. In some embodiments, the excision rate is about 21.1%.

[0096] In some embodiments, the hyperactive transposase protein comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the hyperactive transposase protein is TnPB_hyL43A. In some embodiments, the hyperactive transposase protein has an excision rate of between about 40% and about 55% as measured seamless excision assay performed in Nicotiana benthamiana. In some embodiments, has an excision rate of about 51.5%.

[0097] In some embodiments, the hyperactive transposase protein comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the hyperactive transposase protein is TnPB_hyL43A. In some embodiments, the hyperactive transposase protein has an integration rate of between about 15% and about 25% as measured using a seamless integration assay performed in yeast. In some embodiments, the integration rate is about 20.4%.Methods of reversibly integrating a transposon

[0098] Also provided herein are method of use for the hyperactive transposase proteins described herein. The hyperactive transposase proteins can be used for reversibly integrating a transposon into a genome of a cell. Use of the hyperactive transposase proteins allows for seamless integration and excision of a transposon as described herein.

[0099] Provided herein are methods of reversibly integrating a transposon into the genome of a cell, comprising providing the transposon to the cell; and providing a hyperactive transposase protein described herein to the cell. In some embodiments, the hyperactive transposase protein comprises the amino acid sequence of any one of SEQ ID NO: 2-6. In some embodiments, the hyperactive transposase protein comprises the amino acid sequence22MF-366023694Attorney Docket No: 230702000640 of SEQ ID NO: 3. In some embodiments, the hyperactive transposase protein comprises the amino acid sequence of SEQ ID NO: 6.

[0100] In some embodiments, the transposon comprises a nucleic acid encoding the hyperactive transposase protein. In some embodiments, providing the hyperactive transposase protein comprises expressing the transposon. In some embodiments, providing the transposon and providing the hyperactive transposase protein comprises providing a single nucleic acid to the cell.

[0101] In some embodiments, providing the hyperactive transposase protein comprises expressing a nucleic acid vector comprising a nucleic acid encoding the hyperactive transposase protein. In some embodiments, the transposon is a first nucleic acid and the nucleic acid vector comprising the nucleic acid encoding the hyperactive transposase protein is a second nucleic acid.

[0102] In some embodiments, the transposon comprises a nucleic acid construct flanked by inverted terminal repeat (ITR) sites. In some embodiments, the transposon comprises a nucleic acid construct flanked by a first inverted repeat site and / or a second inverted repeat site. In some embodiments, the nucleic acid construct comprises a reporter system. In some embodiments, the nucleic acid construction comprises a protein encoding gene.

[0103] The transposon and / or the hyperactive transposase protein (e.g., a nucleic acid encoding the hyperactive transposase protein) can be provided to the cell using transformation methods know in the art. The methods may comprise Agrobacterium tumefaciens, biolistics components, agroinfiltration components or plant virus vectors.

[0104] In some embodiments, reversibly integrating the transposon comprises scarless excision of the transposon. In some embodiments, the method comprises excising the transposon from the cell. In some embodiments, the transposon is excised using a hyperactive transposase protein as described herein. In some embodiments, following excision of the transposon, the genome of the cell has not been modified compared to the genome of the cell before integration of the transposon.

[0105] In some embodiments, the method comprises reversibly transforming a cell. In some embodiments, the cell is a plant cell. In some embodiments, the plant cell is a plant cell from Nicotiana benthamiana (Nb), Citrus reticulata, a citrus fruit, a stone fruit, a tree fruit, an 23MF-366023694Attorney Docket No: 230702000640 edible plant, an ornamental plant, model plant, and / or any other plant. In some embodiments, the plant cell is a cell from a citrus fruit species, such as but not limited to Citrus sinensis, Citrus reticulata, Citrus aurantium, Citrus bergamia, Citrus limon, Citrus medica, Citrus maxima, Citrus hystrix, Citrus aurantiifolia, Citrus x sinensis, or Citrus x paradisi.Recombinant nucleic acids, vectors, and host cells

[0106] Also provided herein are nucleic acids, vectors and host cells. In some embodiments, the nucleic acids, vectors and host cells are for use according to the methods described herein.

[0107] Provided herein are recombinant nucleic acid molecule encoding the hyperactive transposase proteins described herein. In some embodiments, the recombination nucleic acid molecules encode the amino acid sequence of any one of SEQ ID NO: 2-6. In some embodiments, the recombination nucleic acid molecules encode the amino acid sequence of SEQ ID NO: 3. In some embodiments, the recombination nucleic acid molecules encode the amino acid sequence of SEQ ID NO: 6.

[0108] Provided herein are vectors comprising the recombinant nucleic acid molecules described herein. In some embodiments, the vectors comprise recombinant nucleic acid molecules encoding the amino acid sequences of any of one SEQ ID NO: 2-6. In some embodiments, the vectors comprise recombinant nucleic acid molecules encoding the amino acid sequence of any SEQ ID NO: 3. In some embodiments, the vectors comprise recombinant nucleic acid molecules encoding the amino acid sequence of any SEQ ID NO: 6.

[0109] Also provided herein are host cells comprising any of the recombinant nucleic acid molecules or vectors described herein. In some embodiments, the host cell is a yeast cell. In some embodiments, the host cell is a plant cell. In some embodiments, the plant cell is a plant cell of any of the plants described herein.4. Exemplary Embodiments

[0110] Among the provided embodiments are:

[0111] Embodiment 1. A hyperactive transposase protein comprising an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO: 3 and / or SEQ ID NO:6.24MF-366023694Attorney Docket No: 230702000640

[0112] Embodiment 2. The hyperactive transposase protein of embodiment 1, wherein the hyperactive transposase protein comprises a core catalytic domain comprising a DDD motif.

[0113] Embodiment 3. The hyperactive transposase protein of embodiment 1 or 2, wherein the hyperactive transposase protein has excision and / or integration activity.

[0114] Embodiment 4. The hyperactive transposase protein of any of embodiments 1-3, wherein the hyperactive transposase protein comprises one or more nuclear localization signal (NLS).

[0115] Embodiment 5. The hyperactive transposase protein of embodiment 4, wherein the one or more NLS are located at the N and / or the C terminus of the hyperactive transposase protein.

[0116] Embodiment 6. The hyperactive transposase protein of embodiment 4 or 5, wherein presence or absence of the one or more NLS does not affect excision activity of the hyperactive transposase protein.

[0117] Embodiment 7. The hyperactive transposase protein of any of embodiments 1-6, wherein the amino acid sequence has at least 95% identity to the amino acid sequence of SEQ ID NO: 3.

[0118] Embodiment 8. The hyperactive transposase protein of any of embodiments 1-7, wherein the hyperactive transposase protein has between about 2-fold about 6-fold improved excision activity compared to a transposase isolated from Trichoplusia ni without one or more amino acid substitutions.

[0119] Embodiment 9. The hyperactive transposase protein of embodiment 8, wherein the transposase isolated from Trichoplusia ni without one or more amino acid substitutions comprises the amino acid of SEQ ID NO: 1.

[0120] Embodiment 10. The hyperactive transposase protein of any of embodiments 1-7, wherein the hyperactive transposase protein has between about 2-fold about 6-fold improved excision activity compared to a transposase comprising the amino acid sequence of SEQ ID NO: 7.25MF-366023694Attorney Docket No: 230702000640

[0121] Embodiment 11. The hyperactive transposase protein of any of embodiments 8-10, wherein the excision activity is measured using a seamless excision assay.

[0122] Embodiment 12. The hyperactive transposase protein of embodiment 11, wherein the seamless excision assay is performed in yeast.

[0123] Embodiment 13. The hyperactive transposase protein of any of embodiments 1-12, wherein the hyperactive transposase has an excision rate between about 15% and about 25%.

[0124] Embodiment 14. The hyperactive transposase protein of any of embodiments 1-13, wherein the hyperactive transposase has an excision rate of about 21.1%.

[0125] Embodiment 15. The hyperactive transposase protein of embodiment 11, wherein the seamless excision assay is performed in Nicotiana benthamiana.

[0126] Embodiment 16. The hyperactive transposase protein of embodiment 15, wherein the hyperactive transposase has an excision rate between about 40% and about 55%.

[0127] Embodiment 17. The hyperactive transposase protein of embodiment 15 or 16, wherein the hyperactive transposase has an excision rate of about 51.5%.

[0128] Embodiment 18. The hyperactive transposase protein of any of embodiments 13-17, wherein the excision rate is the percent of transformed cells wherein excision occurred in a seamless excision assay.

[0129] Embodiment 19. The hyperactive transposase protein of any of embodiments 1-6, wherein the hyperactive transposase protein has excision and integration activity.

[0130] Embodiment 20. The hyperactive transposase protein of any of embodiments 1-6 and 19, wherein the amino acid sequence has at least 95% identity to the amino acid sequence of SEQ ID NO: 6.

[0131] Embodiment 21. The hyperactive transposase protein of embodiment 19 or 20, wherein the hyperactive transposase protein has similar or increased integration activity compared to a transposase isolated from Trichoplusia ni without one or more amino acid substitutions or a transposase comprising the amino acid sequence of SEQ ID NO: 7.26MF-366023694Attorney Docket No: 230702000640

[0132] Embodiment 22. The hyperactive transposase protein of embodiment 21, wherein the transposase isolated from Trichoplusia ni without one or more amino acid substitutions comprises the amino acid of SEQ ID NO: 1.

[0133] Embodiment 23. The hyperactive transposase protein of embodiment 19 or 20, wherein the hyperactive transposase protein has similar or increased integration activity compared to a transposase comprising the amino acid sequence of SEQ ID NO: 7.

[0134] Embodiment 24. The hyperactive transposase protein of any of embodiments 21-23, wherein the integration activity is measured using a seamless integration assay.

[0135] Embodiment 25. The hyperactive transposase protein of embodiment 24, wherein the seamless integration assay is performed in yeast.

[0136] Embodiment 26. The hyperactive transposase protein of any of embodiments 19-25, wherein the hyperactive transposase has an integration rate between 15% and about 25%.

[0137] Embodiment 27. The hyperactive transposase protein of any of embodiments 19-26, wherein the hyperactive transposase has an integration rate of 20.4%.

[0138] Embodiment 28. The hyperactive transposase protein of embodiment 26 or 27, wherein the integration rate is percent of transformed cells wherein integration occurred in a seamless integration assay.

[0139] Embodiment 29. The hyperactive transposase protein of any of embodiments 9 - 18, wherein the seamless excision assay comprises an excision reporter system.

[0140] Embodiment 30. A hyperactive transposase protein, wherein the hyperactive transposase protein has at least 49% sequence identity to SEQ ID NO: 1, and comprises an amino acid substitution relative to SEQ ID NO: 1 in at least one amino acid position of SEQ ID NO: 1, wherein the amino acid position of SEQ ID NO: 1 is selected from the group consisting of: position 2, position 3, position 4, position 7, position 9, position 12, position 13, position 14, position 15, position 17, position 22, position 23, position 24, position 25, position 30, position 31, position 34, position 35, position 41, position 42, position 44, position 45, position 46, position 49, position 50, position 51, position 52, position 53, position 54, position 57, position 58, position 60, position 62, position 63, position 65, position 66, position 67, position 68, position 69, position 70, position 72, position 75,27MF-366023694Attorney Docket No: 230702000640 position 78, position 81, position 83, position 85, position 103, position 105, position 107, position 109, position 112, position 114, position 139, position 161, position 162, position 163, position 165, position 177, position 179, position 180, position 184, position 185, position 188, position 189, position 194, position 198, position 202, position 204, position 206, position 207, position 226, position 231, position 235, position 240, position 244, position 253, position 256, position 263, position 264, position 282, position 289, position 296, position 309, position 315, position 318, position 322, position 333, position 339, position 340, position 352, position 357, position 367, position 371, position 372, position 375, position 381, position 397, position 411, position 424, position 429, position 434, position 436, position 438, position 449, position 450, position 457, position 469, position 470, position 474, position 478, position 482, position 486, position 490, position 496, position 497, position 500, position 501, position 504, position 505, position 507, position 509, position 512, position 513, position 514, position 533, position 538, position 542, position 546, position 553, position 557, position 566, position 568, position 570, position 571, position 572, position 580, and position 583.

[0141] Embodiment 31. The hyperactive transposase protein of embodiment 30, wherein the hyperactive transposase protein has between 49% and 80% sequence identity to SEQ ID NO:1.

[0142] Embodiment 32. The hyperactive transposase protein of embodiment 30, wherein the hyperactive transposase protein comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acid sequence of SEQ ID NO: 2.

[0143] Embodiment 33. The hyperactive transposase protein of embodiment 30, wherein the hyperactive transposase protein comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acid sequence of SEQ ID NO: 3.

[0144] Embodiment 34. The hyperactive transposase protein of embodiment 30, wherein the hyperactive transposase protein comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acid sequence of SEQ ID NO: 4.28MF-366023694Attorney Docket No: 230702000640

[0145] Embodiment 35. The hyperactive transposase protein of embodiment 30, wherein the hyperactive transposase protein comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acid sequence of SEQ ID NO: 5.

[0146] Embodiment 36. The hyperactive transposase protein of embodiment 27, wherein the hyperactive transposase protein comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acid sequence of SEQ ID NO: 6.

[0147] Embodiment 37. A recombinant nucleic acid molecule encoding the hyperactive transposase protein of any one of embodiments 1-36.

[0148] Embodiment 38. A vector comprising the recombinant nucleic acid molecule of embodiment 37.

[0149] Embodiment 39. A host cell comprising the recombinant nucleic acid molecule of embodiment 37 or the vector of embodiment 38.

[0150] Embodiment 40. The host cell of embodiment 39, wherein the host cell is a yeast cell.

[0151] Embodiment 41. The host cell of embodiment 40, wherein the host cell is a plant cell.

[0152] Embodiment 42. A method of reversibly integrating a transposon into the genome of a cell, comprising: providing the transposon to the cell; and providing a hyperactive transposase protein of any of embodiments 1-36 to the cell.

[0153] Embodiment 43. The method of embodiment 42, wherein the transposon comprises a nucleic acid encoding the hyperactive transposase protein.

[0154] Embodiment 44. The method of embodiment 42 or 43, wherein providing to the hyperactive transposase protein comprises expressing the transposon.

[0155] Embodiment 45. The method of embodiment 42, wherein providing to the hyperactive transposase protein comprises expressing a nucleic acid vector comprising a nucleic acid encoding the hyperactive transposase protein.29MF-366023694Attorney Docket No: 230702000640

[0156] Embodiment 46. The method of any of embodiments 42-45, wherein the transposon comprises a nucleic acid construct flanked by a first inverted repeat site and / or a second inverted repeat site.

[0157] Embodiment 47. The method of any of embodiments 42-46, wherein providing the transposon and / or providing the hyperactive transposase protein to the cell comprises Agrobacterium tumefaciens, biolistics components, agroinfiltration components or plant virus vectors.

[0158] Embodiment 48. The method of any of embodiments 42 - 47, wherein reversibly integrating the transposon comprises scarless excision of the transposon.

[0159] Embodiment 49. The method of any of embodiments 42-47, further comprising excising, the transposon from the cell, using the hyperactive transposase.

[0160] Embodiment 50. The method of any of embodiments 42-49, wherein following excision of the transposon, the genome of the cell has not been modified compared to the genome of the cell before integration of the transposon.

[0161] Embodiment 51. The method of any of embodiments 42-50, wherein the cell is a plant cell.

[0162] Embodiment 52. The method of embodiment 51, wherein the plant cell is from a Nicotiana benthamiana (Nb) plant, a citrus fruit, a stone fruit, a tree fruit, an edible plant, an ornamental plant, and / or a model plant.

[0163] Embodiment 53. The method of embodiment 52, wherein the citrus fruit is Citrus reticulata.5. ExamplesExample 1: Rational Engineering of PiggyBac Transposase Variants Through Phylogenetic and Structural Analysis

[0164] A rational design approach was developed to generate novel PiggyBac transposase variants with preserved catalytic activity while maintaining sequence divergence from the native Trichoplusia ni enzyme. The native PiggyBac transposase sequence (TnPB) was used as a query for a comprehensive BLASTp search of protein databases. Retrieved sequences 30MF-366023694Attorney Docket No: 230702000640 were filtered according to strict criteria: sequence identity between 34-90% compared to TnPB and length within ±20% of the native enzyme (475-712 amino acids). Synthetic sequences were excluded from the analysis.

[0165] The filtered sequences were subjected to multiple sequence alignment using Geneious software to generate a distance matrix. Two sequence sets were constructed based on identity thresholds: TnPB_C57 comprising 57 sequences with 40-100% identity, and TnPB_C100 containing 100 sequences with 34-100% identity. For each set, consensus sequences were derived through a systematic process. Gaps in the consensus were filled with corresponding native TnPB residues, insertions relative to TnPB were removed, and ambiguous positions (X / Z / J / B) were replaced with amino acids chemically similar to those in the native sequence.

[0166] Critical catalytic and structural residues were identified through analysis of available crystal structures (PDB: 6x67 and 6x68) and conserved across all variants. The consensus sequences were then modified to incorporate seven specific mutations previously associated with hyperactivity: S103P, M194V / T, 122 IT, and others. These mutations were introduced while maintaining the overall sequence divergence from TnPB.

[0167] Structural validation was performed using AlphaFold predictions for each variant. The predicted structures were aligned with available crystal structures of the native enzyme to calculate root mean square deviation (RMSD) values. The TnPB_C57 variant demonstrated particularly characteristics, with 77.6% sequence identity to native TnPB and an RMSD of 0.365 A relative to the 6x67 crystal structure, indicating preservation of the critical structural elements.

[0168] Excision activity was evaluated using a dual-plasmid system in yeast cells. The transposase variants were expressed from a constitutive promoter, while a separate reporter plasmid contained inverted terminal repeats flanking a marker gene. Quantification of excision events demonstrated that the engineered variants maintained catalytic activity comparable to the native enzyme.

[0169] This systematic engineering approach successfully generated functional PiggyBac transposase variants with sufficient sequence divergence relative to known transposases while maintaining catalytic activity and structural integrity. The methodology described herein31MF-366023694Attorney Docket No: 230702000640 provides a robust framework for the development of novel transposase variants for biotechnology applications.

[0170] The engineered variants represent significant improvements over existing technologies, offering new tools for genetic. These variants maintain the essential biochemical properties of the native enzyme while exhibiting unique sequence characteristics, as demonstrated by both computational and experimental analyses.Example 2: Excision-Based Quantitative Reporter System in Yeast for Characterization of Novel Transposase Variants Using URA3-mCherry Selection

[0171] The experimental system utilizes a reporter construct that combines multiple genetic elements on a single plasmid (FIG 1). The construct contains the URA3 gene, which functions as a selective marker for uracil biosynthesis in yeast, and this gene is interrupted by the insertion of a transposon construct. The transposon carries the mCherry fluorescent protein gene and is flanked by terminal inverted repeat (ITR) sequences that serve as recognition sites for the transposase. The plasmid includes a G418 antibiotic resistance gene that enables selection of transformed yeast cells. The designed transposase variant is expressed from the same plasmid under the control of a strong constitutive promoter, ensuring consistent protein production throughout the experiment.

[0172] The experimental protocol consists of transforming yeast cells with these reporter plasmids containing different transposase variants. After transformation, the cells undergo a three-hour growth period during which the transposase proteins are expressed and can catalyze transposon excision. The experimental design implements a dual selection strategy to quantify transposase activity. The transformed cell population is plated on two different selective media: one containing G418 antibiotic, which selects for all transformed cells regardless of transposition activity, and another containing both G418 and lacking uracil, which selects for cells where transposon excision has occurred and restored a functional URA3 gene. The results from two independent experiments demonstrate the reproducibility of this assay system (FIG 2). Furthermore, representative plate images comparing the designed variant C57a with the reference transposase PBex show clear differences in colony formation under selective conditions (FIG 3).

[0173] A key feature of this system is the verification of scarless excision of the transposon. When excision occurs correctly, the URA3 gene is reconstructed without nucleotide32MF-366023694Attorney Docket No: 230702000640 alterations or "scars," as demonstrated by cell growth on uracil-deficient medium. This characteristic is relevant for applications requiring precise genetic modifications. The system provides both quantitative data about excision efficiency and confirmation of the molecular precision of the excision event.

[0174] This experimental platform functions as a system for characterizing novel transposase variants. It generates quantifiable results while incorporating multiple selection steps. The design enables direct comparison of different transposase variants under standardized conditions, serving as a tool for the development and optimization of new transposase enzymes for genetic engineering applications.Example 3: Rational Engineering of TnPB_hyC57a through site-directed mutagenesis.

[0175] Other works have shown the potential of site-directed mutagenesis in the improvement of transposase activity. The newly developed TnPB_hyC57a variant sequence (SEQ ID NO:3) was aligned and compared with the original T. ni PB transposase. Two amino acid sites from TnPB_C57a were chosen for restoration of the original amino acid residue from T. ni, given their location in the predicted integration domain, and given their stark difference in amino acid polarity. Thus, mutations E381V (TnPB_hyC57a E381V, SEQ ID NO: 9), S411A (TnPB_hyC57a S411A, SEQ ID NO: 10), and the combined E381V / S411A (TnPB_hyC57a E381V-S411A, SEQ ID NO: 11) were generated in TnPB_hyC57a through site-directed mutagenesis.

[0176] The TnPB_hyC57a mutants were compared to their parental transposase in a yeast transposon excision assay (FIG 1). When results were analyzed relative to the parental TnPB_hyC57a, all three mutants exhibited up to 2-fold increase in excision rate (FIG 4).Example 4: Transposon Integration Assay in Yeast for Characterization of Novel Transposase Variants Using G418-mCherry Selection

[0177] Designed transposases were evaluated for their ability to excise transposons from donor plasmids, and integrate them into genomic DNA. A reporter system was established in S. cerevisiae yeast using two types of plasmids: First, yeast replicating plasmids harboring the transposase variants under the control of a strong constitutive promoter, and containing an URA3 selection marker and CEN / ARS sequences for selection and replication in yeast respectively. Second, a transposon donor plasmid that replicates in Escherichia coli, but is unable to replicate in S. cerevisiae. The transposon contains a G418 resistance gene, linked 33MF-366023694Attorney Docket No: 230702000640 by a T2A ribosomal skipping peptide to the mCherry red fluorescent protein, and both genes are under the control of a strong constitutive promoter and flanked by ITR sequences (FIG 5).

[0178] Yeast cells were first transformed with each plasmid containing transposon variants, and selected for growth in the absence of uracil. Transformant cells were then grown, and subsequently transformed with the transposon donor plasmid, incubated for 1.5 hours, and plated for G418 selection. A control yeast replicating plasmid with G418 resistance was also transformed in cells that expressed the transposases to estimate total transformation efficiency for each yeast line. After two days of incubation at 28°C, colonies were confirmed for mCherry fluorescence using epifluorescence microscopy to calculate the number of integration events. The integration rate was calculated as the number of G418 resistant and mCherry(+) colonies in the non-replicating plasmid transformation divided by the number of G418 resistant colonies in the transformation efficiency control.

[0179] The reference transposase hyPB exhibited strong integration activity, with an average of 31% of transformed cells showing integration events (FIG 6). The reference transposase hyPB (also called hyPBase, SEQ ID NO: 7), is a variant of SEQ ID 1, see Yusa, Kosuke, et al. "A hyperactive piggyBac transposase for mammalian applications." Proceedings of the National Academy of Sciences 108.4 (2011): 1531-1536.

[0180] As previously reported, the reference hyPB R372A / K375A / D450N mutant (SEQ ID NO: 8) was unable to integrate transposons into the yeast genome. When designed transposases were tested, varied results were obtained: TnPB_hyC57a was unable to generate genomic integration of the transposon, even though it showed high seamless excision activity in previous experiments, resembling the activities of the excision able and integration-impaired hyPB R372A / K375A / D450N mutant. The designed TnPB_L43a variant exhibited an average 20.4% integration rate, although obtained colonies were slightly smaller in size compared to reference hyPB transposase (FIG 7).

[0181] Since the TnPB_hyC57a variant was devoid of integration activity, the TnPB_hyC57a E381V (SEQ ID NO: 9), TnPB_hyC57a S411A (SEQ ID NO: 10), and TnPB_hyC57a E381V / S411A (SEQ ID NO: 11) mutants which previously showed enhanced excision activity were tested for integration activity (FIG 6). Of these, the TnPB_hyC57a34MF-366023694Attorney Docket No: 230702000640 E381V / S411A showed integration activity in yeast assays, averaging 2.1% transposon integration rate across experiments.Example 5: Activity of designed transposases in plants: Transposon excision assay in Nicotiana benthamiana.

[0182] Designed transposases were evaluated for their ability to excise transposons in a plant model system using transposase expressing and seamless excision reporter plasmids (FIG 8).Two types of plasmids were constructed to express the reference and designed transposases, one set of plasmids containing each transposase variant, expressed using a strong plant viral promoter. A second set of plasmids contained transposase variants flanked by nuclear localization signals (NLS), expressed using a strong plant viral promoter. The excision reporter plasmid contained a reporter cassette consisting of the Neongreen fluorescent gene interrupted by the transposon, containing the mScarlet fluorescent protein gene flanked by ITR sites. Both fluorescent protein encoding genes contained nuclear localization signals. All plasmids have elements to allow replication in Agrobacterium tumefaciens, and genes of interest were flanked by left border (LB) and right border (RB) sequences to allow transformation of plant cells by Agrobacterium T-DNA.

[0183] Transposase expression and excision reporter plasmids were transformed into A. tumefaciens GV31O3 cells and selected for kanamycin resistance. Then, A. tumefaciens cultures were grown, induced with acetosyringone, and syringe infiltrated into N. benthamiana leaves. After 5 days, leaf discs were cut and analyzed by epifluorescence microscopy. Transformed plant cells fluoresce red by expression of the mScarlett gene. If the designed transposase has excisionase activity, it excises the transposon in a scarless manner, reestablishing the Neongreen gene, generating cells that have nuclei with red and green fluorescence.

[0184] The rate of excision for each condition was calculated as the number of Neongreen (+) nuclei divided by the number of mScarlet (+) nuclei (FIG 9). When the reporter construction is infiltrated without a transposase, no Neongreen fluorescence is visible. When the reporter was co-infiltrated with the reference hyPB expressing construction, a 9.3% average excision rate was observed. A drastic increase in excision rate was observed when nuclear localization signals were added to the hyPB transposase (SEQ ID NO: 12), and similar rates of excision were seen with the reference hyPB R372A / K375A / D450N mutant (SEQ ID NO: 13). When the designed transposases were tested, TnPB_hyC57a (with NLS:35MF-366023694Attorney Docket No: 230702000640 SEQ ID NO: 14, without NLS: SEQ ID NO: 3). showed a 51.5% average excision rate, and no significant difference was observed with the addition of NLS to this variant. This same behaviour can be observed in the representative microscopy images (FIG 10).

[0185] These results show that designed transposases are functional in plants and have similar activity rates to published reference transposases of the PiggyBac family.Table 1: Exemplary transposase sequences36MF-366023694Attorney Docket No: 23070200064037MF-366023694Attorney Docket No: 23070200064038MF-366023694Attorney Docket No: 23070200064039MF-366023694Attorney Docket No: 23070200064040MF-366023694

Claims

Attorney Docket No: 230702000640CLAIMSWhat is claimed is:

1. A hyperactive transposase protein comprising an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO: 3 and / or SEQ ID NO:6.

2. The hyperactive transposase protein of claim 1, wherein the hyperactive transposase protein comprises a core catalytic domain comprising a DDD motif.

3. The hyperactive transposase protein of claim 1 or 2, wherein the hyperactive transposase protein has excision and / or integration activity.

4. The hyperactive transposase protein of any of claims 1-3, wherein the hyperactive transposase protein comprises one or more nuclear localization signal (NLS).

5. The hyperactive transposase protein of claim 4, wherein the one or more NLS are located at the N and / or the C terminus of the hyperactive transposase protein.

6. The hyperactive transposase protein of claim 4 or 5, wherein presence or absence of the one or more NLS does not affect excision activity of the hyperactive transposase protein.

7. The hyperactive transposase protein of any of claims 1-6, wherein the amino acid sequence has at least 95% identity to the amino acid sequence of SEQ ID NO: 3.

8. The hyperactive transposase protein of any of claims 1-7, wherein the hyperactive transposase protein has between about 2-fold about 6-fold improved excision activity compared to a transposase isolated from Trichoplusia ni without one or more amino acid substitutions.

9. The hyperactive transposase protein of claim 8, wherein the transposase isolated from Trichoplusia ni without one or more amino acid substitutions comprises the amino acid of SEQ ID NO: 1.41MF-366023694Attorney Docket No: 230702000640 10. The hyperactive transposase protein of any of claims 1-7, wherein the hyperactive transposase protein has between about 2-fold about 6-fold improved excision activity compared to a transposase comprising the amino acid sequence of SEQ ID NO: 7.

11. The hyperactive transposase protein of any of claims 8-10, wherein the excision activity is measured using a seamless excision assay.

12. The hyperactive transposase protein of claim 11, wherein the seamless excision assay is performed in yeast.

13. The hyperactive transposase protein of any of claims 1-12, wherein the hyperactive transposase has an excision rate between aboutl5% and about 25%.

14. The hyperactive transposase protein of any of claims 1-13, wherein the hyperactive transposase has an excision rate of about 21.1%.

15. The hyperactive transposase protein of claim 11, wherein the seamless excision assay is performed in Nicotiana benthamiana.

16. The hyperactive transposase protein of claim 15, wherein the hyperactive transposase has an excision rate between about 40% and about 55%.

17. The hyperactive transposase protein of claim 15 or 16, wherein the hyperactive transposase has an excision rate of about 51.5%.

18. The hyperactive transposase protein of any of claims 13-17, wherein the excision rate is the percent of transformed cells wherein excision occurred in a seamless excision assay.

19. The hyperactive transposase protein of any of claims 1-6, wherein the hyperactive transposase protein has excision and integration activity.

20. The hyperactive transposase protein of any of claims 1-6 and 19, wherein the amino acid sequence has at least 95% identity to the amino acid sequence of SEQ ID NO: 6.42MF-366023694Attorney Docket No: 23070200064021. The hyperactive transposase protein of claim 19 or 20, wherein the hyperactive transposase protein has similar or increased integration activity compared to a transposase isolated from Trichoplusia ni without one or more amino acid substitutions or a transposase comprising the amino acid sequence of SEQ ID NO: 7.

22. The hyperactive transposase protein of claim 21, wherein the transposase isolated from Trichoplusia ni without one or more amino acid substitutions comprises the amino acid of SEQ ID NO: 1.

23. The hyperactive transposase protein of claim 19 or 20, wherein the hyperactive transposase protein has similar or increased integration activity compared to a transposase comprising the amino acid sequence of SEQ ID NO: 7.

24. The hyperactive transposase protein of any of claims 21-23, wherein the integration activity is measured using a seamless integration assay.

25. The hyperactive transposase protein of claim 24, wherein the seamless integration assay is performed in yeast.

26. The hyperactive transposase protein of any of claims 19-25, wherein the hyperactive transposase has an integration rate between 15% and about 25%.

27. The hyperactive transposase protein of any of claims 19-26, wherein the hyperactive transposase has an integration rate of 20.4%.

28. The hyperactive transposase protein of claim 26 or 27, wherein the integration rate is percent of transformed cells wherein integration occurred in a seamless integration assay.

29. The hyperactive transposase protein of any of claims 9 - 18, wherein the seamless excision assay comprises an excision reporter system.43MF-366023694Attorney Docket No: 230702000640 30. A hyperactive transposase protein, wherein the hyperactive transposase protein has at least 49% sequence identity to SEQ ID NO:1, and comprises an amino acid substitution relative to SEQ ID NO: 1 in at least one amino acid position of SEQ ID NO: 1, wherein the amino acid position of SEQ ID NO: 1 is selected from the group consisting of: position 2, position 3, position 4, position 7, position 9, position 12, position 13, position 14, position 15, position 17, position 22, position 23, position 24, position 25, position 30, position 31, position 34, position 35, position 41, position 42, position 44, position 45, position 46, position 49, position 50, position 51, position 52, position 53, position 54, position 57, position 58, position 60, position 62, position 63, position 65, position 66, position 67, position 68, position 69, position 70, position 72, position 75, position 78, position 81, position 83, position 85, position 103, position 105, position 107, position 109, position 112, position 114, position 139, position 161, position 162, position 163, position 165, position 177, position 179, position 180, position 184, position 185, position 188, position 189, position 194, position 198, position 202, position 204, position 206, position 207, position 226, position 231, position 235, position 240, position 244, position 253, position 256, position 263, position 264, position 282, position 289, position 296, position 309, position 315, position 318, position 322, position 333, position 339, position 340, position 352, position 357, position 367, position 371, position 372, position 375, position 381, position 397, position 411, position 424, position 429, position 434, position 436, position 438, position 449, position 450, position 457, position 469, position 470, position 474, position 478, position 482, position 486, position 490, position 496, position 497, position 500, position 501, position 504, position 505, position 507, position 509, position 512, position 513, position 514, position 533, position 538, position 542, position 546, position 553, position 557, position 566, position 568, position 570, position 571, position 572, position 580, and position 583.

31. The hyperactive transposase protein of claim 30, wherein the hyperactive transposase protein has between 49% and 80% sequence identity to SEQ ID NO: 1.

32. The hyperactive transposase protein of claim 30, wherein the hyperactive transposase protein comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acid sequence of SEQ ID NO: 2.44MF-366023694Attorney Docket No: 23070200064033. The hyperactive transposase protein of claim 30, wherein the hyperactive transposase protein comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acid sequence of SEQ ID NO: 3.

34. The hyperactive transposase protein of claim 30, wherein the hyperactive transposase protein comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acid sequence of SEQ ID NO: 4.

35. The hyperactive transposase protein of claim 30, wherein the hyperactive transposase protein comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acid sequence of SEQ ID NO: 5.

36. The hyperactive transposase protein of claim 27, wherein the hyperactive transposase protein comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to amino acid sequence of SEQ ID NO: 6.

37. A recombinant nucleic acid molecule encoding the hyperactive transposase protein of any one of claims 1-36.

38. A vector comprising the recombinant nucleic acid molecule of claim 37.

39. A host cell comprising the recombinant nucleic acid molecule of claim 37 or the vector of claim 38.

40. The host cell of claim 39, wherein the host cell is a yeast cell.

41. The host cell of claim 40, wherein the host cell is a plant cell.45MF-366023694Attorney Docket No: 230702000640 42. A method of reversibly integrating a transposon into the genome of a cell, comprising:providing the transposon to the cell; andproviding a hyperactive transposase protein of any of claims 1-36 to the cell.

43. The method of claim 42, wherein the transposon comprises a nucleic acid encoding the hyperactive transposase protein.

44. The method of claim 42 or 43, wherein providing to the hyperactive transposase protein comprises expressing the transposon.

45. The method of claim 42, wherein providing to the hyperactive transposase protein comprises expressing a nucleic acid vector comprising a nucleic acid encoding the hyperactive transposase protein.

46. The method of any of claims 42-45, wherein the transposon comprises a nucleic acid construct flanked by a first inverted repeat site and / or a second inverted repeat site.

47. The method of any of claims 42-46, wherein providing the transposon and / or providing the hyperactive transposase protein to the cell comprises Agrobacterium tumefaciens, biolistics components, agroinfiltration components or plant virus vectors.

48. The method of any of claims 42 - 47, wherein reversibly integrating the transposon comprises scarless excision of the transposon.

49. The method of any of claims 42-47, further comprising excising, the transposon from the cell, using the hyperactive transposase.

50. The method of any of claims 42-49, wherein following excision of the transposon, the genome of the cell has not been modified compared to the genome of the cell before integration of the transposon.

51. The method of any of claims 42-50, wherein the cell is a plant cell.46MF-366023694Attorney Docket No: 230702000640 52. The method of claim 51, wherein the plant cell is from a Nicotiana benthamiana (Nb) plant, a citrus fruit, a stone fruit, a tree fruit, an edible plant, an ornamental plant, and / or a model plant.

53. The method of claim 52, wherein the citrus fruit is Citrus reticulata.47MF-366023694

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