System for integration and controlled excision of genes

Abstract

The present discloses a reversible transgenesis system for reversible integration into a plant genome comprising a transposable element module comprising, a transgene expression cassette; and a transient expression module comprising, a plurality of integration components and a nucleic acid encoding an excision-only transposase.

Description

Attorney Docket No: 230702000440 SYSTEM FOR INTEGRATION AND CONTROLLED EXCISION OF GENES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The application claims the priority benefit of U.S. Provisional Application No. 63 / 657,664, filed June 7, 2024, the entire contents of which is incorporated herein by reference for all purposes. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (230702000440SEQLIST.xml; Size: 33,040 bytes; and Date of Creation: June 4, 2025) is herein incorporated by reference in its entirety. BACKGROUND 1. Field of The Invention

[0003] The field of invention encompasses methods, systems, and compositions for enhancing precision and efficiency in genome engineering using transposase systems. This field of invention includes the development of modified versions of transposases, strategies for reversible transgenesis, precise genome editing in plants, and temporary expression of gene- editing machinery for targeted mutagenesis. It encompasses both the methodologies and the tools involved in leveraging a transposase system to enhance precision and efficiency in genome engineering in agriculturally important crops. 2. Description of the related art

[0004] Various methods for gene editing in plants have been explored, showcasing the versatility and innovation in this field. The CRISPR / Cas9 system stands out as a powerful tool for targeted gene modification, employing guide RNA (gRNA) to direct the Cas9 endonuclease to specific DNA sequences for editing. This method has been successfully applied across diverse plant species, including Arabidopsis, tobacco, sorghum, rice, wheat, maize, apple, and grapevine. Delivery of the CRISPR / Cas9 components into plant cells is achieved through methods such as biolistic delivery, where genetic material is propelled into cells using microparticles, and lipofection-mediated delivery utilizing lipid-based carriers like liposomes. Page 1 out of 41sf-6759494Attorney Docket No: 230702000440 Nanoparticle-mediated delivery, employing particles like gold or silica nanoparticles, and viral vector-mediated delivery using systems like Barley stripe mosaic virus (BSMV), further expand the toolbox for efficient gene editing in plants, as demonstrated in wheat and maize. Additionally, techniques involving transposons and piggyBac-like transposases offer another avenue for gene editing. Transposons are DNA elements that can move within the genome, and piggyBac-like transposases are enzymes capable of facilitating this movement. These approaches highlight the ongoing efforts to optimize and diversify gene editing techniques, paving the way for advancements in crop improvement and biotechnology.

[0005] 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.

[0006] Research has extensively explored the potential of the piggyBac transposase system in various genome engineering applications. The works by Li, X., et al (2013) and Chen, Q., et al (2020) 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 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.

[0007] In the realm of plant genome engineering, the work by Nishizawa-Yokoi, A. and Toki, S. (2023) 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. Page 2 out of 41sf-6759494Attorney Docket No: 230702000440

[0008] Expanding on the utility of the piggyBac system, the work by Nishizawa-Yokoi, A. and Toki, S. (2023) 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.

[0009] 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.

[0010] Transcriptional-based systems for gene circuits in plants had greatly improved in recent years. Work by Lloyd, J. (2022) developed recombinase-based genetic switches to enable inducible activation or repression of a circuit output to transform a lowly expressed input signal into a strong activation signal taking advantage of the analog-to-digital conversion feature of recombinases. These results may be of great utility in the need for temporary precise control of gene expression events. SUMMARY

[0011] The disclosed invention presents an integrated system for genetic engineering in plants, featuring precise transgene integration and scarless removal. It involves a transposon-based approach facilitated by transposase and excisionase enzymes. The system allows for controlled expression of transgenes through inducible promoters and recombinase enzymes, ensuring efficient genetic modifications without leaving transgenic traces. The invention includes a recombinase-based genetic switch for regulating gene expression and employs specific genetic elements for traceable transgene expression and removal. The system's components include modules for transgene integration, recombinase-based genetic switch regulation, and transient expression, all coordinated to achieve footprint-free controlled excision of the transgene cassette. The transgene expression cassette is highly flexible, enabling the expression of multiple genes tailored to specific applications. Additionally, the system can be delivered using Page 3 out of 41sf-6759494Attorney Docket No: 230702000440 various methods such as Agrobacterium tumefaciens, direct delivery, biolistics or viral vectors. An exemplary embodiment utilizes PiggyBac (PB) transposase for integration and a modified version (called in this invention PBEx) for excision, achieving efficient gene editing with no detectable transgenic remnants. Further enhancements, like codon optimization, virus-induced silencing suppressors, introns, and cellular localization tags, improve performance and precision. This integrated approach provides a powerful tool for precise genetic modifications in plants while resulting in a transgene-free background.

[0012] In some embodiments, provided herein is the reversible transgenesis system for plant genomes, enabling the integration and subsequent excision of transgenes. The system comprises two main modules: a transferable or transposable element module containing a transgene expression cassette, and a transient expression module with integration components and an excision-only transposase. The transgene expression cassette comprises nucleic acids coding genes of interest (such as genes for gene editing), regulated by promoters, and can be integrated into the plant genome via transposase or integrase. The system's excision is activated by the excision-only transposase, ensuring reversibility. A kit is also provided, containing the transgenesis system, a transformation agent (such as Agrobacterium tumefaciens, biolistics, agroinfiltration components, or plant virus vectors), and a buffer. This kit facilitates the reversible integration of the system into plant cells. The system so disclosed, can be used in a method which involves obtaining plasmids with the transgenesis system, integrating them into the plant genome, and activating the excision to remove the system when desired. This reversible approach offers flexibility in genetic modifications, making it ideal for research and agricultural applications where temporary gene expression is advantageous. BRIEF DESCRIPTION OF FIGURES

[0013] FIGURE 1. Main components of the system disclosed in this invention. Some of the components are optional (see Detailed Description of the Invention).

[0014] FIGURE 2. Components of the module 103 of the system, accounting for the Transgene Cargo to be expressed transiently.

[0015] FIGURE 3. Components of the module 102 of the system, accounting for the optional recombinase-based genetic switch (memory circuit), responsible for the efficient excision of the transposon from the genome. Page 4 out of 41sf-6759494Attorney Docket No: 230702000440

[0016] FIGURE 4. Components of the module 106 of the system, accounting optional accessory components aimed to improve the delivery of the entire system into a plant cell.

[0017] FIGURE 5A and 5B. Figure 5A shows a description of the whole system, performing the steps of integration into the plant genome, activation of transgene expression, activation of the optional recombinase-based genetic switch (memory circuit), and the excision process to leave a transgene-free genome. Figure 5B shows an enlarged version of the system being inserted into the plant genome according to embodiments described herein.

[0018] FIGURE 6. Exemplary integration module (Citrus integration plasmid) and transposase variants modules (PB variants plasmids) used in Example 1 using protoplast transfection. Sc: Scarlet fluorescent protein; NG: NeonGreen fluorescent protein; NptII: aminoglycoside 3'-phosphotransferase II.

[0019] FIGURE 7. Epifluorescence microscopy of integration experiment in citrus protoplasts five weeks post-transformation. Upper panel: Bright-field. Mid panel: Scarlet fluorescence field. Lower panel: NeonGreen fluorescence field. CRTL: control.

[0020] FIGURE 8. Exemplary excision module (NISIN plasmid) used in Example 2.

[0021] FIGURE 9. Excision efficiency rates for excision-only PB variants in Nicotiana benthamiana (Nb). PBEx-WT: Wild type excision-only PiggyBac co-transformed with Excision reporter; PBEx-OPT: Optimised PiggyBac variant co-transformed with Excision reporter; Ex-Rep: Excision reporter only (without any PiggyBac variant); Panel: Graph for the quantification of fluorescence ratios; Ordinate: fluorescent protein excision reporter intensity normalised by fluorescent protein transformation reporter.

[0022] FIGURE 10A and 10B. Excision efficiency rates for excision-only PB variants in Citrus. PBEx-WT: Wild type excision-only PiggyBac co-transformed with Excision reporter; PBEx-OPT: Optimised PiggyBac variant co-transformed with Excision reporter; Ex-Rep: Excision reporter only (without any PiggyBac variant). Figure 10A shows epifluorescence microscopy of protoplasts five days post-transformation. Figure 10B shows a graph for the quantification of fluorescence ratios. Ordinate: fluorescent protein excision reporter intensity normalised by fluorescent protein transformation reporter.

[0023] FIGURE 11. Exemplary recombinase-based genetic switch used in Example 3.

[0024] FIGURE 12A and 12B. Induction rates for recombinase-based genetic switch in Nicotiana benthamiana (Nb). DEX: Dexamethasone; HS: Heat-Shock. Figure 12A shows leaf Page 5 out of 41sf-6759494Attorney Docket No: 230702000440 section epifluorescence microscopy. Figure 12B shows a graph for the quantification of fluorescence ratios. Ordinate: fluorescent protein excision reporter intensity normalised by fluorescent protein transformation reporter.

[0025] FIGURE 13A and 13B. Induction rates for recombinase-based genetic switch in Nicotiana benthamiana (Nb). Figure 13A shows Leaf section epifluorescence microscopy. Figure 13B shows a graph for the quantification of fluorescence ratios.

[0026] FIGURE 14A and 14B. Schematic of PBEx excisionase variants with different combinations of nuclear localization signals (NLS) and an excision reporter system. Figure 14A depicts the PBEx_Opt excisionase with NLS and the excision reporter system, consisting of ITR-L and ITR-R sequences flanking a transposon that interrupts the mNeon green fluorescent protein, and the mScarlet red fluorescent protein as a transformation marker. The excision process is indicated with arrows, showing how the transposon excision reconstitutes the functional expression of mNeon. Figure 14B details twelve PBEx_Opt variants generated with different combinations of nuclear localization signals in the N-terminal and C-terminal regions, using SV40 bipartite NLS (BP), SV40 monopartite large T-antigen NLS (SV40), and Nucleoplasmin NLS (NP).

[0027] FIGURE 15A and 15B. Excision efficiency results for different PBEx_Opt variants in Nicotiana benthamiana. Figure 15A shows the percentage of green nuclei (mNeon, indicating transposon excision) relative to red nuclei (mScarlet, indicating transformed cells) for each PBEx_Opt variant. The variants Opt.2 and Opt.4 exhibit approximately a 5-fold increase in excision efficiency compared to the original PBEx_Opt protein, with excision values of 67- 69% for these variants. Figure 15B shows representative fluorescence microscopy images for three selected variants (PBEx_Opt2, PBEx_Opt3, and PBEx_Opt5), showing nuclei expressing mScarlet (left) and mNeon (right) in the same infiltrated areas.

[0028] FIGURE 16A-16C. Transposon integration system in Nicotiana benthamiana using PB integrase variants and a reporter plasmid. Figure 16A schematically shows two constructs: (1) plasmids with integrase variants (PB_Opt_b and PB_Opt_c) flanked by T-DNA borders (LB and RB), and (2) the reporter plasmid containing mScarlet and a transposon consisting of ITR- L and ITR-R flanking a fusion of hygromycin resistance gene, T2A peptide, and mNeon green fluorescent protein. Figure 16B shows the experimental process: infiltration of N. benthamiana, in vitro introduction of leaf disks, hygromycin selection, and obtaining rooted plantlets in jars. Figure 16C shows fluorescence microscopy images of two types of observed events: T-DNA- Page 6 out of 41sf-6759494Attorney Docket No: 230702000440 mediated integration (62.5% of cases) with expression of both mNeon and mScarlet, and PB- mediated integration (37.5% of cases) with expression of mNeon only.

[0029] FIGURE 17. Design of the integration reporter plasmid used in citrus embryogenic callus. The schematic illustrates the structure of a single plasmid containing both the integrase gene and the reporter cassette. The integrase region contains the PB_Opt gene, followed by the T2A peptide and the NeonGreen fluorescent protein. The reporter cassette region contains the hygromycin resistance gene (Hyg) followed by the T2A peptide and the mScarlet red fluorescent protein.

[0030] FIGURE 18A and 18B. Experimental process and results of transposon integration in citrus embryogenic callus using the PB system. Figure 18A shows the workflow: preparation of citrus embryogenic callus, agroinfiltration, co-culture, selection and enrichment of transformed cells, and analysis by genome walking PCR and Nanopore sequencing. The images show the callus and the observed fluorescence (at 50x and 100x magnification). Figure 18B illustrates four integration events (I-IV) detected in the Citrus reticulata genome, showing the transposon sequence (ITR-R), the TTAA insertion site (characteristic of PB-mediated integration), and the adjacent genomic sequences with their respective locations in genome scaffolds. (TTAATGGTCAAAGATTAAAGCCATA (SEQ ID NO: 18); TTAAGGCTTTAAGCAAAGACAATAA (SEQ ID NO: 19); TTAAGCAATCAGCAATAAGCAGTAA (SEQ ID NO: 20); TTAAACACACGGCCACAAAACTAA (SEQ ID NO: 21) DESCRIPTION OF THE EMBODIMENTS 1. General description

[0031] This invention discloses methods and systems for reversible transgenesis in plant genomes, allowing temporary integration and subsequent excision of transgenes. The system includes a transferable element with a transgene expression cassette and a transient module with an excision-only transposase. Integration methods such as Agrobacterium tumefaciens or biolistics facilitate the process. A kit comprising the system, a transformation agent, and a buffer is also provided. This novel system has the ability to reversibly modify plant genomes, enabling controlled, temporary genetic modifications wherein the excision leaves no trace of modification, ideal for research and agricultural applications. Page 7 out of 41sf-6759494Attorney Docket No: 230702000440

[0032] The description of the various embodiments disclosed here should not limit the present invention but rather instruct a skilled person in the art to carry out the different embodiments of the system explained here.

[0033] The present invention is directed to an integrated system that involves the integration of a transposon into a plant genome. This integration is facilitated by a transposase enzyme, allowing for the introduction of transgenes into the genome. Following integration, components of interest are allowed to be expressed, and then controllable or induced expression of excisionase enzymes enables removal of the transposon, without any traceable scars. A recombinase-based genetic switch provides precise control over the excision processes, with inducible promoters and recombinase enzymes regulating timing and execution, and mainly increasing the excision efficiency. Transgene expression can be traceable using specific genetic elements, facilitating clear tracking within the genome (e.g marker and / or reporter genes). This system enables, for example, efficient gene editing in plants, with editing enzymes allowing precise modifications and markers allowing cell / tissue selection and phenotypic screening. After editing, plants exhibit desired traits without retaining transgenes or any genetic scars, ensuring a transgene-free background. Through coordinated action, these transposases and excisionases achieve temporary integration and controllable scarless excision of transgenes, providing flexibility and reliability in genetic engineering efforts. 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 here.

[0035] A transgene is a segment of DNA that has been artificially introduced into the genome of an organism from a different species or from the same species but at a different location. Transgenes are often engineered to confer specific traits or characteristics to the organism, such as disease resistance, enhanced productivity, or the production of a desired protein.

[0036] A transposase is an enzyme responsible for catalyzing the movement of genetic elements called transposons within a genome. Transposons, also known as "jumping genes," are DNA sequences that have the ability to change their position within the genome. They typically contain terminal inverted repeat (ITR) sequences at their ends, which serve as recognition sites for transposase enzymes. ITR sequences are short, inverted DNA repeats that flank the transposon and are essential for its mobilization. Transposons can disrupt genes, alter Page 8 out of 41sf-6759494Attorney Docket No: 230702000440 gene expression, or cause genetic rearrangements, and their movement is facilitated by transposase enzymes binding to and acting upon the ITR sequences. Transposons play important roles in genome evolution, genetic diversity, and genetic engineering applications.

[0037] An excisionase is an enzyme that catalyzes the removal of specific DNA sequences, such as transposons, from a genome. Excisionase activity is crucial for excising transposons or other genetic elements that have been integrated into the genome, allowing for precise control over genetic modifications.

[0038] An inducible promoter is a DNA sequence that controls the expression of a gene in response to specific external signals or environmental conditions. Inducible promoters are typically inactive under normal conditions but become active in the presence of inducers, triggering gene expression. This property allows for precise regulation of gene expression in response to changing environmental cues.

[0039] A constitutive promoter is a DNA sequence that drives the continuous, uninterrupted expression of a gene under normal physiological conditions. Unlike inducible promoters, constitutive promoters are active at all times and do not require external signals or inducers to initiate gene expression. Constitutive promoters are commonly used to drive the expression of essential genes that are required for basic cellular functions.

[0040] A recombinase is an enzyme that catalyzes the rearrangement of DNA molecules by promoting recombination events between DNA sequences. Recombinases play a key role in various genetic processes, such as DNA repair, homologous recombination, and site-specific DNA integration or excision. Recombinases are often used in genetic engineering to manipulate DNA sequences with precision.

[0041] For the pursuit of this invention, a recombinase-based genetic switch refers to a synthetic genetic circuit engineered to trigger a sustained output response after an input signal activates the system. Another term used to denote the recombinase-based genetic switch is a memory circuit.

[0042] A reporter gene is a gene that encodes a protein whose expression can be easily detected and measured, allowing researchers to monitor gene expression or track cellular processes. Reporter genes are often fused to regulatory sequences of interest to study gene expression patterns, promoter activity, or protein localization in cells or organisms.

[0043] Gene editing refers to the precise modification of DNA sequences within the genome of an organism. Gene editing techniques, such as CRISPR-Cas9, allow researchers to make Page 9 out of 41sf-6759494Attorney Docket No: 230702000440 targeted changes to specific genes by introducing, removing, or altering nucleotide sequences. Gene editing has numerous applications in basic research, biotechnology, and medicine, including the correction of genetic disorders, the development of genetically modified organisms, and the study of gene function.

[0044] A negative selection gene is a gene that confers a disadvantageous phenotype to cells or organisms expressing it, typically resulting in their death or elimination. Negative selection genes are often used in genetic engineering to select against undesired events, such as the retention of transgenes or unintended genomic modifications. Cells or organisms that fail to undergo the desired genetic modification, such as transgene removal, are eliminated through the expression of negative selection genes.

[0045] Integration refers to the incorporation or insertion of foreign DNA, such as transgenes or genetic constructs, into the genome of an organism. Integration can occur through various mechanisms, including homologous recombination, non-homologous end-joining, or the activity of transposons or viral vectors. Integration of foreign DNA into the genome allows for stable inheritance of genetic modifications and long-term expression of transgenes.

[0046] A transgene-free genome refers to the absence of any artificially introduced DNA sequences, such as transgenes or genetic constructs, in the genome of an organism. Achieving a transgene-free genome is often desirable in genetic engineering applications to ensure the removal of unwanted transgenic elements or to produce genetically clean organisms with natural genomic backgrounds. This can be achieved through various methods, including the excision of transgenes using site-specific recombination systems or the elimination of transgenic lines through breeding strategies.

[0047] A reversible transgenesis system refers to a genetic engineering technique aimed at reverting a transgenic organism back to its previous, partial or complete, transgene-free genotype state. This reversion process is facilitated by the expression of a transgene-removal enzyme. This enzyme can be introduced into the organism through re-transformation or by controlled activation of an enzyme.

[0048] Agrobacterium tumefaciens is a soil-dwelling bacterium known for its ability to transfer DNA between itself and plant hosts. This bacterium is a natural genetic engineer and is widely utilized in biotechnology for the creation of transgenic plants. The use of Agrobacterium tumefaciens in transgenics involves the delivery of foreign DNA, such as transgenes or genetic constructs, into the genome of plants. In addition to Agrobacterium tumefaciens, other bacteria Page 10 out of 41sf-6759494Attorney Docket No: 230702000440 utilized for plant transformation include certain strains of both pathogenic and non-pathogenic bacteria capable of transferring DNA into plant cells. These bacteria play essential roles in alternative methods of genetic engineering in plants. Examples of such bacteria include Agrobacterium rhizogenes, which induces hairy root formation; various strains of Rhizobium, known for symbiotic relationships with legumes and DNA transfer capabilities; Sinorhizobium meliloti, another useful bacterium for plant transformation; Rhizobium radiobacter (formerly Agrobacterium radiobacter), closely related to A. tumefaciens; and certain strains of Pseudomonas fluorescens, particularly used in tobacco transformation. Each bacterium offers unique advantages and applications, expanding the repertoire of tools available for researchers in plant biotechnology and genetic modification endeavours.

[0049] Protein P19 is a viral suppressor of RNA silencing (VSR) encoded by tombusviruses, a family of positive-sense RNA viruses. P19 functions by binding to small interfering RNA (siRNA) molecules, thereby preventing them from silencing viral RNA. This interference with the RNA silencing pathway enhances viral replication and contributes to the pathogenicity of tombusviruses. P19 is commonly used in plant molecular biology research as a tool to suppress RNA silencing. By expressing P19 in plants, researchers can study the effects of RNA silencing suppression on viral infection, as well as on endogenous gene expression. Additionally, P19 has been utilized in biotechnology applications, such as enhancing transgene expression and improving virus-induced gene silencing (VIGS) efficiency in plants. 3. Detailed description

[0050] The present invention pertains to a system designed for enabling reversible transgenesis. Its primary functions include the introduction of one or more transgenes, programmable or controlled expression of the one or more transgenes (e.g. through inducible expression of a genetic switch), and subsequent removal of the one or more transgenes without leaving any traceable scars. This system consists of a module featuring an expression cassette equipped with a recombinase-based genetic switch to stimulate gene expression, along with a transient expression cassette (refer to Figure 1). The system offers flexibility by either integrating into a single expression vector or being delivered through distinct vectors for each module. The system described herein can produce a rate of integration two-fold higher than integration of a transgene a control system, such as a T-DNA dependent system.

[0051] The integrated system outlined in this patent application, as illustrated in Figure 1, encompasses a minimum of three modules along with additional accessory components. Each Page 11 out of 41sf-6759494Attorney Docket No: 230702000440 module executes specific functions vital to the overall functionality of the system. Integration into vector 100 is achievable, with module 101 housing a transferable or transposable element, optionally flanked by components 104 and 105 containing ITR sites. Module 101 further subdivides into (sub)modules 102 and 103. The former comprises components of an optional recombinase-based genetic switch intended to regulate the controlled expression (e.g. through chemical inputs) of (sub)module 103, housing the transgene expression cassette. Beyond the transferable or transposable element, module 106 confers upon the system the capacity for selection under specific conditions or pressures during integration into a plant genome (e.g., via Agrobacterium tumefaciens, viral delivery, biolistics or direct delivery). Components 107 and 108 serve as optional sites recognized by the integration machinery of Agrobacterium tumefaciens.

[0052] In a particular embodiment of the invention, as mentioned earlier, module 101 contains a transferable or transposable element, which includes submodule 103 housing the transgene expression cassette (refer to Figure 2). This submodule, in further detail, consists of components 103a, 103b, and 103c. Component 103a functions as a promoter or initiator sequence, instigating the expression of transgene 103b, which is then terminated by signal 103c. Optionally, within this transgene expression cassette, additional sets of components such as 103a2, 103b2, and 103c2; 103a3, 103b3, and 103c3; and so forth, up to 103aN, 103bN, and 103cN (each fulfilling corresponding functions) may be included subsequent to the initial components. This flexible configuration of the transgene expression cassette facilitates the expression of multiple genes tailored to specific application requirements. For instance, while 103b could encompass an editing enzyme, 103b2 might house the corresponding sgRNA, 103b3 could carry a resistance marker for selection purposes, and 103b4 might contain a reporter gene.

[0053] As a pivotal aspect of the primary embodiment of the invention, the system also integrates module 102, corresponding to the recombinase-based genetic switch (depicted in Figure 3). This recombinase-based genetic switch comprises components 102a, 102b, 102c, 102e, and 102f. Component 102a operates as a promoter or initiator sequence, instigating the expression of component 102e, which is then terminated by component 102f. Within this sequence, interruptions by components 102b, 102c, and 102d occur. Specifically, component 102c acts as a termination sequence positioned ahead of component 102e, encoding an enzyme with excisionase activity, culminating with its corresponding termination sequence, component 102f. Moreover, within the recombinase-based genetic switch and following component 102e, Page 12 out of 41sf-6759494Attorney Docket No: 230702000440 component 102h encodes an enzyme featuring recombinase activity, regulated by inducible promoter 102g and concluding with termination component 102i. Notably, components 102b and 102d, surrounding termination sequence 102c, serve as recognition sites for enzyme 102h.

[0054] In an alternative embodiment of the invention, as previously alluded to, the system incorporates module 106 for transient expression (refer to Figure 4), with its primary component being 106b, encoding an enzyme with transposase activity capable of recognizing sites within components 104 and 105 (as depicted in Figure 1). Surrounding component 106b are components 106a and 106c, housing a promoter or initiation sequence and a termination sequence, respectively. Moreover, module 106 may optionally include components 106e and 106h, encoding a visible reporter and a negative selection gene, respectively. The presence of component 106h is particularly significant for ensuring integration via ITR sites (components 104 and 105 in Figure 1) rather than LB / RB sites (components 107 and 108 in Figure 1). Additionally, components 106d / 106g and 106f / 106i serve as promoter / initiation sequences and termination sequences, respectively, flanking components 106e and 106h.

[0055] An integral aspect of this invention lies in the interactive capabilities of the system's modules, as illustrated in Figure 5A and Figure 5B. Once the assembled system 200 (in the form of a vector or plasmid) is obtained, it can be utilized for plant cell transformation via various methods such as protoplasts, Agrobacterium tumefaciens, biolistics, agroinfiltration, or plant virus vectors, among others. Within the plant cell, the enzyme with transposase activity encoded by component 206b is transiently expressed, typically under the control of a constitutive promoter (component 206a in Figure 5A and Figure 5B). Subsequently, the transposase enzyme facilitates the excision of the entire module 201 from system 200, leading to the integration of module 201 into the plant genome. Upon activation of constitutive promoters (encoded by components 203a, 203a2, 203a3, and so forth, up to 203aN in Figure 5A), the transgenes encoded within (sub)module 203 are expressed. These transgenes may encompass various elements such as editing enzymes, corresponding sgRNAs, and resistance and / or reporter genes. Following a period, an optional recombinase-based genetic switch can be triggered in a controllable manner, through induction by chemical or external stimuli (e.g. heat shock). The inducible promoter (component 202g in Figure 5A) within (sub)module 202 is activated, prompting the expression of the enzyme with recombinase activity encoded by component 202h. This enzyme is capable of excising the intervening component 202c through recognition of sites 202b and 202d, thereby allowing the expression of the excisionase enzyme encoded by component 202e. The excision process occurs, wherein the excisionase enzyme Page 13 out of 41sf-6759494Attorney Docket No: 230702000440 removes the entire integrated module 201b from the plant genome following the expression of the encoded transgenes. This process leaves no trace of any transgene within the plant genome (after their expression), ensuring the absence of any discernible traces from the presence of transgenes. It is worth mentioning, that the recombinase-based genetic switch is an optional component of the system, since a reversible transgenesis can be achieved by using only transposase and excisionase enzymes under inducible promoters, although using the recombinase-based genetic switch makes the system more reliable.

[0056] By integrating multiple input signals, the recombinase-based genetic switch functions as a genetic processing unit, controlling the expression of an output gene in a user-defined manner. One example of such a switch involves the use of recombinases to modulate gene expression. These recombinases can either activate a gene by removing a terminator sequence between the promoter and coding sequence of the output gene or repress gene expression by excising a segment of the output gene. This recombinase-based design ensures long-term and stable transitions between transcriptional states, eliminating the need for continuous addition of the activating signal. As a result, the genetic recombinase-based genetic switch maintains its response over extended periods, making it suitable for applications requiring sustained changes in gene expression, even with limited external inputs. In this invention, the system disclosed herein comprises a recombinase which can be selected from a group consisting of (but not limited to) B3, Cre, Vika, Flp recombinase or bacteriophage ϕC31 serine integrase (Int), among others.

[0057] As a particular embodiment of this invention, an essential aspect involves integrating a transposon into a plant genome. This integration is facilitated by an enzyme with transposase activity, enabling the transient and programmable integration of transgenes. Subsequently, excisionase enzymes are employed to remove the transposon from the plant genome, ensuring the seamless elimination of any traceable scars associated with the presence of transgenes. Within the system, a recombinase-based genetic switch provides programmable functionality, allowing precise control over genetic processes. Inducible promoters and enzymes with recombinase activity enable the timing and execution of these processes within plant cells. Furthermore, transgene expression within the transposon is traceable using specific genetic elements. Promoters or initiation sequences, along with the transgene expression cassette, ensure clear and trackable expression of desired genetic elements within the plant genome. With the capability for transient integration of transgenes, the system becomes a powerful tool for genetic editing in plants. Editing enzymes, sgRNAs, and resistance markers within the Page 14 out of 41sf-6759494Attorney Docket No: 230702000440 transposon enable precise modification of the plant genome. Upon completion of the editing process and transposon excision, edited plants exhibit the desired genetic modifications without retaining any transgenes, thereby ensuring the production of plants with desired traits while maintaining a transgene-free background. Through the coordinated action of transposases and excisionases, the system achieves transient and programmable integration of transgenes into the plant genome. This dynamic capability allows for precise control over genetic modifications, ensuring both flexibility and reliability in plant engineering endeavours.

[0058] In an exemplary embodiment of this invention, the integrated system utilises PiggyBac (PB) as the transposase enzyme and a modified version solely for excisionase activity (in this invention is generally called “PBEx”). In some embodiments, PB comprises an amino acid sequence as set forth in any one of SEQ IDs: 1 and 15-17. In some embodiments, PB comprises an amino acid sequence with at least 80%, at least 90%, at least 95%, or at least 100% identify to the amino acid sequence of SEQ ID NO:2. In some embodiments, the excision-only transposase comprises one or more nuclear localization signals fused to PBEx and comprises an amino acid sequence having at least 80%, at least 90%, at least 95% or at least 100% identity to an amino acid selected from the group consisting of SEQ IDs: 3-14.

[0059] Figure 5A can be used as exemplary by naming the components of this innovative system. Upon introduction of the construct or plasmid into the plant cell, PB 206b is expressed, facilitating the excision of transposon 201 via sites 204 and 205 for integration into the plant genome. Subsequently, once segment 201 integrates into the genome, the editor 203b, sgRNA- encoding segment 203e, resistance marker 203h and reporter 203k are expressed within the plant cell. The editor and corresponding sgRNA operate to perform directed editing events on specific genes within the plant genome. Following the editing process, the inducible promoter 202g can be programmatically activated, leading to the expression of recombinase 202h. This recombinase removes terminal sequence 202c by recognizing and binding to sites 202b and 202d. With the terminal sequence removed, PBEx 202e is expressed. As PBEx solely possesses excisionase activity, it can effectively remove the entire transposon 201b from the plant genome, leaving no trace of any transgene. Thus, this system offers a means to perform editing events in the plant genome without leaving any detectable remnants of the editor and its accessories in the now “edited genome”.

[0060] In another exemplary embodiment of this invention, the system can comprise additional components that can significantly enhance its performance. This includes how codon optimization and harmonization can improve translational efficiency and protein expression Page 15 out of 41sf-6759494Attorney Docket No: 230702000440 based on host organism preferences (e.g., matching codon usage). Additionally, a virus- induced silencing suppressor like P19 can be integrated to counteract gene silencing effects. Introns can boost gene expression levels when components are intronized within the construct (e.g., incorporating potato introns). Furthermore, cellular localization tags such as SV40 NLS and Nucleoplasmin NLS can direct enzymes or transgenes to specific cellular compartments (e.g., nucleus or plastids) for optimal function. In some embodiments, the systems comprise a nuclear localization signal (NLS) at the c-terminus and / or n-terminus. In some embodiments, the NLS is a SV40 bipartite NLS (BP) or combinations of BP, SV40 monopartite large T- antigen NLS (SV40), and Nucleoplasmin NLS (NP). The system can also feature key cis / trans regulatory elements like promoters, terminators, enhancers, inducible promoters, UTR’s for precise gene regulation and expression control. Among the promoters, this invention comprises promoters which can be selected from a group consisting of (but not limited to) HSP, GR / HAP- Gal-VP, CUP, Tet, MT, XVE / LexA or aclR / alcA promoters, as well as constitutive promoters, selected from (but not limited to) constitutive promoter is selected from a group consisting of (but not limited to) CaMV 35S, Ubiquitin, Actin, Tubulin, NOS, EF1α, RPS5a, rbcS, CmYLCV, p35S, pNos promoters, among others. In the case of terminators, the system disclosed herein comprises one or more termination sites which can be selected from a group consisting of (but not limited to) OCSt, 35S, Actin, mas, nos, hsp, RBCS, U6, Ubiquitin, rbcS, G7, pea3A or ags terminators. Amino acid sequence modifications like mutations, deletions, fusions, shuffled domains, domain duplications and / or AI-assisted protein design, can be implemented to tailor protein function (e.g., enhancing stability and / or activity). Cytoplasmic sequestration domains can be employed so that specific proteins can be sequestered in targeted cellular regions. Innovative features include bridge RNAs (e.g., PS) for targeted interactions and protein domain linkers (e.g., SGGS (SEQ ID NO: 22), XTEN) to optimize protein folding and stability. Additionally, negative selection modules and transformation enhancers for protoplasts and biolistics can be integrated to facilitate efficient genetic manipulation and transformation processes. The use of viruses and specialized Agrobacterium strains as delivery vehicles further enhances how the system can facilitate genetic material transfer and integration into host genomes. In this invention, the system herein disclosed comprises Agrobacterium strains selected from a group consisting of (but not limited to) LBA4404, GV2260, C58C1, GV3100, A136, GV3101, GV3850, GV3101::pMP90, GV3101::pMP90RK, EHA101, EHA105, AGL-1 strains, among others Page 16 out of 41sf-6759494Attorney Docket No: 230702000440

[0061] In some embodiments, the system can comprise a transient expression module comprising a plurality of integration components and a nucleic acid encoding an excision-only transposase. In some embodiments, the excision-only transposase comprises an excision-only variant of a piggyBac transposase (PBEx). In some embodiments, the excision-only transposase further comprises a nuclear localization signal (NLS) fused to the N-terminus or the C-terminus of the excision only variant of the piggBac transposase.

[0062] In some embodiments, the excision-only transposase comprises a PBEx with a NLS fused to the N-terminus of the excision only variant of the piggBac transposase (NLS-PBEx). In some embodiments, the excision-only transposase comprises a PBEx with a NLS fused to the C-terminus of the excision only variant of the piggBac transposase(PBEx-NLS). In some embodiments, the excision-only transposase comprises a PBEx with a NLS fused to the N- terminus and C-terminus of the excision only variant of the piggBac transposase (NLS-PBEx- NLS). In some embodiments, the excision-only transposase comprises an excision only variant of piggyBac transposase fused to one or more NLS, optionally comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-14.

[0063] In some embodiments, the excision-only transposase comprises a PBEx with a NLS fused to the N-terminus and the C-terminus of the excision only variant of the piggBac transposase (NLS-PBEx-NLS). In some embodiments, the NLS sequence on the N-terminus is SV40 and the NLS on the C-terminus is BP. In some embodiments, the PBEx has an amino acid sequence having at least 80%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 4.

[0064] In some embodiments, the excision-only transposase comprises a PBEx with a NLS fused to the N-terminus and the C-terminus of the excision only variant of the piggBac transposase (NLS-PBEx-NLS). In some embodiments, the NLS sequence on the N-terminus and on the C-terminus is SV40. In some embodiments, the PBEx has an amino acid sequence having at least 80%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 6.

[0065] In some embodiments, the excision-only transposase comprises a PBEx with a NLS fused to the N-terminus and the C-terminus of the excision only variant of the piggBac transposase (NLS-PBEx-NLS). In some embodiments, the NLS sequence on the N-terminus and on the C-terminus is NP. In some embodiments, the PBEx has an amino acid sequence Page 17 out of 41sf-6759494Attorney Docket No: 230702000440 having at least 80%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 7.

[0066] In another embodiment of this invention, the system can further comprise modules that enable phenotypic tracking of events, including the monitoring of transiently expressed components or stable integrated components. For this purpose the disclosed system includes reporters, proteins, pigments or metabolites selected from (but not limited to) fluorescent proteins, chromophores (chromoproteins, Ruby reporter (tyrosine to betalain), β-glucuronidase (GUS), anthocyanins) or luciferases, among others. Additionally, the system disclosed herein can further comprises selectable markers genes, which can be selected from a group consisting of (but not limited to) Neomycin, Phosphotransferases, ATP-binding cassette, Aminoglycoside-N-acetyltransferase, Aminoglycoside-300 -adenyltransferase, Streptomycin phosphotransferase, Hygromycin phosphotransferase, Bleomycin resistance, Dihydropteroate synthase, Acetyl transferase, Chloramphenicol acetyl transferase, Phosphinothricin acetyl transferase, 5-Enolpyruvylshikimate-3-phosphate synthase, Glyphosate oxidoreductase, Acetolactate synthase, Bromoxynil nitrilase, Glutamate-1-semialdehyde aminotransferase, Cyanamide hydratase, Cytochrome P450, Phytoene desaturase, a-Tubulin, Protoporphyrinogen oxidase, 2-Deoxyglucose-6-phosphate phosphatase, Betaine aldehyde dehydrogenase, Dihydropicolinate synthase, Octopine synthase, Tryptophan decarboxylase, Dihydrofolate reductase, Phosphinothricin acetyl transferase, Anthranilate synthase, Mutant anthranilate synthase, Tryptophan synthase beta, Threonine deaminase, Cytosine deaminase, Xylose isomerase, Phosphomannose isomerase, b-Glucuronidase, Isopentyl transferase, Indole acetic acid, UK (‘Hairy root’ phenotype), D-Amino acid oxidase, Arabitol dehydrogenase, D-Serine ammonia lyase, Trehalose-6-phosphate synthase or Diphtheria Toxin A Chain - DT-A, among others.

[0067] In a preferred embodiment, the disclosed system for transgenes of module 103 (Figure 1) includes Cas proteins selected from (but not limited to) AsCas12a, Cas12a (Cpf1), Cas12b, Cas12e (CasX), Cas13a, Cas13b, Cas13d, Cas14a, Cas9, Cas9-NGv1, CasX, CasY, CasΦ-2, CjCas9, eCas9, enAsCas12a, enAsCas12a-HF1, enLbCas12a, ErCas12a, eSpCas9, eSpCas9- 1.1, FnCas12a, FnCas9, HF-nCAS9, HF2-CAS9, HypaCas9, LbCas12a, MbCas12a, NmCas9, SaCas9, SaurCas9, SauriCas9, ScCas9, SpCas9, SpCas9-HF1, SpCas9-NG, SpG, SpRY, SpyoCas9, St1Cas9, St3Cas9, StCas9, ttLbCas12a, xCas9, xCas9(3.6), xCas9(3.7), and their corresponding nickase and dead versions. Similarly, the disclosed system can include nucleotide modifying enzymes such as APOBEC, APOBEC-like enzymes, AID, AID-like Page 18 out of 41sf-6759494Attorney Docket No: 230702000440 enzymes, A3A, DNMT3A, MQ3, LSD1, RrA3F, RrA3F-F130L, AmA1, SsA3B, SsA3B- R54Q, PpA1, PpA1-H122A, PpA1-R33A, Anc-rA1, rA1, pmCDA1, A3A, eA3A, rA1-YE1, rA1-EE, rA1-YE2, rA1-YEE, A3A-Y130F, A3A-Y132D, A3B, AIDmono, TadA-7.10, CGBE1, hA3G-CTD, evoAPOBEC1, evoCDA1, evoFERNY, TadA-8e, and TadA-9. In turn, module 103 can also incorporate different components to enhance the system performance, including further accessory or modifying enzymes, event-triggering enzymes, additional linkers, and useful sequence motifs for post-processing events. Among other modifying and event-triggering enzymes, this invention includes uracil-DNA glycosylase inhibitors (UGI), fluorescent proteins, FokI, methyltransferases (e.g., m5U like TrmA, 2′-O like SpoU, DNMT3A, or MQ3), Ψ synthases (like TruA), demethylases (e.g., LSD1), proteins involved in the plant abscisic acid (ABA) stress response pathway (e.g., PYL1 or ABI1), heterodimerizing leucine zippers, Kruppel-associated Box (KRAB), transcriptional activators (e.g., VP64, P65, Rta, P65-HSF1 transactivation complex, or MCP-fused VP64), and repressive domains (e.g., SRDX). Additionally, it may include additional APOBEC or APOBEC-like enzymes and AID or AID-like enzymes. The additional linkers and sequence motifs may include a Hisx6 purification tag with TEV protease cleavage site, an SV40 nuclear localization sequence, an XTEN peptide linker, a flexible linker such as a GGS(xN) repetition (SEQ ID NO: 23), more rigid linkers including polyproline motifs, an all α-helical linker A(EAAAK)nA (SEQ ID NO: 24), or a nucleoplasmin nuclear localization sequence.

[0068] The systems, methods, kits and composition of the current application are for reversible transgenesis systems for reversible integration in a plant genome. The reversible transgenesis system can be used for reversible integration in any plant genome or in a yeast genome. In some embodiments, the plant genome is a genome of Nicotiana benthamiana (Nb), Citrus reticulata, a citrus fruit, a stone fruit, a tree fruit, an edible plant, an ornamental plant, model plant, and / or any other plant. In some embodiments, the plant genome is a genome of a Citrus sp., a Rubus sp., a Prunus sp., a Fragaria sp., or a Vaccinium sp. In some embodiments, the plant genome is a dicot plant genome. In some embodiment, the plant genome is a monocot plant genome. In some embodiments, the plant genome is a genome of a pant including, but not limited to, blueberry, blackberry, watermelon, muskmelon, tomato, tomatillo, pepper, eggplant, grape, orange, lemon, lime, grapefruit, cucumbers, squash, gourd, pumpkin, apple, pear, kiwi, pomegranate, mango, guava, papaya, avocado, stone fruit, date, fig, alfalfa, tobacco, cotton, clover, strawberry, currant, cranberry, gooseberry, boysenberry, raspberry, bilberry, lingonberry, cowberry, huckleberry, dewberry, caneberry, loganberry, marionberry, tayberry, Page 19 out of 41sf-6759494Attorney Docket No: 230702000440 banana, plantain, red banana, latundan banana, Cavendish banana, artichoke, beets, potato, sweet potato, potato, yams, radish, horseradish, turnip, parsnip, rutabaga, yucca, onion, shallot, leek, scallion, garlic, chives, peanut, asparagus, sugarcane, cassava, Brussels sprouts, cabbage, collards, kale, lettuce, chard, spinach, bok choy, okra, cashew nuts, pineapple, celery, birch, rapeseed, mustard, tea, hemp, safflower seed, cedar, quinoa, chickpea, citron, satsuma, tangerine and mandarin, clementine, coffee, cola, hazelnut, saffron, melon and cantaloupe, carrot, oil palms, teff, rubber rabbit brush, eucalyptus, fir, soybean, sunflower, hemlock tree, rubber tree, kenaf, hop, walnut, larch, lentil, flax, maple, miscanthus, basil, olive, millet, pennycress, avocado, green bean, bean, ground cherry, pine, pistachio nut, pea, turf grass, poplar, apricot, plum and prune, almond, nectarine, peach, cherry, rose, rubus, sesame, sorghum, spruce, switchgrass, Russian dandelion, cacao, durum wheat, spelt, broad bean, cowpea, ginger, kohlrabi, broccoli, cauliflower, maize, wheat, rice, barley, oat, rye, bamboo, ryegrass, lawn grass, or ornamental grass. In some embodiments, the plant genome is a genome of Citrus spp., Citrus sinensis, Citrus reticulata, Citrus aurantium, Citrus bergamia, Citrus limon, Citrus medica, Citrus maxima, Citrus hystrix, Citrus aurantiifolia, Citrus × sinensis, Citrus × paradisi, Cucumis spp., Cucumis sativus, Cucurbita spp., Cucurbita argyrosperma, Cucurbita ficifolia, Cucurbita maxima, Cucurbita moschata, C. pepo, Lagenaria spp., Malus spp., Malus domestica, Pyrus spp., Pyrus communis, Pyrus pyrifolia, Pyrus sinkiangensis, Pyrus pashia, Actinidia spp., Actinidia deliciosa, Punica spp., Punica granatum, Mangifera spp., Mangifera indica, Mangifera foetida, Psidium spp., Psidium guajava, Carica spp., Carica papaya, Persea spp., Persea americana, Prunus spp., Phoenix spp., Phoenix dactylifera, Ficus spp., Ficus carica, Medicago spp., Medicago sativa, Nicotiana spp., Nicotiana tabacum, Nicotiana rustica, Gossypium spp., Gossypium hirsutum, Gossypium barbadense, Gossypium arboretum, Gossypium herbaceum, Trifolium spp., Trifolium repens, Trifolium pretense, Fragaria spp., Fragaria virginiana Fragaria chiloensis, Fragaria vesca, Fragaria × ananassa, Ribes spp., Cynara spp., Cynara cardunculus, Beta spp., Beta vulgaris, Beta vulgaris subsp. vulgaris, Ipomoea spp., Ipomoea batatas, Canna spp., Canna indica, Pachyrhizus spp., Pachyrhizus ahipa, Arracacia spp., Arracacia xanthorrhiza, Lepidium spp., Lepidium meyenii, Mirabilis spp., Mirabilis expansa, Oxalis spp., Oxalis tuberosa, Ullucus spp., Ullucus tuberosus, Smallanthus spp., Smallanthus sonchifolius, Dioscorea spp., Dioscorea rotundata, Dioscorea cayennensis, Dioscorea alata, Dioscorea polystachya, Dioscorea bulbifera, Dioscorea esculenta, Dioscorea dumetorum, Dioscorea trifida, Raphanus spp. Raphanus raphanistrum, Armoracia spp., Armoracia rusticana, Brassica spp., Brassica rapa, Brassica rapa subsp. chinensis, Brassica napus, Brassica oleracea, Brassica Page 20 out of 41sf-6759494Attorney Docket No: 230702000440 oleracea, Brassica oleracea var. oleracea, Brassica oleracea var. italica, Pastinaca spp., Pastinaca sativa, Yucca spp., Allium spp., Allium cepa, Allium ampeloprasum, Allium chinense, Allium fistulosum, Allium × proliferum, Allium sativum, Allium schoenoprasum, Arachis spp., Arachis hypogaea, Asparagus spp., Asparagus officinalis, Saccharum spp., Saccharum officinarum, Saccharum barberi, Saccharum robustum, Saccharum spontaneum, Manihot spp., Manihot esculenta, Lactuca spp., Lactuca sativa, Spinacia spp., Spinacia oleracea, Solanum spp., Solanum chacoense, Solanum tuberosum, Solanum lycopersicum, Solanum melongena, Zea spp., Zea mays, Musa spp., Musa acuminata, Musa balbisiana, Vaccinium spp., Vaccinium darrowii, Vaccinium corymbosum, Vaccinium erythrocarpum, Vaccinium macrocarpon, Vaccinium microcarpum, Vaccinium oxycoccos Rubus spp., Rubus fruticosus, Rubus idaeus, Rubus fruticosus, Rubus aboriginum, Rubus × loganobaccus, Rubus idaeus and Rubus strigosus Rubus occidentalis, Citrullus spp., Citrullus lanatus, Cucumis spp., Cucumis melo, Physalis spp., Physalis philadelphica, Physalis ixocarpa, Capsicum spp., Capsicum annuum, Capsicum baccatum, Capsicum chinense, Capsicum frutescens, Capsicum pubescens, Vitis spp., Vitis vinifera, Vitis amurensis, Vitis labrusca, Vitis riparia, Vitis rotundifolia,. In some embodiments, the plant genome is a genome of a citrus fruit species. In some embodiments, the citrus fruit species is 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. In some embodiments, the plant genome is a genome of a Prunus sp, Prunus africana, Prunus aitchisonii, Prunus alaica, Prunus albicaulis, Prunus amplifolia, Prunus andersonii, Prunus angustifolia, Prunus annularis, Prunus antioquensis, Prunus aquifolioides, Prunus arabica, Prunus arborea, Prunus argentea, Prunus austrosinensis, Prunus axitliana, Prunus balansae, Prunus barbata, Prunus beccarii, Prunus bifrons, Prunus brachybotrya, Prunus brachypetala, Prunus brachypoda, Prunus brachystachya, Prunus brigantina, Prunus buergeriana, Prunus campanulata, Prunus canescens, Prunus caroliniana, Prunus cerasoides, or Prunus cyclamina. In some embodiments, the plant genome is a genome of a Rubus sp., Rubus aboriginum, Rubus aciodontus, Rubus acridentulus, Rubus acuminatissimus, Rubus acuminatus, Rubus acutipetalus, Rubus adenoleucus, Rubus adenotrichos, Rubus adscitus, Rubus aggregatus, Rubus alceifolius, Rubus allanderi, Rubus angustisetus, Rubus antonii, Rubus apetalus, Rubus appropinquatus, Rubus apricus, Rubus arabicus, Rubus archboldianus, Rubus arcticus, Rubus arduennensis, Rubus arvinus, Rubus asirensis, Rubus atrovirens, Rubus australis, Rubus austroslovacus, Rubus axillaris, Rubus azuayensis, Rubus bakerianus, or Rubus banghamii. In some embodiments, the plant genome is a genome of a Fragaria sp., Fragaria bifera, Fragaria Page 21 out of 41sf-6759494Attorney Docket No: 230702000440 bucharica, Fragaria emeiensis, Fragaria gracilis, Fragaria nilgerrensis, Fragaria nubicola, Fragaria pentaphylla, Fragaria corymbosa, Fragaria orientalis, Fragaria iinumae, Fragaria daltoniana, Fragaria bracteata, Fragaria californica, Fragaria platypetala, Fragaria moupinensis, Fragaria mandschurica, Fragaria tibetica, Fragaria hayatai, Fragaria yezoensis, or Fragaria iturupensis. In some embodiments, the plant genome is a genome of a Vaccinium sp. Vaccinium absconditum, Vaccinium acrobracteatum, Vaccinium acutissimum, Vaccinium albidens, Vaccinium alvarezii, Vaccinium amblyandrum, Vaccinium amplexicaule, Vaccinium angiense, Vaccinium appendiculatum, Vaccinium arctostaphylos, Vaccinium ardisioides, Vaccinium artum, Vaccinium bancanum, Vaccinium banksii, Vaccinium barbatum, Vaccinium bartlettii, Vaccinium besagiense, Vaccinium blepharocalyx, Vaccinium brachybotrys, Vaccinium bracteatum, Vaccinium brevipedunculatum, Vaccinium caesariense, Vaccinium calycinum, Vaccinium camiguinense, Vaccinium caudatum, Vaccinium cercidifolium, Vaccinium cereum, Vaccinium cespitosum, Vaccinium chimantense, or Vaccinium chlaenophyllum.

[0069] Included herein are plant cells comprising a recombinant DNA construct, wherein the recombinant DNA construct comprises reversible transgenesis system for reversible integration in a plant genome. In some embodiments, the plant cell is a plant cell of with any of the plant genomes described herein. 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 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.

[0070] Finally, the embodiments of the system here disclosed can be implemented, in whole or in part, through a machine that is configured to receive a computer-readable medium containing the necessary instructions. These instructions can be executed by computer- executable components integrated within various platforms such as applications, applets, hosts, servers, networks, websites, communication services, communication interfaces, and the hardware, firmware, or software elements of personal computers or mobile devices. Additionally, other embodiments encompass systems and methods that can be embodied, at least partially, within a machine that is configured to receive a computer-readable medium storing the required instructions. These instructions can be executed by computer-executable components integrated with apparatuses and networks similar to those described earlier. The Page 22 out of 41sf-6759494Attorney Docket No: 230702000440 computer-readable medium can be stored on a range of suitable storage devices, including RAMs, ROMs, flash memory, EEPROMs, optical devices (such as CD, DVD, Blu-Ray), hard drives, floppy drives, cloud storage, or any other appropriate device. While the computer- executable component is typically a processor, it is also possible for the instructions to be executed by any dedicated hardware device that is suitable for the task. 4. Examples Example 1: Integration of transposon

[0071] The activity of PB variants was evaluated on citrus protoplasts. A transfection was carried out with Citrus integration plasmid and PB variants plasmids (e.g., SEQ ID NOs: 1, 15, 16 and 17 detailed in Figure 6. The system allowed for screening and selection of protoplast integration events. The Citrus integration plasmid contains two fluorescent proteins (Sc: Scarlet; NG: NeonGreen). Sc transcription unit is located outside the transposon and its expression is dependent on plasmidial DNA while NG is contained in the transposon beside the selection marker and expression of both transcription units are dependent on plasmid or genome-integrated DNA. Five weeks after transfection (when plasmid-encoded proteins transient expression is no longer detected and cell division already starts), protoplasts were subjected to fluorescence microscopy in order to detect integration events (NG+ / Sc-). Representative images of positive integration events are shown in Figure 7. Example 2: Excision of transposon

[0072] The activity of excision-only PB variants (SEQ ID NOs: 2) was determined by utilisation of the NISIN plasmid detailed in Figure 8. Upon excision of the transposon containing a transcription unit coding for a first fluorescent protein - Scarlet in this case - in a scarless fashion, the reading frame of a second fluorescent protein - NeonGreen in this case - is reestablished. The post excision construct is shown in Figure 8.

[0073] For determination of activity in Nb, agrobacterium strains harbouring NISIN plasmid were co-infiltrated with other strains harbouring the excision-only PB variants in Nb leaves using standard procedures.

[0074] Five to seven days post infiltration, leaf disks were subjected to fluorescence microscopy. Using fluorescent cell count ratios data, excision efficiencies were determined. Excision efficiency up to ~20% was obtained with one of the variants (Figure 9), in contrast to ~0% with the wild type. Page 23 out of 41sf-6759494Attorney Docket No: 230702000440

[0075] For determination of activity in citrus, PEG-mediated transformation of protoplasts was performed using the NISIN plasmid along others containing excision-only PB variants.

[0076] Five to seven days post transformation, protoplast cultures were subjected to fluorescence microscopy. Using fluorescent cell count ratios data, excision efficiencies were determined. Excision efficiency up to ~80% was obtained with one of the variants (Figure 10A and 10B), in contrast to ~0% with the wild type. Example 3: Recombinase-based genetic switch and programmability

[0077] A recombinase-based genetic switch for conditional activation was prototyped in Nb. For this purpose, the MCC plasmid comprising three modules main elements was built: 1) a constitutive / heat-inducible - p35S / pHSP70B - promoter regulating the expression of a Flp recombinase alternatively coupled with a GR domain for nucleus protein translocation control using Dexamethasone glucocorticoid; 2) a recombinase activity detector consisting on a constitutive pNos promoter blocked by a Flp recombinase-removable (FTR flanked) OCSt terminator regulating the expression of a NeonGreen fluorescent protein; and, 3) a transformation reporter conferred by a constitutive promoter regulating expression of a second fluorescent protein like Scarlet. A representation of the MCC plasmid is shown in Figure 11.

[0078] In order to determine the proper function of this recombinase-based genetic switch, agrobacterium strains harbouring MCC plasmid were infiltrated in Nb leaves using standard procedures.

[0079] Five to seven days post infiltration, leaf disks were subjected to fluorescence microscopy. Using fluorescent cell count ratios data, efficiency of the genetic switch was determined. Results for induction ratios under both single and combined controls (dexamethasone and heat-shock) (Figure 12A and 12B) and basal expression under no- induced condition (Figure 13A and 13B) are shown. Data show low basal expression (comparable to WT) and high activation rate (up to 12 fold). Example 4 - PBEx variants with combinatorial nuclear localization signals to improve transposon excision efficiency.

[0080] Based on the PBEx_Opt sequence, SEQ ID NO: 3, additional PBEx variants were generated (SEQ ID NOs: 4-14), the variants contained different combinations of nuclear localization signal (NLS) amino-acid sequences in their N-terminal (NLS-PBEx), C-terminal (PBEx-NLS), or N- and C-terminal regions (NLS-PBEx-NLS); using SV40 bipartite NLS (BP) or combinations of BP, SV40 monopartite large T-antigen NLS (SV40), and Nucleoplasmin Page 24 out of 41sf-6759494Attorney Docket No: 230702000440 NLS (NP) (Figure 14A). Eleven new PBEx variants were generated, transformed into the Agrobacterium tumefaciens GV3101 strain, and each construction co-infiltrated into Nicotiana benthamiana leaves together with the excision reporter (Figure 14A and 14B), which allowed first the detection of transformed cells by the red fluorescent protein mScarlet, and also to follow seamless excision events when the transposon interrupting the green fluorescent protein mNeon is excised, reconstituting the transcription and translation of the fully functional fluorescent marker. After five days of incubation with the different construct combinations, leaf disks were cut from infiltrated areas and analyzed by fluorescence microscopy. Ratios of green nuclei (transposon excised) to red nuclei (transformed cells) were calculated to estimate excision rates in the infiltrated leaves. The experiment was repeated in two independent experimental replicates, and the collected data are shown in Figure 15A and 15B with representative images. Analysis of results revealed that variants Opt.4 (SEQ ID NO: 6) and Opt.2 (SEQ ID NO:4) had an approximately 5-fold increase in excision efficiency compared to the original PBEx_Opt protein. Example 5: Transposon integration, antibiotic selection, and plant regeneration in Nicotiana benthamiana.

[0081] Transgenic Nicotiana benthamiana plants containing stable integration of a transposon via PB integration activity were generated. Plasmids, summarized in Figure 16A, were constructed: One group, encoding different PB variants expressed via a p35S cauliflower mosaic virus promoter flanked by left (LB) and right (RB) T-DNA border recognition sequences (SEQ ID NOs: 16 and 17). The reporter construct was also harbored in a plasmid between LB and RB sequences, and consisted of: (1) A mScarlet (Sc) red fluorescent protein driven by a constitutive plant promoter, and, (2) the transposon, containing a left inverse terminal repeat sequence (ITR-L), a translational fusion consisting of the hygromycin resistance gene, the T2A peptide and a mNeon green (NG) fluorescent protein driven by a constitutive promoter, and finally the right inverse terminal repeat sequence (ITR-R). All constructs were transformed into the Agrobacterium tumefaciens GV3101 strain, and cultures of bacteria harboring PB variant plasmids and reporter construct plasmids were infiltrated into N. benthamiana leaves using different combinations (Figure 16B). After five days of incubation and verification of positive expression from fluorescent reporter proteins by epifluorescence microscopy, leaves were cut into small disks and subjected to hygromycin selection in solid plant MS media. Regenerating plantlets were transferred to jars of solid MS2 media with hygromycin, and leaves of growing, rooted plantlets were analyzed by microscopy, Page 25 out of 41sf-6759494Attorney Docket No: 230702000440 and non-fluorescent plants were discarded. Plants exhibiting fluorescence showed two main types of patterns: positive red (Sc) and green (NG) fluorescence, corresponding to plants regenerated from T-DNA mediated unspecific insertion events; and positive green (NG) fluorescence only, corresponding to true transposon integration events (Figure 16C). In plants transformed with the reporter construct only, all plants (100%) showed green and red fluorescence, corresponding to T-DNA insertions. Plants transformed with reporter plasmid plus PB variant plasmids showed, on average, green and red fluorescence in 62.5% of the regenerated plants, while 37.5% of these showed green fluorescence only, indicative of PB- dependent transposon insertion in the plant genome. These results showed that PB transposases can be used to stably integrate transgenes into the N. benthamiana plant genome at a 1:2 rate compared with T-DNA-dependent events. Example 6: Transposon integration and antibiotic selection in Citrus embryogenic callus.

[0082] The functionality of transposon integration with PB was tested in citrus plants. Embryogenic calluses were generated from nucellar embryos extracted from mandarin seeds, and co-cultured with Agrobacterium tumefaciens AGL1 strains carrying the plasmids depicted in Figure 17: the first plasmid contains the PB_Opt gene (SEQ ID NO: 15) under the control of a P35S constitutive promoter, linked through a class 2A peptide to a Neon green fluorescent protein, next to the reported transposon consisting of a hygromycin gene, linked through a class 2A peptide to a mScarlet fluorescent protein, flanked by inverse terminal repeat sequences. The second plasmid contains the PBEx_Opt (SEQ ID NO: 3) gene under the control of a P35S constitutive promoter, linked through a class 2A peptide to a Neon green fluorescent protein, next to the same transposon as the first plasmid. After seven days of co-culture, calluses were transferred to solid media for hygromycin selection. Five days later (day 12), the tissues were analyzed by fluorescence microscopy, which revealed multiple transformation events for the PB_Opt plasmid, and only two events for the PBEx_Opt plasmid (Figure 18A). Regions of the callus showing only red fluorescence were dissected and propagated, and total DNA extracted. Callus DNA was subjected to genome walking PCR analysis to map transposon integration events in the citrus genome, using the wristwatch approach developed by Wang et al. (Frontiers in Bioengineering and Biotechnology 10 (2022): 792848), using the wild type mandarin DNA and the PB_Opt plasmids as negative and positive controls respectively. Amplified PCR products were subjected to Oxford Nanopore sequencing with a MinION device, and obtained reads were mapped against the Citrus reticulata genome, as well as against the PB_Opt plasmid. Page 26 out of 41sf-6759494Attorney Docket No: 230702000440 This analysis revealed that 25 total integration sites could be identified above threshold read levels in the genomic analysis. When analyzed for the presence of T-DNA elements in these integrated sequences, it was found that 5 of the 25 events (20%) were devoid of T-DNA or plasmid sequences and contained the transposon ITR border inserted in a TTAA integration site in the Citrus reticulata genome, representing true transposition events (Figure 18B). These results show that PB_Opt-dependent transposition events can be generated in a fruit tree species of commercial interest. Exemplary Transposase Variant Sequences: SEQ ID NO: Name Protein Sequence SEQ ID NO:1 PB variant #1 MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDD VQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLA SNRILTLPQRTIRGKNKHCWSTSKPTRRSRVSALNIVRS QRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISL KRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMS TDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRP TLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLL GFRGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGM PYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDN WFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSR SRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDA SINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSR KTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSR KKFMRNLYMGLTSSFMRKRLEAPTLKRYLRDNISNILP KEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKASASCK KCKKVICREHNIDMCQSCFSG SEQ ID NO:2 PBEx-WT MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDD VQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLA SNRILTLPQRTIRGKNKHCWSTSKPTRRSRVSALNIVRS QRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISL KRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMS TDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRP TLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLL GFRGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGM PYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDN WFTSIPLAKNLLQEPYKLTIVGTVASNAREIPEVLKNSR SRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDA SINESTGKPQMVMYYNQTKGGVDTLNQMCSVMTCSR KTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSR Page 27 out of 41sf-6759494Attorney Docket No: 230702000440Page 28 out of 41sf-6759494Attorney Docket No: 230702000440Page 29 out of 41sf-6759494Attorney Docket No: 230702000440 GDKRPAATKKAGQAKKKK SEQ ID NO:8 PBEX-OPT.6 MDKRPAATKKAGQAKKKKMGSSLDDEHILSALLQSDDPage 30 out of 41sf-6759494Attorney Docket No: 230702000440Page 31 out of 41sf-6759494Attorney Docket No: 230702000440Page 32 out of 41sf-6759494Attorney Docket No: 230702000440Page 33 out of 41sf-6759494

Claims

Attorney Docket No: 230702000440 CLAIMS 1. A reversible transgenesis system for reversible integration into a plant genome comprising: a transposable element module comprising, a transgene expression cassette; and a transient expression module comprising, a plurality of integration components and a nucleic acid encoding an excision-only transposase.

2. The system of claim 1, wherein the transferable or transposable element is transferable by means of a transposase and / or an integrase.

3. The system of claim 1 or claim 2, wherein the transgene expression cassette comprises one or more nucleic acids encoding components of a gene editing system.

4. The system of claim 2, wherein the transgene expression cassette comprises a one or more promoter upstream of each of the one or more nucleic acids encoding the components of the gene editing system.

5. The system of claim 4, wherein the one or more promoters is selected from a group consisting of CaMV 35S, Ubiquitin, Actin, Tubulin, NOS, EF1α, RPS5a, rbcS, CmYLCV, HSP, GR / HAP-Gal-VP, CUP, Tet, MT, XVE / LexA and aclR / alcA promoters.

6. The system of any of claims 2-5, wherein the transgene expression cassette comprises a one or more termination sites downstream of each of the one or more nucleic acids encoding the components of the gene editing system.

7. The system of claim 6, wherein the one or more termination sites is selected from a group consisting of OCS, 35S, Actin, mas, nos, hsp, RBCS, U6, Ubiquitin, rbcS, G7, pea3A and ags terminators.

8. The system of claim 7, wherein one of the one or more termination sites is a OCSt terminator.

9. The system of any of claims 3-8, wherein the components of the gene editing system comprise a nucleic acid encoding a gene editing enzyme and a plurality of nucleic acids encoding gene editing enzyme accessory components. Page 34 out of 41sf-6759494Attorney Docket No: 230702000440 10. The system of claim 9, wherein gene editing enzyme is a cas protein.

11. The system of claim 9, wherein the gene editing enzyme is a nickase version of a Cas protein.

12. The system of claim 9, wherein the gene editing enzyme is a nuclease-dead version of a Cas protein.

13. The system of any of claims 10-12, wherein the cas protein is selected from a group consisting of AsCas12a, Cas12a (Cpf1), Cas12b, Cas12e (CasX), Cas13a, Cas13b, Cas13d, Cas14a, Cas9, Cas9-NGv1, CasX, CasY, CasΦ-2, CjCas9, eCas9, enAsCas12a, enAsCas12a- HF1, enLbCas12a, ErCas12a, eSpCas9, eSpCas9-1.1, FnCas12a, FnCas9, HF-nCAS9, HF2- CAS9, HypaCas9, LbCas12a, MbCas12a, NmCas9, SaCas9, SaurCas9, SauriCas9, ScCas9, SpCas9, SpCas9-HF1, SpCas9-NG, SpG, SpRY, SpyoCas9, St1Cas9, St3Cas9, StCas9, ttLbCas12a, xCas9, xCas9(3.6), and xCas9(3.7) 14. The system of any of claims 9- 13, wherein the gene editing accessory components comprise a sgRNA.

15. The system of any of claims 9- 14, wherein the gene editing accessory components comprise a selectable marker gene, selected from a group consisting of Neomycin, Phosphotransferases, ATP-binding cassette, Aminoglycoside-N-acetyltransferase, Aminoglycoside-300 -adenyltransferase, Streptomycin phosphotransferase, Hygromycin phosphotransferase, Bleomycin resistance, Dihydropteroate synthase, Acetyl transferase, Chloramphenicol acetyl transferase, Phosphinothricin acetyl transferase, 5- Enolpyruvylshikimate-3-phosphate synthase, Glyphosate oxidoreductase, Acetolactate synthase, Bromoxynil nitrilase, Glutamate-1-semialdehyde aminotransferase, Cyanamide hydratase, Cytochrome P450, Phytoene desaturase, a-Tubulin, Protoporphyrinogen oxidase, 2-Deoxyglucose-6-phosphate phosphatase, Betaine aldehyde dehydrogenase, Dihydropicolinate synthase, Octopine synthase, Tryptophan decarboxylase, Dihydrofolate reductase, Phosphinothricin acetyl transferase, Anthranilate synthase, Mutant anthranilate synthase, Tryptophan synthase beta, Threonine deaminase, Cytosine deaminase, Xylose isomerase, Phosphomannose isomerase, b-Glucuronidase, Isopentyl transferase, Indole acetic acid, UK (‘Hairy root’ phenotype), D-Amino acid oxidase, Arabitol dehydrogenase, D-Serine ammonia lyase, Trehalose-6-phosphate synthase and Diphtheria Toxin A Chain - DT-A. Page 35 out of 41sf-6759494Attorney Docket No: 230702000440 16. The system of any of claims 9- 15, wherein the gene editing accessory components comprise a reporter selected from a group consisting of fluorescent proteins, chromophores, chromoproteins, Ruby reporter (tyrosine to betalain), β-glucuronidase (GUS), anthocyanins and luciferases.

17. The system of any of claims 9-16, wherein the gene editing system comprises a nucleotide modifying enzyme 18. The system of any of claims 1-17, wherein the transferable or transposable element module comprises native or engineered transposase-specific recognition sequences flanking the transgene(s).

19. The system of any of claims 1-18, wherein excision only transposase facilitates excision of the transposable element.

20. The system of claim 19, wherein the excision of the transposable element by the excision only transposase is footprint free.

21. The system of any of claims 1-20, wherein the excision only transposase comprises an excision only variant of a piggBac transposase.

22. The system of claim 21, wherein the excision-only variant of a piggyBac transposase is PBEx, optionally wherein the excision-only variant of the piggyBac transposase has an amino acid sequence having at least 80%, at least 90%, at least 95%, or 100% identity to the amino acid sequence set forth in SEQ ID NO 2.

23. The system of claim 21 or 22, wherein the excision-only transposase further comprises a nuclear localization signal (NLS) fused to the N-terminus or the C-terminus of the excision-only variant of the piggBac transposase.

24. The system of claim 23, wherein the NLS is selected from a group consisting of a SV40 bipartite NLS (BP), a combinations of BP, SV40 monopartite large T-antigen NLS (SV40), and Nucleoplasmin NLS (NP).

25. The system of claim 23 or 24, wherein the excision-only transposase comprises an amino acid sequence having at least 80%, at least 90%, at least 95%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-14. Page 36 out of 41sf-6759494Attorney Docket No: 230702000440 26. The system of any of claims 1-25, wherein plurality of integration components comprises a selection element and two integration recombination sites.

27. The system of claim 26, wherein the selection element comprises nucleic acids encoding an antibiotic or herbicide resistance transgene.

28. The system of claim 26 or claim 27, wherein the two integration recombination sites comprise two agrobacterium recognition sites.

29. The system of claim 28, wherein the two agrobacterium recognition sites facilitate integration of the reversible transgenesis system into the plant genome.

30. The system of any of claims 1-29, wherein the plant genome is the genome of Nicotiana benthamiana (Nb), a citrus fruit, a stone fruit, a tree fruit, an edible plant, an ornamental plant, and / or a model plant, optionally wherein the citrus fruit is Citrus reticulata.

31. The system of any of claims 1-29, wherein the plant genome is Nb or a citrus fruit, optionally wherein the citrus fruit is Citrus reticulata.

32. The system of any of claims 1-29, wherein the plant genome is a genome of a Citrus sp., a Rubus sp., a Prunus sp., a Fragaria sp., or a Vaccinium sp.

33. The system of any of claims 1-32, wherein the reversible transgenesis system further comprises a constitutive expression module comprising the constitutive promoter linked to a gene encoding a recombinase.

34. The system of claim 33, wherein the constitutive promoter is selected from a group consisting of CaMV 35S, Uniquitin, Actin, Tubulin, NOS, EF1α, RPS5a, rbcS, CmYLCV promoters, p35S, and pNos.

35. The system of claim 33, wherein the constitutive promoter is p35S or pNos.

36. The system of any of claims 1-35, wherein the reversible transgenesis system further comprises a recombinase based genetic switch module.

37. The system of claim 36, wherein the genetic switch module comprises an inducible promoter linked to a gene encoding a recombinase. Page 37 out of 41sf-6759494Attorney Docket No: 230702000440 38. The system of claim 37, wherein the inducible promoter is a nucleic acid element that induces expression of a gene in response to a stimulus.

39. The system of claim 38, wherein the stimulus is an environmental stimulus, comprising change in temperature, change in light, or change in the concentration of a chemical 40. The system of any of claims 37-39 wherein the inducible promoter is selected from a group consisting of (but not limited to) HSP, pHSP70B, GR / HAP-Gal-VP, CUP, Tet, MT, XVE / LexA and aclR / alcA promoters.

41. The system of claim 39, wherein the heat shock promoter is pHSP70B.

42. The system of any of claims 37-41, wherein the recombinase is selected from a group consisting of B3, Cre, Vika, Flp recombinase and bacteriophage ϕC31 serine integrase (Int).

43. The system of any of claims 37-41, wherein the recombinase is a Flp recombinase.

44. The system of claim 36, wherein the genetic switch module comprises a GR domain to control nucleus protein translocation with Dexamethasone Glucocorticoid.

45. The system of any of claims 1-44, wherein the reversible transgenesis system further comprise one or more performance enhancing components.

46. The system of claim 45, wherein the one or more performance enhancing components comprise a codon optimization element, virus-induced silencing suppressors, introns, cellular localization tags.

47. The system of any of claims 1-46, wherein the reversible transgenesis system further comprises one or more and regulatory elements for precise gene regulation and expression control.

48. A kit for reversible integration into a plant genome comprising: the reversible transgenesis system of any of claims 1-47; a transformation agent. Page 38 out of 41sf-6759494Attorney Docket No: 230702000440 49. The kit of claim 48, wherein the transformation agent is used to transform the reversible transgenesis system into a plant cell.

50. The kit of claim 49, wherein the plant cell comprises the plant genome.

51. The kit of any of claim 48-50, wherein the transformation agent is selected from a group consisting of protoplast, Agrobacterium tumefaciens, biolistics components, agroinfiltration components, plant virus vectors.

52. The kit of claim 51, wherein the biolistics components comprise a particle delivery system used to transform plant tissue with DNA and / or RNA and / or Protein coated particles 53. The kit of claim 51, wherein the agrobacterium tumefaciens transformation components comprise an agrobacterium strain selected from a group consisting of LBA4404, GV2260, C58C1, GV3100, A136, GV3101, GV3850, GV3101::pMP90, GV3101::pMP90RK, EHA101, EHA105, AGL-1 strains, among others; used to transform plant tissue by infiltration, co-culture or micro-injection methods.

54. A method for reversible integration into a plant genome comprising: obtaining a library of plasmids comprising the reversible transgenesis system of any of claims 1-47; integrating the library of plasmids into the plant genome by employing a genome integration method; and activating excision of the reversible transgenesis system by activating the excision only transposase.

55. The method of claim 54, wherein the genome integration method comprises Agrobacterium tumefaciens, biolistics components, agroinfiltration components or plant virus vectors.

56. A vector comprising the reversible transgenesis system of any of claims 1-47.

57. A host cell comprising the vector of claim 56.

58. A recombinant DNA construct comprising the system of any of claims 1-47. Page 39 out of 41sf-6759494Attorney Docket No: 230702000440 59. A plant cell comprising the recombinant DNA construct of claim 58.

60. A plant genome comprising the recombinant DNA construct of claim 58. Page 40 out of 41sf-6759494

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