TRUSTing transposable elements to create ultra-clean transgene insertions

Abstract

The molecular improvement of crops necessitates the placement of custom DNA sequences, known as ‘genes of interest’ (GOI), into their genomes. A GOI can be foreign DNA (transgenic) or sequences copied from that same crop genome (cisgenic). The process of adding the GOI into the new genome, called transformation, is inefficient and relies on the use of selectable marker genes, so the majority of plant material that did not successfully undergo transformation is culled. However, after transformation and selection, these selectable marker genes become a liability. They present a significant hurdle in the regulatory process because they introduce foreign (transgenic) materials and have the potential to spread herbicide or antibiotic resistance to wild plant relatives. Marker-free DNA insertion is important to the field of plant biotechnology because it minimizes the potential impact to the environment, reduces the regulatory burden, and improves consumer acceptance (Singh et al., 2022). At the same time, because transformation occurs at a low success rate and requires selection, efficient marker-free transgenesis is currently not feasible (Kausch et al., 2021). In practice, today's crop improvement transgenes possess both a resistance gene and a GOI (Fig. 1a), for example, both an antibiotic resistance gene and a stress tolerance trait. Methods have been developed to remove the selectable marker after transgenesis with the goal of leaving only the remaining GOI (Nishizawa-Yokoi et al., 2015). For example, Cre-lox is a recombination system used to remove the selectable marker from the transgene after transgenesis and selection, leaving a small 34 bp LoxP footprint adjacent to the GOI (Fig. 1b; Yarmolinsky & Hoess, 2015). Cre-lox has been used extensively for commercial crop production; however, the system has significant challenges, such as inefficient excision of the marker gene and the lack of total elimination of foreign DNA.

‘For plants or lines that are recalcitrant to transformation, this reduces the number of transformed individuals required to generate the user's desired final GOI event.’

Fig. 1

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Transgenesis and transposition combined in transposon-mediated ultra-clean selectable transformation (TRUST). (a) Canonical plant transformation of a gene of interest (GOI) on the same transgene as a resistance gene necessary for the selection of successful transformation events. (b) After transformation and selection, Cre-lox site-specific recombination removes the resistance gene and leaves only the GOI and LoxP site remaining. (c) Natural Ac/Ds transposition is controlled by the expression of the Ac transposase protein, which binds the terminal inverted repeats (triangles), excising the element and inserting it into a new chromosomal location. The Ac transposase can mobilize full Ac elements, as well as nonautonomous Ds elements that share the same terminal inverted repeats. (d) In the TRUST system, the GOI is placed inside of a Ds element and mobilized to a new location away from the resistance gene necessary for the selection of successful transformation events. Excision of the Ds + GOI from the parent transgene is marked by restoration of the C1 gene's coding frame, generating a purple kernel. Segregation away of the parent transgene is marked by the loss of red fluorescence.

Due to their natural ability to cleanly excise from and insert into genomes, transposable elements have been increasingly used for genome engineering applications. They have been applied for decades as tools to mutate and simultaneously tag genes (Hancock et al., 2011; Cui et al., 2013), to insert custom DNA (Nishizawa-Yokoi & Toki, 2023) including activation tags (Johnson et al., 2021), and more recently, to perform targeted insertion in combination with CRISPR-Cas (Liu et al., 2024). Specifically, the Ac/Ds transposable element family from maize has several features that make it attractive for engineering purposes, such as clean and efficient excision, movement to new positions in the genome, and the ability to generate multiple distinct insertion sites from a single-donor position (Lazarow et al., 2013). The Ac transposase protein controls the transposable element family by recognizing, excising, and mobilizing Ac elements and their related but nonprotein-coding (nonautonomous) Ds elements (Fig. 1c). The Ac/Ds family has been previously engineered as launching pads to generate nearby linked insertions (Conrad & Brutnell, 2005) and to randomly mobilize enhancer and gene traps to explore patterns of gene expression (Carter et al., 2013).

In a paper by Ma et al. (2025; doi: 10.1111/nph.70017), recently published in New Phytologist, a system they call ‘TRUST’ (transposon-mediated ultra-clean selectable transformation) was created that transposes the GOI away from the rest of the ‘parent transgene’ (pp. ABC–XYZ). This parent transgene encodes the selectable marker (herbicide resistance) necessary for plant transformation. The parent transgene also has the GOI (the cargo) within a Ds transposable element (Ds sequence on either side of the GOI). After successful transgenesis, selection and regeneration of the plants containing the parent transgene, the first generation of maize plants are crossed to an established Ac active line that stimulates transposition. Only once exposed to active Ac does transposition of the compound Ds + GOI portion occur, separating the GOI from the parent transgene. This transposition is tracked in the TRUST system via the expression of the anthocyanin regulator C1, which is initially interrupted by the Ds + GOI in the parent transgene. The excised Ds + GOI transposes away from the parent transgene that includes the selectable marker, C1 and a red fluorescent protein (Fig. 1d). Kernels are screened for the lack of red fluorescent protein (segregation away of the parent transgene), and the presence of the GOI, which in this study by Ma et al., was endosperm-expressed green fluorescent protein (GFP). The new location of the transposed Ds + GOI is then determined by a combination of PCR, short-read and long-read sequencing to identify plants with desired insertion sites. The final products are ‘ultra-clean’ plant lines that lack the parent transgene and have the Ds + GOI at a favorable insertion site.

Four strengths of the TRUST system make it attractive compared to other methods. First, it takes advantage of multiple markers for both the selection of transgenesis (herbicide resistance) and each stage of transposition. This includes seed reporter genes that mark the presence/absence of the parent transgene (dsRED fluorescent protein), kernel color that marks plants with Ds + GOI transposition (C1), and the presence of the GOI itself that is a reporter gene (endosperm-expressed GFP). A second strength was the choice of transposable element system, as there are well-established Ac active lines to induce transposition (Lazarow et al., 2013). Third, the Ds DNA that is a necessary footprint at the GOI insertion site originates from the maize genome. This differs from the commonly used Cre-lox system, which will always remain transgenic because the DNA footprint after selectable marker excision (LoxP) originates from the bacteriophage P1 (Yarmolinsky & Hoess, 2015). Although this idea goes beyond what has been demonstrated to date, if the GOI used in TRUST originates from the maize genome, the resulting final product would be composed entirely of maize DNA. This cisgenic, rather than transgenic, final product may reduce the regulatory hurdles required for commercialization. Last, TRUST separates the initial transgenesis from the final integration of the GOI. This allows a small number of transformants that carry the parent transgene to each serve as ‘launching pads’ to generate a large number of marker-free GOI insertions at different locations (Fig. 2). For plants or lines that are recalcitrant to transformation, this reduces the number of transformed individuals required to generate the user's desired final GOI insertion event.

Fig. 2

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Launching pad concept. Transposon-mediated ultra-clean selectable transformation (TRUST) can be used to disassociate the process of transgenesis from downstream production and selection of gene of interest (GOI) insertion sites. A single plant that comes out of the inefficient transgenesis, selection, and regeneration pipeline can have transposition induced to generate many unique Ds + GOI insertion sites. The progeny can be screened to identify a desirable Ds + GOI insertion site.

Several details of the TRUST system could be improved in the future to make the system more widely applicable. The process established by Ma et al. takes many generations of screening, crossing, and testing to generate the final product. The timeline could be reduced by encoding the Ac transposase in the parent transgene, under the control of an inducible promoter, similar to how Cre is controlled in the Cre-lox system. Second, the Ac/Ds system is known to favor insertion at linked chromosomal sites (Conrad & Brutnell, 2005; Lazarow et al., 2013), which will make segregation of the GOI away from the parent transgene more difficult. A different transposable element system could be used that does not transpose to linked sites. Third, TRUST will be more difficult when the GOI is not a fluorescent reporter gene. PCR and digital PCR will be used to identify plants with a single-copy parent transgene and GOI transposition events. This will need to be continually monitored because, in theory, the Ds + GOI element could duplicate in copy number and/or excise from its desired insertion site. Last, the generational stability of GOI expression will need to be monitored, as a known consequence of using transposable elements is their triggering of chromatin modification and epigenetic silencing pathways.