Trait stacking via targeted genome editing

TLDR

Modern agriculture demands crops with multiple traits, yet random transgene integration and segregation create severe breeding challenges. The authors propose a versatile approach that couples high‑efficiency targeted genome editing with modular trait landing pads to enable on‑demand transgene integration and trait stacking. They first inserted a herbicide‑resistance gene pat into maize using WHISKERS‑mediated transformation, then delivered a second gene aad1 into the same landing pad via microparticle bombardment of immature embryos with donor DNA and a ZFN expression construct. Up to 5% of embryo‑derived transgenic events integrated aad1 precisely at the landing pad, and both herbicide‑resistance traits cosegregated in subsequent generations, demonstrating successful linkage and the method’s applicability across crops.

Abstract

Modern agriculture demands crops carrying multiple traits. The current paradigm of randomly integrating and sorting independently segregating transgenes creates severe downstream breeding challenges. A versatile, generally applicable solution is hereby provided: the combination of high-efficiency targeted genome editing driven by engineered zinc finger nucleases (ZFNs) with modular 'trait landing pads' (TLPs) that allow 'mix-and-match', on-demand transgene integration and trait stacking in crop plants. We illustrate the utility of nuclease-driven TLP technology by applying it to the stacking of herbicide resistance traits. We first integrated into the maize genome an herbicide resistance gene, pat, flanked with a TLP (ZFN target sites and sequences homologous to incoming DNA) using WHISKERS™-mediated transformation of embryogenic suspension cultures. We established a method for targeted transgene integration based on microparticle bombardment of immature embryos and used it to deliver a second trait precisely into the TLP via cotransformation with a donor DNA containing a second herbicide resistance gene, aad1, flanked by sequences homologous to the integrated TLP along with a corresponding ZFN expression construct. Remarkably, up to 5% of the embryo-derived transgenic events integrated the aad1 transgene precisely at the TLP, that is, directly adjacent to the pat transgene. Importantly and consistent with the juxtaposition achieved via nuclease-driven TLP technology, both herbicide resistance traits cosegregated in subsequent generations, thereby demonstrating linkage of the two independently transformed transgenes. Because ZFN-mediated targeted transgene integration is becoming applicable across an increasing number of crop species, this work exemplifies a simple, facile and rapid approach to trait stacking.