Trait stacking via targeted genome editing

Journal Article / 2013

Ainley, William M.; Sastry-Dent, Lakshmi; Welter, Mary E.; Murray, Michael G.; Zeitler, Bryan; Amora, Rainier; Corbin, David R.; Miles, Rebecca R.; Arnold, Nicole L.; Strange, Tonya L.; Simpson, Matthew A.; Cao, Zehui; Carroll, Carley; Pawelczak, Katherine S.; Blue, Ryan; West, Kim; Rowland, Lynn M.; Perkins, Douglas; Samuel, Pon; Dewes, Cristie M.; Shen, Liu; Sriram, Shreedharan; Evans, Steven L.; Rebar, Edward J.; Zhang, Lei; Gregory, Phillip D.; Urnov, Fyodor D.; Webb, Steven R.; Petolino, Joseph F.

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.