Trait stacking via targeted genome editing

Ainley, W.M. and Sastry-Dent, L. and Petolino, J.F. and et al, . (2013) Trait stacking via targeted genome editing. Plant Biotechnology Journal. pp. 1-9.

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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.

Item Type: Article
Uncontrolled Keywords: gene targeting, designed zinc finger nucleases, transgene stacking.
Author Affiliation: Dow AgroSciences LLC, Indianapolis, IN, USA
Subjects: Crop Improvement > Genetics/Genomics
Crop Improvement > Biotechnology
Divisions: General
Depositing User: Mr Siva Shankar
Date Deposited: 20 Aug 2013 14:29
Last Modified: 20 Aug 2013 14:31
Official URL:

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