A Modular Gene Targeting System for Sequential Transgene Stacking in Plants

The demands of modern agriculture are becoming increasingly complex whereby multiple traits with various modes of action are typically required for optimal crop performance. This not only involves the introduction and expression of multiple transgenes in a single product but also requires flexibility for fine-tuning existing products by adding, deleting and/or replacing transgenes. In the present study, a modular, selection-based method was developed for site-specific integration of transgenes into a genomic locus to create multi-gene stacks. High frequency gene targeting was obtained using zinc finger nuclease (ZFN)-mediated double strand break (DSB) formation at a pre-defined target genomic location using a unique intron directly downstream of a promoter driving a selectable marker gene to facilitate homology between target and donor sequences. In this system, only insertion into the target locus leads to a functional selectable marker and random insertions of the promoterless donor construct are eliminated on selection media. A new stack of transgenes can be loaded with each successive cycle of gene targeting by exchanging the selectable marker gene using the intron homology. This system was tested in maize using the pat selectable marker gene whereby up to 30% of the plants regenerated on Bialaphos-containing medium were observed to have the donor construct integrated into the target locus. Unlike previous gene targeting methods that utilize defective or partial genes to select for targeted events, the present method exchanges fully functional genes with every cycle of targeting thereby allowing the recycling of selectable marker genes for multiple generations of gene targeting. The gene targeting strategy described here in maize should be of general applicability across all crop species where transformation is possible.