Non-transgenic approach to deliver ZFNs in seeds for targeted genome engineering

Abstract

In the post-genomic era, the efficient exploitation of the available information for plant breeding is a pressing problem. The discoveries that DNA double-stranded breaks (DSBs) are both recombinagenic and mutagenic have fuelled the development of targetable zinc-finger nucleases (ZFNs), which act as molecular scissors for the induction of controlled DSBs. These powerful tools are used by researchers to accelerate mutagenesis of the normal gene loci toward the development of useful traits in plants. Seeds contain the embryo, which is a multicellular system representing a micrography of a plant. Therefore, they can serve as a foundation for applying targeted genome engineering techniques. The following single-step method describes how to deliver and express transiently ZFNs in tomato (Solanum lycopersicum) seeds using electroporation. Unlike methods that rely on tissue culture and plant regeneration after transformation, the direct delivery of ZFNs to seeds provides a high-throughput breeding technology for safe and site-specific mutagenesis. Tomato is a leading crop in the world and biotechnological advances in this species have great impact.