Enhancing Homologous Recombination in Plants Through the Use of Custom Endonucleases

Homologous recombination (HR) permits the precise insertion, deletion, or substitution of genetic material in the genome of an organism. HR offers numerous opportunities for modifying plant genomes to enhance our understanding of plant gene function and regulation, especially with respect to enhancing the production of biofuels. Although HR normally occurs at very low frequencies, approximately one in ten transformed plant cells will undergo HR at a target locus if the target sustains a double-stranded DNA break. This project will test a novel class of custom endonucleases for their ability to create targeted chromosomal breaks in rapeseed (hereafter collectively referred to as canola), thereby enhancing HR in a crop of interest to the DOE, because of its emerging importance in the production of biodiesel. Phase I will test a series of well characterized custom endonucleases for their ability to mediate HR in canola by correcting a defective marker gene that has recognition sites for the endonucleases. In Phase II, a custom endonuclease will be engineered to modify an endogenous canola gene by HR, thereby creating a germplasm of commercial value. Commercial Applications and other Benefits as described by the awardee: An efficient method for HR in plants should incur significant broad-based economic and societal benefits. HR would make it possible to more fully direct a plant¿s natural biosynthetic capacity to produce biofuels and other plant-derived products of value. Simultaneously, agricultural inputs, along with the negative environmental impact of agriculture, would be reduced; fewer pesticides, herbicides, or fertilizers, would be required.