High-frequency modification of plant genes using engineered zinc-finger nucleases.

Abstract

An efficient method for making directed DNA sequence modifications to plant genes (gene targeting) is presently lacking, thereby frustrating efforts to dissect plant gene function and engineer crop plants that better produce the world's burgeoning need for food, fiber and fuel. Zinc finger nucleases (ZFNs) - enzymes engineered to create DNA double strand breaks at specific loci - are potent stimulators of gene targeting,1,2 including at engineered reporter genes in plants.3,4 Here we demonstrate high frequency ZFN-stimulated gene targeting at endogenous plant genes, namely the tobacco acetohydroxyacid synthase (SuRA and SuRB) genes, for which specific mutations are known to confer resistance to imidazolinone and sulfonylurea herbicides.5 Herbicide-resistance mutations were introduced into SuR loci by ZFN-mediated gene targeting at frequencies exceeding 2% of transformed cells for mutations as far as 1.3 kb from the ZFN cleavage site. More than 40% of recombinant plants had modifications in multiple SuR alleles. The observed high frequency of gene targeting indicates that it is now possible to efficiently make targeted sequence changes in endogenous plant genes.