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

Abstract

The scope for improvement of yield and disease resistance of crop plants by genetic engineering has been limited by the lack of an efficient method for targeted gene modification. Zinc-finger protein technology looks set to fill the gap. This relies on the use of designed zinc-finger nucleases, artificial chimaeric proteins that exploit the natural recognition mechanism of cellular DNA repair machinery, to make sequence-specific double-stranded DNA breaks at a target locus. In this issue two groups report the successful application of this emerging technique. Shukla et al. modify the maize gene IPK1, thereby introducing both herbicide tolerance and modified phytate metabolism into this important crop plant. Townsend et al. target the SuR loci in tobacco plants, conferring resistance to imidazolinone and sulphonylurea herbicides. The method achieves a high frequency of gene targeting and should be suitable for the routine modification of endogenous plant genes. An efficient method for gene targeting in plants has been lacking until now, frustrating efforts to engineer crop plants. Here it is demonstrated that zinc-finger nucleases—enzymes engineered to create DNA double-strand breaks at specific loci—can be used for gene targeting, in this case inducing mutations that confer resistance to herbicides in tobacco plants. An efficient method for making directed DNA sequence modifications to plant genes (gene targeting) is at present lacking, thereby frustrating efforts to dissect plant gene function and engineer crop plants that better meet the world’s burgeoning need for food, fibre and fuel. Zinc-finger nucleases (ZFNs)—enzymes engineered to create DNA double-strand breaks at specific loci—are potent stimulators of gene targeting1,2; for example, they can be used to precisely modify engineered reporter genes in plants3,4. Here we demonstrate high-frequency ZFN-stimulated gene targeting at endogenous plant genes, namely the tobacco acetolactate synthase genes (ALS SuRA and SuRB), for which specific mutations are known to confer resistance to imidazolinone and sulphonylurea herbicides5. 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 kilobases 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.