1. Department of Genetics, Development & Cell Biology, Iowa State University, Ames, Iowa 50011, USA
2. Molecular Pathology Unit and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA
3. Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA
4. Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA
5. Department of Genetics, Cell Biology & Development,
6. Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455, USA
7. These authors contributed equally to this work.

Correspondence to: Daniel F. Voytas^5,6 Correspondence and requests for materials should be addressed to D.F.V. (Email: voytas@umn.edu).

Abstract

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 targeting^{1, 2}; for example, they can be used to precisely modify engineered reporter genes in plants^{3, 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.

1. Department of Genetics, Development & Cell Biology, Iowa State University, Ames, Iowa 50011, USA
2. Molecular Pathology Unit and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA
3. Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA
4. Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA
5. Department of Genetics, Cell Biology & Development,
6. Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455, USA
7. These authors contributed equally to this work.

Correspondence to: Daniel F. Voytas^5,6 Correspondence and requests for materials should be addressed to D.F.V. (Email: voytas@umn.edu).

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