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

Jeffrey A. Townsend, David A. Wright, Ronnie J. Winfrey, Fengli Fu, Morgan L. Maeder, J. Keith Joung, Daniel F. Voytas

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463 Scopus citations

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 worlds burgeoning need for food, fibre and fuel. Zinc-finger nucleases (ZFNs)enzymes engineered to create DNA double-strand breaks at specific lociare potent stimulators of gene targeting; for example, they can be used to precisely modify engineered reporter genes in plants. 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 herbicides. 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.

Original languageEnglish (US)
Pages (from-to)442-445
Number of pages4
JournalNature
Volume459
Issue number7245
DOIs
StatePublished - May 21 2009

Bibliographical note

Funding Information:
Acknowledgements We thank M. Eichtinger for help in making ZFA reagents. This work was supported by grants to D.F.V. from the National Science Foundation and to J.K.J. from the National Institutes of Health and the Massachusetts General Hospital Department of Pathology.

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