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

High-frequency modification of plant genes using engineered zinc-finger nucleases 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 ; 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. ZFNs were engineered that recognize SuR loci, using publicly available resources provided by the Zinc Finger Consortium . The Consortium-sponsored http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nature Springer Journals

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

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Publisher
Springer Journals
Copyright
Copyright © 2009 Macmillan Publishers Limited. All rights reserved
ISSN
0028-0836
eISSN
1476-4687
D.O.I.
10.1038/nature07845
Publisher site
See Article on Publisher Site

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 ; 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. ZFNs were engineered that recognize SuR loci, using publicly available resources provided by the Zinc Finger Consortium . The Consortium-sponsored

Journal

NatureSpringer Journals

Published: Apr 29, 2009

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