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

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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 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.

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Figure 1: The tobacco SuRB locus.
Figure 2: Activity of engineered ZFAs and ZFNs.

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GenBank/EMBL/DDBJ

Data deposits

DNA sequence of the SuRB locus has been deposited with GenBank under accession number FJ649655.

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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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Author notes

  1. Jeffrey A. Townsend and David A. Wright: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Genetics, Development & Cell Biology, Iowa State University, Ames, Iowa 50011, USA,

    Jeffrey A. Townsend, David A. Wright, Ronnie J. Winfrey & Fengli Fu

  2. Molecular Pathology Unit and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA ,

    Morgan L. Maeder & J. Keith Joung

  3. Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA ,

    Morgan L. Maeder & J. Keith Joung

  4. Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA,

    J. Keith Joung

  5. Department of Genetics, Cell Biology & Development,

    Daniel F. Voytas

  6. Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455, USA ,

    Daniel F. Voytas

Authors
  1. Jeffrey A. Townsend
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  2. David A. Wright
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  3. Ronnie J. Winfrey
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  4. Fengli Fu
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  5. Morgan L. Maeder
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  6. J. Keith Joung
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  7. Daniel F. Voytas
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Corresponding author

Correspondence to Daniel F. Voytas.

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Townsend, J., Wright, D., Winfrey, R. et al. High-frequency modification of plant genes using engineered zinc-finger nucleases. Nature 459, 442–445 (2009). https://doi.org/10.1038/nature07845

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