Letter | Published:

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

Nature volume 459, pages 442445 (21 May 2009) | Download Citation


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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


Primary accessions


Data deposits

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


  1. 1.

    , , & Enhancing gene targeting with designed zinc finger nucleases. Science 300, 764 (2003)

  2. 2.

    & Chimeric nucleases stimulate gene targeting in human cells. Science 300, 763 (2003)

  3. 3.

    , , & Targeted mutagenesis using zinc-finger nucleases in Arabidopsis. Proc. Natl Acad. Sci. USA 102, 2232–2237 (2005)

  4. 4.

    et al. High-frequency homologous recombination in plants mediated by zinc-finger nucleases. Plant J. 44, 693–705 (2005)

  5. 5.

    et al. The molecular basis of sulfonylurea herbicide resistance in tobacco. EMBO J. 7, 1241–1248 (1988)

  6. 6.

    et al. Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol. Cell 31, 294–301 (2008)

  7. 7.

    et al. Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly. Nature Protocols 1, 1637–1652 (2006)

  8. 8.

    et al. Unexpected failure rates for modular assembly of engineered zinc fingers. Nature Methods 5, 374–375 (2008)

  9. 9.

    , , & Toward controlling gene expression at will: Selection and design of zinc finger domains recognizing each of the 5′-GNN-3′ DNA target sequences. Proc. Natl Acad. Sci. USA 96, 2758–2763 (1999)

  10. 10.

    , , , & Comparison of zinc finger nucleases for use in gene targeting in mammalian cells. Mol. Ther. 16, 707–717 (2008)

  11. 11.

    et al. DNA-binding specificity is a major determinant of the activity and toxicity of zinc-finger nucleases. Mol. Ther. 16, 352–358 (2008)

  12. 12.

    , & A sequencing method based on real-time pyrophosphate. Science 281, 363–365 (1998)

  13. 13.

    & Resistance of weeds to ALS-inhibiting herbicides: What have we learned? Weed Sci. 50, 700–712 (2002)

  14. 14.

    , & Epigenetic aspects of somoclonal variation in plants. Plant Mol. Biol. 43, 179–188 (2000)

  15. 15.

    et al. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nature Biotechnol. 25, 786–793 (2007)

  16. 16.

    et al. An improved zinc-finger nuclease architecture for highly specific genome editing. Nature Biotechnol. 25, 778–785 (2007)

  17. 17.

    et al. A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells. Nucleic Acids Res. 31, 2952–2962 (2003)

  18. 18.

    et al. Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nature Biotechnol. 26, 702–708 (2008)

Download references


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.

Author information

Author notes

    • Jeffrey A. Townsend
    •  & David A. Wright

    These authors contributed equally to this work.


  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


  1. Search for Jeffrey A. Townsend in:

  2. Search for David A. Wright in:

  3. Search for Ronnie J. Winfrey in:

  4. Search for Fengli Fu in:

  5. Search for Morgan L. Maeder in:

  6. Search for J. Keith Joung in:

  7. Search for Daniel F. Voytas in:

Corresponding author

Correspondence to Daniel F. Voytas.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Figures 1-2 with Legends and Supplementary Tables 1-7.

About this article

Publication history






Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.