Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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 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.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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

Accession codes

Primary accessions


Data deposits

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


  1. Bibikova, M., Beumer, K., Trautman, J. K. & Carroll, D. Enhancing gene targeting with designed zinc finger nucleases. Science 300, 764 (2003)

    CAS  Article  Google Scholar 

  2. Porteus, M. H. & Baltimore, D. Chimeric nucleases stimulate gene targeting in human cells. Science 300, 763 (2003)

    Article  Google Scholar 

  3. Lloyd, A., Plaisier, C. L., Carroll, D. & Drews, G. N. Targeted mutagenesis using zinc-finger nucleases in Arabidopsis . Proc. Natl Acad. Sci. USA 102, 2232–2237 (2005)

    ADS  CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  9. Segal, D. J., Dreier, B., Beerli, R. R. & Barbas, C. F. 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)

    ADS  CAS  Article  Google Scholar 

  10. Pruett-Miller, S. M., Connelly, J. P., Maeder, M. L., Joung, J. K. & Porteus, M. H. Comparison of zinc finger nucleases for use in gene targeting in mammalian cells. Mol. Ther. 16, 707–717 (2008)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  12. Ronaghi, M., Uhlen, M. & Nyren, P. A sequencing method based on real-time pyrophosphate. Science 281, 363–365 (1998)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  14. Kaeppler, S. M., Kaeppler, H. F. & Rhee, Y. Epigenetic aspects of somoclonal variation in plants. Plant Mol. Biol. 43, 179–188 (2000)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

Authors and Affiliations


Corresponding author

Correspondence to Daniel F. Voytas.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-2 with Legends and Supplementary Tables 1-7. (PDF 574 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

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.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing