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

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.

References

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

    Article  CAS  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)

    Article  ADS  CAS  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)

    Article  CAS  Google Scholar 

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

    Article  CAS  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)

    Article  CAS  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)

    Article  CAS  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)

    Article  ADS  CAS  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)

    Article  CAS  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)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  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)

    Article  CAS  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)

    Article  CAS  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)

    Article  CAS  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)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

Download references

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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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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