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Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease

Nature Biotechnology volume 31, pages 691–693 (2013)Cite this article

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To the Editor:

Sustainable intensification of crop production is essential to ensure food demand is matched by supply as the human population continues to increase1. This will require high-yielding crop varieties that can be grown sustainably with fewer inputs on less land. Both plant breeding and genetic modification (GM) methods make valuable contributions to varietal improvement, but targeted genome engineering promises to be critical to elevating future yields. Most such methods require targeting DNA breaks to defined locations followed by either nonhomologous end joining (NHEJ) or homologous recombination2. Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be engineered to create such breaks, but these systems require two different DNA binding proteins flanking a sequence of interest, each with a C-terminal FokI nuclease module. We report here that the bacterial clustered, regularly interspaced, short palindromic repeats (CRISPR) system, comprising a CRISPR-associated (Cas)9 protein and an engineered single guide RNA (sgRNA) that specifies a targeted nucleic acid sequence3, is applicable to plants to induce mutations at defined loci.

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Figure 1: Targeted mutagenesis in planta using the Cas9 RNA-guided endonuclease.
Figure 2: Measurement of the mutation rate induced by Cas9 RNA-guided endonuclease and transgenic N. benthamiana plants carrying mutations in the PDS gene.

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Acknowledgements

We thank S. Marillonnet and Icon Genetics, Halle, Germany for providing plasmid vectors, J. Win and S. Dong for help with figure preparation, and M. Smoker for help with the plant transformation. This work was supported by the Gatsby Charitable Foundation, the European Research Council (ERC), and the Biotechnology and Biological Sciences Research Council (BBSRC).

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  1. Jonathan D G Jones and Sophien Kamoun: These authors contributed equally to this work.

Authors and Affiliations

  1. The Sainsbury Laboratory, Norwich Research Park, Norwich, UK

    Vladimir Nekrasov, Jonathan D G Jones & Sophien Kamoun

  2. Department of Plant and Microbial Biology, University of California, Berkeley, California, USA

    Brian Staskawicz

  3. Max Planck Institute for Developmental Biology, Tübingen, Germany

    Detlef Weigel

Authors
  1. Vladimir Nekrasov
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  2. Brian Staskawicz
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  3. Detlef Weigel
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  4. Jonathan D G Jones
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  5. Sophien Kamoun
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Correspondence to Jonathan D G Jones or Sophien Kamoun.

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V.N., J.D.G.J. and S.K. have filed a patent application based on this work.

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Supplementary Material and Methods, Supplementary Figures 1–8 and Supplementary Tables 1–3 (PDF 3277 kb)

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Nekrasov, V., Staskawicz, B., Weigel, D. et al. Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotechnol 31, 691–693 (2013). https://doi.org/10.1038/nbt.2655

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