Skip to main content

Thank you for visiting nature.com. 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.

  • Correspondence
  • Published:

Multiplex and homologous recombination–mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9

Nature Biotechnology volume 31, pages 688–691 (2013)Cite this article

Subjects

To the Editor:

Elucidation and manipulation of human, animal and plant genomes is key to basic biology research, medical advances and crop improvement. The development of targeted genome editing, particularly homologous recombination–based gene replacement, is of great value in all organisms. Recent advances in engineered nucleases with programmable DNA-binding specificities, such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have provided valuable means to create targeted mutations in metazoan and plant genomes with high specificity1,2,3,4,5,6. However, these technologies demand elaborate design and assembly of individual DNA-binding proteins for each DNA target site1,2,3,4,5,6. Recently, a simple, versatile and efficient genome engineering technology has been developed based on the bacterial clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein (Cas) adaptive immune systems7. In a type II CRISPR-Cas system from Streptococcus pyogenes, a single Cas9 endonuclease guided by a duplex of mature CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) cleaves trespassing DNA from bacteriophage or plasmids in a sequence-specific manner7. By reconstitution of the S. pyogenes Cas9 (SpCas9) and an artificial chimera of crRNA and tracrRNA called synthetic-guide RNA (sgRNA) in eukaryotic cells, including yeast, zebrafish, mouse and human cells, targeted genome editing has been achieved through either error-prone nonhomologous end joining (NHEJ) or homology-directed repair (HDR) of the intended cleavage site7,8,9,10,11,12,13,14. Here, we show the feasibility and efficacy of sgRNA:Cas9–based genome editing technology in the model plants Arabidopsis thaliana and Nicotiana benthamiana.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Targeted plant genome editing by sgRNA:pcoCas9.
Figure 2: Multiplex and HDR-mediated genome editing by sgRNA:pcoCas9 in plant cells.

References

  1. Zhang, F. et al. Proc. Natl. Acad. Sci. USA 107, 12028–12033 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Zhang, Y. et al. Plant Physiol. 161, 20–27 (2013).

    Article  CAS  PubMed  Google Scholar 

  3. Li, T. et al. Nat. Biotechnol. 30, 390–392 (2012).

    Article  CAS  PubMed  Google Scholar 

  4. Gaj, T. et al. Trends Biotechnol. 31, 397–405 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Mussolino, C. & Cathomen, T. Nat. Biotechnol. 31, 208–209 (2013).

    Article  CAS  PubMed  Google Scholar 

  6. Streubel, J. et al. Nat. Biotechnol. 30, 593–595 (2012).

    Article  CAS  PubMed  Google Scholar 

  7. Jinek, M. et al. Science 337, 816–821 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Cong, L. et al. Science 339, 819–823 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Mali, P. et al. Science 339, 823–826 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Hwang, W.Y. et al. Nat. Biotechnol. 31, 227–229 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Cho, S.W. et al. Nat. Biotechnol. 31, 230–232 (2013).

    Article  CAS  PubMed  Google Scholar 

  12. Jinek, M. et al. eLIFE 2, e00471 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  13. DiCarlo, J.E. et al. Nucleic Acids Res. 41, 4336–4343 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Wang, H. et al. Cell 153, 910–918 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Yoo, S.D. et al. Nat. Protoc. 2, 1565–1572 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Qi, L.S. et al. Cell 152, 1173–1183 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Jiang, W. et al. Nat. Biotechnol. 31, 233–239 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. de Pater, S. et al. Plant Biotechnol. J. 11, 510–515 (2013).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank F. Ausubel for critical reading of the article, D. Voytas for discussion on the HDR strategy, Y. Xiong for the CYCD3 expression plasmid. J.F.L. is supported by the MGH ECOR Postdoctoral Fellowship for Medical Discovery. The Research is supported by the Department of Energy grant DE-FG02-02ER63445 to G.M.C., the National Science Foundation grant IOS-0843244 and the National Institutes of Health grants R01 GM60493 and R01 GM70567 to J.S.

Author information

Authors and Affiliations

  1. Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts, USA

    Jian-Feng Li, Matthew McCormack, Dandan Zhang, Jenifer Bush & Jen Sheen

  2. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA

    Jian-Feng Li, Julie E Norville, John Aach, Matthew McCormack, Dandan Zhang, Jenifer Bush, George M Church & Jen Sheen

  3. Wyss Institute for Biological Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA

    Julie E Norville, John Aach & George M Church

Authors
  1. Jian-Feng Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Julie E Norville
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. John Aach
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Matthew McCormack
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dandan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jenifer Bush
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. George M Church
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jen Sheen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jen Sheen.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Sequences, Supplementary Database, Supplementary Methods, Supplementary Figures 1–5 and Supplementary Table 1 (PDF 3448 kb)

Supplementary Dataset (ZIP 23806 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Li, JF., Norville, J., Aach, J. et al. Multiplex and homologous recombination–mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol 31, 688–691 (2013). https://doi.org/10.1038/nbt.2654

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.2654

This article is cited by

Search

Advanced search

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