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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

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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.

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

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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.

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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

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  1. Jian-Feng Li
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  2. Julie E Norville
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Correspondence to Jen Sheen.

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

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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

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