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Targeted Plant Genome Editing via the CRISPR/Cas9 Technology

  • Jian-Feng LiEmail author
  • Dandan Zhang
  • Jen Sheen
Part of the Methods in Molecular Biology book series (MIMB, volume 1284)

Abstract

Targeted modification of plant genome is key for elucidating and manipulating gene functions in basic and applied plant research. The CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated protein (Cas) technology is emerging as a powerful genome editing tool in diverse organisms. This technology utilizes an easily reprogrammable guide RNA (gRNA) to guide Streptococcus pyogenes Cas9 endonuclease to generate a DNA double-strand break (DSB) within an intended genomic sequence and subsequently stimulate chromosomal mutagenesis or homologous recombination near the DSB site through cellular DNA repair machineries. In this chapter, we describe the detailed procedure to design, construct, and evaluate dual gRNAs for plant codon-optimized Cas9 (pcoCas9)-mediated genome editing using Arabidopsis thaliana and Nicotiana benthamiana protoplasts as model cellular systems. We also discuss strategies to apply the CRISPR/Cas9 system to generating targeted genome modifications in whole plants.

Key words

Plant genome editing CRISPR/Cas9 Protoplast transient expression assay Chromosomal mutagenesis Homologous recombination 

Notes

Acknowledgements

The authors thank the Church lab at Harvard Medical School for generating the Arabidopsis gRNA target database. This research was supported by the MGH ECOR Postdoctoral Fellowship for Medical Discovery to J.F.L. and by the National Science Foundation grant ISO-0843244 and the National Institutes of Health grants R01 GM60493 and R01 GM70567 to J.S.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Massachusetts General HospitalBostonUSA

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