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Efficient Genome Editing in Rice Protoplasts Using CRISPR/CAS9 Construct

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Rice Genome Engineering and Gene Editing

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2238))

Abstract

Genome editing technologies, mainly CRISPR/CAS9, are revolutionizing plant biology and breeding. Since the demonstration of its effectiveness in eukaryotic cells, a very large number of derived technologies has emerged. Demonstrating and comparing the effectiveness of all these new technologies in entire plants is a long, tedious, and labor-intensive process that generally involves the production of transgenic plants and their analysis. Protoplasts, plant cells free of their walls, offer a simple, high-throughput system to test the efficiency of these editing technologies in a few weeks’ time span. We have developed a routine protocol using protoplasts to test editing technologies in rice. Our protocol allows to test more than 30 constructs in protoplasts prepared from leaf tissues of 100, 9–11-day-old seedlings. CRISPR/CAS9 construct effectiveness can be clearly established within less than a week. We provide here a full protocol, from designing sgRNA to mutation analysis.

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Acknowledgments

This work was partly supported by the French government through the program “Investissements d’Avenir” and by the French National Research Agency, in the frame of the research project called “GENIUS” (Genome ENgineering Improvement for Useful plants of a Sustainable agriculture), ANR11-BTBR-0001-GENIUS. Thibault Mounier Ph.D. is partly funded by CRP-Rice and University of Montpellier. Léo Herbert is the recipient of a CIRAD and INRA Ph.D. fellowship. Martine Bes, Leo Herbert, and Thibault Mounier contributed equally to this work.

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Author notes

  1. Martine Bes, Leo Herbert and Thibault Mounier contributed equally with all other contributors.

Authors and Affiliations

  1. CIRAD, UMR AGAP, Montpellier Cedex 5, France

    Martine Bes, Leo Herbert, Thibault Mounier, Anne-Cécile Meunier, Emmanuel Guiderdoni & Christophe Périn

  2. Université de Montpellier, CIRAD, INRA, Montpellier SupAgro, Montpellier, France

    Martine Bes, Leo Herbert, Thibault Mounier, Anne-Cécile Meunier, Emmanuel Guiderdoni & Christophe Périn

  3. IAGE Company, Montpellier Cedex 5, France

    Franz Durandet

Authors
  1. Martine Bes
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  2. Leo Herbert
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  3. Thibault Mounier
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  4. Anne-Cécile Meunier
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  5. Franz Durandet
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  6. Emmanuel Guiderdoni
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  7. Christophe Périn
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Corresponding author

Correspondence to Christophe Périn .

Editor information

Editors and Affiliations

  1. Synthetic Biology, Biofuel and Genome Editing R&D, Reliance Industries Ltd, Navi Mumbai, Maharashtra, India

    Anindya Bandyopadhyay

  2. Crop Improvement and Genetics Research Unit, USDA-ARS, Albany, CA, USA

    Roger Thilmony

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Bes, M. et al. (2021). Efficient Genome Editing in Rice Protoplasts Using CRISPR/CAS9 Construct. In: Bandyopadhyay, A., Thilmony, R. (eds) Rice Genome Engineering and Gene Editing. Methods in Molecular Biology, vol 2238. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1068-8_11

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  • DOI: https://doi.org/10.1007/978-1-0716-1068-8_11

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1067-1

  • Online ISBN: 978-1-0716-1068-8

  • eBook Packages: Springer Protocols

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