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. 2021:2238:173-191.
doi: 10.1007/978-1-0716-1068-8_11.

Efficient Genome Editing in Rice Protoplasts Using CRISPR/CAS9 Construct

Martine Bes #  1   2 , Leo Herbert #  1   2 , Thibault Mounier #  1   2 , Anne-Cécile Meunier  1   2 , Franz Durandet  3 , Emmanuel Guiderdoni  1   2 , Christophe Périn  4   5
Affiliations

Efficient Genome Editing in Rice Protoplasts Using CRISPR/CAS9 Construct

Martine Bes et al. Methods Mol Biol. 2021.

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.

Keywords: CRISPR/CAS9; Genome editing; Protoplast; Rice.

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