Efficient genome editing in rice protoplasts using CRISPR/CAS9 construct
Bes, Martine | Herbert, Leo | Mounier, Thibault | Meunier, Anne Cecile | Durandet, Franz | Guiderdoni, Emmanuel | Périn, Christophe
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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