The sonication-assisted whisker method enables CRISPR-Cas9 ribonucleoprotein delivery to induce genome editing in rice

• Published in: Scientific reports Vol. 13; no. 1; p. 14205
• Main Authors: Nakamura, Akiyoshi, Yano, Tsubasa, Mitsuda, Nobutaka, Furubayashi, Maiko, Ito, Seiichiro, Sugano, Shigeo S., Terakawa, Teruhiko
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 07.09.2023; Nature Publishing Group; Nature Portfolio
• Subjects: 631/449/447; 631/449/711; Cell walls; CRISPR; Gene transfer; Genome editing; Genomes; gRNA; Humanities and Social Sciences; Industrial applications; multidisciplinary; Plant breeding; Plant cells; Rice; Science; Science (multidisciplinary); Sonication; Transgenes; Vibrissae
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-023-40433-w

CRISPR/Cas9-based genome editing represents an unprecedented potential for plant breeding. Unlike animal cells, plant cells contain a rigid cell wall, genome editing tool delivery into plant cells is thus challenging. In particular, the delivery of the Cas9-gRNA ribonucleoprotein (RNP) into plant cells is desired since the transgene insertion into the genome should be avoided for industrial applications in plants. In this study, we present a novel RNP delivery approach in rice. We applied the sonication-assisted whisker method, conventionally developed for DNA delivery in plants, for RNP delivery in rice. Combined with marker gene delivery, we successfully isolated Os LCYβ genome-edited lines generated by RNPs. The calli and regenerated shoot of the  Os LCYβ mutant showed abnormal carotenoid accumulation. In addition, we also detected, although at a low frequency, genome editing events in rice calli cells by RNP delivery using the sonication-assisted whisker method without any additional. Therefore, the sonication-assisted whisker method could be an attractive way to create RNP-based genome-edited lines in plants.