CRISPR/Cas9-mediated genome editing of RsGL1a and RsGL1b in radish (Raphanus sativus L.)

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a powerful tool widely used for genome editing in various organisms, including plants. It introduces and facilitates the study of rare genetic mutations in a short time and is a potent tool to assist in plant molecular breeding. Radish ( Raphanus sativus L.) is an important Brassicaceae vegetable cultivated and consumed worldwide. However, the application of the CRISPR/Cas9 system is limited by the absence of an efficient transformation system in radish. This study aimed to establish a CRISPR/Cas9 system in radish employing the Agrobacterium -mediated genetic transformation system reported recently. For this purpose, we performed genome editing using the CRISPR/Cas9 system targeting the GLABRA1 ( GL1 ) orthologs, RsGL1a and RsGL1b , that induces leaf trichome formation in radish. A Cas9/single guide RNA (sgRNA) vector with a common sgRNA corresponding to RsGL1a and RsGL1b was transferred. A total of eight T 0 plants were analyzed, of which six (editing efficiency 75%) had a mutated RsGL1a , five (62.5%) had a mutated RsGL1b , and five showed mutations in both RsGL1a and RsGL1b . Most mutations in T 0 plants were short (<3 bp) deletions or insertions, causing frameshift mutations that might produce non-functional proteins. Chimeric mutations were detected in several T 0 generation plants. In the T 1 generation, the hairless phenotype was observed only in plants with knockout mutations in both RsGL1a and RsGL1b . The majority of mutant alleles in T 0 plants, with the exception of the chimeric mutant plants detected, were stably inherited in the T 1 generation. In conclusion, we successfully knocked out RsGL1a and RsGL1b using the CRISPR/Cas9 system and demonstrated that both RsGL1a and RsGL1b independently contribute to the induction of leaf trichome formation in radish. In this study, genome-edited plants without T-DNA, which are useful as breeding material, were obtained. The findings prove the feasibility of genome editing in radish using a CRISPR/Cas9 system that could accelerate its molecular breeding to improve agronomically desirable traits.

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