AUTHOR=Muto Naoki , Matsumoto Takashi 

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

JOURNAL=Frontiers in Plant Science

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2022.951660

DOI=10.3389/fpls.2022.951660

ISSN=1664-462X

ABSTRACT=<p>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 (<italic>Raphanus sativus</italic> 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 <italic>Agrobacterium</italic>-mediated genetic transformation system reported recently. For this purpose, we performed genome editing using the CRISPR/Cas9 system targeting the <italic>GLABRA1</italic> (<italic>GL1</italic>) orthologs, <italic>RsGL1a</italic> and <italic>RsGL1b</italic>, that induces leaf trichome formation in radish. A Cas9/single guide RNA (sgRNA) vector with a common sgRNA corresponding to <italic>RsGL1a</italic> and <italic>RsGL1b</italic> was transferred. A total of eight T<sub>0</sub> plants were analyzed, of which six (editing efficiency 75%) had a mutated <italic>RsGL1a</italic>, five (62.5%) had a mutated <italic>RsGL1b</italic>, and five showed mutations in both <italic>RsGL1a</italic> and <italic>RsGL1b</italic>. Most mutations in T<sub>0</sub> plants were short (&lt;3 bp) deletions or insertions, causing frameshift mutations that might produce non-functional proteins. Chimeric mutations were detected in several T<sub>0</sub> generation plants. In the T<sub>1</sub> generation, the hairless phenotype was observed only in plants with knockout mutations in both <italic>RsGL1a</italic> and <italic>RsGL1b</italic>. The majority of mutant alleles in T<sub>0</sub> plants, with the exception of the chimeric mutant plants detected, were stably inherited in the T<sub>1</sub> generation. In conclusion, we successfully knocked out <italic>RsGL1a</italic> and <italic>RsGL1b</italic> using the CRISPR/Cas9 system and demonstrated that both <italic>RsGL1a</italic> and <italic>RsGL1b</italic> 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.</p>