CRISPR/Cas9-mediated mutation of 5-oxoprolinase gene confers resistance to sulfonamide compounds in Arabidopsis

Weeds are important biotic constraints to agricultural production, so herbicides are widely used with agronomic crops as the primary method of weed control. Accordingly, extensive efforts to develop herbicide-resistant (HR) crops have been made using various methods, including clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated gene editing. Thus far, most of the HR crops developed using gene-editing methods have adopted site-specific mutations of endogenous genes, but rarely by insertion or deletion (indel) mutations. In this study, we attempted to confer herbicide resistance in plants via indel mutations using the CRISPR/Cas9-mediated editing of 5-oxoprolinase 1 ( OXP1 ) gene as a target in Arabidopsis . OXP1 is an enzyme that is involved in 5-oxoproline metabolism and the glutathione recycling pathway. Here, we generated the indel mutants of Arabidopsis using OXP1 editing ( oxp1 /CRISPR), and verified a reduced sensitivity to sulfamethoxazole (Smex), a sulfonamide herbicide that suppresses the growth of cells by inhibiting the folic acid biosynthetic pathway. In addition, we successfully obtained OXP1 -edited plants by screening the transgenic plants on Smex-containing media, suggesting the possible use of OXP1 as a marker for plant gene editing. Subsequent molecular analyses demonstrated the indel mutations on OXP1 in the Smex-resistant Arabidopsis plants. Moreover, the oxp1 /CRISPR plants exhibited tolerance to heavy metals, such as cadmium, and another sulfonamide compound, amisulbrom. Therefore, the present study provides a way to confer resistance to sulfonamide compounds in plants by generating indel mutations.