Application of the CRISPR/Cas9 system in Dioscorea zingiberensis

Dioscorea zingiberensis is a major pharmaceutical plant that produces diosgenin, an important starting material for steroidal hormones. To date, no genome editing approach in D. zingiberensis has been reported. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system has proven to be an efficient targeted genome modification tool and has been successfully applied in many plants, including rice, soybean, wheat, and Arabidopsis. Here, we report CRISPR/Cas9-mediated targeted mutagenesis in D. zingiberensis using an Agrobacterium tumefaciens-mediated transformation method. The target guide RNA was designed in the first exon of the farnesyl pyrophosphate synthase gene (Dzfps), which is a critical gene involved in the synthesis of secondary metabolites. The single guide RNA expression cassette was driven by the OsU3 promoter, and Cas9 was driven by the 35S promoter. High frequencies of mutants were detected in T0 plants. Among 15 transformed plants, nine mutants that contained five types of mutations at the predicted double-stranded break site were identified. The transcript levels of Dzfps and the content of squalene in isolated mutants were significantly decreased compared with those in wild-type plants. Overall, our research provides a rapid and efficient approach for targeted genome modification in D. zingiberensis.