Application of protoplast-based system to optimize multiple CRISPR/Cas9 editing tools in Setaria italica

Genome editing technologies have been widely applied in various plants for gene function research and genetic improvement. However, high-efficient genome editing tools adopted in Setaria italica are still rare. Here, we developed a protoplast-based system with the deep amplicon sequencing to rapidly test and optimize multiple clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated protein (Cas9) system for high-efficient genome editing in S. italica. Using this method, the tRNA-based system with endogenous Cas9 expressing cassettes has been screened out as a high-efficient editing tool with 51.23% total mutation frequency that could simultaneously target multiple sites with a mutagenesis frequency of 3.23%. CRISPR/Cas RNP complexes have been also proven to work efficiently in genome editing of S. italica protoplasts. In summary, our protoplast-based system is a convenient and effective way to optimize CRIPR/Cas9 genome editing tools for gene functional research and genetic improvement before stable genetic transformation of S. italica.