A novel CRISPR/Cas9 system for efficiently generating Cas9-free multiplex mutants in .

The CRISPR/Cas9 genome-editing system has emerged as a popular powerful tool for biological research. However, the process of selecting efficiently edited Cas9-free plants is usually laborious and time consuming. Here, we demonstrated P2A to be the most efficient self-cleaving peptide for fusing Cas9 and GFP in and then used Cas9-P2A-GFP to develop a novel CRISPR/Cas9 system. Additionally, a pair of isocaudomer restriction enzymes were selected to conveniently assemble multiple sgRNAs. In this system, the GFP fluorescence intensity in T1 transgenic plants indicates the expression level of the Cas9 protein, which correlates well with the editing efficiency. Furthermore, Cas9-free plants can be easily selected by examining GFP fluorescence in T2 transgenic plants. The efficient knockout of , and demonstrated the robustness of our new system. Thus, we designed a novel CRISPR/Cas9 system that can generate Cas9-free multiplex mutants efficiently in and possibly in other plant species.