Efficient Multiplex Genome Editing Induces Precise, and Self-Ligated Type Mutations in Tomato Plants.

Several expression systems for multiple guide RNA (gRNAs) have been developed in the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system to induce multiple-gene modifications in plants. Here, we evaluated mutation efficiencies in the tomato genome using multiplex CRISPR/Cas9 vectors consisting of various expression promoters with multiple gRNA expression combinations. In transgenic tomato calli induced with these vectors, mutation patterns varied depending on the promoters used to express . By using the tomato α (α) promoter to drive , occurrence of various types of mutations with high efficiency was detected in the tomato genome. Furthermore, sequence analysis showed that the majority of mutations using the multiplex system with the α promoter corresponded to specific mutation pattern of deletions produced by self-ligation at two target sites of CRISPR/Cas9 with low mosaic mutations. These results suggest that optimizing the expression promoter used in CRISPR/Cas9-mediated mutation improves multiplex genome editing, and could be used effectively to disrupt functional domains precisely in the tomato genome.