AUTHOR=Hashimoto Ryosuke , Ueta Risa , Abe Chihiro , Osakabe Yuriko , Osakabe Keishi 

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

JOURNAL=Frontiers in Plant Science

VOLUME=9

YEAR=2018

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2018.00916

DOI=10.3389/fpls.2018.00916

ISSN=1664-462X

ABSTRACT=<p>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 <italic>Cas9</italic> 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 <italic>Cas9</italic>. By using the tomato <italic>ELONGATION FACTOR-1</italic>α (<italic>SlEF1</italic>α) promoter to drive <italic>Cas9</italic>, 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 <italic>SlEF1</italic>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 <italic>Cas9</italic> 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.</p>