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Front. Plant Sci. | doi: 10.3389/fpls.2018.00916

Efficient multiplex genome editing induces precise, and self-ligated type mutations in tomato plants

  • 1Graduate School of Advanced Technology and Science, Tokushima University, Japan
  • 2Graduate School of Technology, Industrial and Social Sciences, Tokushima University, Japan

Several expression systems for multiple 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 Cas9 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 Cas9. By using the tomato ELONGATION FACTOR-1α (SlEF1α) promoter to drive Cas9, 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 SlEF1α 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 Cas9 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.

Keywords: CRISPR/Cas9, Multiplex genome editing, SlEF1α promoter, Tomato, tRNA

Received: 17 Dec 2017; Accepted: 08 Jun 2018.

Edited by:

Ian D. Godwin, The University of Queensland, Australia

Reviewed by:

Michael Palmgren, University of Copenhagen, Denmark
Sergey Morozov, Lomonosov Moscow State University, Russia  

Copyright: © 2018 Hashimoto, Ueta, Abe, Osakabe and Osakabe. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Prof. Keishi Osakabe, Tokushima University, Graduate School of Technology, Industrial and Social Sciences, 2-1 Minamijyosannjima-cho, Tokushima, 770-8513, Tokushima, Japan, kosakabe@tokushima-u.ac.jp