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

Frontiers in Plant Science
Ryosuke HashimotoKeishi Osakabe

Abstract

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 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.

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Citations

Oct 28, 2019·Horticulture Research·Jiemeng XuZhaobo Lang
Mar 20, 2020·Plant & Cell Physiology·Shunya HayashiTsubasa Shoji
Jan 28, 2020·Plant Biotechnology·Ryota AkiyamaMasaharu Mizutani
Dec 14, 2018·Frontiers in Plant Science·Willian Batista-SilvaWagner L Araújo
Mar 16, 2019·Frontiers in Bioengineering and Biotechnology·María Santos-MerinoDaniel C Ducat
Jun 27, 2019·Horticulture Research·Tian WangHongliang Zhu
Nov 26, 2020·Scientific Reports·Johan HunzikerHiroshi Ezura
Apr 10, 2021·Plant Biotechnology Journal·Rim LassouedStuart J Smyth
Jun 8, 2021·Frontiers in Plant Science·Chihiro Abe-HaraYuriko Osakabe
Jan 12, 2021·Briefings in Bioinformatics·Hui PengJinyan Li

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Methods Mentioned

BETA
transgenic
PCR
electrophoresis
gene knock-in

Software Mentioned

Cas
OT
focas

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