Optimizing glyphosate tolerance in rapeseed by CRISPR/Cas9-based geminiviral donor DNA replicon system with Csy4-based single-guide RNA processing

文献类型： 外文期刊

第一作者： Wang, Zhuanrong

作者： Wang, Zhuanrong;Wan, Lili;Wang, Zhuanrong;Wan, Lili;Xin, Qiang;Zhang, Xiaohui;Song, Yixian;Wang, Pengfei;Hong, Dengfeng;Yang, Guangsheng;Fan, Zhixiong

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关键词： BnaC04EPSPS; CRISPR/Cas9; Csy4; geminiviral replicon; gene replacement; glyphosate tolerance; homologyr cted DNA repair; rapeseed

期刊名称：JOURNAL OF EXPERIMENTAL BOTANY （ 影响因子：6.992； 五年影响因子：7.86 ）

ISSN： 0022-0957

年卷期： 2021 年 72 卷 13 期

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收录情况： SCI

摘要： Rapeseed (Brassica napus L.) is an important oil crop worldwide, and effective weed control can protect its yield and quality. Farmers can benefit from cultivars tolerant to herbicides such as glyphosate. Amino acid substitutions in enolpyruvylshikimate-3-phosphate synthase (EPSPS) render the plant less sensitive to glyphosate. Therefore, we aimed to optimize the glyphosate tolerance trait in rapeseed via endogenous EPSPS modification. To achieve effective gene replacement in B. napus L., we employed a CRISPR/Cas9 system expressing single-guide RNAs (sgRNAs) cleaved by the CRISPR-associated RNA endoribonuclease Csy4 from Pseudomonas aeruginosa, for targeted induction of double-strand breaks. Both the donor template and a geminiviral replicon harbouring an sgRNA expression cassette were introduced into plant cells. Using sgRNAs targeting adjacent donor DNA template containing synonymous mutations in sgRNA sites, we achieved precise gene replacements in the endogenous B. napus EPSPS gene, BnaC04EPSPS, resulting in amino acid substitutions at frequencies up to 20%. Rapeseed seedlings harbouring these substitutions were glyphosate-tolerant. Furthermore, modifications in BnaC04EPSPS were precisely transmitted to the next generation. Our genome editing strategy enables highly efficient gene targeting and the induction of glyphosate tolerance in oilseed rape.

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