数字农科院2.0

Directed evolution rice genes with randomly multiplexed sgRNAs assembly of base editors

文献类型： 外文期刊

作者： Zhang, Ao;Shan, Tiaofeng;Sun, Yan;Chen, Zhipeng;Hu, Jianjian;Hu, Zhichao;Ming, Ziheng;Zhu, Zhitao;Li, Xue;He, Jun;Liu, Shijia;Jiang, Ling;Dong, Xiaoou;Wu, Yufeng;Wang, Yanpeng;Liu, Yuqiang;Li, Chao;Wan, Jianmin

作者机构：

关键词： directed evolution;dual base editor;herbicide resistance;multiplexed and orthogonal base editor;randomly multiplexed sgRNAs assembly

期刊名称： PLANT BIOTECHNOLOGY JOURNAL

ISSN： 1467-7644

年卷期： 2023 年

页码：

收录情况： SCIE（2023版）

摘要： CRISPR-based directed evolution is an effective breeding biotechnology to improve agronomic traits in plants. However, its gene diversification is still limited using individual single guide RNA. We described here a multiplexed orthogonal base editor (MoBE), and a randomly multiplexed sgRNAs assembly strategy to maximize gene diversification. MoBE could induce efficiently orthogonal ABE (<36.6%), CBE (<36.0%), and A & CBE (<37.6%) on different targets, while the sgRNA assembling strategy randomized base editing events on various targets. With respective 130 and 84 targets from each strand of the 34th exon of rice acetyl-coenzyme A carboxylase (OsACC), we observed the target-scaffold combination types up to 27 294 in randomly dual and randomly triple sgRNA libraries. We further performed directed evolution of OsACC using MoBE and randomly dual sgRNA libraries in rice, and obtained single or linked mutations of stronger herbicide resistance. These strategies are useful for in situ directed evolution of functional genes and may accelerate trait improvement in rice.

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