High-throughput genome editing in rice with a virus-based surrogate system

文献类型： 外文期刊

第一作者： Tian, Yifu

作者： Tian, Yifu;Zhong, Dating;Li, Xinbo;Shen, Rundong;Dai, Yuqin;Yao, Qi;Zhang, Xuening;Cao, Xuesong;Zhu, Jian-Kang;Lu, Yuming;Tian, Yifu;Li, Xinbo;Shen, Rundong;Zhu, Jian-Kang;Tian, Yifu;Li, Xinbo;Shen, Rundong;Zhu, Jian-Kang;Zhong, Dating;Han, Han;Yao, Qi;Zhang, Xuening;Lu, Yuming;Zhu, Jian-Kang

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关键词： genome editing; high-throughput; protein tagging; rice

期刊名称：JOURNAL OF INTEGRATIVE PLANT BIOLOGY （ 影响因子：9.106； 五年影响因子：8.241 ）

ISSN： 1672-9072

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收录情况： SCI

摘要： With the widespread use of clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) technologies in plants, large-scale genome editing is increasingly needed. Here, we developed a geminivirus-mediated surrogate system, called Wheat Dwarf Virus-Gate (WDV-surrogate), to facilitate high-throughput genome editing. WDV-Gate has two parts: one is the recipient callus from a transgenic rice line expressing Cas9 and a mutated hygromycin-resistant gene (HygM) for surrogate selection; the other is a WDV-based construct expressing two single guide RNAs (sgRNAs) targeting HygM and a gene of interest, respectively. We evaluated WDV-Gate on six rice loci by producing a total of 874 T-0 plants. Compared with the conventional method, the WDV-Gate system, which was characterized by a transient and high level of sgRNA expression, significantly increased editing frequency (66.8% vs. 90.1%), plantlet regeneration efficiency (2.31-fold increase), and numbers of homozygous-edited plants (36.3% vs. 70.7%). Large-scale editing using pooled sgRNAs targeting the SLR1 gene resulted in a high editing frequency of 94.4%, further demonstrating its feasibility. We also tested WDV-Gate on sequence knock-in for protein tagging. By co-delivering a chemically modified donor DNA with the WDV-Gate plasmid, 3xFLAG peptides were successfully fused to three loci with an efficiency of up to 13%. Thus, by combining transiently expressed sgRNAs and a surrogate selection system, WDV-Gate could be useful for high-throughput gene knock-out and sequence knock-in.

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