J Integr Plant Biol. ›› 2023, Vol. 65 ›› Issue (3): 646-655.DOI: 10.1111/jipb.13381

• New Technology • Previous Articles     Next Articles

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

Yifu Tian1,2,3†, Dating Zhong1,4†, Xinbo Li1,2,3†, Rundong Shen1,2,3, Han Han4, Yuqin Dai1, Qi Yao1,4, Xuening Zhang1,4, Qi Deng2, Xuesong Cao1, Jian-Kang Zhu1,2,3,5* and Yuming Lu1,4*   

  1. 1. Shanghai Center for Plant Stress Biology, Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 201602, China;
    2. Center for Advanced Bioindustry Technologies, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China;
    3. Hainan Yazhou Bay Seed Lab, Sanya 572024, China;
    4. Shanghai Collaborative Innovation Center of Agri-Seeds, Joint Center for Single Cell Biology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China;
    5. Institute of Advanced Biotechnology, School of Life Sciences, Southern University of Science and Technology, Shenzhen 518055, China
    These authors contributed equally.
    *Correspondences: Jian‐Kang Zhu (zhujk@sustech.edu.cn); Yuming Lu (luymin@sjtu.edu.cn, Dr. Lu is fully responsible for the distribution of the materials associated with this article)
  • Received:2022-05-10 Accepted:2022-10-10 Online:2022-10-11 Published:2023-03-01

Abstract: 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 T0 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.

Key words: genome editing, high-throughput, protein tagging, rice

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