J Integr Plant Biol.

• Breakthrough Report •    

Simultaneous gene editing of three homoeoalleles in self-incompatible allohexaploid grasses

Liang Zhang1,3† , Tao Wang5† , Guangyang Wang1,6, Aoyue Bi1,3, Misganaw Wassie1,3, Yan Xie1,2, Liwen Cao1,2, Huawei Xu5 , Jinmin Fu6 , Liang Chen1,2*, Yang Zhao4 * and Tao Hu1,2*   

  1. 1 CAS Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, The Innovative Academy of Seed Design, The Chinese Academy of Sciences, Wuhan 430074, China
    2 Center of Economic Botany, Core Botanical Gardens, The Chinese Academy of Sciences, Wuhan 430074, China
    3 University of Chinese Academy of Sciences, Beijing 100049, China
    4 Shanghai Center for Plant Stress Biology, and CAS Center of Excellence in Molecular Plant Sciences, The Chinese Academy of Sciences, Shanghai 200032, China
    5 College of Agriculture, Henan University of Science and Technology, Luoyang 471023, China
    6 School of Resources and Environmental Engineering, Ludong University, Yantai 264011, China

    These authors contributed equally to this work.
    *Correspondences: Liang Chen (chenliang888@wbgcas.cn); Yang Zhao (zhaoyang@sibs.ac.cn); Tao Hu (Hut420@wbgcas.cn, Dr. Hu is fully responsible for the distritution of the materials associated with this article)
  • Received:2021-02-10 Accepted:2021-04-18 Online:2021-04-29

Abstract: Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been widely used for precise gene editing in plants. However, simultaneous gene editing of multiple homoeoalleles remains challenging, especially in self-incompatible polyploid plants. Here, we simultaneously introduced targeted mutations in all three homoeoalleles of two genes in the self-incompatible allohexaploid tall fescue, using both CRISPR/Cas9 and LbCas12a (LbCpf1) systems. Loss-of-function mutants of FaPDS exhibited albino leaves, while knockout of FaHSP17.9 resulted in impaired heat resistance in T0 generation of tall fescue. Moreover, these mutations were inheritable. Our findings demonstrate the feasibility of generating loss-of-function mutants in T0 generation polyploid perennial grasses using CRISPR/Cas systems.

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