J Integr Plant Biol. ›› 2023, Vol. 65 ›› Issue (9): 2194-2203.DOI: 10.1111/jipb.13543

• Molecular Physiology • Previous Articles     Next Articles

Artificial evolution of OsEPSPS through an improved dual cytosine and adenine base editor generated a novel allele conferring rice glyphosate tolerance

Chen Zhang1, Xue Zhong1,2, Shaoya Li1,2,3, Lei Yan1, Jingying Li1,2,3, Yubing He1,2,3, Yong Lin4, Yangjun Zhang4 and Lanqin Xia1,2,3*   

  1. 1. Institute of Crop Sciences (ICS), Chinese Academy of Agricultural Sciences (CAAS), Beijing 100081, China;
    2. CAAS/Hainan Yazhou Bay Seed Laboratory, National Nanfan Research Institute (Sanya), Sanya 572024, China;
    3. Key Laboratory of Gene Editing Technologies (Hainan), Ministry of Agricultural and Rural Affairs, Sanya 572024, China;
    4. Beijing Dabeinong Technology Group Co., Ltd, Beijing 10080, China
    *Correspondence: Lanqin Xia (xialanqin@caas.cn)
  • Received:2023-03-27 Accepted:2023-06-29 Online:2023-07-04 Published:2023-09-01

Abstract: Exploiting novel endogenous glyphosate-tolerant alleles is highly desirable and has promising potential for weed control in rice breeding. Here, through fusions of different effective cytosine and adenine deaminases with nCas9-NG, we engineered an effective surrogate two-component composite base editing system, STCBE-2, with improved C-to-T and A-to-G base editing efficiency and expanded the editing window. Furthermore, we targeted a rice endogenous OsEPSPS gene for artificial evolution through STCBE-2-mediated near-saturated mutagenesis. After hygromycin and glyphosate selection, we identified a novel OsEPSPS allele with an Asp-213-Asn (D213N) mutation (OsEPSPS-D213N) in the predicted glyphosate-binding domain, which conferred rice plants reliable glyphosate tolerance and had not been reported or applied in rice breeding. Collectively, we developed a novel dual base editor which will be valuable for artificial evolution of important genes in crops. And the novel glyphosate-tolerant rice germplasm generated in this study will benefit weeds management in rice paddy fields.

Key words: artificial evolution, dual cytosine and adenine base editor, nCas9-NG, rice (Oryza sativa L), near-saturated mutagenesis

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