Affiliation: | 1. Institute of Crop Sciences (ICS), Chinese Academy of Agricultural Sciences (CAAS), Beijing, 100081 China;2. Institute of Crop Sciences (ICS), Chinese Academy of Agricultural Sciences (CAAS), Beijing, 100081 China CAAS/Hainan Yazhou Bay Seed Laboratory, National Nanfan Research Institute (Sanya), Sanya, 572024 China;3. Institute of Crop Sciences (ICS), Chinese Academy of Agricultural Sciences (CAAS), Beijing, 100081 China CAAS/Hainan Yazhou Bay Seed Laboratory, National Nanfan Research Institute (Sanya), Sanya, 572024 China 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 |
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. |