Generating Broad-Spectrum Resistance to ALS-Inhibiting Herbicides in Rice by CRISPR/Cas9-Mediated NHEJ

作者：Chao Ouyang, Xiongxia Jin, Huimin Zhao, Silan Chen, Guangmiao Zhao, Dan Li, Wei Liu, Xiuying He, Yongzhong Wu, Jing Yang, Baoguang An · 发表于：Rice · 年份：2025 · DOI：10.1186/s12284-025-00845-w · 被引用次数：7 · 研究领域：CRISPR and Genetic Engineering、Plant tissue culture and regeneration、GABA and Rice Research

Herbicides are pivotal for modern agriculture, but challenges like weed resistance and crop rotation issues necessitate the development of herbicide-resistant genetic resources. This study focused on acetolactate synthase (ALS), a key enzyme targeted by numerous herbicides. Using CRISPR/Cas9-mediated non-homologous end joining (NHEJ) and combining with whole-stage selection, we induced mutations in the OsALS gene of indica rice and identified novel in-frame mutations at the P171 and S627 sites, respectively. Among them, one mutation at the P171 site, the triple mutation P171T/R172G/M174L (ALS-TM) conferred broad-spectrum resistance to Imidazolinones Pyrimidinylthiobenzoates Sulfonylaminocarbonyltriazolinones and Sulfonylureas herbicides. Compared to wild-type (WT) rice, ALS-TM showed 1153-fold higher resistance to imazethapyr (IMT) than WT based on GR 50 values (The herbicide dose causing a 50% reduction in growth), with minimal growth inhibition at 10-fold IMT treatment. Enzymatic assays revealed that ALS-TM maintained catalytic efficiency while reducing herbicide binding, which validated the resistance at the protein level. Field trials showed that ALS-TM mutant retained normal agronomic traits even after IMT spraying, indicating no yield penalty. Additionally, ALS mutations were validated as effective transgenic selection markers, enabling efficient rice transformation under different selection systems. These results demonstrated that ALS-TM could also serve as a reliable ...