Multiplex Gene Editing Creates Triple-Resistant Rice Against Both Insect Herbivores and Pathogens

Rice (Oryza sativa) production faces serious threats from multiple biotic stresses, particularly the brown planthopper, rice blast, and bacterial blight. Developing resistant cultivars is the most sustainable control strategy. Compared to race-specific resistance genes, disrupting susceptibility genes often confers broader and potentially more durable resistance. However, engineering broad-spectrum resistance against both insect pests and pathogens by editing susceptibility genes remains challenging. In this study, we employed multiplex CRISPR/Cas9 editing to simultaneously disrupt key susceptibility genes involved in distinct defense pathways: ACS2 (for brown planthopper), Bsr-D1, ERF922 or Pi21 (for fungal blast), and Xa5 (for bacterial blight). Three triple-mutant lines (abx, aex, and apx) were successfully generated, and all exhibited significantly enhanced resistance to brown planthopper, blast, and bacterial blight without compromising major agronomic traits compared to the wild type. Our work demonstrates the feasibility of multiplex susceptibility gene editing as a precise and efficient strategy for breeding rice varieties with synchronized, broad-spectrum resistance to both insect pests and pathogenic diseases.