<scp>RNase III</scp> ‐deficient <scp> <i>Bacillus thuringiensis</i> </scp> enables stable <scp>dsRNA</scp> production and enhances <scp>HD1</scp> activity against <scp> <i>Spodoptera frugiperda</i> </scp>

Abstract BACKGROUND Spodoptera frugiperda is one of the most important invasive agricultural pests in China. Integrating RNA interference (RNAi) with Bacillus thuringiensis (Bt)‐based biocontrol represents a promising strategy for pest control. However, efficient dsRNA production in Bt remains challenging as endogenous RNase activity limits dsRNA stability and accumulation. RESULTS In this study, an RNase III ‐deficient Bt strain, DBΔ rnc , was generated using an optimized CRISPR ‐Cas9 system. Deficiency of RNase III markedly enhanced intracellular dsRNA accumulation. A hairpin dsRNA construct exhibited higher transcript levels than the dual‐promoter construct, especially in the DBΔ rnc background. Oral delivery of Bt expressed hairpin dsRNA targeting the endochitinase gene Sf CHI of S. frugiperda induced effective gene silencing and caused pupation defects. Moreover, co‐application with Bt HD1 strain significantly increased larval mortality in both neonate and 2 nd ‐instar larvae. CONCLUSION Collectively, our findings highlight that the RNase III‐deficient strain DBΔ rnc is an efficient chassis for dsRNA expression. Furthermore, the enhanced insecticidal activity from co‐application of DBΔ rnc (hpds CHI ) and HD1 demonstrates the feasibility of integrating RNAi with Bt‐based pest control. © 2026 Society of Chemical Industry.