Using Transcriptome Diversity to Evaluate the Off-Target Effects of miRNA-Mediated Transgenic Rice

RNAi technology is an innovative tool for pest control in agriculture. The RNAi-based rice event, Csu260-16, expressing Chilo suppressalis endogenous miRNA Csu-novel-260 targeting the Csdib gene shows great potential for managing this pest. However, off-target effects limit its long-term sustainable utility. Thus, developing scientific evaluation methods for environmental risk of RNAi rice is necessary. Here, we performed comparative transcriptome analysis of C. suppressalis (target) and Cnaphalocrocis medinalis (nontarget), after feeding on Csu260-16 and its nontransgenic control (ZH11), to explore potential cross-species off-target effects at transcriptome level. Identified unintended differentially expressed genes (DEGs) fell into three categories: those biologically associated with miRNA-targeted genes, those potentially binding to Csu-novel-260, and unexpected DEGs. Transcriptome diversity indices, Shannon entropy (Hj) and normalized Shannon entropy (Hs), showed no significant differences between RNAi and the control treatments. Moreover, Kullback-Leibler divergence (Dj) increased in C. suppressalis after 21 days of feeding on Csu260-16, indicating significantly altered gene expression patterns compared to the reference transcriptome. Our findings demonstrate that Csu-novel-26-mediated off-target effects may occur at individual gene levels without compromising overall transcriptome complexity. This study establishes transcriptome diversity metrics as a novel tool for quantifying RNAi risks, advancing the environmental safety evaluation of RNAi-based crops, and supporting eco-friendly pest control strategies.