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 <i>Chilo suppressalis</i> endogenous miRNA <i>Csu-novel-260</i> targeting the <i>Csdib</i> 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 <i>C. suppressalis</i> (target) and <i>Cnaphalocrocis medinalis</i> (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 (<i>H</i>_<i>j</i>) and normalized Shannon entropy (<i>H</i>_<i>s</i>), showed no significant differences between RNAi and the control treatments. Moreover, Kullback-Leibler divergence (<i>D</i>_<i>j</i>) increased in <i>C. suppressalis</i> after 21 days of feeding on Csu260-16, indicating significantly altered gene expression patterns compared to the reference transcriptome. Our findings demonstrate that <i>Csu-novel-26</i>-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.