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 endogenous miRNA targeting the 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 (target) and (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 () and normalized Shannon entropy (), showed no significant differences between RNAi and the control treatments. Moreover, Kullback-Leibler divergence () increased in after 21 days of feeding on Csu260-16, indicating significantly altered gene expression patterns compared to the reference transcriptome. Our findings demonstrate that -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.