A Genetic Compensation Phenomenon and Global Gene Expression Changes in Sex-miR-2766-3p Knockout Strain of Spodoptera exigua Hübner (Lepidoptera: Noctuidae)

SIMPLE SUMMARY: miRNAs are endogenous, small, non-coding RNA species that can be crucial regulators in a variety of biological processes, such as development, immunity, and metamorphosis. Because one miRNA may regulate hundreds of targets, nearly all genes are expected to be regulated by miRNAs to some degree. Since this is the case, miRNA knockouts may show obvious symptoms, such as developmental defects and embryonic mortality. In this study, we reported that Sex-miR-2766-3p knockout in S. exigua was non-lethal, confirming the theory that miRNAs function redundantly with other miRNAs or other pathways. ABSTRACT: MicroRNAs (miRNAs) drive the post-transcriptional repression of target mRNAs and play important roles in a variety of biological processes. miR-2766-3p is conserved and abundant in Lepidopteran species and may be involved in a variety of biological activities. In this study, Sex-miR-2766-3p was predicted to potentially bind to the 3′ untranslated region (UTR) of cap ‘n’ collar isoform C (CncC) in Spodoptera exigua, and Sex-miR-2766-3p was confirmed to regulate the expression of SeCncC through screening with a luciferase reporter system. Although CRISPR/Cas9 has been extensively utilized to examine insect gene function, studies of miRNA function are still relatively uncommon. Thus, we employed CRISPR/Cas9 to knock out Sex-miR-2766-3p from S. exigua. However, the expression of SeCncC was not significantly altered in the knockout strain (2766-KO) compared with that of the WHS strain. This result suggested that a miRNA knockout might lack phenotypes because of genetic robustness. Additionally, we used transcriptome analysis to examine how the global gene expression patterns of the Sex-miR-2766-3p knockout strain varied. RNA-seq data revealed 1746 upregulated and 2183 downregulated differentially expressed genes (DEGs) in the 2766-KO strain, which might be the result of Sex-miR-2766-3p loss or DNA lesions as the trigger for transcriptional adaptation. GO function classification and KEGG pathway analyses showed that these DEGs were enriched for terms related to binding, catalytic activity, metabolic process, and signal transduction. Our findings demonstrated that S. exigua could compensate for the missing Sex-miR-2766-3p by maintaining the expression of SeCncC by other pathways.