Efficient CRISPR/Cas9-mediated genome editing in the European corn borer, Ostrinia nubilalis

The European corn borer (Ostrinia nubilalis) is an agricultural pest and burgeoning model for research on speciation, seasonal adaptation and insect resistance management. Although previous work in O. nubilalis has identified genes associated with differences in life cycle, reproduction, and resistance to Bt toxins, the general lack of a robust gene-editing protocol for O. nubilalis has been a barrier to functional validation of candidate genes. Here, we demonstrate an efficient and practical methodology for heritable gene mutagenesis in O. nubilalis using the CRISPR/Cas9 genome editing system. Precise loss-of-function (LOF) mutations were generated at two circadian clock genes, period (per) and pigment-dispersing factor receptor (pdfr), and a developmental gene, prothoracicotropic hormone (ptth). Precluding the need for a visible genetic marker, gene-editing efficiency remained high across different single guide RNAs (sgRNA) and germline transmission of mutations to F-1 offspring approached 100%. When single or dual sgRNAs were injected at a high concentration, gene-specific phenotypic differences in behaviour and development were identified in F-0 mutants. Specifically, F-0 gene mutants demonstrated that PER, but not PDFR, is essential for normal timing of eclosion. PTTH F-0 mutants were significantly heavier and exhibited a higher incidence of diapause. This work will accelerate future studies of gene function in O. nubilalis and facilitate the development of similar screens in other Lepidopteran and non-model insects.