Genome-wide CRISPR screening in Spodoptera frugiperda reveals the determinants of insecticide activation and sensitivity

Insecticide resistance poses a growing threat to global human health and food security. Addressing this challenge requires a comprehensive understanding of the molecular determinants that govern insecticide action and insect sensitivity. However, identifying and functionally validating the specific genes - among the tens of thousands present in a typical insect genome - that contribute to insecticide mode of action and resistance remains a major challenge. Here, we present a genome-wide CRISPR knockout screening system in a cell line derived from Spodoptera frugiperda, a highly damaging insect pest, and demonstrate its utility for dissecting the molecular basis of insecticide activity and resistance. We construct a library of 90,759 single guide RNAs (sgRNAs) targeting 14,434 genes and conduct a three-round screen under successively increasing concentrations of the insecticide pyridalyl. This enriches for the cytochrome P450 gene CYP333B4. Subsequent P450 inhibitor assays, gene knockout in S. frugiperda, and ectopic expression in Drosophila melanogaster confirm that members of the CYP333B subfamily are key mediators of pyridalyl activation and serve as potential resistance targets. Collectively, our findings establish a versatile and scalable platform for genome-wide interrogation of insecticide mode of action and resistance, while uncovering the long-elusive determinants of pyridalyl activity and sensitivity in insects. New tools to investigate the molecular determinants of insect sensitivity to insecticides are needed. Here, a new genome-wide CRISPR-based screening platform is established and used to identify genes involved in activating the insecticide pyridalyl.