Integrated transcriptomic and variant analysis reveals molecular mechanisms of pyrethroid resistance in a genetically homogenized Cas9 strain of Aedes aegypti

Abstract Insecticide resistance, a central problem in insecticide toxicology, in Aedes aegypti continues to undermine vector control efforts worldwide. Although transcriptomic studies have identified numerous resistance-associated genes, differences in genetic background between resistant and susceptible strains often obscure which changes are directly linked to resistance. To reduce background variability, we generated a genetically homogenized pyrethroid-resistant Cas9 strain (PRCas9) by repeatedly backcrossing a susceptible Cas9 strain (exu-Cas9 line) with a highly permethrin-resistant Puerto Rico (PR) strain. PRCas9 retained extreme resistance comparable to the parental PR strain (> 18,000-fold resistance ratio) and was further analyzed alongside the PR and Cas9 strains using RNA-seq. Using this controlled genetic background, transcriptomic profiling identified > 70 differentially expressed genes between resistant and susceptible mosquitoes, revealing reproducible resistance-associated detoxification in insecticide toxicology signatures dominated by cytochrome P450s and carboxylesterases. qRT-PCR validated elevated expression of key detoxification candidates. Variant and promoter sequencing further revealed unique SNPs in detoxification genes, including CYP9J23 , CYP9J28 , and venom carboxylesterase-6, as well as distinct variants in NOX4-art, a NADPH oxidase involved in reactive oxygen species (ROS) production. Gene ontology analysis supported enrichment of detoxification-related molecular functions. Increased NOX4-art expression, together with these regulatory variants, suggests a potential role for ROS-linked regulatory pathways in coordinating detoxification gene activation. This study demonstrates the value of a genetically homogenized resistant Cas9 background for dissecting resistance mechanisms while identifying candidate genes and regulatory elements contributing to pyrethroid resistance. PRCas9 provides a powerful platform for future CRISPR-based functional analyses in insect toxicology aimed at improving vector control strategies.