Dual-line Genome-scale CRISPR Screening Enables Robust Target Gene Discovery.

Pooled CRISPR screening enables genome-scale functional gene discovery by linking genetic perturbations to cellular phenotypes. However, its application in insects remains limited, as systematic differences often obscure the universal functional signals and compromise the reliable identification of genetic regulators. We found that Cas9 genomic integration was associated with distinct transcriptional states, editing activity, and screening outcomes. To address this limitation, we developed a dual-line CRISPR screening framework that prioritizes genes with concordant effects across independently derived Cas9-expressing insect cell lines. In Bactrocera dorsalis, dual-line consensus screening increased in vivo validation of candidate genes from 60% in single-line screens to 90%. Under cold stress, this framework identified candidate regulators of cold tolerance, including a previously uncharacterized gene whose perturbation altered embryonic survival at low temperature. These results show that dual-line screening can filter cell line-specific artifacts and improve functional genomic screening for reliable genetic target discovery in insects.