CRISPR screening unravels genome‐wide regulators of juvenile hormone signaling in Bombyx mori

Abstract Insect metamorphosis constitutes a sophisticated endocrine cascade governed by the dynamic interplay between juvenile hormone (JH) and 20‐hydroxyecdysone (20E). Although JH has been established as a critical developmental regulator, its intracellular signaling mechanisms remain incompletely characterized. In this study, we implemented a genome‐wide CRISPR screening strategy in the lepidopteran model Bombyx mori to systematically identify components of JH signaling. First, we developed a JH‐responsive reporter cell line with EGFP expression under the control of JH‐sensitive promoters, enabling real‐time monitoring of pathway activation. Then we established a genome‐scale CRISPR knockout library specifically optimized for silkworm cells, and performed screening through integration of high‐throughput flow cytometry and next‐generation sequencing. Systematic analysis revealed a total of 387 candidate genes that significantly modulated JH signaling activity (231 enriched vs. 156 depleted). Functional annotation clustering revealed predominant enrichment in membrane biogenesis processes and small molecule metabolism. It is worth noting that through integrated genomic screening and experimental verification, we found that the tyrosine kinase receptor BmALK is an important regulatory node for JH signal transduction. This study establishes the first comprehensive genetic landscape of JH signaling in a holometabolous insect, providing a robust platform for functional mining of JH signaling pathways, and molecular evidence for membrane‐associated JH signal transduction in Lepidoptera.