Doublesex gene in Bombyx mori: CRISPR-mediated female-to-male sex reversal through exon skipping

Abstract

BACKGROUND: Sex control technology has great potential to improve the efficiency of economic insect utilization and integrated pest management by enabling targeted population suppression or elimination. The silkworm (Bombyx mori), a lepidopteran model, holds immense economic importance attributable to its unique silk production capabilities. Male silkworms favored over females as a result of their higher yield and quality silk. Doublesex (dsx) is a key gene for sex determination in insects. Previously, inactivation of Bmdsx, among other sex determination-related genes, resulted in intersex individuals rather than a complete female-to-male transformation. RESULTS: We used CRISPR/Cas9 technology to edit the splice sites of Bmdsx at female-specific exons 3 and 4 to induce transformation of BmdsxF to BmdsxM. The edited genome produced only male-specific BmdsxM. The results revealed that these genetic modifications had significant effects on genital development in female individuals. The edited genome led to the emergence of male-specific gonads in females, causing genital developmental defects. However, it is worth noting that males remained unaffected by these genetic alterations. Furthermore, we conducted a comprehensive analysis of dsx gene structure and explored the potential applicability of this method for inducing female-to-male transgenic transformation in other insects. CONCLUSIONS: This study demonstrates that precision editing of dsx splice sites can enforce unidirectional sex conversion in insects, disrupting female reproductive capacity without affecting males. Overall, these findings provide valuable insights into our understanding of gonadal development and insect population management. © 2025 Society of Chemical Industry.