An Inducible CYP19A1 Excision Model for Sexual Differentiation in Chicken (Gallus gallus) via the CRISPR/Cas9 System

Aromatase, a crucial enzyme for estrogen synthesis, plays a vital role in gender determination and differentiation. This study aimed to establish an inducible knockout model of the chicken <i>CYP19A1</i> gene, which encodes aromatase, to support gender control in chickens. We selected the most efficient sgRNA target site and constructed an inducible knockout model based on the Tet-on system. The knockout efficiency reached 80% with 20 μg/mL DOX induction in vitro. The encapsulation of the plasmid with PEI and injection into eggs achieved a knockout efficiency of 45% in ovo. qRT-PCR analysis revealed a significant downregulation of female-related genes (<i>CYP19A1</i>, <i>FOXL2</i>, <i>ESR1</i>) and upregulation of male-related genes (<i>DMRT1</i>, <i>SOX9</i>, <i>AMH</i>) in female chicken embryos after induction. Western blotting showed decreased protein expression of CYP19A1 and FOXL2, and increased SOX9 expression in female embryos post-DOX induction. Elisa detection further confirmed lower estradiol levels in the gonads of induced female embryos compared to normal and non-induced females. These findings demonstrate the successful establishment of an inducible knockout system for the <i>CYP19A1</i> gene in chickens, providing theoretical and technical support for the creation of new breeding materials for gender control.