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 CYP19A1 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 (CYP19A1, FOXL2, ESR1) and upregulation of male-related genes (DMRT1, SOX9, AMH) 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 CYP19A1 gene in chickens, providing theoretical and technical support for the creation of new breeding materials for gender control.