Sex Chromosome–Targeted Cas9 Knock-in and Functional Validation in Chicken Primordial Germ Cells

ABSTRACT Sex-specific control of genome editing remains a significant challenge in birds. Chickens exhibit a ZW sex-determination system in which the Z and W chromosomes encode genes essential for sex differentiation and germline development, providing a rationale for sex-linked genome engineering. In this study, we established the sex chromosome–linked knock-in system for Cas9 in chicken primordial germ cells (PGCs). Donor constructs carrying Cas9-GFP were engineered to integrate into either the Z chromosome ( DMRT1 – DMRT 3 intergenic region) or the W chromosome (5’ region of HINTW locus). The targeting strategy was validated in DF-1 fibroblasts and PGCs, where site-specific integration was confirmed by junction PCR and sequencing. Functionality of the integrated Cas9 was verified by targeting two different loci, demonstrating efficient genome cleavage at NHEJ1 loci and indel-associated loss of GFP fluorescence following GFP targeting. The knock-in PGCs expressed Cas9 protein while retaining germ cell markers and migration capacity, demonstrating preservation of germline identity. Collectively, our findings establish a sex chromosome–linked Cas9 knock-in system in chicken PGCs and demonstrate that these sites support stable Cas9 expression without compromising germline characteristics, thereby providing a practical foundation for controlled, sex-specific genome engineering in avian research.