Sex chromosome–linked Cas9 knock-in and functional validation in chicken primordial germ cells

Authors: JUNG, KYUNG MIN - University Of Missouri; MONY, SABRINA - University Of Missouri; Chen, Paula; LEE, KIHO - University Of Missouri; LEE, HONG JO - University Of Missouri

Submitted to: Poultry Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/1/2026
Publication Date: N/A
Citation: N/A

Interpretive Summary: The ability to express genes in a sex-specific manner is important in poultry to solve various issues in the industry, such as culling of male chicks. However, insertion of genes into sex chromosomes can have deleterious effects that prevent proper development of the cells that eventually become sperm or oocytes. In this study, two regions on the chicken sex chromosomes were validated for their ability to harbor and allow for expression of inserted genes. This provides a new platform for genome editing in the cells that contribute to subsequent generations and can be used to direct expression of target genes based on sex.

Technical Abstract: Sex-specific control of genome editing in birds remains a significant challenge for both basic research and biotechnological applications. Chickens exhibit a ZW sex-determination system in which the Z and W chromosomes encode genes essential for sexual 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 the Z chromosome (DMRT1–DMRT3 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.