CRISPR/Cas9-mediated TOP1 knockout in chicken DF-1 cells reveals its critical role in apoptosis regulation and genomic stability.

The role of topoisomerase I (encoded by TOP1) in avian cell survival and apoptosis regulation remains unclear, limiting its potential application in poultry biotechnology. This study aimed to establish a CRISPR/Cas9-mediated TOP1 knockout platform in chicken DF-1 cells and evaluate its functional impact on apoptosis. Three sgRNAs targeting TOP1 were designed and delivered via liposome vectors, achieving knockout efficiencies up to 50% as confirmed by T7 Endonuclease I (T7E1) assay and Sanger sequencing, with no detectable off-target effects. Functional analysis revealed that TOP1 knockout significantly increased apoptosis rates and upregulated DNA damage markers (γH2AX) and apoptotic genes (Caspase 8 and BRCA1). These results demonstrate that TOP1 is essential for maintaining genomic stability in avian somatic cells, and its depletion triggers apoptosis through DNA damage accumulation. Although synthetic lethality-based sex control was not directly tested here, our findings provide critical evidence that TOP1 dysfunction could theoretically enable selective elimination of specific cell populations (e.g., primary germ cells) via CRISPR editing. Notably, developing targeted delivery systems for PGCs-a focus of future research not addressed in this study-will be essential to achieve such selectivity in vivo, highlighting a significant technological hurdle to overcome.