High genotoxicity of CRISPR/Cas9 versus limited efficacy of CRISPRi in chicken primordial germ cells

CRISPR/Cas9 technology has transformed genome editing across species; however, its application in avian germ cells remains constrained-not only by editing efficiency, but also by limited evaluation of potential genotoxic effects. In this study, we systematically assessed the performance and genomic safety of CRISPR/Cas9 and CRISPR interference (CRISPRi) in chicken primordial germ cells (PGCs). While CRISPR/Cas9 achieved high editing efficiency, it simultaneously induced substantial DNA damage, Apoptosis, and sex-specific cell cycle arrest, revealing the pronounced genotoxic sensitivity of PGCs. In contrast, CRISPRi was well tolerated but failed to achieve effective gene repression in chicken cells. Comparative experiments showed that CRISPRi functioned efficiently in human 293T cells but not in chicken PGCs or somatic DF-1 cells, suggesting species-dependent limitations of mammalian-optimized repression systems. Together, these findings reveal a fundamental trade-off-"efficient but toxic" versus "safe but ineffective"-when applying CRISPR tools to avian germ cells. Our results highlight the need for species-adapted, low-toxicity genome-editing platforms in poultry and provide a framework for evaluating editing strategies in developmentally sensitive cell types.

CRISPR interference; CRISPR/Cas9; Chicken; Genotoxicity; Primordial germ cell.