DNA damage hypersensitivity in chicken primordial germ cells limits CRISPR-Cas9 editing and supports an shRNA-based screening approach

Abstract Chickens (Gallus gallus) are uniquely suited for germline studies because their primordial germ cells (PGCs) can be propagated long-term in vitro and used for germline transmission. To develop a loss-of-function screening platform in chicken PGCs, we compared three perturbation methods: CRISPR/Cas9 knockout, CRISPR interference (CRISPRi), and shRNA-mediated knockdown. We found that CRISPR/Cas9 editing causes severe toxicity in PGCs, with DNA damage hypersensitivity over 20-fold greater than in somatic cells, and with distinct DNA damage checkpoint responses between male (ZZ) and female (ZW) lines. CRISPRi using dCas9-KRAB was ineffective in chicken—likely because of species-specific epigenetic constraints—whereas shRNA knockdown produced robust, nontoxic gene silencing. These results identify DNA damage hypersensitivity as a major barrier to nuclease-based editing in the germline and establish RNAi as a feasible platform for genome-wide functional screening in chicken PGCs.