PGC-based cryobanking, regeneration through germline chimera mating, and CRISPR/Cas9-mediated TYRP1 modification in indigenous Chinese chickens

Abstract

Abstract Primordial germ cells (PGCs) are essential precursors for sperm and eggs. PGCs are highly reliable for conserving chicken germplasm and producing genetically modified chickens. Currently, efforts to conserve native chicken germplasm through PGCs and research on genetically modified chickens using CRISPR/Cas9 are limited. In this study, we successfully established 289 PGC lines from eight Chinese chicken populations with an 81.6% derivation rate. Furthermore, we successfully regenerated Piao chickens derived from these PGCs by repropagating cryopreserved PGCs and transplanting them into recipient chickens, resulting in a 12.7% efficiency rate. The regenerated chickens carried mitochondrial DNA from the female PGC donor and the rumplessness (Rp) mutation from male and female PGC donors in a homozygous condition. As a further application, we utilized CRISPR/Cas9 genome editing to disrupt the TYRP1 gene and created KO PGC lines. Transplanting KO cells into male recipients and mating them with wild-type hens enabled the successful generation of four TYRP1 KO chickens. Molecular analysis confirmed a 73 bp deletion in these four chicks, resulting in a frameshift mutation in the TYRP1 target region. These KO chickens had brown plumage due to reduced eumelanin production, observed in both homozygous and hemizygous mutations, compared to wild-type chickens. In conclusion, we have efficiently cultured and cryopreserved chicken PGCs, used them to regenerate chickens, and generated gene-edited chickens with TYRP1 dysfunction. These achievements will greatly impact the conservation of chicken genetic diversity, create new mutation models, and have potential applications in biomedical and avian research and breeding advancements.