PGC-based cryobanking, regeneration through germline chimera mating, and CRISPR/Cas9-mediated TYRP1 modification in indigenous Chinese chickens
Keiji Kinoshita, Kumiko Tanabe, Yoshiaki Nakamura, Ken‐ichi Nishijima, Takayuki Suzuki, Yuya Okuzaki, Shusei Mizushima, Mingshan Wang, Samiullah Khan, Kaixiang Xu, Muhammad Ameen Jamal, Taiyun Wei, Heng Zhao, Yanhua Su, Feizhou Sun, Gang Liu, Fangxian Zhu, Hong‐Ye Zhao, Hong‐Jiang Wei, Hong‐Ye Zhao, Hong‐Jiang Wei
Abstract
Primordial germ cells (PGCs) are vital for producing sperm and eggs and are crucial for conserving chicken germplasm and creating genetically modified chickens. However, efforts to use PGCs for preserving native chicken germplasm and genetic modification via CRISPR/Cas9 are limited. Here we show that we established 289 PGC lines from eight Chinese chicken populations with an 81.6% success rate. We regenerated Piao chickens by repropagating cryopreserved PGCs and transplanting them into recipient chickens, achieving a 12.7% efficiency rate. These regenerated chickens carried mitochondrial DNA from female donor PGC and the rumplessness mutation from both male and female donors. Additionally, we created the TYRP1 (tyrosinase-related protein 1) knockout (KO) PGC lines via CRISPR/Cas9. Transplanting KO cells into male recipients and mating them with wild-type hens produced four TYRP1 KO chickens with brown plumage due to reduced eumelanin production. Our work demonstrates efficient PGC culture, cryopreservation, regeneration, and gene editing in chickens. The combination of efficient PGC culture, practical cryopreservation, regeneration from recipient chickens, and CRISPR/Cas9 gene-editing to create knockout chickens for conserving and genetically improving rare native breeds.