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The CRISPR/Cas9 system is a simple and powerful tool for genome editing in various organisms including livestock animals. However, the system has not been applied to poultry because of the difficulty in accessing their zygotes. Here we report the implementation of CRISPR/Cas9-mediated gene targeting in chickens. Two egg white genes, ovalbumin and ovomucoid, were efficiently (>90%) mutagenized in cultured chicken primordial germ cells (PGCs) by transfection of circular plasmids encoding Cas9, a single guide RNA, and a gene encoding drug resistance, followed by transient antibiotic selection. We transplanted CRISPR-induced mutant-ovomucoid PGCs into recipient chicken embryos and established three germline chimeric roosters (G0). All of the roosters had donor-derived mutant-ovomucoid spermatozoa, and the two with a high transmission rate of donor-derived gametes produced heterozygous mutant ovomucoid chickens as about half of their donor-derived offspring in the next generation (G1). Furthermore, we generated ovomucoid homozygous mutant offspring (G2) by crossing the G1 mutant chickens. Taken together, these results demonstrate that the CRISPR/Cas9 system is a simple and effective gene-targeting method in chickens.

In avian species, primordial germ cells (PGC) use the vascular system as a vehicle to transport them to the future gonadal region. The aim of this study was to elucidate the details of migration system and size of the PGC population in the early chicken embryo. We analyzed whole chicken embryos during stages X and 2 to 17 by immunohistochemical staining using specific antibody raised against chicken vasa homolog. At stage X, PGC were dense in the central zone of the area pellucida. Following the formation of the primitive streak, PGC moved anteriorly to the edge of the extraembryonic region. The size of the PGC population increased gradually during stages X (130.4 +/- 31.9) to 10 (439.3 +/- 93.6). At stage 10, PGC began to accumulate in the region anterior to the head, and then we could observe that PGC invaded into the vascular system in this region. At stage 11, the number of PGC decreased in the region anterior to the head (129.8 +/- 42.5 to 46.7 +/- 4.2) and increased in the blood vessels (194.0 +/- 41.6 to 285.0 +/- 7.5). No PGC could be recognized in the intermediate mesoderm, the future gonadal region, until stage 14, but they first appeared there at stage 15. The number of PGC recognized in the intermediate mesoderm increased from stage 15 to 17. Interestingly, the number of PGC between the left and right sides of this region was consistently and significantly different (P < 0.05) in females and males. The present study mainly clarified that chicken PGC continue to proliferate throughout early development, many PGC invaded into the vascular system from the region anterior to the head in stage 11, and PGC actively left the blood vessels and migrated to the intermediate mesoderm from stage 15.