title: Targeted mutagenesis in chicken using CRISPR/Cas9 system.
abstract: 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.
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, 1-8-31, Midorioka, Ikeda, Osaka 563-8577, Japan.
authors.forename: Isao
authors.initials: I
authors.lastname: Oishi
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, 1-8-31, Midorioka, Ikeda, Osaka 563-8577, Japan.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Yoshii
authors.affiliation: Faculty of Agriculture, Shinshu University, 8304 Minamiminowa, Nagano 399-4598, Japan.
authors.forename: Daichi
authors.initials: D
authors.lastname: Miyahara
authors.affiliation: Faculty of Agriculture, Shinshu University, 8304 Minamiminowa, Nagano 399-4598, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kagami
authors.affiliation: Animal Breeding and Reproduction Research Division, National Agriculture and Food Research Organization, Institute of Livestock and Grassland Science, 2 Ikenodai, Tsukuba, Ibaraki 305-0901, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Tagami
mesh_terms: D000818:Animals; D030161:Avian Proteins / Q000235:genetics*; D064113:CRISPR-Cas Systems; D002478:Cells, Cultured; D002642:Chick Embryo; D002645:Chickens / Q000235:genetics*; D005260:Female; D018390:Gene Targeting / Q000379:methods; D005854:Germ Cells / Q000166:cytology / Q000378:metabolism; D008297:Male; D016296:Mutagenesis; D009154:Mutation; D010047:Ovalbumin / Q000235:genetics*; D010059:Ovomucin / Q000235:genetics*; D015203:Reproducibility of Results
chemical_list: D030161:Avian Proteins; D010059:Ovomucin; D010047:Ovalbumin