title: Site-Directed Genome Knockout in Chicken Cell Line and Embryos Can Use CRISPR/Cas Gene Editing Technology.
abstract: The present study established an efficient genome editing approach for the construction of stable transgenic cell lines of the domestic chicken (Gallus gallus domesticus). Our objectives were to facilitate the breeding of high-yield, high-quality chicken strains, and to investigate gene function in chicken stem cells. Three guide RNA (gRNAs) were designed to knockout the C2EIP gene, and knockout efficiency was evaluated in DF-1 chicken fibroblasts and chicken ESCs using the luciferase single-strand annealing (SSA) recombination assay, T7 endonuclease I (T7EI) assay, and TA clone sequencing. In addition, the polyethylenimine-encapsulated Cas9/gRNA plasmid was injected into fresh fertilized eggs. At 4.5 d later, frozen sections of the embryos were prepared, and knockout efficiency was evaluated by the T7EI assay. SSA assay results showed that luciferase activity of the vector expressing gRNA-3 was double that of the control. Results of the T7EI assay and TA clone sequencing indicated that Cas9/gRNA vector-mediated gene knockdown efficiency was approximately 27% in both DF-1 cells and ESCs. The CRISPR/Cas9 vector was also expressed in chicken embryos, resulting in gene knockdown in three of the 20 embryos (gene knockdown efficiency 15%). Taken together, our results indicate that the CRISPR/Cas9 system can mediate stable gene knockdown at the cell and embryo levels in domestic chickens.
authors.affiliation: Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China.
authors.forename: Qisheng
authors.initials: Q
authors.lastname: Zuo
authors.affiliation: Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China.
authors.forename: Yinjie
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China.
authors.forename: Shaoze
authors.initials: S
authors.lastname: Cheng
authors.affiliation: Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Lian
authors.affiliation: Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China.
authors.forename: Beibei
authors.initials: B
authors.lastname: Tang
authors.affiliation: Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Wang
authors.affiliation: Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China.
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Lu
authors.affiliation: Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China.
authors.forename: Yanqing
authors.initials: Y
authors.lastname: Ji
authors.affiliation: Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China.
authors.forename: Ruifeng
authors.initials: R
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China.
authors.forename: Wenhui
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China.
authors.forename: Kai
authors.initials: K
authors.lastname: Jin
authors.affiliation: Animal and Avian Sciences, University of Maryland, College Park, Maryland 20741.
authors.forename: Jiuzhou
authors.initials: J
authors.lastname: Song
authors.affiliation: Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China yubcli@yzu.edu.cn ynzhang@yzu.edu.cn.
authors.forename: Yani
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China yubcli@yzu.edu.cn ynzhang@yzu.edu.cn.
authors.forename: Bichun
authors.initials: B
authors.lastname: Li
mesh_terms: D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D002460:Cell Line; D002642:Chick Embryo; D002645:Chickens; D003001:Cloning, Molecular; D003850:Deoxyribonuclease I / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods; D015870:Gene Expression; D055786:Gene Knockout Techniques; D023061:Gene Order; D018390:Gene Targeting; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D016678:Genome; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D003850:Deoxyribonuclease I
keywords: CRISPR/Cas; chicken; gene knockout