title: CRISPR/Cas9 mediated chicken Stra8 gene knockout and inhibition of male germ cell differentiation.
abstract: An efficient genome editing approach had been established to construct the stable transgenic cell lines in the domestic chicken (Gallus gallus domesticus) at present. Our objectives were to investigate gene function in the differentiation process of chicken embryonic stem cells (ESCs) into spermatogonial stem cells(SSCs). Three guides RNA (gRNAs) were designed to knockout the Stra8 gene, and knockout efficiency was evaluated in domestic chicken cells using cleavage activity of in vitro transcription of gRNA, Luciferase-SSA assay, T7 endonuclease I assay(T7E1) and TA clone sequence. In addition, the Cas9/gRNA plasmid was transfected into ESCs to confirm the function of Stra8. SSA assay results showed that luciferase activity of the vector expressing gRNA-1 and gRNA- 2 was higher than that of gRNA-3. TA clone sequencing showed that the knockdown efficiency was 25% (10/40) in DF-1 cells, the knockdown efficiency was 23% (9/40) in chicken ESCs. T7E1 assay indicated that there were cleavage activity for three individuals, and the knockdown efficiency was 12% (3/25). Cell morphology, qRT-PCR, immunostaining and FCS indicated that Cas9/gRNA not only resulted in the knockout of Stra8 gene, but also suggested that the generation of SSCs was blocked by the Stra8 gene knockdown in vitro. Taken together, our results indicate that the CRISPR/Cas9 system could mediate stable Stra8 gene knockdown in domestic chicken's cells and inhibit ECSs differentiation into SSCs.
authors.affiliation: College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu province, P.R. China.|Key Laboratory for Animal Genetics, Breeding, Reproduction, and Molecular Design of Jiangsu Province, Yangzhou, Jiangsu province, P.R. China.
authors.forename: Yani
authors.identifier: http://orcid.org/0000-0002-0222-8389
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu province, P.R. China.|Key Laboratory for Animal Genetics, Breeding, Reproduction, and Molecular Design of Jiangsu Province, Yangzhou, Jiangsu province, P.R. China.
authors.forename: Yingjie
authors.initials: Y
authors.lastname: Wang
authors.affiliation: College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu province, P.R. China.|Key Laboratory for Animal Genetics, Breeding, Reproduction, and Molecular Design of Jiangsu Province, Yangzhou, Jiangsu province, P.R. China.
authors.forename: Qisheng
authors.initials: Q
authors.lastname: Zuo
authors.affiliation: College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu province, P.R. China.|Key Laboratory for Animal Genetics, Breeding, Reproduction, and Molecular Design of Jiangsu Province, Yangzhou, Jiangsu province, P.R. China.
authors.forename: Dong
authors.initials: D
authors.lastname: Li
authors.affiliation: College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu province, P.R. China.|Key Laboratory for Animal Genetics, Breeding, Reproduction, and Molecular Design of Jiangsu Province, Yangzhou, Jiangsu province, P.R. China.
authors.forename: Wenhui
authors.initials: W
authors.lastname: Zhang
authors.affiliation: College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu province, P.R. China.|Key Laboratory for Animal Genetics, Breeding, Reproduction, and Molecular Design of Jiangsu Province, Yangzhou, Jiangsu province, P.R. China.
authors.forename: Fei
authors.initials: F
authors.lastname: Wang
authors.affiliation: College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu province, P.R. China.|Key Laboratory for Animal Genetics, Breeding, Reproduction, and Molecular Design of Jiangsu Province, Yangzhou, Jiangsu province, P.R. China.
authors.forename: Yanqin
authors.initials: Y
authors.lastname: Ji
authors.affiliation: College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu province, P.R. China.|Key Laboratory for Animal Genetics, Breeding, Reproduction, and Molecular Design of Jiangsu Province, Yangzhou, Jiangsu province, P.R. China.
authors.forename: Jing
authors.initials: J
authors.lastname: Jin
authors.affiliation: College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu province, P.R. China.|Key Laboratory for Animal Genetics, Breeding, Reproduction, and Molecular Design of Jiangsu Province, Yangzhou, Jiangsu province, P.R. China.
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Lu
authors.affiliation: College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu province, P.R. China.|Key Laboratory for Animal Genetics, Breeding, Reproduction, and Molecular Design of Jiangsu Province, Yangzhou, Jiangsu province, P.R. China.
authors.forename: Man
authors.initials: M
authors.lastname: Wang
authors.affiliation: College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu province, P.R. China.|Key Laboratory for Animal Genetics, Breeding, Reproduction, and Molecular Design of Jiangsu Province, Yangzhou, Jiangsu province, P.R. China.
authors.forename: Chen
authors.initials: C
authors.lastname: Zhang
authors.affiliation: College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu province, P.R. China.|Key Laboratory for Animal Genetics, Breeding, Reproduction, and Molecular Design of Jiangsu Province, Yangzhou, Jiangsu province, P.R. China.
authors.forename: Bichun
authors.initials: B
authors.lastname: Li
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D030801:Animals, Genetically Modified; D064113:CRISPR-Cas Systems; D002454:Cell Differentiation; D002645:Chickens; D003001:Cloning, Molecular; D053595:Embryonic Stem Cells / Q000166:cytology; D055786:Gene Knockout Techniques; D005822:Genetic Vectors; D005854:Germ Cells / Q000166:cytology*; D008297:Male; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D013093:Spermatogonia / Q000166:cytology
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000094704:RNA, Guide, CRISPR-Cas Systems