CRISPR/Cas9 mediated chicken Stra8 gene knockout and inhibition of male germ cell differentiation

Yani Zhang; Yingjie Wang; Qisheng Zuo; Dong Li; Wenhui Zhang; Fei Wang; Yanqin Ji; Jing Jin; Zhenyu Lu; Man Wang; Chen Zhang; Bichun Li

doi:10.1371/journal.pone.0172207

CRISPR/Cas9 mediated chicken Stra8 gene knockout and inhibition of male germ cell differentiation

PLoS One. 2017 Feb 24;12(2):e0172207. doi: 10.1371/journal.pone.0172207. eCollection 2017.

Authors

Yani Zhang^{1

2}, Yingjie Wang^{1

2}, Qisheng Zuo^{1

2}, Dong Li^{1

2}, Wenhui Zhang^{1

2}, Fei Wang^{1

2}, Yanqin Ji^{1

2}, Jing Jin^{1

2}, Zhenyu Lu^{1

2}, Man Wang^{1

2}, Chen Zhang^{1

2}, Bichun Li^{1

2}

Affiliations

¹ College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu province, P.R. China.
² Key Laboratory for Animal Genetics, Breeding, Reproduction, and Molecular Design of Jiangsu Province, Yangzhou, Jiangsu province, P.R. China.

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.

MeSH terms

Adaptor Proteins, Signal Transducing / metabolism*
Animals
Animals, Genetically Modified
CRISPR-Cas Systems*
Cell Differentiation*
Chickens
Cloning, Molecular
Embryonic Stem Cells / cytology
Gene Knockout Techniques*
Genetic Vectors
Germ Cells / cytology*
Male
RNA, Guide, CRISPR-Cas Systems / genetics*
Spermatogonia / cytology

Substances

Adaptor Proteins, Signal Transducing
RNA, Guide, CRISPR-Cas Systems

Grants and funding

This work was supported by the National Natural Science Foundation of China (31301959), Natural Science Foundation of Jiangsu Province (BK20161331), science and technology projects fund of Yangzhou City (YZ2015105), Priority Academic Program Development of Jiangsu Higher Education Institutions. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.