Identification of Two Potential Gene Insertion Sites for Gene Editing on the Chicken Z/W Chromosomes

文献类型： 外文期刊

第一作者： Wu, Gaoyuan

作者： Wu, Gaoyuan;Liang, Youchen;Chen, Chen;Chen, Guohong;Zuo, Qisheng;Niu, Yingjie;Jin, Kai;Li, Bichun;Wu, Gaoyuan;Liang, Youchen;Chen, Chen;Chen, Guohong;Zuo, Qisheng;Niu, Yingjie;Jin, Kai;Li, Bichun;Wu, Gaoyuan;Liang, Youchen;Chen, Chen;Chen, Guohong;Zuo, Qisheng;Niu, Yingjie;Jin, Kai;Li, Bichun;Niu, Yingjie;Jin, Kai;Song, Jiuzhou;Han, Wei;Li, Bichun

作者机构：

关键词： CRISPR/Cas9; chicken; sex chromosomes; genome engineering; gene insertion sites

期刊名称：GENES （ 影响因子：2.8； 五年影响因子：3.3 ）

ISSN：

年卷期： 2024 年 15 卷 7 期

页码：

收录情况： SCI

摘要： The identification of accurate gene insertion sites on chicken sex chromosomes is crucial for advancing sex control breeding materials. In this study, the intergenic region NC_006127.4 on the chicken Z chromosome and the non-repetitive sequence EE0.6 on the W chromosome were selected as potential gene insertion sites. Gene knockout vectors targeting these sites were constructed and transfected into DF-1 cells. T7E1 enzyme cleavage and luciferase reporter enzyme analyses revealed knockout efficiencies of 80.00% (16/20), 75.00% (15/20), and 75.00% (15/20) for the three sgRNAs targeting the EE0.6 site. For the three sgRNAs targeting the NC_006127.4 site, knockout efficiencies were 70.00% (14/20), 60.00% (12/20), and 45.00% (9/20). Gel electrophoresis and high-throughput sequencing were performed to detect potential off-target effects, showing no significant off-target effects for the knockout vectors at the two sites. EdU and CCK-8 proliferation assays revealed no significant difference in cell proliferation activity between the knockout and control groups. These results demonstrate that the EE0.6 and NC_006127.4 sites can serve as gene insertion sites on chicken sex chromosomes for gene editing without affecting normal cell proliferation.

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