遗传 ›› 2016, Vol. 38 ›› Issue (7): 651-657.doi: 10.16288/j.yczz.16-058

• 研究报告 • 上一篇    下一篇

利用CRISPR/Cas9技术构建CTCF蛋白降解细胞系

谢德健, 师明磊, 张彦, 王天艺, 沈文龙, 叶丙雨, 李平, 何超, 张香媛, 赵志虎   

  1. 军事医学科学院生物工程研究所,北京 100071
  • 收稿日期:2016-02-18 出版日期:2016-07-20 发布日期:2016-07-20
  • 作者简介:谢德健,硕士研究生,专业方向:遗传学。E-mail: ncuskxiedejian@163.com
  • 基金资助:
    国家自然科学基金(编号:31370762)和国家重点基础研究发展规划项目(编号:2013CB966802)资助[Supported by the Nationa Natural Foundation of China (No.31370762) and the Major State Basic Research Development Program of China (No.2013CB966802)]

Construction of CTCF degradation cell line by CRISPR/Cas9 mediated genome editing

Dejian Xie, Minglei Shi, Yan Zhang, Tianyi Wang, Wenlong Shen, Bingyu Ye, Ping Li, Chao He, Xiangyuan Zhang, Zhihu Zhao   

  1. Beijing Institute of Biotechnology, Beijing 100071, China
  • Received:2016-02-18 Online:2016-07-20 Published:2016-07-20

摘要: CTCF是脊椎动物关键的绝缘子蛋白,在细胞生命过程中发挥重要作用,敲除CTCF基因会导致小鼠胚胎死亡。为进一步探讨CTCF的功能,本文利用CRISPR/Cas9介导的同源重组,在内源性CTCF表达框上游敲入一个有丝分裂期降解结构域(Mitosis-special degradation domain, MD),该结构域可以带动CTCF融合蛋白在M期降解。作为对照,将MD结构域的第42位的精氨酸突变为丙氨酸,形成无降解活性的MD*,可使MD*-CTCF融合蛋白始终稳定存在。将嘌呤霉素与融合蛋白同时表达,即可利用抗生素筛选,高效地筛选到纯合克隆。利用蛋白印迹技术和免疫荧光检测3种细胞在不同细胞周期的CTCF蛋白变化情况,发现MD-CTCF细胞系CTCF蛋白含量约为野生型细胞的10%,MD*-CTCF细胞系的CTCF含量与野生型没有显著差别;通过流式细胞术观测降解CTCF对细胞的影响,发现MD-CTCF细胞系G1期明显延长。总之,利用CRISPR/Cas9技术在CTCF表达框上游高效地插入MD,首个CTCF特异性降解的人类细胞系获得成功构建。

关键词: CRISPR/Cas9, 基因组编辑, CTCF, 蛋白质稳定性, 蛋白降解

Abstract: The CCCTC-binding factor (CTCF) is the main insulator protein described in vertebrates. It plays fundamental roles during diverse cellular processes. CTCF gene knockout mice led to death during embryonic development. To further explore the functions of CTCF, we employed a CRISPR/Cas9-based genome engineering strategy to in-frame insert the mitosis-special degradation domain (MD) of cyclin B into the upstream open reading frame of CTCF gene. Fusion protein is designed to degrade during mitosis leaded by MD. As a control group, mutation of a single arginine (R42A) within the destruction box inactivates the MD leading to constitutive expression of MD*-CTCF. The homozygous clones were obtained via the screening by puromycin when coexpressed with puromycin resistence gene. The protein level of CTCF in MD-CTCF cell line was about 10% of wild-type cells throughout cell cycles by the analyses of Western blotting and immunofluorescence. There was no significant difference between MD*-CTCF cell line and wild type. Flow cytometry results showed prolonged G1 phase in MD-CTCF cell line. Taken together, we demonstrated the feasibility of efficiently inserting MD domain into genome with the CRISPR/Cas9 technology and reported the first CTCF-specific degradation human cell line.

Key words: CRISPR/Cas9, genome editing, CTCF, protein stability, protein degradation