title: Construction of CTCF degradation cell line by CRISPR/Cas9 mediated genome editing.
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 G1phase 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.
authors.affiliation: Beijing Institute of Biotechnology, Beijing 100071, China.
authors.forename: De-jian
authors.initials: DJ
authors.lastname: Xie
authors.affiliation: Beijing Institute of Biotechnology, Beijing 100071, China.
authors.forename: Ming-lei
authors.initials: ML
authors.lastname: Shi
authors.affiliation: Beijing Institute of Biotechnology, Beijing 100071, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Beijing Institute of Biotechnology, Beijing 100071, China.
authors.forename: Tian-yi
authors.initials: TY
authors.lastname: Wang
authors.affiliation: Beijing Institute of Biotechnology, Beijing 100071, China.
authors.forename: Wen-long
authors.initials: WL
authors.lastname: Shen
authors.affiliation: Beijing Institute of Biotechnology, Beijing 100071, China.
authors.forename: Bing-yu
authors.initials: BY
authors.lastname: Ye
authors.affiliation: Beijing Institute of Biotechnology, Beijing 100071, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Li
authors.affiliation: Beijing Institute of Biotechnology, Beijing 100071, China.
authors.forename: Chao
authors.initials: C
authors.lastname: He
authors.affiliation: Beijing Institute of Biotechnology, Beijing 100071, China.
authors.forename: Xiang-yuan
authors.initials: XY
authors.lastname: Zhang
authors.affiliation: Beijing Institute of Biotechnology, Beijing 100071, China.
authors.forename: Zhi-hu
authors.initials: ZH
authors.lastname: Zhao
mesh_terms: D000076246:CCCTC-Binding Factor; D064113:CRISPR-Cas Systems / Q000502:physiology*; D002455:Cell Division; D045744:Cell Line, Tumor; D016193:G1 Phase; D000072669:Gene Editing; D006801:Humans; D012097:Repressor Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000076246:CCCTC-Binding Factor; C000619697:CTCF protein, human; C000619698:Ctcf protein, mouse; D012097:Repressor Proteins