JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE) ›› 2021, Vol. 41 ›› Issue (3): 297-301.doi: 10.3969/j.issn.1674-8115.2021.03.002

• Basic research • Previous Articles     Next Articles

Construction of inducible CRISPR/Cas9 system for studying gene function in mouse

Yan-na ZHAO1(), Rong QIU2(), Nan SHEN1, Yuan-jia TANG1()   

  1. 1.Shanghai Institute of Rheumatology, Department of Rheumatology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China
    2.Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences, Shanghai 200031, China
  • Received:2020-03-11 Online:2021-03-28 Published:2021-04-06
  • Contact: Yuan-jia TANG;;
  • Supported by:
    National Natural Science Foundation of China(81871287);Innovative Research Team of High-Level Local Universities in Shanghai(SSMU-ZDCX20180100)

Abstract: Objective

·To construct inducible CRISPR/Cas9 system for studying gene function in mouse immune cells, combining Dox-inducible single guide RNA (sgRNA) expression vector with Cas9 transgenic mice.


·U6-TetO-sgRNA and EF1α-T2A-Puro-BFP-T2A-TetR fragments were obtained by gene synthesis. The two synthetic fragments were assembled into the retroviral vector backbone by using homologous recombination. sgRNA targeting protein coding region of F4/80 and non-targeting control (NC) were designed. Bone marrow cells were isolated from Cas9 transgenic mice and transfected with retrovirus expressing sgRNA. The experimental conditions were divided into Dox-added group (Dox +) and non Dox-added group (Dox-). The knockout effect was tested by flow cytometry and T7 endonuclease Ⅰ (T7EⅠ) experiments.


·①Dox-inducible sgRNA retroviral vector and Cas9 transgenic mice were successfully constructed. ② The result of flow cytometry showed that F4/80 was only knocked out in the F4/80Dox+ population, but not in NC Dox-, NC Dox+ and F4/80 Dox- populations. ③ T7EⅠ results showed that the DNA was cut into two bands in the F4/80Dox+ group, while the DNA band was intact in the F4/80 Dox- group.


·An inducible CRISPR/Cas9 system combining Dox-inducible sgRNA retroviral vector with Cas9 transgenic mice are successfully constructed. With this system, inducible gene knockout in mouse immune cells are successfully achieved.

Key words: inducible CRISPR/Cas9 system, Dox-inducible sgRNA expression vector, hematopoietic stem cell, gene editing, macrophage

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