%A Yan-na ZHAO, Rong QIU, Nan SHEN, Yuan-jia TANG %T Construction of inducible CRISPR/Cas9 system for studying gene function in mouse %0 Journal Article %D 2021 %J Journal of Shanghai Jiao Tong University (Medical Science) %R 10.3969/j.issn.1674-8115.2021.03.002 %P 297-301 %V 41 %N 3 %U {https://xuebao.shsmu.edu.cn/CN/abstract/article_12927.shtml} %8 2021-03-28 %X 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.

Methods

·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.

Results

·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.

Conclusion

·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.