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

doi:10.3969/j.issn.1674-8115.2021.03.002

Abstract

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.

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.

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

CLC Number:

R394.4

Yan-na ZHAO, Rong QIU, Nan SHEN, Yuan-jia TANG. Construction of inducible CRISPR/Cas9 system for studying gene function in mouse[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(3): 297-301.

Figures/Tables 5

Fig 1 Construction strategy of Dox-inducible sgRNA retroviral vector

Fig 2 Retrovirus packaging and NIH3T3 cell infection

Fig 3 Flow cytometry detection of F4 / 80 expression in bone marrow-derived macrophage

Fig 4 Detect DNA mutations of F4/80 locus by T7EⅠ

Fig 5 Retrovirus infection of mouse hematopoietic stem cells

TrendMD

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