Journal of Shanghai Jiao Tong University (Medical Science) >

2021 , Vol. 41 >Issue 3: 297 - 301

DOI: https://doi.org/10.3969/j.issn.1674-8115.2021.03.002

Basic research

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

  • Yan-na ZHAO ,
  • Rong QIU ,
  • Nan SHEN ,
  • Yuan-jia TANG
Expand
  • 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 date: 2020-03-11

  Online published: 2021-04-06

Supported by

National Natural Science Foundation of China(81871287);Innovative Research Team of High-Level Local Universities in Shanghai(SSMU-ZDCX20180100)

Fold

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

Cite this article

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 Jiao Tong University (Medical Science), 2021 , 41(3) : 297 -301 . DOI: 10.3969/j.issn.1674-8115.2021.03.002

References

1 Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J]. Science, 2012, 337(6096): 816-821.
2 Makarova KS, Haft DH, Barrangou R, et al. Evolution and classification of the CRISPR-Cas systems[J]. Nat Rev Microbiol, 2011, 9(6): 467-477.
3 Makarova KS, Grishin NV, Shabalina SA, et al. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action[J]. Biol Direct, 2006, 1(1): 1-26.
4 Deltcheva E, Chylinski K, Sharma CM, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase Ⅲ[J]. Nature, 2011, 471(7340): 602-607.
5 Davis AJ,Chen DJ. DNA double strand break repair via non-homologous end-joining[J]. Transl Cancer Res, 2013, 2(3):130-143.
6 Butt H, Eid A, Ali Z, et al. Efficient CRISPR/Cas9-mediated genome editing using a chimeric single-guide RNA molecule[J]. Front Plant Sci, 2017, 8: 1441.
7 Liang PP, Xu YW, Zhang XY, et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes[J]. Protein Cell, 2015, 6(5): 363-372.
8 Pellagatti A, Dolatshad H, Valletta S, et al. Application of CRISPR/Cas9 genome editing to the study and treatment of disease[J]. Arch Toxicol, 2015, 89(7): 1023-1034.
9 Platt RJ, Chen S, Zhou Y, et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling[J]. Cell, 2014, 159(2): 440-455.
10 Yin WX, Xiang P, Li QL. Investigations of the effect of DNA size in transient transfection assay using dual luciferase system[J]. Anal Biochem, 2005, 346(2): 289-294.
11 Adli M. The CRISPR tool kit for genome editing and beyond[J]. Nat Commun, 2018, 9(1): 1911.
12 Zhang XH, Tee LY, Wang XG, et al. Off-target effects in CRISPR/Cas9-mediated genome engineering[J]. Mol Ther Nucleic Acids, 2015, 4: e264.
13 Lin YN, Cradick TJ, Brown MT, et al. CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences[J]. Nucleic Acids Res, 2014, 42(11): 7473-7485.
14 Aubrey BJ, Kelly GL, Kueh AJ, et al. An inducible lentiviral guide RNA platform enables the identification of tumor-essential genes and tumor-promoting mutations in vivo[J]. Cell Rep, 2015, 10(8): 1422-1432.
15 Cao J, Wu LZ, Zhang SM, et al. An easy and efficient inducible CRISPR/Cas9 platform with improved specificity for multiple gene targeting[J]. Nucleic Acids Res, 2016, 44(19): e149.
16 Das AT, Tenenbaum L, Berkhout B. Tet-on systems for doxycycline-inducible gene expression[J]. Curr Gene Ther, 2016, 16(3): 156-167.
17 Herold MJ, van den Brandt J, Seibler J, et al. Inducible and reversible gene silencing by stable integration of an shRNA-encoding Lentivirus in transgenic rats[J]. PNAS, 2008, 105(47): 18507-18512.
18 Polstein LR, Gersbach CA. A light-inducible CRISPR-Cas9 system for control of endogenous gene activation[J].Nat Chem Biol, 2015, 11(3):198-200.
19 Dow LE, Fisher J, O'Rourke KP, et al. Inducible in vivo genome editing with CRISPR-Cas9[J]. Nat Biotechnol, 2015, 33(4): 390-394.
20 Lu J, Zhao C, Zhao YZ, et al. Multimode drug inducible CRISPR/Cas9 devices for transcriptional activation and genome editing[J]. Nucleic Acids Res, 2018, 46(5): e25.
21 de Solis CA, Ho A, Holehonnur R, et al. The development of a viral mediated CRISPR/Cas9 system with doxycycline dependent gRNA expression for inducible in vitro and in vivo genome editing[J]. Front Mol Neurosci, 2016, 9: 70.
22 LaFleur MW, Nguyen TH, Coxe MA, et al. A CRISPR-Cas9 delivery system for in vivo screening of genes in the immune system[J]. Nat Commun, 2019, 10: 1668.
Options
Outlines

/

〈 〉