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Establishing a dual knock-out cell line by lentivirus based combined CRISPR/Cas9 and Loxp/Cre system

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Abstract

The clustered regulatory interspersed short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system has been widely used for gene knock-out. Lentiviral vectors have been commonly used as a delivery method for this system, however, prolonged Cas9/sgRNA expression due to lentiviral integration can lead to accumulating off-target mutations. To solve this issue in engineering a gene knock-out cell line, this study established a novel system, which was composed of two lentiviral vectors. One lentiviral vector carried simultaneously sgRNAs and CRISPR/Cas9 expression cassettes targeting single or multiple gene(s); the other lentiviral vector carried Cre that could remove excess sgRNAs and Cas9 expression cassettes in the genome after gene targeting was achieved. To prove the principle, two candidate genes, extracellular matrix protein 1 (ECM1) and progranulin (PGRN), both highly expressed in MDA-MB-231 cells, were selected for testing the novel system. A dual knock-out of ECM1 and PGRN was successfully achieved in MDA-MB-231 cell line, with the sgRNAs and Cas9 expression cassettes being removed by Cre. This system should have great potential in applications for multiple genes knock-out in vitro.

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Acknowledgements

This study was supported by the research grants to H.X. from the National Natural Science Foundation of China (No. 81471772 and No. 81773265) and Key research and development plan of Shaanxi Province (No.2018SF-106).

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Authors and Affiliations

  1. Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, 199 South Chang’an Road, Xi’an, 710062, Shaanxi, People’s Republic of China

    Ya Li, Weifeng Zhang, Junli Zhao, Sai Li, Linlin Shan, Jiuling Zhu, Yan Li, He Zhu & Haibin Xia

  2. Department of Pathology, Northwestern University Feinberg School of Medicine, 303 E. Chicago Avenue, Ward 3-140, Chicago, IL, 60611, USA

    Qinwen Mao

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  1. Ya Li
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  2. Weifeng Zhang
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  4. Sai Li
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  5. Linlin Shan
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  6. Jiuling Zhu
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  7. Yan Li
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  8. He Zhu
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  9. Qinwen Mao
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  10. Haibin Xia
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Correspondence to Haibin Xia.

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Li, Y., Zhang, W., Zhao, J. et al. Establishing a dual knock-out cell line by lentivirus based combined CRISPR/Cas9 and Loxp/Cre system. Cytotechnology 70, 1595–1605 (2018). https://doi.org/10.1007/s10616-018-0252-2

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