, Volume 70, Issue 6, pp 1595–1605 | Cite as

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

  • Ya Li
  • Weifeng Zhang
  • Junli Zhao
  • Sai Li
  • Linlin Shan
  • Jiuling Zhu
  • Yan Li
  • He Zhu
  • Qinwen Mao
  • Haibin XiaEmail author


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.


CRISPR/Cas9 Gene editing Lentivirus Loxp/Cre system Off-target 



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).

Compliance with ethical standards

Conflict of interest

The authors declares that they have no conflicts of interest.

Supplementary material

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Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Laboratory of Gene Therapy, Department of Biochemistry, College of Life SciencesShaanxi Normal UniversityXi’anPeople’s Republic of China
  2. 2.Department of PathologyNorthwestern University Feinberg School of MedicineChicagoUSA

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