CRISPR/Cas9, a new approach to successful knockdown of ABCB1/P-glycoprotein and reversal of chemosensitivity in human epithelial ovarian cancer cell line

Document Type : Original Article


1 Department of Molecular Medicine, School of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran

2 Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran

3 Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

4 Department of Biochemistry and Genetic, School of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran


Objective(s): Multidrug resistance (MDR) is a major obstacle in the successful chemotherapy of ovarian cancer. Inhibition of P-glycoprotein (P-gp), a member of ATP-binding cassette (ABC) transporters, is a well-known strategy to overcome MDR in cancer. The aim of this study was to investigate the efficiency and ability of CRISPR/Cas9 genome editing technology to knockdown ABCB1 gene expression in adriamycin resistant (A2780/ADR) ovarian cancer cell line and evaluate the sensitivity changes to doxorubicin.
Materials and Methods: Three single-guide RNAs (sgRNAs) targeting the fourth and fifth exons of human ABCB1 gene were designed in this study. Expression level of ABCB1 was detected using quantitative real time PCR (qRT-PCR) after co-transfection of all three sgRNAs into A2780/ADR cell line and subsequent antibiotic selection. Drug sensitivity to doxorubicin was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Results: The results showed that CRISPR/Cas9 system could significantly reduce the expression of P-gp. The dramatic decline in ABCB1 gene expression was associated with increased sensitivity of cells transfected with sgRNAs to doxorubicin.
Conclusion: Based on the results of this study, it is concluded that the CRISPR-based systems, used in the present study, effectively down-regulated the target gene and acted as an ideal and cost-effective tool for gene editing of A2780/ADR cell line resulting in restoration of nonmalignant phenotype.


Main Subjects

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