Evaluation of the CRISPR/Cas9 directed mutant TP53 gene repairing effect in human prostate cancer cell line PC-3


Batir M. B., Sahin E., Cam F. S.

MOLECULAR BIOLOGY REPORTS, vol.46, no.6, pp.6471-6484, 2019 (SCI-Expanded) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 46 Issue: 6
  • Publication Date: 2019
  • Doi Number: 10.1007/s11033-019-05093-y
  • Journal Name: MOLECULAR BIOLOGY REPORTS
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Page Numbers: pp.6471-6484
  • Keywords: CRISPR/Cas9, Prostate cancer, TP53 gene, p53, Homology-directed repair (HDR), P53, THERAPY, APOPTOSIS, MUTATIONS
  • Dokuz Eylül University Affiliated: No

Abstract

Prostate cancer is a common health problem among men worldwide and most of these prostate cancer cases are related to a dysfunctional mutant Tumor Protein p53 (TP53) gene. However, the CRISPR/Cas9 system can be used for repairing of a dysfunctional mutant TP53 gene in combination with donor single-stranded oligodeoxynucleotide (ssODN) via cells' own homology-directed repair (HDR) mechanism. In this study, we aimed to evaluate the CRISPR/Cas9 repairing efficiency on TP53 414delC (p.K139fs*31) null mutation, located in the TP53 gene, of human prostate cancer cell line PC-3 in combination with ssODNs. According to the next-generation sequencing results, TP53 414delC mutation was repaired with an efficiency of 19.95% and 26.0% at the TP53 414delC position with ssODN1 and ssODN2 accompanied by sgRNA2 guided CRISPR/Cas9, respectively. Besides, qPCR and immunofluorescence analysis showed that PC-3 cells, the TP53 414delC mutation of which were repaired, expressed wild type p53 again. Also, significantly increased number of apoptotic cells, driven by the repaired TP53 gene were detected compared to the control cells by flow cytometry analysis. As a result, sgRNA2 guided CRISPR/Cas9 system accompanied by ssODN was shown to effectively repair the TP53 414delC gene region and inhibit the cell proliferation of PC-3 cells. Therefore, the effects of the TP53 414delC mutation repairment in PC-3 cells will be investigated in the in vivo models for tumor clearance analysis in the near future.