Evaluation of the CRISPR/Cas9 directed mutant TP53 gene repairing effect in human prostate cancer cell line PC-3
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.
KeywordsCRISPR/Cas9 Prostate cancer TP53 gene p53 Homology-directed repair (HDR)
This research was financially supported by the Scientific Investigation Department of Manisa Celal Bayar University with the project number FEF 2017-144.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflicts of interest.
- 15.Ihry RJ, Worringer KA, Salick MR, Frias E, Ho D, Theriault K, Kommineni S, Chen J, Sondey M, Ye CY, Randhawa R, Kulkarni T, Yang Z, McAllister G, Russ C, Reece-Hoyes J, Forrester W, Hoffman GR, Dolmetsch R, Kaykas A (2018) p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells. Nat Med 24(7):939CrossRefGoogle Scholar
- 18.Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
- 21.Song F, Stieger K (2017) Optimizing the DNA donor template for homology-directed repair of double-strand breaks. Mol Ther 7:53–60Google Scholar
- 24.Janic A, Valente LJ, Wakefield MJ, Di Stefano L, Milla L, Wilcox S, Yang HY, Tai L, Vandenberg CJ, Kueh AJ, Mizutani S, Brennan MS, Schenk RL, Lindqvist LM, Papenfuss AT, O’Connor L, Strasser A, Herold MJ (2018) DNA repair processes are critical mediators of p53-dependent tumor suppression. Nat Med 24(7):947CrossRefGoogle Scholar
- 33.Maeder ML, Stefanidakis M, Wilson CJ, Baral R, Barrera LA, Bounoutas GS, Bumcrot D, Chao H, Ciulla DM, DaSilva JA, Dass A, Dhanapal V, Fennell TJ, Friedland AE, Giannoukos G, Gloskowski SW, Glucksmann A, Gotta GM, Jayaram H, Haskett SJ, Hopkins B, Horng JE, Joshi S, Marco E, Mepani R, Reyon D, Ta T, Tabbaa DG, Samuelsson SJ, Shen S, Skor MN, Stetkiewicz P, Wang T, Yudkoff C, Myer VE, Albright CF, Jiang H (2019) Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10. Nat Med 25(2):229–233CrossRefGoogle Scholar
- 40.Liu JJ, Orlova N, Oakes BL, Ma E, Spinner HB, Baney KLM, Chuck J, Tan D, Knott GJ, Harrington LB, Al-Shayeb B, Wagner A, Brotzmann J, Staahl BT, Taylor KL, Desmarais J, Nogales E, Doudna JA (2019) CasX enzymes comprise a distinct family of RNA-guided genome editors. Nature 566(7743):218CrossRefGoogle Scholar