Crispr-cas9 directed TP53 gene restoration in human prostate cancer cell line PC-3

daha sonra Aim: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. Material and method:CRISPR/Cas9 backbone carier px459 plasmids were used in this study. Edited TP53 414delC gene region were analysed with next-generation sequencing system. qPCR and immunofluorescence analysis was performed for detection of p53 expression from the edited PC-3 cells. Flow cytometry analysis was also performed for the detection of apoptotic cells. Results: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 ssODN1and 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 weredetected compared to the control cells by flow cytometry analysis. Discussion: As a result, CRISPR/Cas9 system accompanied by ssODN was shown to effectively repair the TP53 414delC gene region doldurulacaktır