hTERT Gene Modification Using CRISPR-dCas9-dnmt3a System as a Therapeutic Approach Against Glioma.

BACKGROUND: Abnormal DNA methylation patterns have been reported in various diseases, including different cancers. CRISPR/Cas9 is a low-cost and highly effective gene editing tool that has lately revolutionized biotechnology. Studies have shown that the CRISPR/Cas9 system can effectively target and correct methylation.

OBJECTIVES: Telomerase plays a survival role for cancer cells. It is encoded by the hTERT gene. The effectiveness of CRISPR/Cas9 in targeting hTERT to treat glioma cancer cells was assessed in this study.

METHODS: EF1a-hsaCas9-U6-gRNA vector carrying sgRNA and Cas9 hybrids were used to transfect U87 glioma cells. Four and eight μg/mL polybrene concentrations were investigated to improve transfection efficiency. The expression level of hTERT that has undergone metabisulfite modification was assessed using real-time PCR. Flow cytometry and Western blotting were also used to determine whether telomerase was present in the cells. High-resolution melting analysis (HRM) was used to examine the hTERT promoter's methylation. Finally, flow cytometry was used to measure the apoptotic rate of transfected U87 cells.

RESULTS: The findings demonstrated that gRNA significantly boosted transfection effectiveness. Significant variations were seen in the expression of hTERT in U87 cells at 4 μg/mL polybrene and 80 μg/mL transfection compared to transfection without gRNA and basal cells. Flow cytometry showed a decrease in hTERT levels in transfected cells. Furthermore, transfection with gRNA increased U87 cell apoptosis compared to transfection without gRNA.

CONCLUSIONS: It appears that the designed CRISPR/Cas9 system can reduce hTERT expression and telomerase activity and thus inhibit glioma cell growth.