<i>htert</i> 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 htertgene. 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 htertthat 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 htertpromoter'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 htertin 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 htertlevels 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 htertexpression and telomerase activity and thus inhibit glioma cell growth.