Enhancing Temozolomide (TMZ) chemosensitivity using CRISPR-dCas9-mediated downregulation of O⁶-methylguanine DNA methyltransferase (MGMT)

Purpose: Glioblastoma (GBM) stands out as the most prevalent and aggressive intracranial tumor, notorious for its poor prognosis. The current standard-of-care for GBM patients involves surgical resection followed by radiotherapy, combined with concurrent and adjuvant chemotherapy using Temozolomide (TMZ). The effectiveness of TMZ primarily relies on the activity of O⁶-methylguanine DNA methyltransferase (MGMT), which removes alkyl adducts from the O⁶ position of guanine at the DNA level, thereby counteracting the toxic effects of TMZ. Method: In this study, we employed fusions of catalytically-inactive Cas9 (dCas9) to DNA methyltransferases (dCas9-DNMT3A) to selectively downregulation MGMT transcription by inducing methylation at MGMT promoter and K-M enhancer. Result: Our findings demonstrate a significant reduction in MGMT expression, leading to intensified TMZ sensitivity in the HEK293T cell line. Conclusion: This study serves as a proof of concept for the utilization of CRISPR-based gene suppression to overcome TMZ resistance and enhance the lethal effect of TMZ in glioblastoma tumor cells.