Epigenetic modification and characterization of theMGMTpromoter region using CRISPRoff in glioblastoma cells

The methylation status of the O6-methylguanine DNA methyltransferase ( MGMT ) promoter region is a critical predictor of response to alkylating agents in glioblastoma. However, current approaches to study the MGMT status focus on analyzing models with non-identical backgrounds. Here, we present an epigenetic editing approach using CRISPRoff to introduce site-specific CpG methylation in the MGMT promoter region of glioma cell lines. Sanger sequencing revealed successful introduction of methylation, effectively generating differently methylated glioma cell lines with an isogenic background. The introduced methylation resulted in reduced MGMT mRNA and protein levels. Furthermore, the cell lines with MGMT promoter region methylation exhibited increased sensitivity to temozolomide, consistent with the impact of methylation on treatment outcomes in patients with glioblastoma. This precise epigenome-editing approach provides valuable insights into the functional relevance of MGMT promoter regional methylation and its potential for prognostic and predictive assessments, as well as epigenetic-targeted therapies.