胶质母细胞瘤是中枢神经系统中最常见和最具侵袭性的原发肿瘤，预后差。目前的黄金标准治疗是手术切除后联合放疗和化疗。硝唑酮（TMZ）的疗效作为主要的化疗药物，取决于O6-甲基鸟嘌呤DNA甲基转移酶（MGMT）的DNA甲基化状态，该酶已被确定为胶质母细胞瘤患者的预后生物标志物。 临床研究表明，MGMT启动子高甲基化的胶质母细胞瘤患者对TMZ治疗有更好的反应和明显改善的总生存期。因此，本研究利用CRISPRoff基因组编辑工具在MGMT启动子区域介导有针对性的DNA甲基化。该系统携带着一个CRISPR禁活化的Cas9（dCas9），与甲基转移酶（Dnmt3A/3L）结合，通过有针对性的DNA甲基化抑制了TMZ耐药的人类胶质母细胞瘤细胞系中的MGMT表达。MGMT表达水平的下调逆转了TMZ耐药性，在TMZ耐药的胶质母细胞瘤细胞系中导致了剂量依赖性的细胞死亡率。 总之，我们展示了以RNA引导的MGMT启动子有针对性甲基化作为克服药物耐药性和改善TMZ对胶质母细胞瘤的细胞毒作用的有希望的工具。版权所有 © 2023 Elsevier Inc. 发布。

Glioblastoma is the most common and aggressive primary tumor of the central nervous system with poor outcome. Current gold standard treatment is surgical resection followed by a combination of radio- and chemotherapy. Efficacy of temozolomide (TMZ), the primary chemotherapeutic agent, depends on the DNA methylation status of the O6-methylguanine DNA methyltransferase (MGMT), which has been identified as a prognostic biomarker in glioblastoma patients. Clinical studies revealed that glioblastoma patients with hypermethylated MGMT promoter have a better response to TMZ treatment and a significantly improved overall survival. In this study, we thus used the CRISPRoff genome editing tool to mediate targeted DNA methylation within the MGMT promoter region. The system carrying a CRISPR-deactivated Cas9 (dCas9) fused with a methyltransferase (Dnmt3A/3L) domain downregulated MGMT expression in TMZ-resistant human glioblastoma cell lines through targeted DNA methylation. The reduction of MGMT expression levels reversed TMZ resistance in TMZ-resistant glioblastoma cell lines resulting in TMZ induced dose-dependent cell death rates. In conclusion, we demonstrate targeted RNA-guided methylation of the MGMT promoter as a promising tool to overcome chemoresistance and improve the cytotoxic effect of TMZ in glioblastoma.Copyright © 2023. Published by Elsevier Inc.