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Background <p>Current standard of care for glioblastoma involves fractionated radiotherapy administered with Temozolomide (TMZ), a DNA-alkylating agent. Inhibition of the DNA repair enzyme, O⁶-methylguanine-DNA methyltransferase (MGMT), promotes sensitivity to TMZ, particularly in tumors that repress <i>MGMT</i> mRNA transcription through promoter methylation. Novel strategies to inhibit <i>MGMT</i> are a promising avenue to improve therapeutic outcomes to TMZ. We hypothesized that CRISPR-Cas13-mediated RNA regulatory silencing of <i>MGMT</i> mRNA enhances response of immortalized and primary patient-derived gliomaspheres to TMZ in vitro.</p> Methods <p>We utilized the Cas13x and Cas13d variants to target <i>MGMT</i> mRNA in the MGMT-expressing LN18 glioma cell line and in two patient-derived gliomasphere lines (GS104, GS081). Cas13-guide RNA ribonucleoproteins were delivered via lipofection, and stable knockdown was achieved using a lentiviral all-in-one system. <i>MGMT</i> mRNA and protein downregulation were assessed by RT-PCR and Western blot, respectively. Cell viability and chemosensitivity to TMZ were evaluated using MTT assays.</p> Results <p>Both Cas13x and Cas13d systems, directed by specific guide CRISPR RNAs, achieved rapid and potent knockdown of <i>MGMT</i> mRNA and protein in all tested cell lines. This downregulation of MGMT expression led to an increase in the cytotoxic effects of TMZ, sensitizing previously resistant glioma cells and patient-derived gliomaspheres to standard chemotherapy. The lentiviral Cas13d system established stable chemosensitization in gliomasphere models.</p> Conclusion <p>CRISPR-Cas13-mediated targeting of <i>MGMT</i> mRNA is an effective strategy for overcoming TMZ resistance in in vitro glioblastoma models. This RNA regulatory editing approach offers a proof-of-principle for CRISPR mediated therapeutics in patients with <i>MGMT</i> unmethylated gliomas.</p>

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MGMT downregulation by CRISPR/Cas13 RNA-guided RNA targeting enhances glioma cell sensitivity to TMZ chemotherapy

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月12日

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  • Terry J. Prins,
  • Thomas J. Lai,
  • Tie Li,
  • Addison Fisher,
  • Blaine S. C. Eldred,
  • Ryan Mostafavi,
  • Linda M. Liau,
  • Robert A. Chong,
  • Phioanh Leia Nghiemphu,
  • Timothy F. Cloughesy,
  • David A. Nathanson,
  • Albert Lai

摘要

Background

Current standard of care for glioblastoma involves fractionated radiotherapy administered with Temozolomide (TMZ), a DNA-alkylating agent. Inhibition of the DNA repair enzyme, O⁶-methylguanine-DNA methyltransferase (MGMT), promotes sensitivity to TMZ, particularly in tumors that repress MGMT mRNA transcription through promoter methylation. Novel strategies to inhibit MGMT are a promising avenue to improve therapeutic outcomes to TMZ. We hypothesized that CRISPR-Cas13-mediated RNA regulatory silencing of MGMT mRNA enhances response of immortalized and primary patient-derived gliomaspheres to TMZ in vitro.

Methods

We utilized the Cas13x and Cas13d variants to target MGMT mRNA in the MGMT-expressing LN18 glioma cell line and in two patient-derived gliomasphere lines (GS104, GS081). Cas13-guide RNA ribonucleoproteins were delivered via lipofection, and stable knockdown was achieved using a lentiviral all-in-one system. MGMT mRNA and protein downregulation were assessed by RT-PCR and Western blot, respectively. Cell viability and chemosensitivity to TMZ were evaluated using MTT assays.

Results

Both Cas13x and Cas13d systems, directed by specific guide CRISPR RNAs, achieved rapid and potent knockdown of MGMT mRNA and protein in all tested cell lines. This downregulation of MGMT expression led to an increase in the cytotoxic effects of TMZ, sensitizing previously resistant glioma cells and patient-derived gliomaspheres to standard chemotherapy. The lentiviral Cas13d system established stable chemosensitization in gliomasphere models.

Conclusion

CRISPR-Cas13-mediated targeting of MGMT mRNA is an effective strategy for overcoming TMZ resistance in in vitro glioblastoma models. This RNA regulatory editing approach offers a proof-of-principle for CRISPR mediated therapeutics in patients with MGMT unmethylated gliomas.