CRISPR Screen Identifies SOX2 as Key Glioblastoma Regulator

Glioblastoma remains one of the most treatment-resistant brain cancers, partly due to glioblastoma stem cells that express the surface marker CD133. These cells can self-renew and drive tumour recurrence after therapy. To understand what controls CD133 expression, researchers performed a CRISPR-Cas9 functional screen in patient-derived glioblastoma stem cell lines.

The screen involved transducing cells with the Toronto Knockout Library version 3, which targets approximately 18,000 human genes with four single guide RNAs per gene. After 12 cell doublings, the researchers sorted cells by CD133 surface expression levels and sequenced the surviving cell populations to identify genes whose knockout affected CD133 levels.

The screen revealed 12 positive regulators of CD133, including SOX2, KEAP1, TADA1, TADA2B, FBXW7 and SIAH1, plus one negative regulator, CUX1. Validation experiments confirmed that SOX2 knockout substantially reduced both CD133 surface expression and total protein levels whilst impairing stem cell self-renewal and proliferation across multiple glioblastoma stem cell lines.

Using CUT&RUN chromatin profiling, the team demonstrated that SOX2 binds directly to the PROM1 promoter and first intron. Comparing glioblastoma stem cells to normal neural stem cells revealed that SOX2 preferentially binds stress response genes – including FOS, SESN2, TXNIP and NES – in cancer cells, suggesting SOX2 helps glioblastoma stem cells tolerate therapeutic stress.

The study was led by Neil Savage and Sheila Singh from McMaster University in Hamilton, Canada. It was published in Scientific Reports on 16 October 2025.