CRISPR Screen Reveals SOX2 as a Critical Regulator of CD133 and Cellular Stress Response in Glioblastoma II

Glioblastoma (GBM) remains a formidable challenge in clinical settings due limited treatments available. The surface protein CD133 marks glioblastoma stem cells (GSCs), cells capable of overcoming therapeutic pressures and correlate with more aggressiveness tumor phenotypes. In this study, we employed a CRISPR-Cas9 functional screen to deconvolute CD133 dynamics in tumors. This led us to establish that SOX2 is a key player in controlling the PROM1 gene, which in turn influences how cells react to stress factors, including those induced by chemoradiation treatment. The discoveries in this study shed light on the complex web of mechanisms that control the survival and resistance of GSCs, offering promising new avenues for targeting and potentially overcoming therapy resistance. The CRISPR screen was established, where 1E8 cells were transduced with the TKOv3 lentivirus library at an MOI of 0.3, representing more than 400-fold coverage per sgRNA after selection with puromycin. Media was changed 24 hours after infection to puromycin-containing medium (1.2µg/mL). 72 hours after transduction, 3E7 puromycin-selected cells were collected for genomic DNA extraction at starting timepoint, and 9E7 cells were evenly divided into triplicates (3E7 cells each, 2.5E6 cells per 15cm dish). Passaging subsequently occurred every 7 days (approximately 3 cell doublings), where 3E7 cells in each replicate were reseeded until the 12th doubling (T12) when sorting was performed. A representative portion of the final population of cells (bulk/unsorted) was collected for sequencing prior to staining for flow cytometry followed by the top and bottom 5th-percentiles of CD133 surface expression being collected in cooled flow tubes. Collected cells were centrifuged and stored at -80 °C until genomic extraction as described below.