bioRxiv2026
BackgroundGlioblastoma (GBM) is the most aggressive primary brain tumor in adults. Radioresistance, partly mediated by glioma stem-like cells, represents a major clinical challenge which could be overcome by the identification of the modulators of radioresistance. Existing CRISPR screens in human GBM models have largely used two-dimensional cultures with short-term viability readouts, failing to capture the long-term clonogenic behaviour underlying tumour recurrence after radiotherapy. MethodWe developed ClonoScreen3D-CRISPRi, combining CRISPRi-mediated gene knockdown with three-dimensional clonogenic survival assays. Two GBM cell lines (G7 and GBML20), differing in MGMT promoter methylation status, were engineered to express the KRAB-dCas9 editor. Nine candidate radiosensitivity modifiers, selected through transcriptomic analysis, pharmacological studies, and literature review, were examined in both lines. Target validation was performed using full radiation dose-response assays and a pharmacological inhibitor. ResultsThe majority of candidate genes significantly altered survival fraction following irradiation in both cell lines. Knockdown of NFKB2, RELB, and CDK9 produced the most potent radiosensitization, with sensitizer enhancement ratios of 1.39-1.70 in validation studies -- exceeding those of established radiosensitizers including PARP and ATM inhibitors. Notably, knockdown of these genes induced no significant cytotoxicity in the absence of radiation. Pharmacological validation using an IKK inhibitor confirmed these findings, implicating non-canonical NF-{kappa}B signalling and CDK9-dependent transcriptional elongation as critical adaptive mechanisms in GBM radioresistance. ConclusionsClonoScreen3D-CRISPRi is a scalable, physiologically relevant platform for identifying genetic modifiers of radioresistance. The non-canonical NF-{kappa}B pathway and CDK9 represent promising radiosensitizing targets, and larger screens could enable systematic prioritisation of candidates for clinical translation. Key PointsO_LIClonoScreen3D-CRISPRi combines gene knockdown with 3D clonogenic survival assays C_LIO_LIWe identified NFKB2, RELB, and CDK9 as modifiers of radioresistance in two GBM cell lines C_LIO_LIValidation experiments show ClonoScreen3D-CRISPRi reliably identifies radiosensitizers in GBM C_LI Importance of the studyGlioblastoma (GBM) remains one of the most lethal human cancers, with radioresistance representing a central barrier to improved patient outcomes. While CRISPR-based screens have begun to illuminate genetic drivers of GBM biology, prior approaches using human models have largely relied on two-dimensional culture systems and short-term viability readouts that inadequately model the disease. This study introduces ClonoScreen3D-CRISPRi, a novel platform that integrates CRISPRi-mediated gene knockdown with three-dimensional clonogenic survival assays in patient-derived GBM cells -- more faithfully recapitulating the long-term clonogenic potential that underlies post-radiotherapy recurrence. Using this platform, we identified NFKB2, RELB, and CDK9 as potent genetic modifiers of radioresistance, with sensitizer enhancement ratios exceeding those of established clinical radiosensitizers such as PARP and ATM inhibitors. Pharmacological validation of the non-canonical NF-{kappa}B pathway demonstrates direct translational relevance, providing a rationale for targeting this axis in combination with radiotherapy to improve GBM treatment.