BackgroundBrain tumours are the leading cause of cancer-related death in children, and there is no effective treatment. A growing body of evidence points to deregulated epigenetics as a tumour driver, particularly in paediatric cancers as they have relatively few genomic alterations, and key driver mutations have been identified in histone 3 (H3). Cancer stem cells (CSC) are implicated in tumour development, relapse and therapy resistance and thus particularly important to target. We therefore aimed to identify novel epigenetic treatment targets in CSC derived from H3-mutated high-grade glioma (HGG) through a CRISPR-Cas9 knockout screen.ResultsThe knockout screen identified more than 100 novel genes essential for the growth of CSC derived from paediatric HGG with H3K27M mutation. We successfully validated 12 of the 13 selected hits by individual knockout in the same two CSC lines, and for the top six hits we included two additional CSC lines derived from H3 wild-type paediatric HGG. Knockout of these genes led to a significant decrease in CSC growth, and altered stem cell and differentiation markers.ConclusionsThe screen robustly identified essential genes known in the literature, but also many novel genes essential for CSC growth in paediatric HGG. Six of the novel genes (UBE2N, CHD4,LSM11, KANSL1, KANSL3andEED)were validated individually thus demonstrating their importance for CSC growth in H3-mutated and wild-type HGG. These genes should be further studied and evaluated as novel treatment targets in paediatric HGG.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13148-023-01498-6.