SRA

High-grade pediatric gliomas often contain histone H3.3 mutations, but open questions remain about oncogenic mechanisms. To address this gap, we performed 'reciprocal gene editing' using CRISPR-Cas9 to introduce H3.3 mutations (K27M, G34R) into H3.3-wildtype brain and glioma cells, while in parallel reverting pre-existing K27M mutations in glioma cells back to wildtype. Analyses of our reciprocally-edited cells indicate that H3.3 mutation leads to specific transcriptomic and epigenetic events, and associated cell biological changes including in xenograft assays. We used these data and the reciprocally-edited cells to screen selected drugs and identify specific putative treatments that are mutant H3.3-specific. Overall, reciprocal gene editing provides new insights into mutant H3.3 oncogenic mechanisms and more broadly may prove useful for studying other cancer-associated mutations. Overall design: We used CRISPR-Cas9 gene editing of H3F3A to precisely revert preexisting K27M mutations to WT in patient-derived glioma cells to identify consistently functionally important effectors. Two replicates of H3K27me3 and two replicates of H3.3 ChIP-Seq were performed in each of the following cell lines: Line XIII, Line XIII WT, Line XVII, Line XVII WT.