BackgroundLow-grade gliomas (LGG) are a type of brain tumor that can be lethal, and it is essential to identify genes that are correlated with patient prognosis. In this study, we aimed to use CRISPR-cas9 screening data to identify key signaling pathways and develop a genetic signature associated with high-risk, low-grade glioma patients.MethodsThe study used CRISPR-cas9 screening data to identify essential genes correlated with cell survival in LGG. We used RNA-seq data to identify differentially expressed genes (DEGs) related to cell viability. Moreover, we used the least absolute shrinkage and selection operator (LASSO) method to construct a genetic signature for predicting overall survival in patients. We performed enrichment analysis to identify pathways mediated by DEGs, overlapping genes, and genes shared in the Weighted correlation network analysis (WGCNA). Finally, the study used western blot, qRT-PCR, and IHC to detect the expression of hub genes from signature in clinical samples.ResultsThe study identified 145 overexpressed oncogenes in low-grade gliomas using the TCGA database. These genes were intersected with lethal genes identified in the CRISPR-cas9 screening data from Depmap database, which are enriched in Hippo pathways. A total of 19 genes were used to construct a genetic signature, and the Hippo signaling pathway was found to be the predominantly enriched pathway. The signature effectively distinguished between low- and high-risk patients, with high-risk patients showing a shorter overall survival duration. Differences in hub gene expression were found in different clinical samples, with the protein and mRNA expression of REP65 being significantly up-regulated in tumor cells. The study suggests that the Hippo signaling pathway may be a critical regulator of viability and tumor proliferation and therefore is an innovative new target for treating cancerous brain tumors, including low-grade gliomas.ConclusionOur study identified a novel genetic signature associated with high-risk, LGG patients. We found that the Hippo signaling pathway was significantly enriched in this signature, indicating that it may be a critical regulator of tumor viability and proliferation in LGG. Targeting the Hippo pathway could be an innovative new strategy for treating LGG.Supplementary InformationThe online version contains supplementary material available at 10.1186/s10020-023-00652-3.