Genome-wide CRISPR-Cas9 screens identify genes/pathways relevant for IgH-CRLF2-r ALL cell survival. (A) Schematic representation of the CRISPR-Cas9 screen. MUTZ5 cells stably expressing Cas9 were transduced with the Brunello library, selected in puromycin, and cultured for 17 days. (B) Genes were significantly enriched after the 17-day incubation period. Genes essential for cell fitness are represented by negative β scores. Genes encoding positive regulators of the RAS (orange) or mTORC1 (purple) pathways were identified as essential genes. Genes encoding proteins involved in JAK/STAT signaling are indicated. (C) MUTZ5 cells stably expressing sgRNAs targeting JAK2 or CRLF2 were generated, and the activation status of signaling factors involved in JAK/STAT, RAS, and mTORC1 pathways was assessed via western blot. (D) Saturation mutagenesis scan was performed by targeting coding exons of indicated genes. TVA1 cells, which harbor the ETV6-ABL1 fusion, served as controls. Read counts from final and initial time points were normalized to nontargeting guides, and log2 fold-change in guide abundance was calculated.