Whole-genome CRISPR depletion screen identifies pathways differentially required for the growth of mutant CALR-transformed BA/F3-MPL cells, validated in a secondary CRISPR pooled screen. GSEA on the whole-genome CRISPR screen showing that genes in the N-glycan biosynthesis pathway (A), the protein secretion pathway (B), as well as the UPR pathway (C) are differentially depleted in CALR^Δ52 −IL3 cells as compared with EV plus IL3 cells. GSEA ranking for the pathways indicated was performed with the genes rank-ordered based on fold change. Genes that are more depleted in EV +IL3 condition are represented on the left and genes that are more depleted in CALR^Δ52 −IL3 condition are represented on the right. The comparison for CALR^Δ52 −IL3 vs CALR^Δ52 +IL3 is shown on the right side of each panel. (D-F) Results of the CRISPR pooled screen for validation of the whole-genome CRISPR screen. (D) Gene ranking, comparing CALR^Δ52 −IL3 with empty vector +IL3. The 10 most differentially depleted genes for CALR^Δ52 (as compared with EV +IL3 cells) are shown, ranked by corrected P values. Genes involved in protein glycosylation are highlighted in dark blue. (E) Volcano plot depicting significance and fold change of depleted genes, separated by the conditions stated, highlighting Dpm2 and important control genes. Log₂ fold change threshold = ±1. FDR adjusted P threshold = .05. (F) Venn diagram depicting the significantly depleted genes, comparing overlapping hits in the comparisons stated. FDR, false discovery rate; neg. LFC, negative log₂ fold change; NES, normalized enrichment score.