Genome-wide CRISPR/Cas9-based LOF screen identifies genetic dependencies of STAT3^Y640F-driven cells. (A) Workflow of a genome-wide LOF CRISPR/Cas9 screen. Clonal Cas9-expressing HPC7 cells (empty vector or STAT3^Y640F) were transduced with the Vienna sgRNA library at low multiplicity of infection (MOI) to ensure single integration. After equal population duplications, cells were harvested and genomic DNA extracted. After library preparation cells were subjected to next-generation sequencing (NGS) to compare sgRNA abundances. (B) Comparative analysis of 2 CRISPR-based LOF screens in empty vector vs STAT3^Y640F-driven HPC7 progenitor cells. Gene effects were depicted with respect to effect size of defined core-essential and nonessential genes. Depletion of core-essential genes (left) and normalized STAT3^Y640F- and empty vector–specific dependencies (right) have been depicted. Dependencies defined as less than –0.7 normLog2FC compared with the plasmid pool and a difference of at least 0.5 normLog2FC. (C) Comparison of gene ontology analyses (Biological Process 2021) (https://maayanlab.cloud/Enrichr/^25-27) of selective genetic dependencies of STAT3^Y640F (red) and selective dependencies of empty vector screen (blue). FC, fold change; NA, not applicable; sgRNA, single guide RNA.