Genome-wide CRISPR/Cas9 screening with InO in the NALM-6 cell line (passage 10). (A) Volcano plots showing InO sensitization (negatively selected, in blue) and resistance (positively selected, in red) genes by CRISPR/Cas9 screening. (B) GSEA of negatively enriched pathways. Magma colors and size of the dots represent the normalized enrichment score (NES) and −log₁₀ false discovery rate (FDR) q-values of the top 20 negatively enriched pathways, respectively. The respective gene set members of each pathway are denoted by a bubble plot. Viridis colors and size of the dots represent running enrichment score and −log₁₀ of CRISPR/Cas9 P value respectively. (C) Log₂ FC values of sgRNAs targeting the top 10 InO resistance genes. Each gray line represents an individual sgRNA and the central dotted line represents the median log₂ FC of the sgRNAs (∼0). Each red line represents the sgRNA of the target gene. Multiple sgRNAs targeting each of the top 10 genes (including CD22) were significantly enriched by inotuzumab, showing an on-target gene knockout by CRISPR/Cas9. (D) DNTT RNA expression in paired patient samples compared with pre- and post-InO. The plot represents the mean ± standard error of the mean for each group. P = .03 by two-tailed paired t test. Samples belonging to the same patient were connected using lines. CPM, counts per million FC, fold change; GSEA, gene set enrichment analysis; sgRNAs, single-guide RNAs.