Use of CRISPR/Cas9 screening to study intrinsic tumor resistance mechanisms to CAR T cells. (A) Schematic showing the CRISPR/Cas9 screening process. (B) The 10 genes with the most significant sgRNA enrichment. (C) Log₂ fold change of normalized counts of each sgRNA targeting CD58, CD81, ICAM1, or ITGA4 in the screening. (D) Efficacy of CD58, CD81, ICAM1, or ITGA4 KO in Nalm6 cells. (E-H) GFP-labeled indicated KO cells and mCherry-labeled WT cells were mixed at a ∼1:1 ratio and cocultured with control T or CAR T cells. The KO (GFP⁺)/WT (mCherry⁺) ratio was calculated over time. CD58^KO/WT Nalm6 ratio (E), CD81^KO/WT Nalm6 ratio (F), ICAM1^KO/WT Nalm6 raito (G), and ITGA4^KO/WT Nalm6 ratio (H) in growth competition assay. KO cell lines referred to sgRNA-1 targeting the indicated genes in panels E-H. A mixture of cells was cocultured with control T or CD19 CAR T cells at a 1:20 E:T ratio (n = 3). Statistical comparisons were performed using a 2-way ANOVA test with multiple comparisons. The values are shown as the means plus or minus SD. ∗P < .05; ∗∗P < .01; ∗∗∗ P < .001. ns, not significant (P > .05); RRA, Robust Rank Aggregation.