CRISPRi/CAR-T screen identifies genes modulating response to BCMA-targeted CAR-T cells. (A-B) CRISPRi AMO1 cells expressing BFP-sgRNA targeted toward BCMA or nontargeting control sgRNA were cocultured at a 1:1 ratio with BCMA– or CD19–GFP CAR-T cells. Fold changes in T-cell activation was determined by analyzing for cell surface expression of CD69 on T cells normalized to CD69 expression on resting T cells. Myeloma cell viability was determined by propidium iodide staining of BFP-positive myeloma cells. (C) Schematic representation of the CRISPRi/CAR-T cell coculture screen. (D) Comparison of BCMA expression phenotype to sensitivity toward BCMA-targeted CAR-T cells indicating different sgRNAs targeted toward BCMA. (E) Volcano plot indicating CAR-T cell sensitivity phenotypes and statistical significance of knockdown of human genes (orange dots) and quasi-genes from negative control sgRNAs (gray dots) in MM cells. Hit genes corresponding to functional categories are color-coded as labeled in the panel. (F) Comparison of BCMA expression phenotype from the CRISPRi primary screen (Figure 1) to sensitivity toward BCMA-targeted CAR T cells. Hit genes corresponding to functional categories are color-coded as labeled in the panel. (G) CRISPRi AMO1 cells expressing 2 independent sgRNAs targeting ICAM1 or nontargeting control sgRNA were cocultured overnight at a 1:1 ratio with BCMA-targeted CAR-T cells. Frequency of viable myeloma cells was determined by using flow cytometry.