Fig. 3

A co-culture CRISPRi screen identified regulators of B7-H3 CAR T cell-mediated cytotoxicity in GBM cells. A The construct for expressing the CRISPRi machinery from the CLYBL safe-harbor locus: catalytically dead Cas9 (dCas9) fused to a blue fluorescent protein (BFP) and the KRAB domain, under the control of the constitutive CAG promoter. B Functional validation of CRISPRi activity in the CRISPRi-U87 cells via qPCR with sgRNAs targeting STAT1, TFRC and IFNAR. A non-targeting sgRNA was used as the control. C Killing effect of B7-H3 and CD19 CAR T cells against U87 MG cells at an E: T ratio of 1:2 or 1:4 after 1-, 2- or 3-day co-culture. D Schematic of the co-culture CRISPRi screen. CRISPRi-U87 cells were transduced with the H1 sgRNA library and co-cultured with CD19 or B7-H3 targeting CAR T cells at an E: T ratio of 1:4 for 36 h, followed by a 48-hour recovery. Frequencies of CRISPRi-U87 cells expressing a given sgRNA were determined in each population by next-generation sequencing. The screens were performed in parallel with CAR T cells derived from 2 donors as biological replicates. E Correlations of sgRNA counts between two donors of B7-H3 (left) and CD19 (right) CAR T screening groups. F Volcano plots summarizing phenotypes and statistical significances of gene perturbations in the screen as determined by the MAGeCK-iNC pipeline. Dashed line indicates the cut-off for hit genes (false discovery rate (FDR) = 0.1). Dots in blue or red represent hit genes whose knockdown enhanced or suppressed CAR T cytotoxicity, respectively. Non-hit genes are shown in orange and negative controls are shown in gray. Gene symbols of the top ten negative hits and the top ten positive hits are shown. G Enrichment analysis of the negative screening hits against gene sets in the Human Molecular Signatures Database (MSigDB). Enriched terms with adjusted P values less than 0.05 are shown. H Screening hits grouped by their biological function. Gene symbols in blue and red represent genes whose knockdown in U87 MG cells represses and enhances CAR T cytotoxicity, respectively