Fig. 1: Genome-wide CRISPR/Cas9 screen identifies Cbfb as a regulator of tumour sensitivity to T cell derived TNF.

A Schematic of CAR-T cell CRISPR/Cas9 screen. T cells from C57BL/6 mice were activated and retrovirally transduced to express CARs targeting hHER2 and cultured with MC38 tumour cells engineered to express hHER2, Cas9 and a genome-wide sgRNA library. Enriched guides were sequenced following 3 rounds of selection. B Lysis of ⁵¹Cr-labelled parental or hHER2-expressing MC38 tumour cells by hHER2-directed CAR-T cells in an 18 h co-culture, measured by ⁵¹Cr at increasing CAR-T to tumour cell ratios, representative plot of n = 3. C, D Enriched sgRNA from screen in A. C Representative plot of duplicate screens. D Shared sgRNA enrichment from each replicate screen. E GO term analysis showing enriched biological processes for top screen hits. F Schematic of CRISPR/Cas9-mediated genetic deletion of Cbfb in tumour cells by electroporation with ribonucleoprotein of Cas9 nuclease complexed to sgRNA targeting Cbfb (sgCbfb), or a non-targeting control (sgNT), and immunoblot validation of CBFβ deletion in MC38 cells. G Lysis of ⁵¹Cr-labelled MC38-hHER2 tumour cells by hHER2-directed CAR-T cells in a 16 h co-culture, measured by ⁵¹Cr release at increasing CAR-T to tumour cell ratios. Relative lysis is calculated as the efficiency of CAR-T cells to achieve an equal percent lysis of tumour cells, unpaired t-test, n = 5. H Shared sgRNA enrichment from screen in A and a screen for MC38-OVA resistance to OT-I T cell killing [5]. I Lysis of ⁵¹Cr-labelled MC38 tumour cells in increasing concentrations of TNF over 16 h. Relative lysis is calculated as the efficiency of TNF to achieve an equal percent lysis of tumour cells, unpaired t test, n = 3. J Representative flow cytometry plots of MC38 tumour cells with death measured by PI uptake following 16 h of treatment with 10 ng/mL TNF (left), n = 4 quantified 2way ANOVA. K Schematic of competition assay to assess relative resistance of MC38 cells to CAR-T cells or TNF. MC38-hHER2-sgNT control cells were labelled with E2-Crimson and co-cultured with unlabelled MC38-hHER2-sgCbfb cells, followed by the addition of CAR-T cells (at a CAR-T to tumour cell ration of 2:1) or 10 ng/mL TNF for 16 h. L Representative flow cytometry plots (left) and n = 3 quantification of competition assay (right). All error bars show +/−SEM, ***P < 0.001, ****P < 0.0001.