Extended Data Fig. 3: Analysis and validation of knockout CAR-T cells.
From: In vivo CRISPR screens identify modifiers of CAR T cell function in myeloma
a. Proportions of each knockout cell type amongst clusters. b. Hallmark gene set enrichment analysis (GSEA) of pseudo-bulk pooled PTPN2 KO (top panel) or CDKN1B KO (bottom panel) CAR-T cells compared to no guide CAR-T cells. c. Construct design for the 4-1BB BCMA CAR (pCAR) and double-guide cassette (pGuide) containing individual gene sgRNAs (variable sgRNA): CDKN1B, IL2RA, PTPN2, and RASA2. d. Quantification of insertions/deletions (indels) in sgRNA target region by next generation sequencing. Data represents target sgRNA 1 and 2 for CDKN1B, PTPN2, and RASA2, respectively (n = 2 samples); presented as mean +/– SEM. e. IL2RA expression by flow cytometry, with IL2RA KO CAR-T cells (before electroporation/CD3 negative selection and after) compared to UTD and intergenic control KO (both IL-2 and IL-7/IL-15-produced, respectively). UTD = untransduced T cells.
