Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of B cell malignancies, and extending this success to other cancers remains an urgent clinical objective. CRISPR activation (CRISPRa) is a gene nomination platform with the potential to direct next-generation cell product development, but genome-wide surveys using this technology in primary T cells have been limited by prohibitive cell requirements and short-lived activity. Here, we describe a CAR-restricted T cell expansion platform generating yields of CRISPRa-CAR T cells far exceeding those required for genome-scale screening (∼109). CRISPRa activity is sustained by incorporating divergent, duplicate elongation factor 1α core/human T cell leukemia virus hybrid promoters and by delivering guide RNAs late by lentiviral transduction to avoid losing perturbations that compromise early T cell activation or expansion. Manufactured CRISPRa-CAR T cells maintain potent on-target gene overexpression (>85% target+) across varied cell subsets, including those associated with T cell exhaustion, and over extended culture periods. When applied to survival-based genome-wide screens, CRISPRa-CAR T cell pools nominate endogenous genes that enhance CAR T cell persistence across varied cytokine levels in the presence of tumor (e.g., IRF4, GFI1, and GSE1). In a proof-of-concept study, we find that the survival effect of GSE1 can be preserved using a clinically relevant transgene overexpression strategy. More broadly, this screening framework establishes a foundation for systematic identification and downstream validation of genes that enhance cellular therapies.
