A genome-scale gain-of-function CRISPR screen identifies proline metabolism in T cells as a means to enhance CAR-T therapy

Chimeric antigen receptor (CAR)-T cell-based immunotherapy for cancer and immunological diseases has made great strides, but it still faces multiple hurdles. Finding the right molecular targets to engineer T cells toward a desired function has broad implications for the armamentarium of T cell-centered therapies. Here, we developed a dead-guide RNA (dgRNA)-based CRISPR activation screen in primary CD8+ T cells, and we identified gain-of-function (GOF) targets for CAR-T engineering. Targeted knock-in of a lead target, PRODH2, enhanced CAR-T-based killing and in vivo efficacy in multiple cancer models. Transcriptomics and metabolomics revealed that augmenting PRODH2 expression in the T cell re-shaped broad and distinct gene expression and metabolic programs. Mitochondrial, metabolic and immunological analyses showed that PRODH2 engineering enhances the metabolic and immune functions of CAR-T cells against cancer. Together these findings provide a system for identification of GOF immune boosters, and demonstrate PRODH2 as a target to enhance CAR-T efficacy.