A versatile CRISPR/Cas13d platform for multiplexed transcriptomic regulation and metabolic engineering in primary human T cells

CRISPR technologies have begun to revolutionize T cell therapies; however, conventional CRISPR/Cas9 genome-editing tools are limited in their safety, efficacy, and scope. To address these challenges, we developed MEGA (Multiplexed Effector Guide Arrays), a platform for programmable and scalable regulation of the T cell transcriptome using the RNA-guided, RNA-targeting activity of CRISPR/Cas13d. MEGA enables quantitative, reversible, and massively-multiplexed gene knockdown in primary human T cells without targeting or cutting genomic DNA. Applying MEGA to a model of CAR T cell exhaustion, we robustly suppressed inhibitory receptor upregulation and uncovered paired regulators of T cell function through combinatorial CRISPR screening. We additionally implemented druggable regulation of MEGA to control CAR activation in a receptor-independent manner. Lastly, MEGA enabled multiplexed disruption of immunoregulatory metabolic pathways to enhance CAR T cell fitness and anti-tumor activity in vitro and in vivo. MEGA offers a versatile synthetic toolkit for applications in cancer immunotherapy and beyond. Overall design: Bulk RNA-seq of in vitro day 10 MEGA HA-28? CAR T cells (L vs NT; T vs NT; LT vs NT; GLY vs NT; 4KO vs AAVS1); Bulk RNA-seq of in vitro day 5 MEGA T cells (B2M vs NT)