ABSTRACT Chimeric Antigen Receptor (CAR) T cell therapy has proven to be a breakthrough treatment with curative potential in hematologic cancer patients as well as in aggressive preclinical models. However, recent studies have shed light on major barriers to progress – many patients that initially respond completely to CAR T cell therapy eventually relapse, and CAR T cells have demonstrated limited clinical efficacy in the treatment of solid tumors. A key phenomenon that has been causally implicated in these failure modes is CAR T cell exhaustion, where tonically-signaling CAR T cells are driven to a distinct and dysfunctional phenotype with restrained antitumor activity. Previous genome-wide perturbation studies using CRISPR-Cas9 have identified a growing list of single gene targets that, when knocked out, help mitigate exhaustion and modestly improve T cell function. However, the resulting individual gene hits from these screens are often context-dependent and disparate across different tumor and CAR models. Altogether, these studies indicate that the exhausted T cell phenotype is largely driven by the upregulation of key gene programs rather than single genes, though the complex network of these genetic interactions remains poorly defined. To address these unmet needs, we propose a synthetic biology-driven approach using CRISPR-Cas13d transcriptome engineering to develop potent, exhaustion-resistant CAR T cells. Cas13d is a small CRISPR RNA- guided RNA endonuclease that can process a single guide RNA array to degrade multiple distinct target RNA transcripts in a highly sequence-specific and robust manner. In AIM 1, we will develop a novel platform using Cas13d to simultaneously downregulate multiple endogenous genes in primary human T cells, with a specific focus on improving exhausted CAR T cell effector function. In AIM 2, we will use this technology to conduct a double knockdown proliferation screen in exhausted CAR T cells targeting pairs of putative negative regulators of T cell antitumor activity. We will utilize an established computational framework to identify highly enriched gene pairings, to map genetic interactions (GI) between genes, and to define a network of exhaustion. We hypothesize that our multimodal screening methodology can be used to identify new synergistic gene pairings that outperform single knockdown phenotypes seen in prior studies. Our proposed project will establish a new platform for multiplexed gene repression and screening in primary human T cells that overcomes limitations faced by state-of-the-art CRISPR-Cas9 and RNAi technologies. Furthermore, our studies will demonstrate a novel strategy to mitigate CAR T cell exhaustion, which will improve upon current immune therapies and enhance their effectiveness. Ultimately, our work will address clinical unmet needs as well as help the broader scientific community 1) better understand the complex network of T cell exhaustion and 2) use this data to inform the developm...