Abstract A026: Deciphering natural and synthetic genetic programs governing human T cell function with combinatorial CRISPR activation and interference screens

Abstract Adoptive immune cell therapies, such as CAR T, have revolutionized how we can approach cancer therapy, however challenges remain to increase their efficacy, response rates, and resistance to dysfunction. The systematic investigation of gene programs governing human T cell function would be advantageous for reprogramming cell products for clinical benefit. Recent studies have demonstrated the power of CRISPR-based forward genetic screens in primary human T cells to nominate candidate perturbations for enhanced T cell function, however combinations of perturbations will likely be necessary to overcome the most challenging tumors. To enable the systematic and high-throughput interrogation of combinations of gain and loss-of-function perturbations, we have developed a bidirectional CRISPR activation and interference (CRISPRai) system, where pairs of genes can be simultaneously activated and repressed in primary human T cells. Leveraging hits from previous genome-wide CRISPRa and CRISPRi screens, we constructed a library of >66,000 sgRNA combinations and screened for regulators of acute T cell stimulation responses, including cytokine production and proliferation. Reassuringly, these screens identified expected gene epistasis interactions (e.g. rescue of PLCG1 repression by PLCG2 activation), while also discovering novel genetic interactions, providing mechanistic insights into the regulatory networks governing stimulation response. To further identify gene “building blocks” for evaluation as combinations, we completed genome-wide CRISPRa and CRISPRi screens for regulators of human CD8+ CAR T cell exhaustion. These screens identified known potential enhancements (e.g. gain of BATF, loss of MED12), and also uncovered novel regulators of T cell function, with CRISPRa discovering genes not normally expressed in T cells but whose overexpression could cause resistance to exhaustion by synthetic mechanisms. Gene expression programs controlled by perturbation of key regulators were mapped by pairing CRISPR with single-cell RNA-seq (Perturb-seq) and arrayed functional assays. Finally, a top gain-of-function candidate was evaluated for in vivo tumor control, using an antigen-inducible Synthetic Notch system. Together, this work demonstrates how advanced forward genetics approaches can discover and elucidate both natural and synthetic programs that regulate T cell states, and the nomination of perturbations to encode in logic-gated synthetic biology designs for cell therapies. Citation Format: Zachary Steinhart, Jeffrey Perera, Dmytro Dorovskyi, Luke Workley, Carinna Tran, Rosmely Hernandez, Zhongmei Li, Max Foisey, Daniel Goodman, Kole Roybal, Brian Shy, Alexander Marson. Deciphering natural and synthetic genetic programs governing human T cell function with combinatorial CRISPR activation and interference screens [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A026.