Precision CRISPR annotation of the functional enhancer landscape in primary human T cells

Precise modulation of T cell function through engineering the non-coding genome holds great promise for advancing next-generation immunotherapies. However, robust high-throughput approaches to annotate functional cis-regulatory elements (CRE) in human T cells remain limited. Here, we developed a simple and highly efficient CRISPR interference (CRISPRi) perturbation platform to systematically annotate CREs in human primary T cells. Using this platform, we identified novel CREs controlling <i>PDCD1</i> , <i>HAVCR2</i> , and <i>TBX21</i> expression. Combinatorial CRE perturbations revealed synergistic CRE pairs that fine-tune <i>PDCD1</i> and <i>HAVCR2</i> expression, while Cas9-indel-based mutagenesis pinpointed the critical nucleotides within each enhancer that are essential for their activity. Functional experiments demonstrated that CRE-edited <i>HAVCR2</i> outperformed conventional total gene knockout in enhancing CAR T cell anti-tumor efficacy. Moreover, CRE editing of <i>PDCD1</i> and <i>HAVCR2</i> repressed PD-1 and TIM-3 expression in human tumor-infiltrating lymphocyte CD8 T cells, highlighting regulatory role of these CREs in disease relevant exhausted T cells. Together, this approach offers a compact CRISPRi platform that enables high-throughput dissection of functionally relevant non-coding genomic regions in T cells, providing insights for mechanistic studies and precision genome engineering of advanced cellular therapies.