Abstract LB289: Single-cell CRISPR screens at scale to understand T cell biology

Abstract CRISPR screens have become the primary discovery engine in modern biology and are widely used to uncover novel targets in immuno-oncology. However, these genetic screens are usually coupled to rather simplistic read-outs such as cell fitness. In contrast, CROP-Seq (or “CRISPR Droplet” Sequencing) screens combine CRISPR perturbation with single-cell transcriptomics, enabling high-content phenotyping at single-cell resolution. Briefly, cells are perturbed with a pooled sgRNA library and transcriptomic profiles of each cell are recorded using conventional single-cell RNA sequencing platforms. Currently, the scale of these screens is limited to the perturbation of a couple of hundred genes, possibly up to 1. 000 genes, because of increased costs for single-cell library preparation and next generation sequencing (NGS). At Myllia, we have built a platform allowing high-content CROP-Seq screens in both CRISPR-ready cancer cell lines as well as primary immune cells, e. g. , human T cells. Using CRISPR KO, CRISPR interference or CRISPR activation workflows, we can help accelerate the identification of target genes and regulatory networks involved in immuno-oncology. Primary T cells are of great interest in the immunotherapy community as they are key players in autoimmune and inflammatory disease. Engineered T-lymphocytes such as CAR-T cells are currently developed as novel cellular medicines, yet many T cell-intrinsic features involved in CAR-T potency remain elusive. To enable the discovery of novel targets, we have performed multiple customized CROP-Seq CRISPR KO screens in primary human T cells investigating T cell stemness and effector phenotypes as well as CD4+ T helper cell differentiation towards Th1, Th2, or Th17 subsets. Here, we present screens for regulators of functional T cell plasticity and Th2 commitment in which we aimed to identify genes determining critical T cell fate decisions, potentially linking gene (dys) function to either autoimmune/inflammatory disease or functional persistence of cytotoxic T cells for cancer immunotherapy. Overall, the advanced CROP-Seq platform combining large-scale perturbations and targeted sequencing (TA) read-outs will catalyse a paradigm shift for CROP-Seq enabling high-throughput functional genomic screens that support the validation of drug targets in autoimmunity, inflammation and immuno-oncology. Citation Format: Nicole Untermoser, Johanna Irnstorfer, Anatoly Vasilyev, Nikola Vinko, Sumit Pawar, Anke Loregger, Adam Krejci, Henrik Schmidt, Tilmann Bürckstümmer. Single-cell CRISPR screens at scale to understand T cell biology abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (7Suppl): Abstract nr LB289.