W177 - Single-cell CRISPR Screens in Primary Human T Cells Identify Regulators of th2 Cell Skewing

Anke Loregger – Myllia Biotechnology GmbH; Johanna Irnstorfer – Myllia Biotechnology GmbH; Nicole Untermoser – Myllia Biotechnology GmbH; Nikola Vinko – Myllia Biotechnology GmbH; Adam Krejci – Myllia Biotechnology GmbH; Tilmann Bürckstümmer – Myllia Biotechnology GmbH

Abstract Text: CRISPR screens have become the primary discovery engine in modern biology. At Myllia, we combine CRISPR screening with single-cell RNA sequencing in primary T cells, leveraging two transformative technologies to enable genetic screening for complex phenotypes. We utilize pooled CRISPR screens to map the impact of thousands of genetic perturbations at single-cell resolution which allows to identify novel drug targets or to elucidate unknown mechanisms of actions of drugs.

Primary human T cells are key players in autoimmunity and other inflammatory diseases, but also represent attractive targets for immunotherapy of cancer. To enable the discovery of novel targets, we built a workflow that utilizes CD4+ T cells from peripheral blood and allows functional genomic screens. Upon activation, naïve CD4+ T cells proliferate and differentiate into specific Th1, Th2, or Th17 cell subsets. Here, we present data of a pooled CRISPR screen for regulators of T cell fates in which we aimed to identify genes whose knockout boosts or attenuates the ability of primary naïve CD4+ T cells to become Th2 cells.

In our screen, the different T cell subsets could be captured using curated transcriptomic signatures. Importantly, several gene KOs introduced in a pooled fashion using CRISPR/Cas9 accumulated in distinct subpopulations, suggesting that these genes regulate the differentiation of naïve T cells into the various T helper cell subsets.

Overall, our pooled CRISPR screening platform enables to decipher primary T cell plasticity and identifies genes that could serve as drug targets in autoimmunity, inflammation and immuno-oncology.