Abstract

CRISPR screens have become the primary discovery engine in modern biology. However, many screening workflows are still performed in cancer cell lines and coupled to simplistic read-outs such as cellular fitness. At Myllia Biotechnology, we combine CRISPR screening with single-cell RNA sequencing, leveraging two transformative technologies to enable genetic screening for complex phenotypes. We utilize the CRISPR screening workflow to map the impact of thousands of genetic perturbations on the global transcriptome at single-cell resolution. Our powerful approach has broad applications in identifying novel drug targets or elucidating unknown mechanisms of actions of drugs. Primary human T cells are currently of great interest in thescientific community. They are not only 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 in these cells. Upon activation, naïve CD4+ T cells proliferate and differentiate into specific Thelper cell subsets, such as Th1, Th2, or Th17 cells. Here, we present data of an experiment in which we screened for regulators of T helper cell differentiation and skewed cells towards the Th2 subset. We aimed to identify genes whose knockout boosts or attenuates the ability of primary naïve CD4+ T cells to become Th2 cells. Th2 cells support the humoral immune response, and their dysfunction has been linked to inflammatory diseases, including asthma. 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 screening approach in primary human T cells allows for novel insights in the plasticity of T cells and identifies genes that could serve as drug targets in autoimmunity, inflammation and immuno-oncology.