Abstract

CRISPR screens have become the primary discovery engine in modern biology. At Myllia, 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 the scientific community. They are not only key players in autoimmunity, but also represent attractive targets for immunotherapy of cancer. To enable the discovery of novel targets in immunology and immunotherapy, we built a workflow that utilizes T cells from peripheral blood. Here, we present a first proof-of-concept experiment in which we screened for regulators of T cell differentiation. Different stages of cellular differentiation could be captured using transcriptomic signatures. Importantly, several gene KOs introduced in a pooled fashion using CRISPR/Cas9 accumulated in distinct subpopulations, suggesting that these genes regulate T cell differentiation. We also compare two screening paradigms, namely screens conducted with the Cas9 nuclease as well as dead Cas9 fused to KRAB (CRISPR interference). To ensure comparability, we perturbed the same set of genes and analyzed downstream consequences by targeted sequencing focusing on the identical marker set. Comparing those two experiments reveals more perturbation/ marker pairs when the Cas9 nuclease is used for perturbation, suggesting that CRISPRi-induced phenotypes are less penetrant. Of note, there is considerable overlap of the gene knockout/ marker pairs revealed with both technologies, suggesting that both technologies can uncover true genotype-phenotype relationships.