Th146 - Pooled CRISPR Screening for Drug Target Discovery in Primary Human Myeloid Cells

Abstract Text: CRISPR screens have become the core discovery engine in modern biology and are widely used to uncover novel targets for immunotherapy. These genetic screens are usually performed in simplistic cell models and often coupled to readouts such as cell fitness. In contrast, Perturb-Seq or CROP-Seq screens combine CRISPR perturbations with single-cell transcriptomics, enabling information-rich phenotyping in physiologically relevant primary cell types. Thereby, cells are typically transduced with lentiviruses comprising a pooled sgRNA library and a Cas effector protein followed by measuring transcriptomic profiles of individual cells.

Macrophages are pivotal in the maintenance of tissue homeostasis and have been directly linked to a wide range of diseases, including infection, inflammation and cancer. This renders them attractive therapeutic targets, but their specific functions in pathogenesis often remain elusive. High-content CRISPR screens can help dissecting their diverse roles, yet the genetic manipulation of primary human macrophages – and especially the delivery of the CRISPR machinery by lentiviruses – has proven extremely challenging.

At Myllia, we have built a technology platform for CROP-Seq screens in primary human macrophages, which were previously inaccessible for genetic perturbation screens at scale. We developed a first-of-its-kind CROP-CITE-Seq screening workflow to assess macrophage identity as well as activation state by measuring transcriptomic and proteomic changes upon pooled perturbation of more than 50 target genes. Our results recapitulate the role of well-established mediators of immune signaling in macrophages. Ultimately, genetic screens in primary cells that reflect human disease biology will lay the foundations for successful drug discovery campaigns