High-Throughput CRISPR Screening Identifies Genes Involved in Macrophage Viability and Inflammatory Pathways

Macrophages are critical effector cells of the immune system, and understanding genes involved in their viability and function is essential for gaining insights into immune system dysregulation during disease. We use a high-throughput, pooled-based CRISPR-Cas screening approach to identify essential genes required for macrophage viability. In addition, we target 3′ UTRs to gain insights into previously unidentified cis-regulatory regions that control these essential genes. Next, using our recently generated nuclear factor κB (NF-κB) reporter line, we perform a fluorescence-activated cell sorting (FACS)-based high-throughput genetic screen and discover a number of previously unidentified positive and negative regulators of the NF-κB pathway. We unravel complexities of the TNF signaling cascade, showing that it can function in an autocrine manner in macrophages to negatively regulate the pathway. Utilizing a single complex library design, we are capable of interrogating various aspects of macrophage biology, thus generating a resource for future studies. [Display omitted] •CRISPR screens to identify genes required for macrophage viability and function•Identification of cis-regulatory elements that control essential genes•TNF can function as a negative regulator of inflammation in a cell-intrinsic manner Covarrubias et al. screen ~21,000 targets, generating a resource guide of genes required for macrophage viability as well as previously unidentified positive and negative regulators of NF-κB signaling. They identify regulatory elements within essential genes and show that membrane-bound TNF primarily functions in macrophages in an autocrine fashion to negatively regulate inflammation.