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

Abstract

SUMMARY 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.

Transcriptional regulatory networks of circulating immune cells in type 1 diabetes: A community knowledgebase

“…We reasoned that if our analysis to this point were valid, ST1DDs would be enriched for known regulators of macrophage activation. To investigate this in a more statistically systematic manner, we sourced a recent genome-wide CRISPR screen by Covarrubias and colleagues that identified genes with essential roles in macrophage viability ( Table S18 ) ( Covarrubias et al., 2020 ). This study assigned Mann-Whitney (MW) scores to 21,379 mouse genes, such that the gene with the lowest MW score was designated the most essential for macrophage viability.…”

What sequencing technologies can teach us about innate immunity*

“…In addition to the inflammatory screen, we performed a viability screen which identified all genes required for viability including macrophage-specific viability genes such as IRF8. 167 We also provided insights into new regulatory elements present in the 3′UTRs of essential genes. We made use of our recently developed NF-kB-GFP reporter system 61 and identified 115 novel regulators of NF-kB as well as showing that TNF can act as a negative regulator of the pathway in a cell intrinsic manner.…”

“…We made use of our recently developed NF-kB-GFP reporter system 61 and identified 115 novel regulators of NF-kB as well as showing that TNF can act as a negative regulator of the pathway in a cell intrinsic manner. 167 The majority of the pooledbased screens have been performed in cell lines, but the technology is now being utilized to move toward in vivo screening as well as screening primary human cells. Lafleur et al, have developed CHIME:…”

High-content CRISPR screening in tumor immunology

“…These factors include the previously uncharacterized oligosaccharyltransferase (OST) complex and PAF transcription complex ( 86 ). Likewise, screens in immortalized and primary murine macrophages have similarly uncovered regulators of LPS-induced pathways ( 52 , 87 ). For instance, Mettl3 -mediated m 6 A RNA modification has been found to positively regulate LPS-induced macrophage activation to control tumor growth ( 87 ).…”

“…As a result, lentiviral vectors serve as the most efficient and well-known means to stably introduce the CRISPR system into target cells. In the case of pooled CRISPR screens, a low multiplicity of infection (MOI) (~0.3) is often used to ensure that each cell is infected with only one virus, and thus only contains one perturbation (50)(51)(52). On the other hand, a high MOI may be used to induce multiple perturbations per cell for the study of genetic interactions (53,54).…”