Description of a genome-wide CRISPR/Cas9 screen in immortalized mouse macrophages aiming at the unbiased identification of gene products involved in Nlrp3 inflammasome activation. We employed a FACS-based screen for Nlrp3-dependent cell death, using the ionophoric compound nigericin as a potassium efflux-inducing stimulus.
FACS-based screening strategy to identify components upstream of Nlrp3 inflammasome activation. A, under steady state conditions Nlrp3 inflammasome activation requires a priming signal (signal 1) that upregulates Nlrp3 expression (left panel). To overcome this requirement, cells stably expressing Nlrp3 were used (right panel). These cells directly respond to nigericin (signal 2) without a previous priming signal. B, Following transduction with a gRNA (additionally encoding for GFP), cells are stimulated with nigericin or left untreated. Cells are then stained with PI and subjected to FACS analysis. Transduced cells that are alive are GFP positive and PI negative, whereas dead cells lose their GFP positivity, while acquiring a PI signal.