A Genome-wide CRISPR Screen Identifies NEK7 as an Essential Component of NLRP3 Inflammasome Activation

The Journal of Biological Chemistry, 291, 103-109.

The mechanisms of NLRP3 activation are still poorly understood. Jonathan Schmid-Burgk and colleagues present new data on the identification of NEK7, which specifically functions upstream of NLRP3 activation. NEK7 was identified in an unbiased genetic screening approach, which employed the CRISPR technology to identify macrophages that were rendered defective in NLRP3 signal transduction.

The Flow Cytometry Core Facility supported the project by providing an optimized workflow for the separation of live cells on a single cell basis.

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.

Similar Posts