title: A Genome-wide CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Screen Identifies NEK7 as an Essential Component of NLRP3 Inflammasome Activation.
abstract: Inflammasomes are high molecular weight protein complexes that assemble in the cytosol upon pathogen encounter. This results in caspase-1-dependent pro-inflammatory cytokine maturation, as well as a special type of cell death, known as pyroptosis. The Nlrp3 inflammasome plays a pivotal role in pathogen defense, but at the same time, its activity has also been implicated in many common sterile inflammatory conditions. To this effect, several studies have identified Nlrp3 inflammasome engagement in a number of common human diseases such as atherosclerosis, type 2 diabetes, Alzheimer disease, or gout. Although it has been shown that known Nlrp3 stimuli converge on potassium ion efflux upstream of Nlrp3 activation, the exact molecular mechanism of Nlrp3 activation remains elusive. Here, we describe 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. Using a genome-wide guide RNA (gRNA) library, we found that targeting Nek7 rescued macrophages from nigericin-induced lethality. Subsequent studies revealed that murine macrophages deficient in Nek7 displayed a largely blunted Nlrp3 inflammasome response, whereas Aim2-mediated inflammasome activation proved to be fully intact. Although the mechanism of Nek7 functioning upstream of Nlrp3 yet remains elusive, these studies provide a first genetic handle of a component that specifically functions upstream of Nlrp3.
authors.affiliation: From the Institute of Molecular Medicine and.
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: Schmid-Burgk
authors.affiliation: From the Institute of Molecular Medicine and.
authors.forename: Dhruv
authors.initials: D
authors.lastname: Chauhan
authors.affiliation: From the Institute of Molecular Medicine and.
authors.forename: Tobias
authors.initials: T
authors.lastname: Schmidt
authors.affiliation: From the Institute of Molecular Medicine and.
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Ebert
authors.affiliation: Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital, University of Bonn, 53127 Bonn and.
authors.forename: Julia
authors.initials: J
authors.lastname: Reinhardt
authors.affiliation: From the Institute of Molecular Medicine and.
authors.forename: Elmar
authors.initials: E
authors.lastname: Endl
authors.affiliation: From the Institute of Molecular Medicine and the Gene Center and Department of Biochemistry, Ludwig-Maximilians-University Munich, 81377 Munich, Germany hornung@genzentrum.lmu.de.
authors.forename: Veit
authors.initials: V
authors.lastname: Hornung
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D016678:Genome; D057809:HEK293 Cells; D006801:Humans; D058847:Inflammasomes / Q000378:metabolism*; D051379:Mice; D000072379:NIMA-Related Kinases; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D002352:Carrier Proteins; D058847:Inflammasomes; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C492716:Nlrp3 protein, mouse; D000072379:NIMA-Related Kinases; C512686:Nek7 protein, mouse; D017346:Protein Serine-Threonine Kinases
keywords: NEK7; NLRP3; caspase 1 (CASP1); cell death; genetic screen; inflammasome; inflammation; pyroptosis