Genome-wide CRISPR Identification of Regulators Governing the NLRP3 Inflammasome 2331537

Abstract Introduction Dysregulated NLRP3 inflammasome activation drives chronic inflammation in autoimmune and autoinflammatory diseases. Sustained NLRP3-dependent IL-1β production promotes tissue damage, yet current therapies rely on broad immunosuppression or cytokine blockade that can impair host defense and provide incomplete disease control. Although direct NLRP3 inhibitors are emerging, their utility is limited by toxicity and an incomplete understanding of inflammasome regulation. Defining genetic networks that tune inflammasome activation is therefore critical for identifying therapeutic targets that selectively limit pathological inflammation while preserving protective innate immune responses. Methods We conducted a genome-wide CRISPR—Cas9 KO screen in human THP-1 macrophages stably expressing ASC-GFP to identify genetic regulators of NLRP3 inflammasome activation. After inflammasome activation, cells are sorted by flow cytometry based on ASC speck formation. Candidate genes were prioritized using enrichment statistics and pathway analysis. Top hits were validated using independent sgRNAs, with readouts including IL-1β ELISA and imaging-based ASC speck quantification. NF-κB phosphorylation was assessed to exclude false-positive regulators affecting inflammasome priming rather than activation. Results The screen robustly identified known NLRP3 pathway components, validating screen performance, and revealed multiple previously uncharacterized regulators spanning cytoskeletal dynamics, mitochondrial homeostasis, and ubiquitin signaling. Genetic perturbation of top candidates significantly altered IL-1β release without affecting NF-κB priming. Conclusion Our study defines a comprehensive genetic landscape governing human NLRP3 inflammasome activation and uncovers novel regulatory nodes that decouple inflammasome assembly from upstream priming. These findings provide mechanistic insight into innate immune regulation and nominate new targets for selective therapeutic modulation of inflammasome-driven disease. Funding Source n/a Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)