I Schmidt1; H Ossia1; A Werner1; T Doll1; T Hoerter1; Z Yang1; G MacAllister1; J Reece-Hoyes1; C Russ1; M Mueller1; H Sparrer1; C Parker1;
1 Novartis AG, Switzerland
AbstractCRISPR-Cas9 technology offers an unprecedented opportunity to use phenotypic screens to identify the genetic nodes regulating the activity of a biological pathway. This report describes the application of this technology to screen for genes regulating nigericin induced pyroptosis mediated by the NLRP3 inflammasome in THP-1 cells (a human cell line, resembling macrophages). Inflammasomes are multiprotein complexes that sense danger or damage-associated molecular patterns, DAMPS, as part of the innate immune system. This recognition leads to the release of cytokines and other signaling molecules that can then lead to cell death. Typically the term inflammasome refers to the complex of proteins including PYCARD (ASC), NLRP3 (NLRC4 or AIM2) and pro-caspase-1. Upon activation by various DAMPS this multi-protein complex promotes activation of caspase-1, which then leads to a cascade of events which cause release of intercellular signals such as IL-1b and IL-18. These danger signals, as well as released intracellular components, can then further activate inflammasomes present in surrounding cells. In addition, the activation of the inflammasome and caspase-1 can also lead to cell death due to the activation of membrane pores such as Gasdermin D. Mutations in components of the inflammasome have demonstrated this as a key pathway regulating autoimmune diseases; e.g. cryopyrin-associated periodic syndrome (CAPS), pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND) and Familia Mediterranean Fever (FMF). A detailed understanding of the constituents of the inflammasome, and the pathway leading to its activation, will have utility in designing treatments for a range of diseases associated with inflammation. This report describes the development of an assay monitoring the induction of inflammasome mediated cell death (pyroptosis). Development of this assay allowed a genome-wide CRISPR-Cas9 screen to identify genes which regulate assembly and activation of the inflammasome. The assay utilized nigericin induction of the NLRP3 inflammasome mediated cell death in PMA differentiated THP-1 cells.The screen successfully identified known components of the inflammasome as well as a number of genes which have not been previously implicated in inflammasome induced cell death. The use of a genome-wide screen allowed a comprehensive evaluation of the pathways controlling inflammasome assembly and activation. The top 1000 genes were identified for the creation of a focused mini-pool library of potential targets. Retesting of this mini-pool of potential targets confirmed the activity of many of these genes as modulating the inflammasome. So a further selection of genes was then made and these genes were knocked out individually using CRISPR-Cas9.