FIGURE 1.

A CRISPR loss-of-function screen identifies Nek7 as a component involved in Nlrp3 signaling. A, Nlrp3-Cas9 macrophages were transduced with a control gRNA or a gRNA targeting Nlrp3, which additionally encoded for GFP at an MOI of 0.01. Following stable transduction, macrophages were either stimulated with nigericin for 5 h or left untreated. Subsequently, cells were labeled with PI and subjected to FACS analysis. Depicted are schematic views of the transduction and stimulation modalities as well as FACS plots of a representative result. Highlighted are the frequencies of cells in the stringent live gate that contains GFP-positive and PI-negative cells. Data are representative of three independent experiments with comparable results. B, schematic view of the genome-wide screening approach that was undertaken to identify factors that confer resistance to nigericin-induced cell death. Please see “Experimental Procedures” for details. Ctrl., control. C, dot plot representation of the screening results. Each dot represents the frequency of cells carrying a specific gRNA that were found in the live gate of mock-treated cells (x axis) or of nigericin-treated cells (y axis). Highlighted are five outlier gRNAs targeting Nlrp3 (x2), Nek7, Fam83c, or Cdkn2a. D, depicted are the gRNA sequences found in the outlier population. E, Nlrp3-Cas9 macrophages were transduced with the same gRNAs identified in the screen and a control gRNA targeting Emc3. Subsequently, cells were subjected to nigericin stimulation, and cell survival was analyzed by FACS. The relative survival benefit was calculated by dividing the gated cell fraction from the stimulated condition by that from the unstimulated condition and is depicted as a mean value ± S.E. of three independent experiments. F, two alternative gRNAs targeting Nek7 and Fam83c were used to transduce Nlrp3-Cas9 macrophages. Nigericin stimulation and subsequent cell survival were analyzed as in E.