Fig. 4: NIS-Seq perturbation screening in primary human macrophages.

a, Lentiviral vector co-expressing an sgRNA and C1C–EGFP for monitoring inflammasome activation (top) and a VPX–VPR expression plasmid for efficient lentiviral transduction of primary human macrophages (bottom). b, Outline of NIS-Seq perturbation screening in primary human macrophages. c, EGFP expression in primary human M-CSF macrophages transduced with CROPseq-iT7 C1C–EGFP and analyzed by FACS. FSC, forward scatter. d, Confocal imaging of C1C–EGFP localization in primary human macrophages. Cells were primed with 20 ng ml⁻¹ LPS for 3 h and activated after 30 min of caspase inhibition (40 µM VX-765 and 50 µM Z-VAD) by 1 h of stimulation with 1 µg ml⁻¹ PA and 10 ng ml⁻¹ PrgI. Nuclei were stained with Hoechst 33342 (blue). Scale bars, 100 µm. Representative data from two experimental replicates are shown. e, Image analysis results of FACS-sorted EGFP⁺ cells stimulated as in c. f, Genome editing efficiencies in primary human macrophages transduced with the lentiviral vector depicted in a and electroporated with SpCas9 protein. Genomic editing rates were quantified by NGS in cells from two healthy donors. g, Reporter activation and four cycles of NIS-Seq in primary human macrophages after stimulation as described in d. Highlighted are two cells assigned to either a non-targeting control or an ANTXR2-targeting sgRNA by NIS-Seq. Scale bars, 50 μm. Representative data from two biological replicates are shown. h, Pooled optical perturbation screen in primary human macrophages. Shown are relative fractions of cells with blunted reporter activation for each of five perturbed genes targeted by four sgRNAs each or eight non-targeting control sgRNAs. Data points indicate results from two independent screening wells, each containing a mixture of macrophages from two healthy donors. In total, 2,663 macrophage cells were mapped to both a library sgRNA and a phenotype. i, Arrayed validation using alternative sgRNAs delivered as RNP complexes to primary human macrophages. Knockout rates were determined by sequencing (blue bars), and PrgI+PA-induced ASC specking rates were determined by anti-ASC antibody staining (red bars). Data from four biological replicates based on different human donors are shown. **P < 0.01; NS P > 0.05; two-sided t-test. NS, not significant.