Fig. 2: Genome-scale optical perturbation screening for mediators of NF-κB activation.

a, Genome-wide NIS-Seq perturbation screening in HeLa–Cas9–p65–mNeonGreen cells stimulated with IL-1β. Fold changes are calculated based on the mean pixel-wise Pearson correlation between mNeonGreen and nuclear staining signals across cells with the same targeted gene versus non-targeting (NT) control cells. Deviation of their correlation value distributions was tested using a two-sided Wilcoxon–Mann–Whitney test followed by the Benjamini–Hochberg procedure. b, Collages of cellular images from a mapped to perturbed genes indicated. Shown is the mNeonGreen signal. c, Arrayed hit validation in HeLa–Cas9–p65–mNeonGreen cells using alternative sgRNA sequences from the TKOv3. Top panel, exemplary mNeonGreen images of IL-1β-stimulated cells. Bottom panel, distribution of activation states, quantified by pixel-wise Pearson correlation between mNeonGreen and nuclear staining signals. Scale bar, 50 µm. Representative data from two experimental replicates are shown. d, Genome-wide NIS-Seq perturbation screening in HeLa–Cas9–p65–mNeonGreen cells stimulated with TNF. Fold changes were calculated based on the mean pixel-wise Pearson correlation between mNeonGreen and nuclear staining signals across cells with the same targeted gene versus NT control cells. Deviation of their correlation value distributions was tested using a two-sided Wilcoxon–Mann–Whitney test followed by the Benjamini–Hochberg procedure. e, Collages of cellular images from d mapped to perturbed genes indicated. Shown is the mNeonGreen signal. f, Arrayed hit validation in HeLa–Cas9–p65–mNeonGreen cells using alternative sgRNA sequences from the TKOv3. Top panel, exemplary mNeonGreen images of TNF-stimulated cells. Bottom panel, distribution of activation states, quantified by pixel-wise Pearson correlation between mNeonGreen and nuclear staining signals. Scale bar, 50 µm. Representative data from two experimental replicates are shown.