Figure 4.

Anticipated results. (a) Example images from a CRISPR knockout screen for regulators of NFkB activation in A549 cells⁴³. Sequencing and phenotyping data (p65 localization) are shown for a single field of view. White outlines in sequencing images represent individual cells; colored outlines in the phenotype image represent clusters of neighboring cells with identical sgRNA assignments (scale bar, 50 μm). (b) Distribution of per-cell nuclear translocation scores after TNFɑ stimulation for non-targeting sgRNAs and sgRNAs targeting TNFRSF1A (TNFa receptor), MAP3K7 (downstream NFkB regulator), and IL1R1 (not involved in TNFa signaling). All sgRNAs were downsampled to a maximum of 100 cells to more easily compare distributions. (c) Kinetics of NFkB activation from a separate live-cell optical pooled screen performed in HeLa cells⁴³. In unperturbed cells, p65 translocates from the cytoplasm to the nucleus ~45 min after stimulation, followed by a slower, partial relaxation back to the cytoplasm. Top, translocation traces for individual cells mapped to sgRNAs targeting TNFRSF1A, RIPK1, and TNFAIP3 (negative regulator of TNFa signaling). The gray curve indicates the average of cells assigned to non-targeting controls. Middle, per-sgRNA averages, with error bars indicating standard error of the mean (data downsampled to 300 cells per sgRNA). Bottom, per-gene distributions at fixed time points of all cells mapped to sgRNAs targeting the respective gene (green) or non-targeting sgRNAs (gray). The single-cell resolution of optical pooled screens reveals distribution features of perturbation effects, including bimodality and distribution width.