Fig. 5. Analysis of knock-out and knock-down perturbation effects in the LPS response.

(a) Distribution of perturbed genes based on their number of significant effects ($\mathrm{q}<0.05$) on downstream genes. (b) Distribution of downstream genes based on how many perturbed genes significantly affect their expression. (c) PCA of perturbed and control cells based on the expression of the top 2,000 most variable genes. Control cells (grey) contain a non-targeting guide only. Perturbed cells (red/blue) contain a guide for one of the following genes. Red: IKBKB, IKBKG, IRAK1, IRAK4, MAP2K1, MAP3K7, MAPK14, MYD88, RELA, TIRAP, TLR1, TLR2, TRAF6. Blue: CISH, CYLD, STAT3, TNFAIP3, TRIB1, ZFP36. Numbers in parentheses indicate percent variance explained by PCs. (d) Heatmaps of perturbation effect sizes (inferred with FR-Perturb) from the knock-out (left) and knock-down (right) screens. Rows: top 50 perturbed genes based on their average magnitude of effects on all downstream genes. Columns: top 2,000 downstream genes based on the average magnitude of effects of all perturbed genes acting on them. Rows and columns are clustered using Leiden clustering. Clusters are labelled based on their GO enrichment terms. All effects with $\mathrm{q}>0.2$ are whited out. (e) (Left) Correlation of knock-out effect sizes (y-axis) between all pairs of perturbed genes (x-axis). Top and bottom gene pairs are labelled. (Top right) Graph of all perturbed genes that physically interact with XPR1 and/or KIDINS220, based on AP-MS data from Bioplex 3.0⁴⁶. Edges represent physical interaction. (Bottom right) Mean effects of perturbed genes from top right on P1-P4. (f) Analysis of genetic interaction effects. (Left) Effect sizes relative to control (y-axis) of cells containing 0, 1, or 2 guides (x-axis) within each perturbation module (lines connecting three dots). Modules with significant effects ($\mathrm{q}<0.05$) are highlighted in color and labeled, with the expected effect of cells containing two guides in the module represented with a dotted line. Error bars represent standard errors obtained from bootstrapping. (Right plots) Violin plots of the mean effects of individual cells containing 0, 1, or 2 guides in the three perturbation modules with significant interaction effects. Dotted line represents the expected effect of cells with 2 guides. Two-sided p-values are computed from permutation testing.