Pooled genome-wide CRISPRi screen for protein transport. (A) Schematic representation of the pooled CRISPRi screening workflow. HeLa TAC-GFP dCas9-KRAB cells (1) were infected with the CRISPRi-v2 library (2). Then, using flow cytometry–based sorting, two cell fractions representing a low and high surface/total TAC expression ratio were collected (3), and the abundance of each sgRNA in the two fractions was assessed by sequencing (4). The scatter plot represents the sgRNA read counts (log2) derived from each cell fraction obtained after DNA sequencing. The color bar indicates the values of phenotype (fold change [log2]) obtained for each individual sgRNA. sgRNAs with a phenotype <0 indicate an enrichment in the lower quartile, and sgRNAs with a phenotype >0 indicate an enrichment in the upper quartile. (B) Volcano plot showing for each gene, the knockdown effect on TAC transport, and P value (−log10) of phenotype. Screen replicates were averaged. Each gene targeted by the library of sgRNA is indicated with a black dot. Genes included in the 100 top ranked genes (inserts) and belonging to functional categories of interest (highlighted in C and D) are indicated with red dots for genes inhibiting TAC transport and with blue dots for genes stimulating TAC transport. Entire datasets are reported in Table S1. (C and D) gene ontology (GO) term enrichment analysis among the 100 top ranked genes whose depletion results in TAC transport inhibition (C) and TAC transport stimulation (D).