Genome-wide CRISPRi screen to reveal proteins and pathways impacting PF8503 toxicity in K562 cells.

(A) Overview of the CRISPRi experiment, carried out using K562 cell lines expressing dCas-KRAB and the hCRISPRi-v2 library. Promoters in the lentiviruses for sgRNA (murine U6, mU6) and Puromycin resistance cassette (EF1α) are shown [17]. After the cells were cultured for 11 days, with or without treatment with PF8503, the quantified genomically-integrated sgRNAs (day 0 and day 11) were used to calculate the effect of each sgRNA on growth without drug (Gamma), with drug (Tau) and the effect of drug treatment (toxin phenotype, Rho). Cells are color-coded as follows: white, no sgRNA; grey, non-targeting sgRNA; green, enriched sgRNA; blue, depleted sgRNA. Right, genes with enriched sgRNAs sensitize cells (green, protein product predicted to enhance PF8503 toxicity) or with depleted sgRNAs protect cells (blue, protein product predicted to decrease PF8503 toxicity), from PF8503 treatment (Rho parameter). (B) Position of the 50 top protective (blue) and sensitizing (green) genes mapped to cellular pathways. The size of the circles are proportional to the PF8503 Rho phenotype shown in (A). (C) Genes known to be involved in translation which sensitize (green) or protect (blue) cells from PF8503 treatment. (D) Genes with opposite effects in the tau and gamma phenotypes. Some of the proteins of interest involved in tRNA maturation (orange), translation quality control (purple), mRNA degradation (blue) or ribosome assembly (green) are highlighted. In panels (B-D), all phenotypes are the average of the 3 best sgRNAs from experiments carried out in biological duplicate.