Unique features in the intracellular transport of typhoid toxin revealed by a genome-wide screen
Fig 2
A genome-wide CRISPR/Cas9-mediated gene inactivation screen identifies genes essential for typhoid toxin intoxication.
(A) A schematic of the workflow for the screen to identify genes involved in typhoid toxin toxicity. HEK293T cells expressing Cas9 were transduced with a lentiviral library encoding sgRNA targeting human genes as described in Material and Methods. After puromycin selection, cells were split and either mock treated or treated with typhoid toxin. Cells that survived the toxin treatment or that were mock treated were subjected to nucleotide sequence analysis as indicated in Materials and Methods. Sequences were aligned to the reference genome and high-quality reads were analyzed with the MAGeCK algorithm to identify positively-selected genes. (B and C) Scatter plots of effect size (log fold change; x axis) versus P value (-log10 raw P-value; y-axis) for all genes. Three replicates were performed for each sub-library and the MAGeCK algorithm was used to compare treated with untreated cells across replicates for the human GeCKO v.2 sub-library A and B as described in Material and Methods. Inactivation of the genes colored in red conferred resistance to typhoid toxin with a P-value cutoff corresponding to 15% FDR. (D) Gene ontology term enrichment analysis of genes whose inactivation conferred toxin resistance. The P-values represent the probability of the identified genes to be annotated to a particular GO term relative to all the annotated human genes. GO terms are shown depicting biological processes in black and cellular components in gray.