Figure 5. Functional Characterization of Indispensable Conserved Apicomplexan Proteins.
(A) Position of analyzed genes within the phenotypic ranking of all T. gondii genes. Known indispensable (red) or dispensable (yellow) genes used as controls are indicated. The ICAPs (blue) and uncharacterized genes predicted to be dispensable (green) are numbered in ascending order according to their rank.
(B–C) Gene disruption observed at a population level three days after transfection with various control constructs. Disruption of the target locus is observed by Surveyor assay comparing, for each locus, the specific sgRNA to an irrelevant sgRNA against the dispensable gene MYOC (B). Loss of the target proteins (red) is observed in samples treated with the targeting sgRNA but not the control, while the loading controls (green) remain unchanged (C).
(D–E) Plaque assays performed immediately following transfection with sgRNAs targeting ICAPs or control genes (D). The number of plaques observed for disruption of each gene relative to the sgRNA against SAG1 (E). Mean ± SEM for n = 2 independent experiments, * FDR-adjusted p < 0.1 relative to the control.
(F) Secondary screen for genes involved in invasion. The period of intracellular growth prior to phenotypic changes was extended by forced release and passaging the day after transfection. Invasion was assayed after the subsequent lysis, and calculated relative to PLP1 disruption. Mean ± SEM for n = 2 independent experiments, * FDR-adjusted p < 0.1 relative to the control. Representative immunofluorescence images are shown.