Figure 5. Functional Characterization of Indispensable Conserved Apicomplexan Proteins
(A) Position of analyzed genes within the phenotypic ranking of allT. gondiigenes. 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.
Mean±SEM for n = 4 independent experiments.
(B and 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 geneMYOC(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 and 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 againstSAG1(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 toPLP1disruption. Mean±SEM for n = 2 independent
experiments; , FDR-adjusted p < 0.1 relative to the control. Representative immunofluorescence images are shown.
See alsoFigure S3andTable S2.
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