Figure 1.
A Genetic Screen Identifies a Putative Regulator of T. gondii Differentiation
(A) Construction of a differentiation reporter strain that constitutively expresses RFP and Cas9 and conditionally expresses mNeonGreen (mNG) under the regulation of the bradyzoite-specific BAG1 promoter. SAG2Y is a bradyzoite-specific surface marker. Images were taken after 48 h of growth under unstressed or alkaline-stressed conditions. Scale bar is 10 μm.
(B) Transfection and selection for a gRNA targeting the surface antigen SAG1 resulted in gene disruption in 98% of the resulting population. The mean was plotted for n = 3 biological replicates; 92–102 vacuoles were scored for each replicate; ∗∗∗∗p < 0.0001 by Student’s two-tailed t test.
(C) Percentage of alkaline-stressed reporter parasites expressing mNG, quantified by FACS. The mean ± SD was plotted for n = 2–4 biological replicates.
(D) RNA sequencing and differential expression (DE) analysis identified 1,311 genes as significantly upregulated (green) and 933 genes as significantly downregulated (red) in bradyzoites (adjusted p < 0.001), with 1,240 and 700 changing 2-fold or more, respectively (dotted lines). The analysis was based on n = 3 independent experiments.
(E) Screening and analysis workflow. The log2-transformed fold changes from the input library to the final unstressed or stressed mNG+ populations are defined as fitness or differentiation scores, respectively.
(F and G) Fitness and differentiation scores at the gene level following screening L1 (F) or L2 (G).
(H) Fitness and differentiation scores for individual gRNAs in L2.