We selected 11 genes from the 85 hits from the genome-wide screen based on the following criteria: RPKM >20, ToxoDB phenotype score > 0. From that list of 43 genes, we mostly picked genes encoding for dense granule proteins but also added some random non-secretory genes. To confirm these 11 hits identified from the screen, RH-Cas9 (wild-type) and RH-Cas9Δgra17 (Δgra17) parasites were transfected with 2 plasmids, each containing a different sgRNA targeting the 11 genes under investigation. Immediately after transfection plaque assays were performed by adding 5,000 parasites into 3 wells of a 6-well plate with HFFs in the presence of 1 μM pyrimethamine (Sigma–Aldrich, Cat#46706) (the plasmid contains a pyrimethamine resistance cassette). Plaque numbers were determined 8 days p.i. As a negative control, we used sgRNAs targeting SAG1, of which the knockout has no phenotype in either background, while as positive controls we used sgRNAs targeting GRA23 (TGGT1_297880), which is synthetically lethal with GRA17 but of which the knockout has no growth effect in wild-type [4 (link)]. sgRNAs targeting CDPK1 (TGGT1_301440, phenotype score = -3.3), which was previously shown to be essential in RH [23 (link)], and TGGT1_223440 (phenotype score = -3.6 [24 (link)]) were used as the control for genes important for fitness in both wild-type and Δgra17 parasites.
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