Figure 2. Using Pooled Screens to Identify genes responsible for drug sensitivity.
(A) Schematic depiction of the pooled CRISPR screen. Cas9-expressing parasites are transfected with the sgRNA library and grown in human fibroblasts (HFFs). At various time-points, sgRNAs are amplified and enumerated by sequencing to determine relative abundance and phenotype scores for individual genes.
(B) Time-line for the generation of mutant populations and subsequent selection in the presence or absence of FUDR. Times at which parasites were passaged (P) are indicated.
(C) Heat-map showing the phenotype score of genes at different time-points following transfection of the library into wild-type (wt) or Cas9-expressing parasites.
(D) Relative abundance of sgRNAs following growth of the population in the presence or absence of FUDR. Mean log2(normalized abundance) for each sgRNA in three independent experiments; sgRNAs against UPRT (blue).
(E) Phenotype score calculated for each gene comparing growth ± FUDR. Mean ± SEM for n = 3 independent experiments; UPRT (blue).
(F) Comparison of phenotypic scores in untreated samples after three (P3) or six (P6) passages. Pearson’s correlation coefficient (r) is shown.