FIG 4.

(A and B) CRISPR-Cas9-mediated gene editing. Dd2 and 3D7 parental parasites were transfected with a plasmid encoding the sgRNA, Cas9 nuclease, and human dihydrofolate reductase (hdhfr) as a selectable marker, and another plasmid encoding a donor sequence containing PfPARE G58C or L82* (A) and PfUba2 Q62L (B). (C to E) Electropherograms showing unmodified and genome-edited parasites. Gray boxes highlight nucleotides that differ from wild-type parasites. PfPARE and PfUBA2 single-letter amino acid substitutions and/or stop mutations are indicated in red. (F) Susceptibility to AN13762 of the parental (WT) lines and genetically modified lines. Lines P and P0 are WT strains. P1, P3, and Q1 were cultured under 150 nM AN13762 drug pressure, and lines P2, P4, Q2, Q3, S1, and S2 were selected with WR29910 and blasticidin. Bars represent mean IC₅₀ values, and error bars are the standard deviations of the results from 3 independent experiments, each performed in triplicate. UTR, untranslated region.