FIG 4 .

Knockout of pfhsp70x does not affect intraerythrocytic growth. (A) Schematic representation showing interruption of the PfHsp70x ORF with the hDHFR cassette. The Cas9-mediated double-stranded break in the pfhsp70x ORF is repaired using homology regions on the template plasmid while inserting an hDHFR cassette into the locus. (B) Southern blot analysis confirming knockout of PfHsp70x. Genomic DNA from independent knockout clones (A3, A7, B3, and B9) was isolated and digested with BamHI and ScaI. The membrane was hybridized with a biotin-labeled probe complementary to the first 800 bp of the pfhsp70x ORF. (C) Western blot analysis demonstrating loss of PfHsp70x protein expression in independent knockout clones. Schizont-stage parasites were purified on a Percoll gradient, and whole-cell lysate was used for analysis. The membrane was probed with antibody raised against PfHsp70x and antibody against plasmepsin V as a loading control. (D) Parental lines and independent PfHsp70x-KO clones (A7 and B3) were seeded at equal parasitemia in triplicate. Parasitemia was measured every 24 h using flow cytometry. Data are fit to an exponential growth equation and are represented as means ± SEM (n = 3).