FIG 1.

Orthologue replacement (OR) of Plasmodium knowlesi ATP4. (A) Schematic of CRISPR-Cas9 genome editing strategy. Integration of atp4 orthologues into the target pkatp4 locus was via homologous recombination. Arrows indicate oligonucleotide positions for diagnostic PCRs. (B) Parasites transfected (TF) with pCas9/sg_ATP4 and pDonor_atp4^OR plasmids were analyzed with diagnostic PCRs. Shown are results from PCRs detecting the wild-type locus (i.e., from the parental A1-H.1 clone) (Wt fwd + rev) and integration locus (Int fwd + rev) as well as a control PCR targeting an unrelated locus (PkMTIP fwd + PkMTIP rev). The WT locus band for the PkATP4^OR clonal line was confirmed as PkATP4^OR by amplicon sequencing. (C) Graph showing fold multiplication of WT, PkATP4^OR, PfATP4^OR, PmATP4^OR, PocATP4^OR, or PvATP4^OR parasites in erythrocytes over one intraerythrocytic growth cycle (27 h). Assays were carried out in technical duplicates in Duffy-positive erythrocytes with three independent biological replicates. A one-way ANOVA revealed there was no statistically significant difference in growth rates between at least two groups: F(5, 12) = [1.966] and P = 0.16.