Generation and verification of MitoRed parasite line. (A) Schematic overview of strategy to generate a parasite line harboring a fluorescent mitochondrial marker. CRISPR-Cas9 is used to create two double-strand breaks at SIL7 (indicated by scissors). A construct containing the promotor and targeting sequence of the mitochondrial protein HSP70-3 (PF3D7_1134000) fused with mScarlet is integrated by double homologous recombination. Once integrated, the mitochondrial-targeted mScarlet is expressed and leads to fluorescent staining of the mitochondrion. (B) Diagnostic PCR of MitoRed parasite line with WT- and integration-specific primer combinations (indicated in panel A) demonstrating successful 5′ and 3′ integration and the absence of WT parasites in the MitoRed line. (C) Growth assay showing similar growth of MitoRed and WT parasites. Three independent cultures were set up from one tightly synchronized parasite culture for both MitoRed and WT. Samples were taken over a 5-day period and parasitemia (corrected for dilution factors) was determined with flow cytometry. Error bars (note they are quite small) indicate standard deviation. Source data are available for this figure: SourceData FS1.