Parasite maintenance, synchronisation and transfection

The DiCre‐expressing P. falciparum B11 line (Perrin et al, 2018) and P. falciparum EXP2‐mNeon line (C. Bisson, manuscript in preparation) were maintained at 37°C in human RBCs in RPMI 1640 containing Albumax II (Thermo Fisher Scientific) supplemented with 2 mM l‐glutamine. The human RBC‐adapted P. knowlesi A1.H1 line (Moon et al, 2013) was maintained as described previously (Mohring et al, 2019). Cultures were routinely microscopically examined using Giemsa‐stained thin blood films and synchronised by standard procedures (Harris et al, 2005). Highly synchronous schizonts were isolated by centrifugation over 70% (v/v) isotonic Percoll (GE Healthcare, Life Sciences) cushions.

Transfections were performed by introducing DNA into ~10⁸ Percoll‐enriched schizonts by electroporation using an Amaxa 4D‐Nucleofector X (Lonza), using program FP158 as previously described (Moon et al, 2013). For Cas9‐based genetic modifications, 20 μg of Cas9 expression plasmid and 60 μg of linearised repair templates were electroporated. Drug selection with 2.5 nM WR99210 was applied 24 h post‐transfection for 4 days. Clonal transgenic lines were obtained by serial limiting dilution in flat‐bottomed 96‐well plates (Thomas et al, 2016) followed by selection of single plaques. For sequential Cas9‐based genetic modifications, established transgenic parasites were treated with 1 μM 5‐fluorocytosine (5‐FC) provided as clinical grade Ancotil® (MEDA) for 7 days prior to the secondary transfection. For transfections with plasmids to be maintained episomally, 10 μg of DNA was electroporated and the growth medium continuously supplemented with 2.5 μg/ml blasticidin‐S from 24 h post‐transfection. DiCre‐mediated excision of DNA was induced by transient RAP treatment of highly synchronous early ring‐stage parasites (2–3 h post‐invasion) as previously described (Collins et al, 2013a). Parasite genomic DNA for genotype analysis was extracted using the QIAGEN DNeasy Bood and Tissue kit and analysed by PCR using GoTaq® G2 Green Master Mix (Promega).