Amaxa 4d electroporator

The tightly synchronized mature schizont-stage parasites of P. knowlesi were transfected using the Amaxa 4D electroporator (Lonza, Basel, Switzerland) and the P3 Primary cell 4D Nucleofector X Kit L (Lonza) following previous reports (Moon et al., 2016 (link)). Briefly, a 20-μg repair template and 20 μg pCas9/sg (Mohring et al., 2019 (link)) containing sgRNA sequences for pkmsp1p were mixed with P3 Primary Cell nucleofector solution, including supplement 1 (Lonza), and transferred to a Nucleocuvette™ Vessel (Lonza), followed by nucleofection with program FP158. Transfected parasites were immediately transferred to complete media with RBCs and incubated at 550 rpm for approximately 30 min at 37°C to allow invasion before transferring to standard culture conditions. After 24 h, transfected parasites were selected by drug pressure with 100 nM pyrimethamine (Sigma-Aldrich), and the medium, including pyrimethamine, was replaced at daily intervals for 5 days. The transfected parasites were cloned out by limiting dilution and confirmed by genotyping with diagnostic primers of extracted genomic DNA (Supplementary Tables 1, 2).

The gap45:ha3:loxP::comp_gap45[G2A] construct was generated through PCR, digest, ligation, and cloning using the gap45:ha3:loxP::comp_gap45 construct and the same guide [19 (link)] and cloned into the pDC2-cam-Cas-9-U6-hDHFRyFCU-plasmid [36 (link),45 (link),46 (link)]. Guide and rescue plasmids were paired and ethanol precipitated prior to transfection. P. falciparum gap45:ha3:loxP (B11 background) [19 (link)] or 3D7 parasites were used. For transfection, mature schizonts were electroporated using the Amaxa 4D electroporator (Lonza, Slough, United Kingdom) and the P3 Primary cell 4D Nucleofector X Kit L (Lonza) and program FP158 [47 (link)], with 60 μg of linearized rescue plasmid and 20 μg of the CRISPR/Cas-9 plasmid carrying the respective guide RNA. Selections were carried out as recently described [36 (link)]; parasites were cultured in the presence of 2.5 nM WR99210 for 5 days to select for parasites with the Cas-9/guide plasmid. Transfected parasites were detected after 22 days, and DNA integration was confirmed by PCR amplification. Parasites were then treated with 1 μM 5-fluorocytosine (Ancotil) to remove residual Cas-9/guide plasmid and cloned by limiting dilution after 37 days [48 (link)]. Individual clones were then screened by PCR amplification to confirm integration of the required DNA sequence.

PF3D7_1476800 and PI-PLC cKO parasites are based on the DiCre-expressing P. falciparum clone B11, derived from the 3D7 parasite line (31 (link)). Two transfections (one per guide RNA) were performed. Mature schizonts enriched using Percoll (GE Healthcare, Chicago, IL, USA) were electroporated with 20 µg of guide plasmid and 60 µg of linearized repair plasmid using an Amaxa 4D electroporator and P3 Primary cell 4D Nucleofector X Kit L (Lonza, Basel, Switzerland) using program FP158 as described (26 (link)). Twenty-four hours post transfection, the culture medium was replaced with fresh medium containing WR99210 (2.5 nM), which was withdrawn after 4 days. Once drug-resistant parasites appeared (in about 2 weeks), they were cloned by limiting dilution using a plaque-based method (57 (link)). Successful integration was confirmed by diagnostic PCR using GOtaq Hot Start Green Master Mix (Promega, Fitchburg, WI, USA). For primer sequences, see Supplemental file 1.