Comparison of in vitro transformation efficiency methods for Plasmodium falciparum.

Poor efficiency plagues conventional methods to transfect Plasmodium falciparum with genetic modifications, impeding research aimed at limiting the damage wrought by this agent of severe malaria. Here, we sought and documented improvements, using fluoresce imaging, cell sorting, and drug selection as means to measure efficiency. Through the transfection of EGFP plasmid, the transfection efficiency of the three methods used in this study was as high as 10. A method that pre-loaded uninfected erythrocytes with plasmids using the Bio-Rad Gene Pulser Xcell achieved the highest efficiency (0.48%±0.06%), twice the efficiency of a method using nuclear transfection of ring stages employing the 4D-Nucleofector X Kit L. We also evaluated an approach using the Nucleofactor system to transform schizont stages. We considered efficiency and the time required to complete drug screening experiments when evaluating transfection methods. Fluorescence measurements confirmed greater efficiencies for the Pre-load method (52.4% vs. 25%; P < 0.0001), but the Nuc-Ring method required less time to complete drug selection experiments following CRISPR/Cas9 editing. These data should benefit future studies seeking to remove or modify genes of P. falciparum.