Malaria remains a globally important cause of morbidity and mortality; a steady reduction in malaria deaths since 2000 has plateaued and, more recently, reversed due to limited access to preventative measures, disruptions during the CoVID-19 pandemic, and evolving vector and pathogen resistance (WHO, 2022). A better understanding of parasite biology and focused examination of drug and vaccine targets is critical for maintaining a pipeline of new antimalarial therapies that could reduce the malaria burden.

DNA transfection of the virulent P. falciparum human pathogen, which accounts for over 90% of malaria deaths, is critical for identification and evaluation of parasite targets (Crabb et al., 2004, Hasenkamp et al., 2012, Ghorbal et al., 2014, Ribeiro et al., 2018). Transfection has also provided foundational insights into unique aspects of parasite biology including characterization of virulence factors, host cell remodeling, immune evasion, drug resistance mechanisms, and developmental progression through the parasite life cycle. Despite its central importance, parasite transfection remains a rate-limiting step that is available to only a fraction of malaria research laboratories. The low transfection efficiency and relatively slow replication of in vitro blood cultures render P. falciparum transfection laborious and time-consuming.

In most molecular biology laboratories, P. falciparum transfection is performed by electroporation of parasite cultures to introduce plasmid DNA (Crabb et al., 2004, Hasenkamp et al., 2012). Plasmid uptake by the intracellular parasite after DNA loading of uninfected erythrocytes has been shown to improve transfection efficiency (Deitsch et al., 2001), presumably through reduced attrition of viable parasites. Other methods, including plasmid loading through hypotonic lysis and nucleofection (Gopalakrishnan et al., 2013, Govindarajalu et al., 2019), have also been reported, but have not yielded improved transfection efficiency or reduced the time required for parasite outgrowth.

Notably, each of these methods has relied, to varying extents, on transfection technologies developed for other cell types. For example, electroporation of both parasite cultures and uninfected erythrocytes continues to rely on a cytomix buffer composition developed for mammalian cell transfection (Crabb et al., 2004). Systematic optimization of this recipe for P. falciparum is warranted because erythrocyte poration and resealing, as well as parasite metabolism and ionic tolerances, differ markedly from those of model mammalian cell lines (Schwoch and Passow, 1973, Nash and Meiselman, 1985, Pillai et al., 2013). Such optimization has not been undertaken, in part because facile methods for rapid and quantitative assessment of buffer composition and electroporation conditions are missing.

Here, we sought to improve transfection efficiency and reduce the reliance on costly resources that are not broadly available to basic malaria researchers. With this aim, we developed and used a sensitive and rapid NanoLuc reporter plasmid to evaluate transfection success after only 48 h. These studies reveal that plasmid loading is inefficient with standard electroporation methods and that inadequate loading compromises transfection outcomes. Using hypotonic lysis and DNA incorporation into resealed erythrocytes, we systematically optimized key variables to achieve significantly greater loading. The variables for examination were based on classical studies of erythrocyte ghosts, which are the resealed cells with reduced hemoglobin content formed after hypotonic lysis (Schwoch and Passow, 1973, Nash and Meiselman, 1985). In our studies, transfection outcomes were significantly affected by changes in lysis conditions, resealing buffer composition and the subsequent incubation. The resulting optimized conditions significantly improved kinetics of NanoLuc expression throughout the transfection time course and yielded faster microscopic parasite outgrowth in episome-based transfections and stable CRISPR-Cas9 genome editing.