Abstract

The recurrent emergence of drug resistance in Plasmodium falciparum increases the urgency to genetically validate drug resistance mechanisms and identify new targets. Reverse genetics have facilitated genome-scale knockout screens in Plasmodium berghei and Toxoplasma gondii, in which pooled transfections of multiple vectors were critical to increasing scale and throughput. These approaches have not yet been implemented in P. falciparum, mainly because the extent to which pooled transfections can be performed in this species still remains unknown. Here we use next-generation sequencing to quantitate uptake of a pool of 94 barcoded vectors. The distribution of vectors in different transfections allowed us to estimate the number of barcodes and DNA molecules taken up by the parasite population. Dilution cloning showed that single transfected parasites routinely carry as many as seven episomal barcodes, revealing an intake of multiple vectors in a highly non-uniform fashion. Transfection of non-overlapping fluorescent proteins, which allowed us to follow the dynamics of the process, confirmed the tendency for parasites to take up multiple vectors from the early stages of transfection. This finding has important implications for how reverse genetics can be scaled in P. falciparum.