CRISPR/Cas9-based gene editing of the malaria parasite Plasmodium falciparum has emerged as a transformative tool for advancing functional studies on parasite biology and identifying new therapeutic targets. Currently applied CRISPR/Cas9 methodologies depend on a limited set of heterologous drug resistance markers for the selection of transgenic parasites, which restricts the potential for iterative genetic modifications. Here, we developed a heterologous marker-free CRISPR/Cas9 gene editing strategy (CRISPR/Cas9pyrR) for P. falciparum based on the simultaneous editing of a gene of interest and introduction of pyrimethamine (PYR) resistance-conferring mutations into the dihydrofolate reductase-thymidylate synthase (pfdhfr-ts) gene. By providing a pfdhfrpyrR donor sequence and the Cas9 expression cassette on separate plasmids, CRISPR/Cas9pyrR ensures that only parasites acquiring both plasmids survive under PYR pressure. As a proof of principle, we applied CRISPR/Cas9pyrR to generate two transgenic parasite lines expressing GFP-tagged versions of the putative nuclear envelope protein PfGEX1 and nuclear pore protein PfNUP116, respectively. We show that PfGEX1-GFP marks the nuclear envelope specifically in gametocytes, but not in asexual blood stage parasites. Similarly, and against previous reports, we find PfNUP116-GFP expression is undetectable in asexual parasites but instead localizes to a distinct perinuclear region in early gametocytes. These results suggest dynamic compositional changes of the nuclear periphery during sexual differentiation. We further demonstrate sequential genetic engineering of the PfNUP116-GFP-expressing line using the human dhfr drug resistance marker combined with WR99210-based selection by additionally tagging PfAP2-G, the master transcriptional regulator of sexual commitment, and the nuclear pore protein PfNUP313. Hence, CRISPR/Cas9pyrR provides a versatile and effective new method that enhances and complements the current genetic toolkit for malaria research.IMPORTANCEMalaria tropica, which is caused by the unicellular parasite Plasmodium falciparum, is one of the most devastating infectious diseases worldwide. The development of urgently needed effective vaccines and new antimalarial drugs with novel modes of action requires a profound understanding of parasite biology. CRISPR/Cas9-based genome engineering is beyond doubt the most important experimental approach to study the function and essentiality of parasite proteins and to identify and validate new vaccine and drug targets. In this study, we developed and successfully applied a modified CRISPR/Cas9 strategy, termed CRISPR/Cas9pyrR, that avoids the use of a heterologous drug resistance marker for the selection of genetically modified parasites. CRISPR/Cas9pyrR thus complements the CRISPR/Cas9 toolbox available for gene editing in P. falciparum and overcomes some of the limitations of currently employed protocols.
Carrington et al. (Thu,) studied this question.