Efficient CRISPR-Cas9-mediated genome editing in Plasmodium falciparum

Malaria is a major cause of global morbidity and mortality, and new strategies for treating and preventing this disease are needed. The protozoan parasite, Plasmodium falciparum, contributes to the annual 219 million cases and 655,000 deaths from malaria. Gene knockouts, while central to studying this parasite, are inefficient and present a substantial bottleneck. Here we show that the Streptococcus pyogenes Cas9 DNA endonuclease and single guide RNAs (sgRNAs) made in situ using T7 RNA polymerase (T7 RNAP) efficiently edit the P. falciparum genome. Targeting the native knob-associated histidine-rich protein (kahrp) and erythrocyte binding antigen 175 (eba-175) genes, we achieve high ( ≥ 50–100%) gene disruption frequencies within the usual timeframe for generating transgenic parasites. Our results demonstrate a straightforward strategy for efficiently manipulating the P. falciparum genome to facilitate basic biological studies, and drug and vaccine development efforts in a human pathogen of major significance to global health. The most commonly used approach for modifying chromosomal loci in P. falciparum relies on spontaneous single- or double- crossover recombination using plasmids containing sequence homologous to the target region 1–3. This is extremely inefficient, and require