Malaria causes ~660,000 deaths per year, and P. falciparum is the protozoan responsible for its most severe form. The lack of effective vaccines and the parasite's ability to develop drug resistance are major barriers to malaria treatment and eradication. Classical genome manipulation in P. falciparum is arduous and not robust. Zinc-finger nucleases that introduce targeted DNA double-strand breaks (DSBs) have been shown to be functional in P. falciparum, providing a useful means to generate targeted mutations 1 . However, although reported to be highly efficient, this technique is not widely used, mainly because of the cost and laborious design process. Recently, an alternative genome-editing strategy was developed based on the clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) system (CRISPR-Cas), a prokaryotic adaptive immune mechanism against invading viruses and plasmids 2 . In vitro reconstitution of the type II Streptococcus pyogenes CRISPR-Cas9 system showed that a single guide RNA (sgRNA) can guide the Cas9 endonuclease to cause DSBs in target DNA sites 3 . The sgRNA carries the Cas9 binding domain and a customizable 20 nucleotides (hereafter referred to as the guide), matching the target-DNA site. The protospacer-adjacent motif (PAM), a sequence immediately downstream from the target region, must be present for cleavage.
DSBs generated by Cas9 (or other genome editing technologies) can be repaired by homologous recombination using donor DNA or error-prone, nonhomologous end-joining (NHEJ), which often introduces mutations at the target site. P. falciparum seems to be naturally deficient in canonical NHEJ and, although an alternative NHEJ has