Genome modification strategies for P. falciparum . (A) Conventional allelic exchange or knockout strategies rely on a rare homology-driven integration event, probably resulting from a stochastic double-strand break (DSB) near the target site (asterisk). This approach requires several weeks to months of continuous culture, and yields a complex genomic locus after crossover-mediated recombination that includes a selectable marker and duplicated gene fragments. By contrast, genome-editing approaches are driven by a directed DSB event, mediated by site-specific nucleases expressed from transfected plasmids, triggering homology-directed repair from a donor template to yield gene disruptions or nucleotide substitutions (asterisk). (B) In ZFN-based editing, heterodimerization of an engineered pair of ZFNs (ZFN-L and ZFN-R) each fused to a split FokI domain (red) yields a functional nuclease that recognizes the specified target site. (C) The two-component CRISPR-Cas system consists of a constant nuclease, Cas9, which is directed to the desired location by RNA-DNA base pairing dictated by an expressed gRNA.