Fig. 2

High-throughput genetic validation of targets. Two methods have been used to determine gene essentiality. a Zhang et al. [36] used a piggyBac transposon system in P. falciparum to determine genes that could be disrupted using culture conditions that were considered ideal for the asexual blood stage [36]. Transfection with the piggyBac plasmid (pLBacII-HDH) was performed in a 96-well plate, and parasites containing the plasmid marker (dhfr) were selected for and regrown in culture. DNA was then extracted and quantitative insertion-site sequencing (QI-seq) was performed to determine the sites of insertion. Mutagenesis index scoring was then used to identify genes with the highest confidence of disruption and nondisruption. b Bushell et al. [58] used barcode vectors to determine which genes were essential for asexual blood stage growth using an in vivo system in mice. The vectors were transfected into P. berghei schizonts, which were inoculated into mice, and growth was determined by measuring parasitemia on subsequent days of infection. Four growth phenotypes were observed, among which “essential genes” and “slow-growing mutants” were determined to be essential or important for asexual blood stage growth