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. 2016 Dec 20;11(12):e0168362. doi: 10.1371/journal.pone.0168362

Fig 1. Schematic representation of improved CRISPR/Cas9 plasmids and selection protocol.

Fig 1

Parasites are transfected with two plasmids (Cas9 construct and sgRNA/donor construct). The Cas9 construct contains the bsd selectable marker. The sgRNA/donor construct contains a fusion of the positive selectable marker hdhfr and the negative selectable marker yfcu genes and two homology regions (HR1 and HR2) that target a gene of interest (GOI) and introduce the donor DNA by homologous recombination. Double positive selection using both BSD and WR99210 is applied from day (d) 1 resulting in the selection of parasites that contain both plasmids within a period of 3 weeks (w). After positive selection, cultures are maintained 2–4 days without drug before negative selection is applied using 5-FC to select parasites free of episomal plasmid DNA. Parasites are genotyped by diagnostic PCR for integration of the donor DNA followed by cloning of the parasites by limiting dilution (w6). Clones are genotyped for the correct genotype by diagnostic PCR and Southern analysis. This transfection and selection protocol can result in the generation of cloned mutant parasites within a period of 11 weeks.