Figure 3.

Genetic engineering using 3D7^cas9. (A) To engineer the gene, 3D7^cas9 was co-transfected with the linear form of donor template DNA and psgRNA1_cen containing the sgRNA. The targeted genomic locus was cleaved by the Cas9-sgRNA complex, followed by HDR with the donor template. (B) pfap2-g was disrupted by insertion of a single adenosine in the open reading frame. The PAM sequence TGG was also mutated by substitution from guanosine to cytosine. (C) The mutations introduced in pfap2-g were confirmed by sequencing. Red indicates the mutation. (D) The 3D7^{cas9-pfap2-g-ko} parasite completely lacks gametocyte production. (E) The gfp gene was integrated at the C-terminus of PfAP2-I. (F) Genotyping PCR of 3D7^{cas9-pfap2-i::gfp} parasites was performed using the p5 and p6 primers to examine the integration of gfp. Full length gel image is included Fig. S4D. (G) PfAP2-I-GFP expression in 3D7^{cas9-pfap2-i::gfp} was uniquely confirmed in trophozoite (T) and schizont (S), not in ring form (R).