Figure 4.

Generation of a reporter parasite line of sexual parasites by double genetic engineering. (A) Two linear donor templates, which contained male- and female-specific reporter cassettes, and the psgRNA2_cen plasmid containing two sgRNAs were co-introduced into 3D7^cas9 parasites. A male-specific reporter cassette with the gfp gene was integrated at the pfcsp locus. A female-specific cassette with the mCherry gene was performed at the pfpalm locus. If the cleaved genomic loci at pfcsp and pfpalm were repaired with donor templates by HDR, the parasite would survive. However, if one of them was not repaired, parasites would die due to instability of the cleaved chromosome. (B) Genotyping PCR was performed using genomic DNA purified from 3D7^{cas9-Pfg_red/green} before and after limiting dilution. The primers used for this analysis are shown in Supplementary data 1. Full length gel image is included in Fig. S4G. (C) GFP and mCherry were expressed in male and female gametocytes of 3D7^{cas9-Pfg_red/green}, respectively.