Generation of Plasmodium yoelii malaria parasite carrying double fluorescence reporters in gametocytes.

Male and female gametocytes are the infectious forms critical for malaria transmission and targets of intervention. Gametocytes are generally produced in relatively small numbers, and it has been difficult to obtain pure male and female gametocytes for various studies. Male and female gametocytes expressing unique fluorescence reporters have been generated for both Plasmodium falciparum and Plasmodium berghei parasites, which allows isolation of large numbers of pure male and female gametocytes and has greatly contributed to our understanding of gametocyte biology. To establish Plasmodium yoelii as another model for studying gametocytogenesis, here we generate a parasite line with male and female gametocytes expressing GFP or mCherry reporter, respectively, using CRISPR/Cas9-mediated gene editing method. We first inserted genes encoding intact fluorescence proteins downstream of parasite coding region of ccp2 and Dhc1 genes, respectively, generating the knockin parasites producing ccp2::mCherry (female) and Dhc1::gfp (male) gametocytes. We next obtained a parasite clone carrying double-fluorescent reporters by genetically crossing the ccp2::mCherry and Dhc1::gfp lines. The resulting double-labeled DFsc7 parasite displays normal development during the whole life cycle and expresses the fluorescence proteins in male and female gametocyte separately. This parasite strain provides a new platform for facilitating studies of gametocyte biology and malaria transmission.