CRISPR/Cas9-engineered inducible gametocyte producer lines as a valuable tool for Plasmodium falciparum malaria transmission research

Abstract

The malaria parasite Plasmodium falciparum replicates inside erythrocytes in the blood of infected humans. During each replication cycle, a small proportion of parasites commits to sexual development and differentiates into gametocytes, which are essential for parasite transmission via the mosquito vector. Detailed molecular investigation of gametocyte biology and transmission has been hampered by difficulties in generating large numbers of these highly specialised cells. Here, we engineer P. falciparum NF54 inducible gametocyte producer (iGP) lines for the routine mass production of synchronous gametocytes via conditional overexpression of the sexual commitment factor GDV1. NF54/iGP lines consistently achieve sexual commitment rates of 75% and produce viable gametocytes that are transmissible by mosquitoes. We also demonstrate that further genetic engineering of NF54/iGP parasites is a valuable tool for the targeted exploration of gametocyte biology. In summary, we believe the iGP approach developed here will greatly expedite basic and applied malaria transmission stage research.

Revisiting the Effect of Pharmaceuticals on Transmission Stage Formation in the Malaria Parasite Plasmodium falciparum

“…The pH-gC_ap2g-3' suicide plasmid targeting the 3' end of pfap2-g has been described previously (Brancucci et al, 2017). The donor plasmid pD_ap2g-mScarlet was generated by assembling (i) the BamHI and SfoI-digested pD_ap2g-gfp plasmid (Brancucci et al, 2017) with a PCR product containing (ii) the mScarlet sequence preceded by nucleotides encoding a GSAG linker using the primers mScarlet-F and mScarlet-R amplified from a P. falciparum codon-optimized synthetic mScarlet sequence (Boltryk et al, 2021), and (iii) the 3' homology region amplified from the pD_ap2g-gfp plasmid (Brancucci et al, 2017) using the primers ap2-g-3'-F and ap2-g-3'-SfoI-R in a Gibson reaction (Gibson et al, 2009). The donor plasmid pD_ap2g-re9h was generated by assembling two PCR products using the primers iso_re9h-F and iso_re9h-R amplified from pD_ap2g-mScarlet, and (Gutman et al, 2009) re9h-F and re9h-R to amplify the re9h fragment (Branchini et al, 2010) from pTRIX2-re9h (Lewis et al, 2014).…”

PMRT1, aPlasmodiumspecific parasite plasma membrane transporter is essential for asexual and sexual blood stage development

“…RNA-seq data suggest Pf PMRT1 is also expressed during other developmental stages, such as gametocytes (56, 57). Therefore, we assessed expression of Pf PMRT1-GFP during gametocytogenesis by re-engineering Pf PMRT1-GFP-glmS in the inducible gametocyte producer (iGP) ‘3D7-iGP’ (58) parasite line, which allows the robust induction of sexual commitment by conditional expression of gametocyte development 1 protein (GDV1) upon addition of shield-1 (58) (Figure S7A). We show that Pf PMRT1 is indeed expressed during all stages of gametocytogenesis and again localizes to the PPM, colocalizing with the PPM-marker Lyn-mCherry (41) (Figure 5A, B).…”

The 3-phosphoinositide–dependent protein kinase 1 is an essential upstream activator of protein kinase A in malaria parasites

“…To obtain the NF54/PKAc cOE parasite line, the pD_pkac_cOE donor plasmid was generated in several cloning steps using Gibson assembly reactions. First, a pD_cOE_DD precursor plasmid was generated using a 2-fragment Gibson assembly joining (1) the plasmid backbone including the glp3 -specific 5 ′ and 3′ HRs amplified from pD_ cg6_cam-gdv1-gfp-glmS [ 84 ] (primers glp3_F and glp3_R); and (2) the cam 5′-gfpdd-hrp2 3′ sequence amplified from pHcamGFP-DD [ 53 ] (primers cam_F and hrp2_R). Second, the subsequent precursor plasmid pD_cOE_glmS was generated by performing a Gibson assembly using 2 fragments (1) the pD_cOE_DD precursor plasmid digested using SalI and AgeI ; and the (2) glmS sequence amplified from pD_ cg6_cam-gdv1-gfp-glmS [ 84 ] (primers glmS_F and glmS_R).…”

“…First, a pD_cOE_DD precursor plasmid was generated using a 2-fragment Gibson assembly joining (1) the plasmid backbone including the glp3 -specific 5 ′ and 3′ HRs amplified from pD_ cg6_cam-gdv1-gfp-glmS [ 84 ] (primers glp3_F and glp3_R); and (2) the cam 5′-gfpdd-hrp2 3′ sequence amplified from pHcamGFP-DD [ 53 ] (primers cam_F and hrp2_R). Second, the subsequent precursor plasmid pD_cOE_glmS was generated by performing a Gibson assembly using 2 fragments (1) the pD_cOE_DD precursor plasmid digested using SalI and AgeI ; and the (2) glmS sequence amplified from pD_ cg6_cam-gdv1-gfp-glmS [ 84 ] (primers glmS_F and glmS_R). To insert a new cloning site and generate the next precursor plasmid pD_cOE, another Gibson assembly reaction was performed joining (1) the pD_cOE_glmS plasmid digested using BamHI and NotI ; and (2) annealed complementary oligonucleotides clon_F and clon_R.…”

“…The final pD_pkac_cOE plasmid was generated by assembling 2 Gibson fragments: (1) the BamHI- and SpeI- digested pD_cOE plasmid; and (2) the pfpkac sequence amplified from NF54 gDNA using primers pka_F and pka_R. The pBF_gC- cg6 suicide plasmid that was cotransfected with pD_pkac_cOE to generate this NF54/PKAc cOE parasite line encodes the sgRNA targeting the glp3 locus [ 84 ]. To obtain the NF54/PKAcT189V cOE parasite line, the pD_pkacT189V_cOE donor plasmid was generated in a Gibson assembly joining 3 fragments: (1) the BamHI- and SpeI- digested pD_cOE plasmid; (2) the 5 ′ fragment of the pfpkac sequence containing the single point mutation resulting in the T189V amino acid change (primers pka_F and T189V_R); and (3) the 3′ fragment of the pfpkac sequence overlapping with the 5 ′ fragment and coding for the same amino acid change (primers T189V_F and pka_R).…”