Fig. 1. Description of the inducible gametocyte producer line 3D7/iGP.

a Schematic of the disrupted cg6 locus carrying a single inducible GDV1-GFP-DD expression cassette. The 5′ and 3′ homology regions used for CRISPR/Cas9-based transgene insertion are shown in orange. b Schematic of the in vitro culture protocol used to quantify sexual commitment rates (SCRs). Synchronous 3D7/iGP ring stage parasites are split at 0–16 hpi and Shield-1 is added to one half of the population to trigger GDV1-GFP-DD expression. SCRs are quantified by determining the proportion of early stage I gametocytes in the total iRBC progeny 36–44 hpi (day 2 of gametocytogenesis) by α-Pfs16 IFAs combined with DAPI staining. Asexual parasites are depicted in grey, sexually committed parasites and gametocytes are depicted in purple. asS/scS, asexual/sexually committed schizont; asR/scR, asexual/sexually committed ring stage; T, trophozoite; I–V, gametocyte stages I–V; D1–D10, days 1–10 of gametocyte maturation. Gen 1/2, generation 1/2. c α-Pfs16 IFA images illustrating the high proportion of early gametocytes in the progeny of Shield-1-treated 3D7/iGP parasites. The white arrow highlights a Pfs16-negative schizont. Nuclei were stained with DAPI. DIC, differential interference contrast. Images are representative of three independent experiments. Scale bar, 10 µm. d Proportion of Pfs16-positive iRBCs (SCRs) in the progeny of 3D7/iGP treated with three different Shield-1 concentrations and the untreated control (−Shield-1) (mean ± SD, n = three biologically independent experiments; two experiments for parasites treated with 337.5 nM Shield-1). Closed circles represent data points for individual experiments (>156 DAPI-positive cells counted per experiment).