Fig. 1

Generation and genotyping of two chimeric P. falciparum parasites (Pf-pvcsp). a Two Pf-pvcsp parasite lines were generated using CRISPR/Cas9 methodology. The coding sequence (CDS) of Pfcsp gene was replaced by insertion of the Pvcsp(vk210) and Pvcsp(vk247) CDS using donor-DNA plasmids pLf0042 and pLf0043. A schematic representation of the Pfcsp locus before and after insertion of the construct showing the location of the restriction sites (A: AvaII, E: EcoRV), sizes (in bp) of restriction fragments (red for Southern blot analysis), location of primers (p), PCR amplicons and sizes (in bp) of the fragments (in black (b, c). HR1, HR2: Pfcsp homology (targeting) regions. The figure is not shown to scale. Primer sequences can be found in Additional file 2. b Diagnostic PCR and long-range PCR (LR-PCR) confirming the correct integration of the Pvcsp CDS into the PfCSP locus. Diagnostic PCR: Pfcsp open reading frame (lane 2; primers p17/p18); Pvcsp open reading frame (lane 3; primers p5/p6); P. falciparum sequestrin gene as a control gene (lane 1; primers p22/p23). LR-PCR: products were run undigested or digested with EcoRV (LR-PCR + E) in order to confirm double cross-over recombination. LR-PCR (lane 4) of cloned parasites of Pf-pvcsp(vk210)(cl7; primers p15/p16), Pf-pvcsp(vk247)(cl5; primers p15/p16) and WT. LR-PCR fragments digested with EcoRV (lane 5) for confirmation of double cross-over integration. c Southern blot analysis of AvaII restricted DNA of WT and chimeric Pf-Pvcsp parasites confirms the specific integration of the Pvcsp genes into the pfcsp gene locus. DNA was hybridized with a probe targeting the homology region 2 of pfcsp (upper panels; HR2; primers p3/p4; see a In addition, to show absence of donor-DNA plasmid and single cross-over events, DNA was hybridized with a probe for the ampicillin gene (lower panels; intermediate donor-DNA plasmid pLf0040 digested with AatII and PvuI). The hybridization pattern observed with the HR2 probe identified the expected different-sized DNA fragments in WT and pf-pvcsp parasites (2057 and 5294 bp)