CRISPR/Cas9 genome editing to generate single variant Plasmodium falciparum lines and enable reverse genetic studies of PfEMP1 function in live parasites

Adhesion interactions between Plasmodium falciparum infected erythrocytes (IEs) and human cells bring about microvascular sequestration and contribute to severe malaria pathology. Parasite adhesion molecules on the IE surface are members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family, encoded by var genes, which interact with receptors on human cells. Progress in understanding PfEMP1-host receptor interaction is hindered by the lack of genetic tools for PfEMP1 functional studies in live parasites and the spontaneous switching of var gene transcription in culture leading to change in adhesion phenotype. We developed a CRISPR/Cas9 genome editing strategy that takes advantage of var gene mutually exclusive expression to generate single variant P. falciparum lines and enable reverse genetic studies of PfEMP1 function. A drug resistance gene and 2A peptide enabling bi-cistronic transcription were inserted between the promoter and exon I of the it4var60 gene encoding a PfEMP1 variant that mediates the virulence-associated rosetting phenotype. After genome editing and drug selection, only it4var60-transcribing parasites survived, and >90% of IEs expressed IT4VAR60-PfEMP1 on their surface and formed rosettes. When drug pressure was removed, switching to other variants occurred. The approach was adapted to generate epitope tagged-PfEMP1 allowing immunofluorescent detection with commercial antibodies, and modifications of the homology directed repair template enabled investigation of PfEMP1 function including point mutations and a gene knockout that abolished adhesion. These methods can be applied to any var gene in any P. falciparum genotype and are potentially transformative for functional studies of multi-gene family members in live parasites.