Abstract
Tackling relapsing Plasmodium vivax and zoonotic Plasmodium knowlesi infections is critical to reducing malaria incidence and mortality worldwide. Understanding the biology of these important and related parasites was previously constrained by the lack of robust molecular and genetic approaches. Here, we establish CRISPR-Cas9 genome editing in a culture-adapted P. knowlesi strain and define parameters for optimal homology-driven repair. We establish a scalable protocol for the production of repair templates by PCR and demonstrate the flexibility of the system by tagging proteins with distinct cellular localisations. Using iterative rounds of genome-editing we generate a transgenic line expressing P. vivax Duffy binding protein (PvDBP), a lead vaccine candidate. We demonstrate that PvDBP plays no role in reticulocyte restriction but can alter the macaque/human host cell tropism of P. knowlesi. Critically, antibodies raised against the P. vivax antigen potently inhibit proliferation of this strain, providing an invaluable tool to support vaccine development.
Highlights
Malaria remains a serious health burden globally, with over 216 million cases annually (WHO, 2018)
Host cell invasion by P. vivax and P. knowlesi relies on the Duffy binding proteins (DBP) P. vivax Duffy binding protein (PvDBP) and PkDBPa, respectively, both ligands for human red blood cell (RBC) Duffy antigen/receptor for chemokines (DARC)
Whilst a variety of approaches have been used in P. falciparum, many of the earlier methods embed these elements into two plasmids, each expressing a different drug-selectable marker (Ghorbal et al, 2014; Crawford et al, 2017; Mogollon et al, 2016)
Summary
Malaria remains a serious health burden globally, with over 216 million cases annually (WHO, 2018). Using an optimised and scalable PCR-based approach for generating targeting constructs we define critical parameters determining effective genome editing and apply the technique to introduce epitope/fluorescent protein tags to a variety of proteins with distinct cellular locations We use these tools to replace the P. knowlesi PkDBPa gene with its PvDBP orthologue, and delete the P. knowlesi DBP paralogues to create a transgenic P. knowlesi line reliant on the PvDBP protein for invasion of RBCs. The additional deletion of the PkDBP paralogues excludes interference through antibody cross-reactivity during growth inhibition assays, and allows us to demonstrate that, in contrast to previous findings (Ovchynnikova et al, 2017), PvDBP plays no role in reticulocyte restriction, but has an effect on macaque/human host cell preference. We have developed a robust and flexible system for genome editing in an important human malaria parasite and generated essential new tools to accelerate both basic and applied malaria research
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