A heterologous marker-free selection approach for CRISPR/Cas9-based gene editing in the malaria parasite Plasmodium falciparum - PubMed
5 hours ago
- #CRISPR/Cas9
- #Gene Editing
- #Malaria Research
- CRISPR/Cas9pyrR is a heterologous marker-free gene editing strategy developed for Plasmodium falciparum.
- The method introduces pyrimethamine resistance mutations into the pfdhfr-ts gene, allowing selection without relying on limited heterologous drug markers.
- It uses separate plasmids for the donor sequence and Cas9 expression, ensuring only parasites with both survive under pyrimethamine pressure.
- Proof-of-principle involved tagging proteins like PfGEX1-GFP and PfNUP116-GFP, revealing dynamic nuclear changes during gametocyte differentiation.
- Sequential engineering demonstrated by tagging PfAP2-G and PfNUP313 in a PfNUP116-GFP line, using human dhfr with WR99210 selection.
- This approach enhances iterative genetic modifications, complementing existing CRISPR/Cas9 tools for malaria research.