CRISPR-Cas9 Genome Editing of Plasmodium knowlesi

Area of Science:

• Malariology
• Parasitology
• Molecular Biology

Background:

• Plasmodium knowlesi is a zoonotic malaria parasite prevalent in Southeast Asia.
• It causes severe and fatal malaria in humans, with increasing reported infections.
• P. knowlesi is the only other malaria parasite, besides P. falciparum, that can be cultured long-term in vitro.

Purpose of the Study:

• To develop a novel, marker-free CRISPR gene-editing system for P. knowlesi.
• To establish a fast and scalable method for generating transfection constructs.
• To analyze transfection efficiencies in P. knowlesi.

Main Methods:

• Generation of marker-free CRISPR gene-edited P. knowlesi parasites.
• Development of a rapid and scalable protocol for producing transfection constructs.
• Analysis of transfection efficiencies using a single, recyclable selection marker.

Main Results:

• Successful generation of marker-free CRISPR gene-edited P. knowlesi.
• Demonstration of fast and scalable production of transfection constructs.
• High transfection efficiencies achieved with the new protocol.

Conclusions:

• The developed protocol allows for rapid, reliable, and unlimited genome editing in P. knowlesi.
• This method offers significant advantages for studying malaria parasite cell and molecular biology.
• The system requires only a single recyclable selection marker for efficient genome editing.