CRISPR/Cas9 system in Plasmodium falciparum using the centromere plasmid

Area of Science:

• Molecular Biology
• Parasitology
• Genetics

Background:

• CRISPR/Cas9 is a key tool for genetic modification of Plasmodium falciparum.
• Current methods using co-transfection of two plasmids show low modification efficiency due to low plasmid frequency.
• Improving gene editing efficiency is crucial for understanding malaria parasite biology.

Purpose of the Study:

• To develop an improved CRISPR/Cas9 system for efficient genetic modification of Plasmodium falciparum.
• To establish a transgenic parasite line, PfCAS9, for stable Cas9 nuclease expression.
• To assess the efficiency of the PfCAS9 system in targeted gene mutagenesis.

Main Methods:

• Development of a transgenic PfCAS9 parasite line expressing Cas9 nuclease via a centromere plasmid.
• Site-directed mutagenesis of the kelch13 gene, associated with artemisinin resistance.
• Co-transfection of the PfCAS9 parasite with a plasmid encoding sgRNA and donor template DNA.
• Treatment with drugs to maintain both centromere and sgRNA plasmids.

Main Results:

• The PfCAS9 parasite line stably expresses Cas9 nuclease.
• Targeted mutation of the kelch13 gene was achieved with nearly 100% efficiency.
• Drug treatment ensured the maintenance of both essential plasmids for high efficiency.

Conclusions:

• The novel PfCAS9 transgenic parasite line significantly enhances CRISPR/Cas9 gene editing efficiency in Plasmodium falciparum.
• This improved system facilitates precise genetic modification for malaria research.
• PfCAS9 offers a valuable tool for studying drug resistance and parasite biology.