A Cas9 transgenic Plasmodium yoelii parasite for efficient gene editing

Area of Science:

• Molecular Biology
• Parasitology
• Genetics

Background:

• The Streptococcus pyogenes Cas9 (SpCas9) is a powerful gene-editing tool.
• Its large size (4.2 kb) hinders its application in malaria parasites requiring plasmid introduction.
• Efficient gene editing in Plasmodium yoelii is crucial for understanding malaria pathogenesis.

Purpose of the Study:

• To establish endogenous and constitutive Cas9 expression in Plasmodium yoelii.
• To create a versatile platform for gene editing in P. yoelii.

Main Methods:

• CRISPR/Cas9-mediated genome editing was used to knock in SpCas9 into the sera1 gene locus.
• The resulting cas9-knockin parasite was named PyCas9ki.
• Gene modifications were achieved by introducing plasmids with sgRNA and homologous templates.

Main Results:

• The PyCas9ki parasite exhibited normal lifecycle progression.
• Cas9 protein was constitutively expressed in asexual blood stages.
• Successful gene deletion and tagging modifications were demonstrated in PyCas9ki parasites.

Conclusions:

• The PyCas9ki parasite offers a new, efficient platform for studying gene function in P. yoelii.
• This tool facilitates malaria parasite research and the development of new interventions.
• Endogenous Cas9 expression overcomes limitations of previous gene-editing methods in Plasmodium.