A genome-scale vector resource enables high-throughput reverse genetic screening in a malaria parasite

Area of Science:

• Malariology
• Genetics
• Molecular Biology

Background:

• Genome-wide identification of gene functions in malaria parasites is challenging due to limited reverse genetic screening tools.
• Developing efficient methods for genetic modification is crucial for understanding Plasmodium berghei biology.

Purpose of the Study:

• To create a large-scale resource of barcoded vectors for modifying the Plasmodium berghei genome.
• To establish a high-throughput screening method for assessing gene function in malaria parasites.

Main Methods:

• Development and utilization of barcoded vectors with long homology arms for Plasmodium berghei genome editing.
• Cotransfection of multiple vectors into haploid blood stages to create pooled barcoded mutants.
• Competitive fitness assessment using barcode sequencing (barseq) during single-mouse infections.
• Rescreening of the Plasmodium berghei kinome and validation of specific kinase targets.

Main Results:

• A resource of barcoded vectors enabling effective Plasmodium berghei genome modification was established.
• Barcode sequencing (barseq) allows for parallel phenotyping and competitive fitness measurement of barcoded mutants.
• Redundant functions of several protein kinases in asexual blood stages were identified.
• Targetability of kinases cdpk1, gsk3, tkl3, and PBANKA_082960 was confirmed through mutant genotyping.

Conclusions:

• Parallel phenotyping of barcoded mutants using barseq unlocks powerful reverse genetic screening for malaria parasites.
• This approach will facilitate the systematic identification of genes critical for in vivo parasite growth and transmission.
• The developed resource and method advance the study of malaria parasite genetics and drug target discovery.