Plasmodium berghei High-Throughput (PbHiT): a CRISPR-Cas9 System to Study Genes at Scale

Area of Science:

• Molecular Biology
• Parasitology
• Genetics

Background:

• Genetic modification is crucial for understanding Plasmodium parasite biology but faces challenges due to low genetic tractability and reliance on homologous recombination.
• Existing methods like PlasmoGEM have limitations in coverage and flexibility for genome-wide knockouts.

Purpose of the Study:

• To develop a scalable CRISPR-Cas9 protocol for efficient genome editing in Plasmodium berghei.
• To create a high-throughput system for generating knockout and tagged parasite lines.
• To provide a reproducible tool for functional studies and genetic screens in rodent malaria parasites.

Main Methods:

• The Plasmodium berghei high-throughput (PbHiT) system utilizes a single cloning step to link guide RNA (gRNA) with short homology arms (100 bp).
• Pooled vector transfections are enabled by gRNA barcoding, allowing mutant identification via downstream gRNA sequencing.
• The protocol supports both knockout and tagging strategies for genome editing.

Main Results:

• The PbHiT system demonstrates efficient and reproducible genome editing in P. berghei.
• It reliably recapitulates known mutant growth phenotypes.
• The method supports high-throughput generation of knockout and tagged parasite lines.

Conclusions:

• The PbHiT system provides a scalable and efficient CRISPR-Cas9 platform for P. berghei genome editing.
• This protocol facilitates targeted functional studies and high-throughput genetic screens.
• An online resource for P. berghei gene construct design is available to support the protocol.