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Plasmodium berghei high-throughput (PbHiT): a CRISPR-Cas9 system to study genes at scale
Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Instituto de Agrobiotecnología y Biología Molecular (IABIMO), INTA-CONICET, Buenos Aires, Hurlingham, Argentina.
Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).ORCID iD: 0000-0002-7745-2844
Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).ORCID iD: 0000-0003-2863-4112
2026 (English)In: Bio-protocol, E-ISSN 2331-8325, Vol. 16, no 2, article id e5572Article in journal (Refereed) Published
Abstract [en]

Genetic modification is essential for understanding parasite biology, yet it remains challenging in Plasmodium. This is partially due to the parasite’s low genetic tractability and reliance on homologous recombination, since the parasites lack the canonical non-homologous end-joining pathway. Existing approaches, such as the PlasmoGEM project, enable genome-wide knockouts but remain limited in coverage and flexibility. Here, we present the Plasmodium berghei high-throughput (PbHiT) system, a scalable CRISPR-Cas9 protocol for efficient genome editing in rodent malaria parasites. The PbHiT method uses a single cloning step to generate vectors in which a guide RNA (gRNA) is physically linked to short (100 bp) homology arms, enabling precise integration at the target locus upon transfection. The gRNA also serves as a unique barcode, allowing pooled vector transfections and identification of mutants by downstream gRNA sequencing. The PbHiT system reliably recapitulates known mutant growth phenotypes and supports both knockout and tagging strategies. This protocol provides a reproducible and scalable tool for genome editing in P. berghei, enabling both targeted functional studies and high-throughput genetic screens. Additionally, we provide an online resource covering the entire P. berghei protein-coding genome and describe a step-by-step pooled ligation approach for large-scale vector production.

Place, publisher, year, edition, pages
2026. Vol. 16, no 2, article id e5572
Keywords [en]
Apicomplexan, CRISPR-Cas9, Gene modification, High-throughput, Malaria, Plasmodium, Plasmodium berghei, Transfection
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-250569DOI: 10.21769/BioProtoc.5572ISI: 001676399900011PubMedID: 41607697Scopus ID: 2-s2.0-105030247594OAI: oai:DiVA.org:umu-250569DiVA, id: diva2:2045788
Funder
Swedish Research Council, 2021-06602Knut and Alice Wallenberg Foundation, 2019.0178Swedish Cancer SocietyAvailable from: 2026-03-13 Created: 2026-03-13 Last updated: 2026-03-13Bibliographically approved

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Jonsdottir, Thorey K.Paoletta, MartinaHenriksson, JohanBushell, Ellen

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Jonsdottir, Thorey K.Paoletta, MartinaHenriksson, JohanBushell, Ellen
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Molecular Infection Medicine Sweden (MIMS)Department of Molecular Biology (Faculty of Medicine)Umeå Centre for Microbial Research (UCMR)
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