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A scalable CRISPR-Cas9 gene editing system facilitates CRISPR screens in the malaria parasite Plasmodium berghei
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, de Los Reseros y Dr. Nicolás Repetto s/n, P.O. Box 25 (B1712WAA),Hurlingham, Buenos Aires, Argentina.
Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Parasitology and Zoology Unit, Department of Infection and Pathology, School of Veterinary Medicine, Rakuno Gakuen University, 582 Bunkyodai-midorimachi, Hokkaido, Ebetsu, Japan.
Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
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2025 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 53, no 2, article id gkaf005Article in journal (Refereed) Published
Abstract [en]

Many Plasmodium genes remain uncharacterized due to low genetic tractability. Previous large-scale knockout screens have only been able to target about half of the genome in the more genetically tractable rodent malaria parasite Plasmodium berghei. To overcome this limitation, we have developed a scalable CRISPR system called P. berghei high-throughput (PbHiT), which uses a single cloning step to generate targeting vectors with 100-bp homology arms physically linked to a guide RNA (gRNA) that effectively integrate into the target locus. We show that PbHiT coupled with gRNA sequencing robustly recapitulates known knockout mutant phenotypes in pooled transfections. Furthermore, we provide an online resource of knockout and tagging designs to target the entire P. berghei genome and scale-up vector production using a pooled ligation approach. This work presents for the first time a tool for high-throughput CRISPR screens in Plasmodium for studying the parasite’s biology at scale.

Place, publisher, year, edition, pages
Oxford University Press, 2025. Vol. 53, no 2, article id gkaf005
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-235698DOI: 10.1093/nar/gkaf005ISI: 001402022200002PubMedID: 39844455Scopus ID: 2-s2.0-85216463244OAI: oai:DiVA.org:umu-235698DiVA, id: diva2:1940329
Funder
Swedish Research Council, 2021-06602Knut and Alice Wallenberg Foundation, 2019.0178Swedish Cancer Society, 23 3102 PjAvailable from: 2025-02-26 Created: 2025-02-26 Last updated: 2025-05-12Bibliographically approved
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Jonsdottir, Thorey K.Paoletta, MartinaIshizaki, TakahiroHernandez, Sophia Raine C.Ivanova, MariaHerrera Curbelo, AliciaSelinger, MartinDas, DebojyotiHenriksson, JohanBushell, Ellen

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Jonsdottir, Thorey K.Paoletta, MartinaIshizaki, TakahiroHernandez, Sophia Raine C.Ivanova, MariaHerrera Curbelo, AliciaSelinger, MartinDas, DebojyotiHenriksson, 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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Nucleic Acids Research
Cell and Molecular Biology

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