Plasmodium berghei high-throughput (PbHiT): a CRISPR-Cas9 system to study genes at scale
2026 (Engelska)Ingår i: Bio-protocol, E-ISSN 2331-8325, Vol. 16, nr 2, artikel-id e5572Artikel i tidskrift (Refereegranskat) 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.
Ort, förlag, år, upplaga, sidor
2026. Vol. 16, nr 2, artikel-id e5572
Nyckelord [en]
Apicomplexan, CRISPR-Cas9, Gene modification, High-throughput, Malaria, Plasmodium, Plasmodium berghei, Transfection
Nationell ämneskategori
Cell- och molekylärbiologi
Identifikatorer
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
Forskningsfinansiär
Vetenskapsrådet, 2021-06602Knut och Alice Wallenbergs Stiftelse, 2019.0178Cancerfonden2026-03-132026-03-132026-03-13Bibliografiskt granskad