| Title: |
Plasmodium berghei high-throughput (PbHiT) : a CRISPR-Cas9 system to study genes at scale |
| Authors: |
Jonsdottir, Thorey K.; Paoletta, Martina; Henriksson, Johan; Bushell, Ellen |
| Publisher Information: |
Umeå universitet, Molekylär Infektionsmedicin, Sverige (MIMS); Umeå universitet, Institutionen för molekylärbiologi (Medicinska fakulteten); Umeå universitet, Umeå Centre for Microbial Research (UCMR); Instituto de Agrobiotecnología y Biología Molecular (IABIMO), INTA-CONICET, Buenos Aires, Hurlingham, Argentina |
| Publication Year: |
2026 |
| Collection: |
Umeå University: Publications (DiVA) |
| Subject Terms: |
Apicomplexan; CRISPR-Cas9; Gene modification; High-throughput; Malaria; Plasmodium; Plasmodium berghei; Transfection; Cell and Molecular Biology; Cell- och molekylärbiologi |
| Description: |
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. |
| Document Type: |
article in journal/newspaper |
| File Description: |
application/pdf |
| Language: |
English |
| Relation: |
Bio-protocol, 2026, 16:2; PMID 41607697; ISI:001676399900011 |
| DOI: |
10.21769/BioProtoc.5572 |
| Availability: |
http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-250569; https://doi.org/10.21769/BioProtoc.5572 |
| Rights: |
info:eu-repo/semantics/openAccess |
| Accession Number: |
edsbas.5918040C |
| Database: |
BASE |