A scalable CRISPR-Cas9 gene editing system facilitates CRISPR screens in the malaria parasite Plasmodium berghei.
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Jonsdottir TK, Paoletta MS, Ishizaki T, Hernandez S, Ivanova M, Herrera Curbelo A, Saiki PA, Selinger M, Das D, Henriksson J, Bushell ESC
Nucleic Acids Res. 53 (2) - [2025-01-11; online 2025-01-23]
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.
NGI Stockholm (Genomics Production) [Service]
National Genomics Infrastructure [Service]
PubMed 39844455
DOI 10.1093/nar/gkaf005
Crossref 10.1093/nar/gkaf005
pmc: PMC11754126
pii: 7973899