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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\u2019s biology at scale.<\/jats:p>","DOI":"10.1093\/nar\/gkaf005","type":"journal-article","created":{"date-parts":[[2025,1,9]],"date-time":"2025-01-09T07:34:47Z","timestamp":1736408087000},"source":"Crossref","is-referenced-by-count":7,"title":["A scalable CRISPR-Cas9 gene editing system facilitates CRISPR screens in the malaria parasite\n                    <i>Plasmodium berghei<\/i>"],"prefix":"10.1093","volume":"53","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0618-4731","authenticated-orcid":false,"given":"Thorey\u00a0K","family":"Jonsdottir","sequence":"first","affiliation":[{"name":"The Laboratory for Molecular Infection Medicine Sweden, Ume\u00e5 University , F\u00f6rs\u00f6rjningsv\u00e4gen 2A, 901\u00a087 Ume\u00e5,","place":["Sweden"]},{"name":"Department of Molecular Biology, Ume\u00e5 University ,\u00a0F\u00f6rs\u00f6rjningsv\u00e4gen 2A, 901\u00a087 Ume\u00e5,","place":["Sweden"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7318-489X","authenticated-orcid":false,"given":"Martina\u00a0S","family":"Paoletta","sequence":"additional","affiliation":[{"name":"The Laboratory for Molecular Infection Medicine Sweden, Ume\u00e5 University , F\u00f6rs\u00f6rjningsv\u00e4gen 2A, 901\u00a087 Ume\u00e5,","place":["Sweden"]},{"name":"Department of Molecular Biology, Ume\u00e5 University ,\u00a0F\u00f6rs\u00f6rjningsv\u00e4gen 2A, 901\u00a087 Ume\u00e5,","place":["Sweden"]},{"name":"Instituto de Agrobiotecnolog\u00eda y Biolog\u00eda Molecular (IABIMO), INTA\u2013CONICET , de Los Reseros y Dr. Nicol\u00e1s Repetto s\/n, P.O. 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