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Inducible ways to interfere with gene expression like the DiCre-lox system are necessary to study the function of these essential genes. However, existing DiCre-lox strategies are not well-suited to be deployed at scale to study several genes simultaneously. To overcome this, we have developed SHIFTiKO (frameshift-based trackable inducible knockout), a novel scaleable strategy that uses short, easy-to-construct, barcoded repair templates to insert loxP sites around short regions in target genes. Induced DiCre-mediated excision of the flanked region causes a frameshift mutation resulting in genetic ablation of gene function. Dual DNA barcodes inserted into each mutant enables verification of successful modification and induced excision at each locus and collective phenotyping of the mutants, not only across multiple replication cycles to assess growth fitness but also within a single cycle to identify specific phenotypic impairments. As a proof of concept, we have applied SHIFTiKO to screen the functions of malarial rhomboid proteases, successfully identifying their blood stage-specific essentiality. SHIFTiKO thus offers a powerful platform to conduct inducible phenotypic screens to study essential gene function at scale in the malaria parasite.<\/jats:p>","DOI":"10.1093\/nar\/gkae1274","type":"journal-article","created":{"date-parts":[[2024,12,12]],"date-time":"2024-12-12T07:25:42Z","timestamp":1733988342000},"source":"Crossref","is-referenced-by-count":7,"title":["A scaleable inducible knockout system for studying essential gene function in the malaria parasite"],"prefix":"10.1093","volume":"53","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9372-5526","authenticated-orcid":false,"given":"Abhinay","family":"Ramaprasad","sequence":"first","affiliation":[{"name":"Malaria Biochemistry Laboratory, The Francis Crick Institute ,\u00a0 1 Midland Road,\u00a0NW1 1AT London ,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael J","family":"Blackman","sequence":"additional","affiliation":[{"name":"Malaria Biochemistry Laboratory, The Francis Crick Institute ,\u00a0 1 Midland Road,\u00a0NW1 1AT London ,","place":["UK"]},{"name":"Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine , Keppel Street, WC1E 7HT London ,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2024,12,31]]},"reference":[{"key":"2025030421270006200_B1","doi-asserted-by":"crossref","first-page":"373","DOI":"10.1038\/nrmicro3450","article-title":"Advances in molecular genetic systems in malaria","volume":"13","author":"de\u00a0Koning-Ward","year":"2015","journal-title":"Nat. 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