Ramaprasad Abhinay, Blackman Michael J
Malaria Biochemistry Laboratory, The Francis Crick Institute, 1 Midland Road, NW1 1AT London, UK.
Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, WC1E 7HT London, UK.
Nucleic Acids Res. 2025 Feb 8;53(4). doi: 10.1093/nar/gkae1274.
The malaria parasite needs nearly half of its genes to propagate normally within red blood cells. 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.
疟原虫在红细胞内正常繁殖需要近一半的基因。像DiCre-lox系统这样可诱导干扰基因表达的方法对于研究这些必需基因的功能是必要的。然而，现有的DiCre-lox策略不太适合大规模应用以同时研究多个基因。为了克服这一问题，我们开发了SHIFTiKO（基于移码的可追踪诱导敲除），这是一种新型的可扩展策略，它使用短的、易于构建的条形码修复模板在靶基因的短区域周围插入loxP位点。诱导的DiCre介导的侧翼区域切除会导致移码突变，从而导致基因功能的遗传消融。插入每个突变体的双DNA条形码能够验证每个位点的成功修饰和诱导切除，并对突变体进行集体表型分析，不仅可以在多个复制周期中评估生长适应性，还可以在单个周期内识别特定的表型损伤。作为概念验证，我们应用SHIFTiKO筛选了疟原虫菱形蛋白酶的功能，成功鉴定了它们在血液阶段的特异性必需性。因此，SHIFTiKO提供了一个强大的平台，可进行诱导性表型筛选，以大规模研究疟原虫中必需基因的功能。
