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Implementation of a tunable t-CRISPRi system for gene regulation in Giardia duodenalis
Department of Genetics and Molecular Biology, Centro de Investigaciones y Estudios Avanzados (CINVESTAV), Ave. IPN #2508, San Pedro Zacatenco, Gustavo A. Madero, 07360 Mexico City, Mexico.ORCID iD: 0000-0001-7730-6452
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2022 (English)In: Plasmid, ISSN 0147-619X, E-ISSN 1095-9890, article id 102641Article in journal, News item (Refereed) In press
Abstract [en]

Giardia duodenalis, is a binuclear and microaerophilic protozoan that causes giardiasis. Up to date, several molecular approaches have been taken to understand the molecular mechanisms of diverse cellular processes in this parasitic protozoan. However, the role of many genes involved in these processes needs further analysis. The CRISPR interference (CRISPRi) system has been widely used, as a constitutive expression system for gene silencing purposes in several parasites, including Giardia. The aim of this work was to implement a tunable t-CRISPRi system in Giardia to silence abundant, moderately and low expressed genes, by constructing an optimized and inducible plasmid for the expression of both gRNA and dCas9. A doxycycline inducible pRan promoter was used to express dCas9 and each gRNA, consistently dCas9 expression and nuclear localization were confirmed by Western-blot and immunofluorescence in transfected trophozoites. The transcriptional repression was performed on α-tubulin (high expression), giardipain-1 (moderate expression) and Sir2 and Sir4 (low expression) genes. The α-tubulin gene knock-down caused by dCas9 doxycycline-induction was confirmed by a decrease in its protein expression which was of 50% and 60% at 24 and 48 h, respectively. This induced morphological alterations in flagella. The giardipain-1 knock down, showed a decrease in protein expression of 40 and 50% at 12 and 24 h, respectively, without affecting trophozoites viability, consistent with this a zymogram analysis on giardipain-1 knock down revealed a decrease in giardipain-1 protease activity. When repressing sirtuins expression, a total repression was obtained but trophozoites viability was compromised. This approach provides a molecular tool for a tailored repression to produce specific gene knockdowns.

Place, publisher, year, edition, pages
2022. article id 102641
Keywords [en]
Giardia, CRISPRi, gene knock-down, doxycycline-induced
National Category
Biochemistry Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-481347DOI: 10.1016/j.plasmid.2022.102641OAI: oai:DiVA.org:uu-481347DiVA, id: diva2:1686219
Available from: 2022-08-09 Created: 2022-08-09 Last updated: 2025-02-20

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Lagunas-Rangel, Francisco Alejandro

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