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Robust and stable transcriptional repression in Giardia using CRISPRi

By Shane G. McInally, KD Hagen, C Nosala, J Williams, K. Nguyen, J Booker, K. Jones, C. Dawson Scott

Posted 28 Jun 2018
bioRxiv DOI: 10.1101/358598 (published DOI: 10.1091/mbc.e18-09-0605)

Giardia is a widely prevalent yet understudied protistan parasite causing significant diarrheal disease worldwide. Despite the availability of several sequenced genomes, basic and applied Giardia research has lagged behind other parasites due to a lack of molecular genetic tools for functional genomic research. While used extensively in other non-model eukaryotes, the adaptation of CRISPR/Cas9-mediated genome editing has been challenging in Giardia due to two transcriptionally-active diploid nuclei, the lack of a non-homologous end-joining (NHEJ), few positive selectable markers, and an inability to target Cas9 to both nuclei. Yet precise, stable, and tunable transcriptional repression is also an essential tool to interrogate gene function. CRISPR interference (CRISPRi) is a modification of the CRISPR/Cas9 system using a catalytically inactive Cas9 (dCas9) to induce gene knockdowns by site-specific transcriptional repression. CRISPRi is stable, modular, inducible, and reversible and thus has significant advantages over existing transient methods of gene disruption in Giardia. To adapt CRISPRi, we first defined a native Giardia nuclear localization signal (NLS) that is necessary and sufficient to target Cas9 or dCas9 to both nuclei. CRISPRi-mediated knockdown using single or multiple gRNAs allowed us to efficiently and stably repress one exogenous reporter gene and three endogenous cytoskeletal genes important for pathogenesis. Each endogenous gene knockdown had significant flagellar or ventral disc defects indicating that Giardia CRISPRi is robust, stable and flexible method of transcriptional repression. This tunable knockdown strategy will transform molecular genetic studies in Giardia, permitting the targeted knockdown of one or more genes, as well genome-scale functional genetic profiling.

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