CRISPR-Cas9-Based Toolkit for Clostridium botulinum Group II Spore and Sporulation Research

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dc.contributor University of Helsinki, Miia Lindström / Principal Investigator en
dc.contributor University of Helsinki, Food Hygiene and Environmental Health en
dc.contributor University of Helsinki, Food Hygiene and Environmental Health en
dc.contributor University of Helsinki, Miia Lindström / Principal Investigator en
dc.contributor.author Mertaoja, Anna
dc.contributor.author Nowakowska, Maria B.
dc.contributor.author Mascher, Gerald
dc.contributor.author Heljanko, Viivi
dc.contributor.author Groothuis, Daphne
dc.contributor.author Minton, Nigel P.
dc.contributor.author Lindström, Miia
dc.date.accessioned 2021-05-04T12:48:01Z
dc.date.available 2021-05-04T12:48:01Z
dc.date.issued 2021-01-27
dc.identifier.citation Mertaoja , A , Nowakowska , M B , Mascher , G , Heljanko , V , Groothuis , D , Minton , N P & Lindström , M 2021 , ' CRISPR-Cas9-Based Toolkit for Clostridium botulinum Group II Spore and Sporulation Research ' , Frontiers in Microbiology , vol. 12 , 617269 . https://doi.org/10.3389/fmicb.2021.617269 en
dc.identifier.issn 1664-302X
dc.identifier.other PURE: 159596902
dc.identifier.other PURE UUID: fb00080d-4943-49eb-b05e-391a4d0350a8
dc.identifier.other WOS: 000616516100001
dc.identifier.uri http://hdl.handle.net/10138/329627
dc.description.abstract The spores of Clostridium botulinum Group II strains pose a significant threat to the safety of modern packaged foods due to the risk of their survival in pasteurization and their ability to germinate into neurotoxigenic cultures at refrigeration temperatures. Moreover, spores are the infectious agents in wound botulism, infant botulism, and intestinal toxemia in adults. The identification of factors that contribute to spore formation is, therefore, essential to the development of strategies to control related health risks. Accordingly, development of a straightforward and versatile gene manipulation tool and an efficient sporulation-promoting medium is pivotal. Our strategy was to employ CRISPR-Cas9 and homology-directed repair (HDR) to replace targeted genes with mutant alleles incorporating a unique 24-nt “bookmark” sequence that could act as a single guide RNA (sgRNA) target for Cas9. Following the generation of the sporulation mutant, the presence of the bookmark allowed rapid generation of a complemented strain, in which the mutant allele was replaced with a functional copy of the deleted gene using CRISPR-Cas9 and the requisite sgRNA. Then, we selected the most appropriate medium for sporulation studies in C. botulinum Group II strains by measuring the efficiency of spore formation in seven different media. The most effective medium was exploited to confirm the involvement of a candidate gene in the sporulation process. Using the devised sporulation medium, subsequent comparisons of the sporulation efficiency of the wild type (WT), mutant and “bookmark”-complemented strain allowed the assignment of any defective sporulation phenotype to the mutation made. As a strain generated by complementation with the WT gene in the original locus would be indistinguishable from the parental strain, the gene utilized in complementation studies was altered to contain a unique “watermark” through the introduction of silent nucleotide changes. The mutagenesis system and the devised sporulation medium provide a solid basis for gaining a deeper understanding of spore formation in C. botulinum, a prerequisite for the development of novel strategies for spore control and related food safety and public health risk management. en
dc.format.extent 12
dc.language.iso eng
dc.relation.ispartof Frontiers in Microbiology
dc.rights en
dc.subject CRISPR-Cas9 en
dc.subject Clostridium botulinum Group II en
dc.subject spo0A en
dc.subject spore en
dc.subject sporulation medium en
dc.subject 11832 Microbiology and virology en
dc.title CRISPR-Cas9-Based Toolkit for Clostridium botulinum Group II Spore and Sporulation Research en
dc.type Article
dc.description.version Peer reviewed
dc.identifier.doi https://doi.org/10.3389/fmicb.2021.617269
dc.type.uri info:eu-repo/semantics/other
dc.type.uri info:eu-repo/semantics/publishedVersion
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