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Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping
Chalmers, Dept Biol & Biol Engn, Gothenburg, Sweden..
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Chalmers, Dept Biol & Biol Engn, Gothenburg, Sweden..
Chalmers, Dept Biol & Biol Engn, Gothenburg, Sweden..
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2016 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 37938Article in journal (Refereed) Published
Abstract [en]

Bacterial plasmids are extensively involved in the rapid global spread of antibiotic resistance. We here present an assay, based on optical DNA mapping of single plasmids in nanofluidic channels, which provides detailed information about the plasmids present in a bacterial isolate. In a single experiment, we obtain the number of different plasmids in the sample, the size of each plasmid, an optical barcode that can be used to identify and trace the plasmid of interest and information about which plasmid that carries a specific resistance gene. Gene identification is done using CRISPR/Cas9 loaded with a guide-RNA (gRNA) complementary to the gene of interest that linearizes the circular plasmids at a specific location that is identified using the optical DNA maps. We demonstrate the principle on clinically relevant extended spectrum beta-lactamase (ESBL) producing isolates. We discuss how the gRNA sequence can be varied to obtain the desired information. The gRNA can either be very specific to identify a homogeneous group of genes or general to detect several groups of genes at the same time. Finally, we demonstrate an example where we use a combination of two gRNA sequences to identify carbapenemase-encoding genes in two previously not characterized clinical bacterial samples.

Place, publisher, year, edition, pages
2016. Vol. 6, article id 37938
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:uu:diva-311501DOI: 10.1038/srep37938ISI: 000388980000001PubMedID: 27905467OAI: oai:DiVA.org:uu-311501DiVA, id: diva2:1060440
Funder
EU, Horizon 2020, 634890Torsten Söderbergs stiftelseÅke Wiberg FoundationSwedish Research Council, 2014-4305 K2013-99X-22208-01-5Available from: 2016-12-28 Created: 2016-12-28 Last updated: 2018-01-13Bibliographically approved

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