title: Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping.
abstract: 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.
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
authors.forename: Vilhelm
authors.initials: V
authors.lastname: Müller
authors.affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.
authors.forename: Fredrika
authors.initials: F
authors.lastname: Rajer
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
authors.forename: Karolin
authors.initials: K
authors.lastname: Frykholm
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
authors.forename: Lena K
authors.initials: LK
authors.lastname: Nyberg
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.|Department of Astronomy and Theoretical Physics, Lund University, Lund, Sweden.
authors.forename: Saair
authors.initials: S
authors.lastname: Quaderi
authors.affiliation: Department of Applied Physics, Chalmers University of Technology, Gothenburg, Sweden.
authors.forename: Joachim
authors.initials: J
authors.lastname: Fritzsche
authors.affiliation: Department of Mathematical Sciences, Chalmers University of Technology/University of Gothenburg, Gothenburg, Sweden.|Centre for Antibiotic Resistance Research (CARe), University of Gothenburg, Gothenburg, Sweden.
authors.forename: Erik
authors.initials: E
authors.lastname: Kristiansson
authors.affiliation: Department of Astronomy and Theoretical Physics, Lund University, Lund, Sweden.
authors.forename: Tobias
authors.initials: T
authors.lastname: Ambjörnsson
authors.affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.
authors.forename: Linus
authors.initials: L
authors.lastname: Sandegren
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
authors.forename: Fredrik
authors.initials: F
authors.lastname: Westerlund
mesh_terms: D001419:Bacteria / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D064113:CRISPR-Cas Systems; D002874:Chromosome Mapping; D004269:DNA, Bacterial / Q000235:genetics; D004352:Drug Resistance, Microbial; D036103:Nanotechnology; D010957:Plasmids / Q000235:genetics*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D000072760:Single Molecule Imaging
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D000094704:RNA, Guide, CRISPR-Cas Systems