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Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping.

Vilhelm Müller (Institutionen för biologi och bioteknik) ; Fredrika Rajer ; Karolin Frykholm ; Lena K Nyberg ; Saair Quaderi ; Joachim Fritzsche ; Erik Kristiansson (Institutionen för matematiska vetenskaper, Tillämpad matematik och statistik) ; Tobias Ambjörnsson ; Linus Sandegren ; Fredrik Westerlund (Institutionen för biologi och bioteknik, Kemisk biologi)
Scientific reports (2045-2322). Vol. 6 (2016), p. 37938.
[Artikel, refereegranskad vetenskaplig]

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.



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Denna post skapades 2016-12-06. Senast ändrad 2017-02-17.
CPL Pubid: 245877

 

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