Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping.
Artikel i vetenskaplig tidskrift, 2016

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.

Författare

Vilhelm Müller

Chalmers, Biologi och bioteknik, Kemisk biologi

Fredrika Rajer

Uppsala universitet

Karolin Frykholm

Chalmers University of Technology

Lena Nyberg

Chalmers University of Technology

Mahmood Saair Quaderi

Lunds universitet

Chalmers University of Technology

Joachim Fritzsche

Chalmers University of Technology

Erik Kristiansson

Chalmers, Matematiska vetenskaper, Tillämpad matematik och statistik

Göteborgs universitet

T. Ambjornsson

Lunds universitet

L. Sandegren

Uppsala universitet

Fredrik Westerlund

Chalmers, Biologi och bioteknik, Kemisk biologi

Scientific Reports

2045-2322 (ISSN)

Vol. 6 37938- 37938

Drivkrafter

Hållbar utveckling

Innovation och entreprenörskap

Styrkeområden

Nanovetenskap och nanoteknik

Livsvetenskaper och teknik

Ämneskategorier

Biomedicinsk laboratorievetenskap/teknologi

Mikrobiologi inom det medicinska området

Kemi

DOI

10.1038/srep37938

PubMed

27905467