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.
Highlights
Slow and/or require existing knowledge of the targeted sequence
We have previously developed a single-step optical DNA mapping assay, that is based on competitive binding between the fluorescent dye YOYO-1 (YOYO) and the AT-selective molecule netropsin, both commercially available at low cost[12,13]
We demonstrate the assay on several important resistance genes including the extended spectrum beta-lactamase (ESBL) gene family blaCTX-M
Summary
Slow (requiring days or weeks for completion for S1/PFGE and conjugational approaches) and/or require existing knowledge of the targeted sequence (as for PCR-based or hybridization-based methods). The enzyme uses a 20 basepair (bp) RNA-sequence to direct the Cas[9] enzyme to a specific sequence and cut the DNA backbone on both strands at this location. This 20 bp sequence is part of the guide RNA (gRNA) that binds to Cas[9] to guide it to the correct sequence. McCaffrey et al used a mutated version of Cas[9] (D10A) that only cuts one of the strands and subsequently repaired the nick(s) with a fluorescent nucleobase to visualize specific genes on DNA stretched in nanochannels[17]. The horizontal lines in the histograms correspond to the mean value for the control experiments (dashed line) and three standard deviations above the mean for all experiments (solid lines) from the balls-in-boxes statistics
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