Journal
ACS SENSORS
Volume 4, Issue 8, Pages 2065-2072Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssensors.9b00686
Keywords
nanopore sensing; Cas9; DNA barcode; single-molecule detection; biosensor
Funding
- Oxford Nanopore Technologies
- Canada UK Foundation
- University of Cambridge Office of Postdoctoral Affairs
- EPSRC
- Cambridge Trust
- Trinity Hall, Cambridge
- ERC Consolidator Grant [647144]
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Decorating double-stranded DNA with dCas9 barcodes to identify characteristic short sequences provides an alternative to fully sequencing DNA samples for rapid and highly specific analysis of a DNA sample. Solid state nanopore sensors are especially promising for this type of single molecule sensing because of the ability to analyze patterns in the ionic current signatures of DNA molecules. Here, we systematically demonstrate the use of highly specific dCas9 probes to create unique barcodes on the DNA that can be read out using nanopore sensors. Single dCas9 probes are targeted to various positions on DNA strands up to 48 kbp long and are effectively measured in high salt conditions typical of nanopore sensing. Multiple probes bound to the same DNA strand at characteristic target sequences create distinct barcodes of double and triple peaks. Finally, double and triple barcodes are used to simultaneously identify two different DNA targets in a background mixture of bacterial DNA. Our method forms the basis of a fast and versatile assay for multiplexed DNA sensing applications in complex samples.
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