Pushing the resolution of dCas9 barcodes for multiplexed DNA identification with nanopore sensors

Double-stranded DNA can rapidly and specifically be identified using dCas9 barcodes that can be read using solid-state nanopore sensors. We have previously shown that multiple highly specific dCas9 probes bound to the same dsDNA strand create distinct double and triple peak barcodes in the ionic current signature as it translocates through a nanopore. The design of these barcodes relies on the identification of distinct peak patterns in the signals caused by neighbouring bound dCas9 probes. Here, we demonstrate the design of multiple pairs of dCas9 probes that create clear double peak barcodes on lambda and T7 DNA. We investigate how closely together dCas9 probes can be bound on dsDNA while still creating resolvable peak signatures measured using glass nanopores. We further explore how varying the spacing (from approximately 200-900 bp) between the dCas9 probes affects the average time between consecutive peaks in the ionic current traces for a given measurement. Our results lay the foundation for future unique barcode designs that form the basis of a fast and versatile assay for multiplexed DNA sensing and identification applications in complex samples.