A universal CRISPR/Cas9-based electrochemiluminescence probe for sensitive and single-base-specific DNA detection

Electrochemiluminescence (ECL) is a sensitive detection technology. Commercial ECL analyzer democratizes ECL as a convenient tool for clinical immunoassay. Still, commercial DNA ECL detection kits are absent, because oligonucleotide-based ECL probes often lack double-stranded DNA (dsDNA) recognition capability and specificity. In this study, we harnessed CRISPR/dCas9 as a universal electrochemiluminescence (ECL) probe for sensitive and single-base-specific DNA detection. Benefiting from the unique characteristics of Cas9, including flexible programmability, dsDNA binding ability and high specificity, the dCas9-ECL probe can directly recognize dsDNA amplification products under constant and mild conditions, and provide higher signal-to-background ratio. Experimental results demonstrated our method can detect 38 cfu‧mL-1 genome of Listeria monocytogenes. Only changing the targeting region of the sgRNA, the probe can be employed to detect 0.1 pg‧μL-1 EGFR L858R mutant with a mutation discrimination selectivity factor of 0.01%. Moreover, the dCas9-ECL probe can be used for DNA detection in complex samples (e.g. milk & cell extracts). In conclusion, the dCas9-ECL probe could be formed into a universal DNA detection kit and be compatible with automated and high-throughput ECL immunoassay analyzers, thus holds a certain application potential in pathogen detection and gene typing.