Bifunctional circular DNA amplifier transforms a classic CRISPR/Cas sensor into an ultrasensitive autocatalytic sensor

The CRISPR/Cas sensors represent a new approach in molecular diagnostics. Based on the programmable trans-cleavage of Type V & VI Cas proteins to degrade single-strand DNA/RNA reporters they detect nucleic acids with single-nucleotide specificity. However, their clinical usefulness is limited by their pM level sensitivity, which is insufficient unless integrated with preamplification. To overcome this limitation, we designed a bifunctional circular DNA amplifier to report nucleic acid detection events and simultaneously facilitate an autocatalytic reaction network. This minimalistic design converts a classic CRISPR/Cas sensor into a DNA amplifier-enhanced CRISPR/Cas autocatalytic sensor (DANCER), without the need for additional amplification reaction or device. DANCER achieved 1 aM level (6 times improvement), within 15 mins, at room temperature, and with 11 orders of magnitude detection range. When combined with a colorimetric lateral flow assay, DANCER realized point-of-care testing of circulating tumor DNA in plasma of a colorectal cancer-mouse.