Hairpin-locker mediated CRISPR/Cas tandem system for ultrasensitive detection of DNA without pre-amplification

Abstract

AbstractAchieving ultra-sensitive detection of DNA is of paramount importance in the field of molecular analytics. Conventional amplification technologies such as polymerase chain reaction (PCR) currently play a leading role in ultrasensitive DNA detection. However, amplicon contamination common in these techniques may lead to false positives. To date, CRISPR-associated nucleases (type V & VI) with their programmable cleavage have been utilised for sensitive detection of unamplified nucleic acids in complex real samples. Nevertheless, without additional amplification strategies, the pM range sensitivity of such CRISPR/Cas sensors is not sufficient for clinical applications. Here, we established a hairpin-locker (H-locker) mediated Cas12-Cas13 tandem biosensing system (Cas12-13 tandem-sensor) for ultrasensitive detection of DNA targets. Without the need for any additional amplification reaction or device, this system is capable of detecting DNA at a notable 1 aM level (<1 copy/uL) sensitivity. In addition, the system was able to distinguish cancer mutations in colorectal cancer (CRC) mice. This is a significant advance for CRISPR/Cas biosensing technology offering simple, highly sensitive, and user-friendly diagnostics for next-generation nucleic acid detection.