Crispr/Cas12a Collateral Cleavage-Driven Transcription Amplification for Direct Nucleic Acid Detection

The CRISPR/Cas system is a powerful tool for nucleic acid detection owing to specific recognition as well as cis- and trans- cleavage capabilities. However, the sensitivity of CRISPR/Cas-based diagnostic approaches is determined by nucleic acid pre-amplification, which has several limitations. Here, we present a method for direct nucleic acid detection without pre-amplification, by combining the CRISPR/Cas12a system with signal enhancement based on light-up aptamer transcription. We first designed two templates to transcribe the light-up aptamer and kleptamer (Kb): the first template encodes a Broccoli RNA aptamer for fluorescence signal generation and the Kb template comprises a dsDNA T7 promoter sequence and a ssDNA sequence that is an antisense strand for the Broccoli aptamer. Hepatitis B virus (HBV) target recognition activates a CRISPR/Cas complex, leading to catalytic cleavage of the ssDNA sequence. Transcription of the added Broccoli aptamer template can then produce several Broccoli aptamer transcripts for fluorescence enhancement. The proposed strategy exhibited excellent sensitivity and specificity with a 22.4 fM detection limit, good accuracy, and stability for determining the target HBV DNA in human serum samples. Overall, this newly designed signal enhancement strategy can be employed as a universal sensing platform for ultrasensitive nucleic acid detection.