Coupling of proteolysis-triggered transcription and CRISPR-Cas12a for ultrasensitive protease detection

The efficient signal amplification capacity of several class 2 CRISPR-Cas systems with trans-cleavage activity has exhibited great value in molecular diagnostics, but its potential application for non-nucleic-acid targets is yet underdeveloped. Here, we deploy CRISPR-Cas system for the ultrasensitive detection of protease biomarkers by the coupling of proteolysis-triggered transcription. In this strategy, a protease-activatable RNA polymerase is adopted for the conversion of each protease-catalyzed proteolysis event into the output of multiple programable RNA sequences by in vitro transcription, and the transcribed RNA subsequently serves as the guide RNA of Cas12a proteins with trans-cleavage activity. The rational design of the transcribed RNA efficiently couples the signal conversion and amplification of proteolysis-triggered transcription and the self-signal amplification of CRISPR-Cas12a, resulting in a two-stage amplified detection of target protease. The versatility of this strategy has been demonstrated in the detection of protease biomarkers including MMP-2 and thrombin with femtomolar sensitivity, which is 5–6 orders of magnitude lower than that of the standard peptide-based methods. Moreover, the proposed method has been further applied in the analysis of MMP-2 secreted by different cancer cell lines as well the assessment of MMP-2 activity in clinical serum samples, providing a generic method for the ultrasensitive detection of protease biomarkers in biochemical research and clinical diagnosis. [graphic not available: see fulltext]