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 Casl2a 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-Casl2a,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.