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

• 作者: Min Yang Kai Shi Fang Liu Wenyuan Kang Chunyang Lei Zhou Nie
• 来源期刊: 中国科学：化学（英文版）
• 年，卷（期）: 2021,（2）
• 所属期刊栏目: ARTICLES
• 页码范围: 330-336
• 页数: 7页
• 语种: 英文

摘要

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.