CRISPR-Cas12a enhanced rolling circle amplification method for ultrasensitive miRNA detection

• Published in: Microchemical journal 2020-11, Vol.158, p.105239, Article 105239
• Main Authors: Zhang, Gong, Zhang, Lei, Tong, Jingtao, Zhao, Xianxian, Ren, Jianlin
• Format: Article
• Language: English
• Subjects: CRISPR-Cas12a; Disease diagnosis; Exosomal microRNAs; Rolling circle amplification
• ISSN: 0026-265X; 1095-9149
• DOI: 10.1016/j.microc.2020.105239

•A high specific method for molecular exosomal miRNAs detection in constant temperature.•CRISPR/Cas12a is responsible for ultrasensitive nucleic acid signal amplification, while ensuring high specificity.•This method provides a new strategy to improve sensitivity of rolling circle amplification (RCA) based methods. MicroRNAs (miRNAs) detection with high specificity and sensitivity received abundant attention because miRNAs have been reported to play a vital role in pathological development of many diseases and regarded as potential biomarkers for the diagnosis and prognosis of diseases. We reported here a highly specific method for molecular exosomal miRNAs detection in constant temperature by integrating the advantages of CRISPR/Cas system and rolling circular amplification (RCA) techniques. Especially, the proposed strategy was demonstrated to obtain a high sensitivity attributed to the dual-specific recognition from miRNA-padlock initiated RCA and CRISPR-Cas12a-triggered specific cleavage. Eventually, the proposed strategy showed a sensitivity of 34.7 fM which was robust enough for exosomal miRNA detection and obtained a high consistency with reverse transcription quantitative polymerase chain reaction (RT-qPCR), revealing the potential of developing a universal molecular detection platform for the screening, diagnosing, and prognosis prediction of multiple diseases.