Accurate detection of lung cancer-related microRNA through CRISPR/Cas9-assisted garland rolling circle amplification.

Abstract

MicroRNA (miRNA) is reported to be closely related to a variety of pathophysiological processes for carcinoma and considered a potential biomarker for the diagnosis of lung cancer with brain metastasis. However, developing an accurate and sensitive miRNA detection method has proven to be a challenge. The aim of the present study was to integrate the advantages of rolling circle amplification (RCA), clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nucleases 9 (Cas9), and catalytic hairpin assembly (CHA) technologies to develop an miRNA detection method. In the present study, we developed a novel approach for the sensitive and accurate detection of miRNA through integrating garland RCA and CRISPR/Cas9-assisted signal generation. In this method, target miRNA cyclized dumbbell padlock and triggered the RCA process to form long single-stranded DNA products with a repeated hairpin structure. Double-stranded DNA sequences (dsDNA) were formed with the addition of complementary sequences. With the assistance of the Cas9 enzyme for specific recognition and cleavage of formed dsDNA, RCA products were disassembled into hairpin probes. The generated hairpin probe could be unfolded by target miRNA to initiate the CHA process for signal generation. Through integration of the RCA and CHA processes, the method demonstrated favorable detection performance. The correlation equation between the signal and concentration of target miRNA was determined to be Y=312.3 × lgC + 2108, with a high correlation coefficient of 0.9786. The approach also exhibited high selectivity to the mismatched miRNAs. Our method could be used in the screening, diagnosis, and prognosis of multiple diseases without complicated thermal cycling instrumentation.