CRISPR–Cas12a with split crRNA for the direct and sensitive detection of microRNA

Abstract

microRNAs (miRNAs) have been identified as potential biomarkers. Despite the prevalence of quantitative PCR in the field of miRNA detection, this technology is encumbered by the complexity of its methodology. This study presents a novel CRISPR/Cas12a-based method for the direct and sensitive detection of miRNA-21 using split crRNA. The system comprises Cas12a protein, crRNA-handle, and activator DNA complementary to the target miRNA. In the presence of the target miRNA, it binds to the activator DNA, forming a duplex. The formed duplex, in conjunction with the crRNA-handle, activates Cas12a's trans-cleavage activity. This leads to cleavage of a fluorescent reporter, generating an enhanced signal. The method enables direct RNA detection without reverse transcription or sample amplification, offering simplicity and efficiency. This method demonstrates high sensitivity with a minimum detectable limit of 5 pM. Furthermore, the method's specificity is substantiated by its capacity to discern point mutations in miRNA. This system has been shown to quantitatively analyse miRNA-21 levels present within serum, as evidenced by the recovery experiment. Therefore, the method's simplicity, stability, and cost-effectiveness render it a powerful tool for nucleic acid detection, with potential for clinical applications.