A sensitive and facile microRNA detection based on CRISPR-Cas12a coupled with strand displacement amplification

Abstract

MicroRNAs (miRNAs) are important biomarkers that are closely associated with certain diseases. The detection of miRNA is critical because it provides the necessary information for Disease Diagnosis. In this study, we achieved miRNA determination by coupling the CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR-associated) system with strand displacement amplification (SDA). In the experiment, miRNA was used as the initiator of SDA, and the activator of Cas12a nuclease activity was amplified by SDA. Subsequently, the unique nuclease activity of Cas12a was exploited to carry out trans cleaving on the ssDNA reporting probe modified with carboxyfluorescein(FAM) and BHQ1(dark Quencher: 480–580 nm) to achieve a signal output. In addition to chain design and reaction simplification, this method is lofty sensitive and selective for the determination of miRNA with a good linear range of 250 fmol·L−1 ∼ 40 pmol·L−1, the detection limit of 150 fmol·L−1 (S/N = 3), and the method showed good recovery in spiked human serum. Overall, this method is expected to be applied to diagnosis with miRNA biomarkers because of its rapidity, high sensitivity, and high selectivity.