Highly sensitive and facile microRNA detection based on target triggered exponential rolling-circle amplification coupling with CRISPR/Cas12a

MicroRNAs (miRNAs) play a crucial role in the regulation of gene expression and have been implicated in many diseases. Herein, we develop a target triggered exponential rolling-circle amplification coupling with CRISPR/Cas12a (T-ERCA/Cas12a) system, which can achieve the ultrasensitive detection with simple operation and no annealing procedure. In this assay, T-ERCA combines the exponential amplification with rolling-circle amplification by introducing a dumb-bell probe with two enzyme recognition sites. miRNA-155 targets are activators that trigger exponential rolling circle amplification to produce large amounts of ssDNA, which is then recognized by CRISPR/Cas12a for further amplification. Compared with single EXPAR or RCA combined with CRISPR/Cas12a, this assay shows higher amplification efficiency. Therefore, benefiting from the excellent amplification effect of T-ERCA and the high recognition specificity of CRISPR/Cas12a, the proposed strategy shows a wide detection range from 1 fM to 5 nM with a LOD (limit of detection) down to 0.31 fM. Moreover, it shows good application ability for assessing miRNA levels in different cells, indicating that the T-ERCA/Cas12a may provide a new guidance for molecular diagnosis and clinical practical application.