Double CRISPR/Cas12a-drived hyperbranched rolling circle amplification with triple signal amplification enables low background miRNA detection

False positives and high background are key problems in nucleic acid amplification-based methods. Herein, a triple amplification and low background sensing platform is developed, termed double CRISPR/Cas12a-drived hyperbranched rolling circle amplification (D-Cas12a/HRCA). By introducing two kinds of CRISPR/Cas12a, we effectively avoid background signal and improve sensitivity. The target miRNA is firstly recognized by a padlock probe to trigger the rolling circle amplification (RCA), which generates mass of long ssDNA and achieves the first amplification. The ssDNA can unlock the cis- and trans-cleavage activity of CRISPR/Cas12a (Cas12a/crRNA-1). Therefore, the cis-cleaved ssDNA and trans-cleaved primer 2 (neighboring C3 inhibitor modification) are used as template and primer for the next hyperbranched rolling circle amplification (HRCA, secondary amplification), respectively. The above amplification products (dsDNA, ssDNA) are recognized again by the CRISPR/Cas12a (third amplification) and output fluorescence or visualization signals. Compared with traditional RCA/Cas12a or HRCA/Cas12a, D-Cas12a/HRCA shows higher sensitivity with lower background. This assay achieves sensitive detection of miRNA-21 low to 10.02 fM. The lateral flow detection strips enable miRNA-21 detection with a LOD of 100 fM, showing the potential of CRISPR/Cas-based technology in POCT detection. More importantly, the platform is consistent with RT-qPCR in detecting miRNA-21 extracted from different cell lines.