A universal two-step strategy for multiple DNA MTase activity: enhancing sensitivity through CRISPR/Cas12a-assisted hyperbranched rolling circle amplification (CA-HRCA).

DNA methyltransferase (DNA MTase) is a valuable target of genetic diseases, and detection of related DNA MTase activity is very important for drug screening, clinical diagnosis and disease treatment. Herein, a universal two-step strategy based on CRISPR/Cas12a-assisted hyperbranched rolling circle amplification (CA-HRCA) for DNA MTase activity detection is constructed, which successfully achieves the detection of Dam MTase and M.SssI MTase. In the presence of DNA MTase and restriction enzymes, the HRCA primer locked in the dumbbell probe will be released and further initiates HRCA. In the first step, DNA methylation, restriction enzyme digestion and HRCA amplification are performed simultaneously, effectively simplifying the reaction process and shortening the detection time. In the second step, the abundant HRCA products (dsDNA) act as activators to induce CRISPR/Cas12a to split fluorescent probes. Compared with ssDNA activators, dsDNA activators can cause higher collateral cleavage of CRISPR/Cas12a. As expected, this strategy presents excellent sensing performance with a detection time of 155 min. The LODs of Dam MTase and M.SssI MTase are calculated to be 7.6 × 10 U mL and 1.8 × 10 U mL, respectively. And the proposed assay possesses extraordinary specificity and reproducibility. Moreover, the practical application ability and drug development potential are proved by the serum spiked test and inhibitor evaluation tests.