An amplification-free CRISPR/Cas12a-based fluorescence assay for ultrasensitive detection of nuclease activity

Nuclease, such as RNase H and DNase I, plays key roles in plenty of cellular processes and could be potential therapeutic target for drug development. It is necessary to establish rapid and simple-to-use methods to detect nuclease activity. Herein, we develop a Cas12a-based fluorescence assay without any nucleic acid amplification steps for ultrasensitive detection of RNase H or DNase I activity. By our design, the pre-assembled crRNA/ssDNA duplex triggered the cleavage of fluorescent probes in the presence of Cas12a enzymes. However, the crRNA/ssDNA duplex was selectively digested with the addition of RNase H or DNase I, which leaded to fluorescence intensity changes. Under optimized conditions, the method exhibited good analytical performance, achieving a limit of detection (LOD) as low as 0.0082 U/mL for RNase H and 0.13 U/mL for DNase I, respectively. The method was feasible for analysis of RNase H in human serum and cell lysates, as well as for screening of enzyme inhibitors. Moreover, it can be adopted to image RNase H activity in living cells. Together, this study provides a facile platform for nuclease detection and could be expanded for other biomedical research and clinical diagnostics.