A universal method for sensitive and cell-free detection of CRISPR-associated nucleases

transcription reaction that produces a fluorescent RNA:small-molecule adduct. We demonstrate that our method is sensitive, detecting activity from low nanomolar concentrations of several families of Cas nucleases, and can be conducted in a high-throughput microplate fashion with a simple fluorescent-based readout. We provide a mathematical framework for quantifying the activities of these nucleases and demonstrate two applications of our method, namely the development of a logic circuit and the characterization of an anti-CRISPR protein. We anticipate our method will be valuable to those studying Cas nucleases and will allow the application of Cas nuclease beyond the field of life sciences.