CRISPR/Cas activity assays and compositions thereof
| DWPI Title: Detecting nuclease activity such as activity of Cas nuclease, involves combining target nuclease with cleavage substrate and synthetic guiding component, incubating reaction mixture, quenching incubated mixture under denaturation condition and measuring detectable signal from reacted substrate |
| Abstract: The present invention relates, in part, to methods for detecting nuclease activity, such as the activity of Cas nucleases. Also described herein are compositions for conducting assays, as well as methods for conducting assays in the presence of test compounds. |
| Use: Method for detecting nuclease activity such as activity of Cas nuclease. |
| Advantage: The method utilizes substrates for quantitative monitoring of Cas9 cleavage activity by fluorescence spectroscopy in a high-throughput microplate format. |
| Novelty: Detecting nuclease activity, involves: combining a target nuclease with a cleavage substrate and a synthetic guiding component, to provide a reaction mixture; incubating the reaction mixture to provide an incubated reaction mixture; quenching the incubated reaction mixture under a denaturation condition configured to denature the target nuclease; and measuring a detectable signal, if any, arising from a reacted cleavage substrate, where the target nuclease is Cas9 homolog or ortholog or SpyCas9 enzyme, the synthetic guiding component is a single guide RNA configured to interact with the target nuclease, and the cleavage substrate comprises a duplex having a target strand and a non-target strand. |
| Filed: 3/21/2019 |
| Application Number: US16360946A |
| Tech ID: SD 14418.1 |
| This invention was made with Government support under Contract No. DE-NA0003525 awarded by the United States Department of Energy/National Nuclear Security Administration. The Government has certain rights in the invention. |
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