RepFluo, a Fast Fluorescent In Vitro Assay of Cas9 Activity Exploiting Melting Curve Analysis

Demand for less labor-intensive in vitro assays of the activity of CRISPR/Cas proteins is rising to extend the potential applications of CRISPR in the field of diagnostics. RNA guided DNA endonucleases of the Cas family generate double-strand breaks in the target DNA, which results in two shorter DNA fragments. We hypothesized that this cleavage event could be studied using melting curve analysis, and using SpyCas9, we demonstrate that it is possible to evaluate the activity of Cas proteins by measuring the melting curves of their products. We present here a novel assay for the in vitro activity of Cas9 that exploits melting curve analysis (MCA) to be fast, inexpensive, and widely accessible. The assay can, in fact, be performed with readily available componentsin its simplest form a real-time thermal cycler and an intercalating dye (SYBR Green I)and produces reliable results with a run-time of 15 min. It does not require external intervention to stop the reaction, which is done by thermal denaturation of the protein directly in the thermal cycler machine. The described advantages, combined with the provided data analysis package, make the assay robust and amenable to high-throughput applications. To increase the accessibility of our assay, we provided an R package that simplifies the analytical process.