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

## TL;DR

This paper introduces RepFluo, a fast and inexpensive in vitro assay for measuring Cas9 activity using melting curve analysis.

## Contribution

A novel, high-throughput Cas9 activity assay using melting curve analysis and an R package for data analysis is introduced.

## Key findings

- The assay uses melting curve analysis to evaluate Cas9 activity in 15 minutes with minimal equipment.
- The method requires no external intervention and uses readily available components like SYBR Green I.
- An R package is provided to simplify data analysis and increase accessibility.

## Abstract

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.