RepFluo, a Fast Fluorescent In Vitro Assay of Cas9 Activity Exploiting Melting Curve Analysis
Filippo Fronza, Roberto Verardo, Claudio Schneider
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.
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 componentsin its simplest form a real-time thermal cycler and an intercalating dye (SYBR Green I)and produces reliable results with a…
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Taxonomy
TopicsCRISPR and Genetic Engineering · RNA Interference and Gene Delivery · PARP inhibition in cancer therapy