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A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases

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

Filippo Fronza¹, Roberto Verardo^2,3, Claudio Schneider^1,2

¹Dipartimento di Medicina (DMED), University of Udine, Piazzale Kolbe 1, Udine 33100, Italy.

|November 24, 2025

Summary

We developed a fast, inexpensive CRISPR-Cas9 assay using melting curve analysis to measure Cas protein activity. This method simplifies diagnostics and is suitable for high-throughput screening.

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Area of Science:

Molecular Biology
Biotechnology
Biochemistry

Background:

CRISPR/Cas systems are powerful tools for genome editing.
There is a growing need for efficient in vitro assays to assess CRISPR/Cas protein activity for diagnostic applications.
Current methods for evaluating CRISPR/Cas activity can be labor-intensive and time-consuming.

Purpose of the Study:

To develop a novel, rapid, and accessible in vitro assay for evaluating CRISPR/Cas9 activity.
To utilize melting curve analysis (MCA) as a method for detecting DNA cleavage by Cas9.
To demonstrate the feasibility of MCA for high-throughput screening of Cas protein activity.

Main Methods:

The assay employs melting curve analysis (MCA) to detect DNA double-strand breaks induced by CRISPR/Cas9.
Cas9 protein activity is measured by observing changes in the melting curves of target DNA products.
The assay utilizes standard laboratory equipment, including a real-time thermal cycler and SYBR Green I dye.

Main Results:

The novel MCA-based assay accurately measures CRISPR/Cas9 activity in vitro.
The assay provides reliable results within a 15-minute run-time.
The method is cost-effective, requires minimal intervention, and is amenable to automation.
An accompanying R package facilitates data analysis and increases assay accessibility.

Conclusions:

Melting curve analysis offers a robust and efficient method for assessing CRISPR/Cas9 activity.
This assay significantly reduces labor and time compared to traditional methods.
The assay's accessibility and speed make it suitable for diagnostic development and high-throughput screening.