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Related Experiment Video

Updated: Jan 25, 2026

A Rapid Method for Modeling a Variable Cycle Engine

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SplitAx: A novel method to assess the function of engineered nucleases.

Richard A Axton¹, Sharmin S Haideri¹, Martha Lopez-Yrigoyen¹

¹MRC Centre for Regenerative Medicine, SCRM Building, The University of Edinburgh, Edinburgh bioQuarter, 5 Little France Drive, Edinburgh, United Kingdom.

|February 18, 2017

Summary

A new SplitAx assay simplifies the evaluation of engineered nuclease activity for genome editing. This method offers a robust readout for assessing nucleases like CRISPR and TALENs, accelerating research in gene therapy.

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Mouse Genome Engineering Using Designer Nucleases

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Published on: April 2, 2014

Area of Science:

Molecular Biology
Biotechnology
Genetics

Background:

Engineered nucleases are crucial for creating disease models and hold therapeutic potential.
Current methods for assessing nuclease activity are often time-consuming and lack sensitivity.

Purpose of the Study:

To develop a simple, efficient, and sensitive assay for evaluating engineered nuclease activity.
To provide a reliable readout for genome editing technologies.

Main Methods:

Developed the SplitAx assay using split GFP (Green Fluorescent Protein) components and a nuclease binding site.
Assessed nuclease activity through restoration of GFP fluorescence after cleavage and non-homologous end joining (NHEJ) or trans-complementation.
Validated the assay with zinc finger nucleases, CRISPR-Cas9, and TALENs, including testing guide RNAs (gRNAs).

Main Results:

The SplitAx assay successfully detected activity of various engineered nucleases, including CRISPR-Cas9 and TALENs.
Functional nucleases identified by SplitAx were confirmed to target endogenous loci (AAVS1, SOX6, Cfms).
The assay demonstrated broad applicability, working with different fluorescent proteins like ZsGreen1.

Conclusions:

SplitAx is a simple, sensitive, and broadly applicable method for assessing engineered nuclease activity.
This assay streamlines the evaluation of genome editing tools, facilitating research and therapeutic development.
SplitAx reduces the need for complex optimization, making it accessible for various applications.