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MegaTevs: single-chain dual nucleases for efficient gene disruption.

Jason M Wolfs¹, Matthew DaSilva¹, Sarah E Meister¹

¹Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON, N6A 5C1, Canada.

Nucleic Acids Research

|July 12, 2014

Summary

Researchers developed a novel MegaTev enzyme for precise gene disruption. This engineered nuclease achieves high gene editing efficiency without DNA repair enzymes, offering a new tool for genome engineering.

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Area of Science:

Molecular Biology
Genetics
Biotechnology

Background:

Gene targeting often relies on non-homologous end-joining (NHEJ) DNA repair, which introduces insertions/deletions (indels) at the break site.
Co-expression of DNA end-processing enzymes with genome-editing nucleases can lead to non-mutagenic repair, reducing gene disruption efficiency.

Purpose of the Study:

To develop a novel genome-engineering strategy that biases repair towards gene disruption.
To create a programmable nuclease platform for highly specific genome modification.

Main Methods:

Fusion of two distinct nuclease active sites (I-TevI and I-OnuI E2) into a single polypeptide chain, creating the MegaTev enzyme.
In vitro characterization of MegaTev activity, including double-strand break generation and fragment excision.
Assessment of MegaTev-mediated gene disruption frequency in HEK 293 cells without co-expressed DNA end-processing enzymes.
Deep sequencing to analyze repair outcomes at target sites.
Off-target cleavage profiling.

Main Results:

The MegaTev enzyme efficiently excises an intervening 30-bp fragment in vitro by generating two double-strand breaks.
High frequency of gene disruption was observed in HEK 293 cells without the need for co-expressed DNA end-processing enzymes.
Deep sequencing indicated minimal DNA processing at disrupted sites, suggesting sequestration of breaks from repair machinery.
Off-target analysis revealed no detectable cleavage at sites lacking the specific spacing of I-TevI and I-OnuI recognition motifs.

Conclusions:

The MegaTev enzyme provides an alternative strategy to enhance gene disruption frequency by bypassing the need for DNA end-processing enzymes.
This engineered nuclease platform demonstrates high specificity and minimal off-target effects.
MegaTev represents a promising tool for precise and efficient genome engineering applications.