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Monomeric site-specific nucleases for genome editing.

Benjamin P Kleinstiver¹, Jason M Wolfs, Tomasz Kolaczyk

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

Proceedings of the National Academy of Sciences of the United States of America

|May 9, 2012

Summary

Researchers developed novel monomeric nucleases from GIY-YIG homing endonucleases for genome editing. These engineered nucleases, Tev-ZFEs and Tev-LHEs, efficiently create double-strand breaks and offer a simpler alternative to existing methods.

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Area of Science:

Molecular Biology
Genetics
Biochemistry

Background:

Targeted genome manipulation relies on site-specific DNA endonucleases to create double-strand breaks.
Existing genome editing tools often require complex designs and multiple components.

Purpose of the Study:

To engineer novel monomeric nuclease domains for genome editing applications.
To assess the efficacy and specificity of these new nucleases in vitro and in vivo.

Main Methods:

Fusion of a GIY-YIG nuclease domain (I-TevI) to zinc-finger DNA binding domains (creating Tev-ZFEs).
Fusion of the I-TevI nuclease domain to an inactive homing endonuclease scaffold (creating Tev-LHEs).
In vitro and in vivo assays (bacterial, yeast) to evaluate nuclease activity and DNA cleavage specificity.

Main Results:

Monomeric GIY-zinc finger endonucleases (GIY-ZFEs) and Tev-LHEs were successfully generated.
These nucleases function as monomers, introduce double-strand breaks, and exhibit specificity for a 5'-CNNNG-3' motif.
Tev-ZFEs demonstrated recombination-inducing activity in yeast comparable to existing homodimeric nucleases.

Conclusions:

Monomeric GIY-YIG derived nucleases offer a simplified approach to genome editing compared to FokI-based systems.
These novel nucleases highlight the biochemical diversity of nuclease domains for genome engineering.
Tev-ZFEs and Tev-LHEs provide versatile tools for targeted DNA manipulation.