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Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases TALENs
Published on: June 20, 2016
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Continuous directed evolution of DNA-binding proteins to improve TALEN specificity.
Basil P Hubbard1, Ahmed H Badran1, John A Zuris1
1Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.
Nature Methods
|August 11, 2015
Summary
We developed DNA-binding phage-assisted continuous evolution (DB-PACE) to improve genome editing tools. This method enhances the specificity of DNA-binding nucleases like TALENs for safer gene therapies.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Programmable nucleases with DNA-binding domains are crucial for genome alteration in model organisms.
- These nucleases hold significant promise for developing novel human therapeutics.
- Enhancing the specificity of these agents is critical for safe and effective therapeutic applications.
Purpose of the Study:
- To introduce DNA-binding phage-assisted continuous evolution (DB-PACE) as a general laboratory evolution method.
- To evolve DNA-binding activity and specificity of nuclease-based genome editing tools.
- To improve the accuracy and safety of genome editing agents.
Main Methods:
- Development and application of the DB-PACE system for protein evolution.
- Laboratory evolution of transcription activator-like effectors nucleases (TALENs) using DB-PACE.
- Assessment of DNA cleavage specificity of evolved TALENs.
Main Results:
- DB-PACE was established as a versatile platform for evolving DNA-binding proteins.
- TALENs with significantly improved DNA cleavage specificity were generated.
- Demonstrated the ability of DB-PACE to enhance the accuracy of genome-editing agents.
Conclusions:
- DB-PACE is a powerful approach for laboratory evolution of DNA-binding activity and specificity.
- The enhanced specificity of TALENs generated via DB-PACE has implications for safer genome editing.
- DB-PACE represents a versatile strategy for improving the precision of gene-editing technologies for therapeutic use.
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