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Rapid screening of endonuclease target site preference using a modified bacterial two-plasmid selection
Jason M Wolfs1, Benjamin P Kleinstiver, David R Edgell
1Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada.
Methods in Molecular Biology (Clifton, N.J.)
|February 11, 2014
Summary
This study presents a new method for rapidly analyzing DNA-sequence specificity of homing endonucleases. The genetic selection efficiently profiles nucleotide substitutions, aiding genome editing applications.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Homing endonucleases are crucial for genome editing but require precise DNA-sequence specificity.
- Understanding endonuclease target site preferences is vital for efficient and accurate genome engineering.
Purpose of the Study:
- To develop a rapid genetic selection method for profiling nucleotide substitutions in endonuclease target sites.
- To enable efficient analysis of DNA-sequence specificity for homing endonucleases and other site-specific enzymes.
Main Methods:
- A modified two-plasmid genetic selection system in Escherichia coli was employed.
- A toxic plasmid encoding a DNA gyrase toxin and the endonuclease target site was used.
- Competent cells with an endonuclease expression plasmid were transformed with target site plasmids, followed by replica plating.
Main Results:
- The selection system allows for rapid profiling of nucleotide substitutions within target sites.
- Cleavage of the toxic plasmid by the endonuclease under selective conditions indicates a functional target site.
- Both cleavable and non-cleavable targets can be identified and analyzed via DNA sequencing.
Conclusions:
- This method facilitates rapid analysis of DNA-sequence specificity for site-specific endonucleases.
- The technique aids in understanding nucleotide preferences, improving genome editing tool design.
- The modified protocol enables efficient screening of numerous target sites for endonuclease activity.
