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Nucleic Acids Research 2005 33(18):e154; doi:10.1093/nar/gni148
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Published online 6 October 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

Methods Online

A highly sensitive selection method for directed evolution of homing endonucleases

Zhilei Chen1 and Huimin Zhao1,2,3,4,5,*

1Center for Biophysics and Computational Biology, University of Illinois Urbana, IL 61801, USA 2Department of Chemical and Biomolecular Engineering, University of Illinois Urbana, IL 61801, USA 3Department of Chemistry, University of Illinois Urbana, IL 61801, USA 4Department of Bioengineering, University of Illinois Urbana, IL 61801, USA 5Institute for Genomic Biology, University of Illinois Urbana, IL 61801, USA

*To whom correspondence should be addressed. Tel: +1 217 333 2631; Fax: +1 217 333 5052; Email: zhao5{at}uiuc.edu

Received June 10, 2005. Revised August 7, 2005. Accepted September 18, 2005.

Homing endonucleases are enzymes that catalyze DNA sequence specific double-strand breaks and can significantly stimulate homologous recombination at these breaks. These enzymes have great potential for applications such as gene correction in gene therapy or gene alteration in systems biology and metabolic engineering. However, homing endonucleases have a limited natural repertoire of target sequences, which severely hamper their applications. Here we report the development of a highly sensitive selection method for the directed evolution of homing endonucleases that couples enzymatic DNA cleavage with the survival of host cells. Using I-SceI as a model homing endonuclease, we have demonstrated that cells with wild-type I-SceI showed a high cell survival rate of 80–100% in the presence of the original I-SceI recognition site, whereas cells without I-SceI showed a survival rate <0.003%. This system should also be readily applicable for directed evolution of other DNA cleavage enzymes.


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