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Nucleic Acids Research 2005 33(20):e178; doi:10.1093/nar/gni175
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Published online 23 November 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

In vivo selection of engineered homing endonucleases using double-strand break induced homologous recombination

Patrick Chames, Jean-Charles Epinat, Sophie Guillier, Amélie Patin, Emmanuel Lacroix and Frédéric Pâques*

CELLECTIS S.A. 102 route de Noisy 93235 Romainville, France

*To whom correspondence should be addressed. Tel: +33 1 41 83 99 00; Fax: +33 1 41 83 99 03; Email: paques{at}cellectis.com

Received September 5, 2005. Revised October 21, 2005. Accepted October 21, 2005.

Homing endonucleases, endonucleases capable of recognizing long DNA sequences, have been shown to be a tool of choice for precise and efficient genome engineering. Consequently, the possibility to engineer novel endonucleases with tailored specificities is under strong investigation. In this report, we present a simple and efficient method to select meganucleases from libraries of variants, based on their cleavage properties. The method has the advantage of directly selecting for the ability to induce double-strand break induced homologous recombination in a eukaryotic environment. Model selections demonstrated high levels of enrichments. Moreover, this method compared favorably with phage display for enrichment of active mutants from a mutant library. This approach makes possible the exploration of large sequence spaces and thereby represents a valuable tool for genome engineering.


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