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Nucleic Acids Research Advance Access published online on November 27, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl720
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© 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences

Julianne Smith, Sylvestre Grizot, Sylvain Arnould, Aymeric Duclert, Jean-Charles Epinat, Patrick Chames, Jesús Prieto1, Pilar Redondo1, Francisco J. Blanco1, Jerónimo Bravo1, Guillermo Montoya1, Frédéric Pâques* and Philippe Duchateau

CELLECTIS S.A., 102 route de Noisy 93235 Romainville France 1 Structural Biology and Biocomputing Programme, Centro Nacional de Investigaciones Oncológicas (CNIO) C/ Melchor Fdez Almagro, 28029 Madrid, Spain

*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 July 18, 2006. Revised September 12, 2006. Accepted September 18, 2006.

Meganucleases, or homing endonucleases (HEs) are sequence-specific endonucleases with large (>14 bp) cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These findings have opened novel perspectives for genome engineering in a wide range of fields, including gene therapy. However, the number of identified HEs does not match the diversity of genomic sequences, and the probability of finding a homing site in a chosen gene is extremely low. Therefore, the design of artificial endonucleases with chosen specificities is under intense investigation. In this report, we describe the first artificial HEs whose specificity has been entirely redesigned to cleave a naturally occurring sequence. First, hundreds of novel endonucleases with locally altered substrate specificity were derived from I-CreI, a Chlamydomonas reinhardti protein belonging to the LAGLIDADG family of HEs. Second, distinct DNA-binding subdomains were identified within the protein. Third, we used these findings to assemble four sets of mutations into heterodimeric endonucleases cleaving a model target or a sequence from the human RAG1 gene. These results demonstrate that the plasticity of LAGLIDADG endonucleases allows extensive engineering, and provide a general method to create novel endonucleases with tailored specificities.


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