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Nucleic Acids Research Advance Access published online on February 14, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn059
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© 2008 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.

Nucleic Acid Enzymes

Computer design of obligate heterodimer meganucleases allows efficient cutting of custom DNA sequences

Emmanuel Fajardo-Sanchez1,2, François Stricher1,2, Frédéric Pâques3, Mark Isalan1,2,* and Luis Serrano1,4

1Structural Biology and Biocomputing Program, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, D-69117 Heidelberg, Germany, 2EMBL-CRG Unit – UPF, Barcelona, Spain, 3Cellectis SA, route de Noisy 102, 93 340 Romainville Cedex, France and 4ICREA Researcher, EMBL-CRG Unit – UPF, Barcelona, Spain

*To whom correspondence should be addressed. Tel: +3 493 316 0100; Fax: +3 493 396 9983; Email: isalan{at}crg.es

Received October 2, 2007. Revised January 25, 2008. Accepted January 30, 2008.

Meganucleases cut long (>12 bp) unique sequences in genomes and can be used to induce targeted genome engineering by homologous recombination in the vicinity of their cleavage site. However, the use of natural meganucleases is limited by the repertoire of their target sequences, and considerable efforts have been made to engineer redesigned meganucleases cleaving chosen targets. Homodimeric meganucleases such as I-CreI have provided a scaffold, but can only be modified to recognize new quasi-palindromic DNA sequences, limiting their general applicability. Other groups have used dimer-interface redesign and peptide linkage to control heterodimerization between related meganucleases such as I-DmoI and I-CreI, but until now there has been no application of this aimed specifically at the scaffolds from existing combinatorial libraries of I-CreI. Here, we show that engineering meganucleases to form obligate heterodimers results in functional endonucleases that cut non-palindromic sequences. The protein design algorithm (FoldX v2.7) was used to design specific heterodimer interfaces between two meganuclease monomers, which were themselves engineered to recognize different DNA sequences. The new monomers favour functional heterodimer formation and prevent homodimer site recognition. This design massively increases the potential repertoire of DNA sequences that can be specifically targeted by designed I-CreI meganucleases and opens the way to safer targeted genome engineering.


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