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Nucleic Acids Research Advance Access originally published online on August 3, 2009
Nucleic Acids Research 2009 37(17):5725-5736; doi:10.1093/nar/gkp643
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Nucleic Acids Research, 2009, Vol. 37, No. 17 5725-5736
© 2009 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.

Genome Integrity, Repair and Replication

Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease

Gijsbert P. van Nierop, Antoine A. F. de Vries, Maarten Holkers, Krijn R. Vrijsen and Manuel A. F. V. Gonçalves*

Virus and Stem Cell Biology Laboratory, Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands

*To whom correspondence should be addressed. Tel: +31 71 5269238; Fax: +31 71 5268270; Email: m.goncalves{at}lumc.nl

Received May 28, 2009. Revised July 17, 2009. Accepted July 19, 2009.

Homologous recombination (HR) is a highly accurate mechanism of DNA repair that can be exploited for homology-directed gene targeting. Since in most cell types HR occurs very infrequently (~10–6 to 10–8), its practical application has been largely restricted to specific experimental systems that allow selection of the few cells that become genetically modified. HR-mediated gene targeting has nonetheless revolutionized genetics by greatly facilitating the analysis of mammalian gene function. Recent studies showed that generation of double-strand DNA breaks at specific loci by designed endonucleases greatly increases the rate of homology-directed gene repair. These findings opened new perspectives for HR-based genome editing in higher eukaryotes. Here, we demonstrate by using donor DNA templates together with the adeno-associated virus (AAV) Rep78 and Rep68 proteins that sequence- and strand-specific cleavage at a native, predefined, human locus can also greatly enhance homology-directed gene targeting. Our findings argue for the development of other strategies besides direct induction of double-strand chromosomal breaks to achieve efficient and heritable targeted genetic modification of cells and organisms. Finally, harnessing the cellular HR pathway through Rep-mediated nicking expands the range of strategies that make use of AAV elements to bring about stable genetic modification of human cells.


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