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

• Published in: Nucleic acids research Vol. 37; no. 17; pp. 5725 - 5736
• Main Authors: van Nierop, Gijsbert P, de Vries, Antoine A.F, Holkers, Maarten, Vrijsen, Krijn R, Gonçalves, Manuel A.F.V
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01-09-2009
• Subjects: Adeno-associated virus; Chromosomes, Human, Pair 19; DNA-Binding Proteins - metabolism; Endodeoxyribonucleases - metabolism; Gene Targeting - methods; Genome Integrity, Repair and; HeLa Cells; Humans; Recombination, Genetic; Transcription, Genetic; Viral Proteins - metabolism
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkp643

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.