Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease.
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 (approximately 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.
van Nierop
Gijsbert P
GP
Virus and Stem Cell Biology Laboratory, Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands.
de Vries
Antoine A F
AA
Holkers
Maarten
M
Vrijsen
Krijn R
KR
Gonçalves
Manuel A F V
MA
D002888:Chromosomes, Human, Pair 19; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D011995:Recombination, Genetic; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism*
D004268:DNA-Binding Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2; D004706:Endodeoxyribonucleases