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

Abstract

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.

Precision genome engineering with programmable DNA-nicking enzymes

“…It has been shown that DNA-nicking enzymes allow homology-directed gene targeting in mammalian cells (Lee et al 2004;van Nierop et al 2009) and that nickases can be created by engineering naturally occurring restriction enzymes (Sanders et al 2009) or meganucleases (McConnell Smith et al 2009). However, these enzymes are not readily reprogrammed to target any predetermined DNA sequence and thus cannot be used to modify DNA sequences at user-defined genomic sites.…”

Targeted gene addition to a predetermined site in the human genome using a ZFN-based nicking enzyme

“…More recently, it was demonstrated that mammalian cells can efficiently utilize HDR to repair single-strand DNA gaps opposite an abasic site or benzo[a]pyrene adduct (Adar et al 2009). DNA nicks generated by adeno-associated virus (AAV) Rep78 and Rep68 proteins can greatly enhance HDR (van Nierop et al 2009). Furthermore, a ''nick-only'' version of the I-Anil homing endonuclease was also shown to stimulate HDR-mediated DNA repair in mammalian cells using a reporter assay system engineered to contain the I-Anil target site (Smith et al 2009;Metzger et al 2011).…”

Concerted nicking of donor and chromosomal acceptor DNA promotes homology-directed gene targeting in human cells

“…Next, the cells were subjected to green fluorescent protein (GFP)-based fluorescence-activated single-cell sorting and clonally expanded as detailed elsewhere (16). …”