AAV-mediated gene editing via double-strand break repair.

Traditionally, the ability to edit the mammalian genome was inhibited by the inherent low efficiency of homologous recombination (HR; approximately <1 in a million events) and the inability to deliver DNA efficiently to dividing and non-dividing cells/tissue. Despite these limitations, creative selections designed over 20 years ago, clearly demonstrated the powerful implications of gene knock-in and knockout technology for the genetic engineering of mice (Doetschman et al. Nat 330(6148): 576-578, 1987; Thomas and Capecchi. Cell 51(3): 503-512, 1987). The development and application of recombinant vectors based on adeno-associated virus (rAAV) have helped to overcome both of the initial limitations regarding DNA delivery and the frequency of HR. Considering DNA delivery, rAAV infects non-dividing and dividing cultured cells as well as most tissues in mouse and larger animal models (including humans). At the DNA editing level, rAAV genomes have been reported to increase the frequency of HR several orders of magnitude by serving as the repair substrate (Russell and Hirata. Nat Genet 18(4): 325-330, 1998). However, reports on the ability of rAAV genomes to stimulate HR, compared to plasmid DNA and oligonucleotides, are variable, and many labs have found it necessary to augment the frequency of rAAV-induced HR using site-specific endonucleases (Ellis et al. Gene Ther, 2012; Hirsch et al. Gene Ther 17(9): 1175-1180, 2010; Porteus et al. Mol Cell Biol 23(10): 3558-3565, 2003; Radecke et al. Mol Ther 14(6): 798-808, 2006). In this protocol, we describe a method to perform rAAV-mediated double-strand break (DSB) repair for precise genetic engineering in human cells.