title: A southern blot protocol to detect chimeric nuclease-mediated gene repair.
abstract: Gene targeting by homologous recombination at chromosomal endogenous loci has traditionally been considered a low-efficiency process. However, the effectiveness of such so-called genome surgery or genome editing has recently been drastically improved through technical developments, chiefly the use of designer nucleases like zinc-finger nucleases (ZFNs), meganucleases, transcription activator-like effector nucleases (TALENs) and CRISPR/Cas nucleases. These enzymes are custom designed to recognize long target sites and introduce double-strand breaks (DSBs) at specific target loci in the genome, which in turn mediate significant improvements in the frequency of homologous recombination. Here, we describe a Southern blot-based assay that allows detection of gene repair and estimation of repair frequencies in a cell population, useful in cases where the targeted modification itself cannot be detected by restriction digest. This is achieved through detection of a silent restriction site introduced alongside the desired mutation, in our particular example using integration-deficient lentiviral vectors (IDLVs) coding for ZFNs and a suitable DNA repair template.
authors.affiliation: School of Biological Sciences, Royal Holloway-University of London, Egham, Surrey, UK.
authors.forename: Céline J
authors.initials: CJ
authors.lastname: Rocca
authors.forename: Hayder H
authors.initials: HH
authors.lastname: Abdul-Razak
authors.forename: Michael C
authors.initials: MC
authors.lastname: Holmes
authors.forename: Philip D
authors.initials: PD
authors.lastname: Gregory
authors.forename: Rafael J
authors.initials: RJ
authors.lastname: Yáñez-Muñoz
mesh_terms: D000818:Animals; D015139:Blotting, Southern / Q000379:methods; D004260:DNA Repair; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D059765:Homologous Recombination; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D014161:Transduction, Genetic; D016335:Zinc Fingers / Q000502:physiology*
chemical_list: D004720:Endonucleases