title: Quantification of designer nuclease induced mutation rates: a direct comparison of different methods.
abstract: Designer nucleases are broadly applied to induce site-specific DNA double-strand breaks (DSB) in genomic DNA. These are repaired by nonhomologous end joining leading to insertions or deletions (in/dels) at the respective DNA-locus. To detect in/del mutations, the heteroduplex based T7-endonuclease I -assay is widely used. However, it only provides semi-quantitative evidence regarding the number of mutated alleles. Here we compared T7-endonuclease I- and heteroduplex mobility assays, with a quantitative polymerase chain reaction mutation detection method. A zinc finger nuclease pair specific for the human adeno-associated virus integration site 1 (AAVS1), a transcription activator-like effector nuclease pair specific for the human DMD gene, and a zinc finger nuclease- and a transcription activator-like effector nuclease pair specific for the human CCR5 gene were explored. We found that the heteroduplex mobility assays and T7-endonuclease I - assays detected mutations but the relative number of mutated cells/alleles can only be estimated. In contrast, the quantitative polymerase chain reaction based method provided quantitative results which allow calculating mutation and homologous recombination rates in different eukaryotic cell types including human peripheral blood mononuclear cells. In conclusion, our quantitative polymerase chain reaction based mutation detection method expands the array of methods for in/del mutation detection and facilitates quantification of introduced in/del mutations for a genomic locus containing a mixture of mutated and unmutated DNA.
authors.affiliation: Department of Human Medicine, Faculty of Health, Center of Biomedical Education and Research (ZBAF), Institute for Virology and Microbiology, Witten/Herdecke University , Witten, Germany.
authors.forename: Eric
authors.initials: E
authors.lastname: Ehrke-Schulz
authors.affiliation: Department of Human Medicine, Faculty of Health, Center of Biomedical Education and Research (ZBAF), Institute for Virology and Microbiology, Witten/Herdecke University , Witten, Germany.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Bergmann
authors.affiliation: Department of Human Medicine, Faculty of Health, Center of Biomedical Education and Research (ZBAF), Institute for Virology and Microbiology, Witten/Herdecke University , Witten, Germany.
authors.forename: Maren
authors.initials: M
authors.lastname: Schiwon
authors.affiliation: Department of Human Medicine, Faculty of Health, Center of Biomedical Education and Research (ZBAF), Institute for Virology and Microbiology, Witten/Herdecke University, Witten, Germany; Department of Surgery II, Helios Klinikum Wuppertal, University Witten/Herdecke, Wuppertal, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Doerner
authors.affiliation: Division of Medical Genetics, University of Washington, Seattle, Washington, USA; Department of Pathology, University of Washington, Seattle, Washington, USA.
authors.forename: Kamola
authors.initials: K
authors.lastname: Saydaminova
authors.affiliation: Division of Medical Genetics, University of Washington, Seattle, Washington, USA; Department of Pathology, University of Washington, Seattle, Washington, USA.
authors.forename: Andre
authors.initials: A
authors.lastname: Lieber
authors.affiliation: Department of Human Medicine, Faculty of Health, Center of Biomedical Education and Research (ZBAF), Institute for Virology and Microbiology, Witten/Herdecke University , Witten, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Ehrhardt