title: An unbiased method for detection of genome-wide off-target effects in cell lines treated with zinc finger nucleases.
abstract: We describe a method for detecting and validating genomic aberrations arising from cell lines exposed to zinc finger nucleases (ZFNs), an important reagent used for targeted genome modifications. This method makes use of cloned cell lines, an approach that adds power when testing variables that may affect gene correction efficiency and evaluating potential side effects on a genome-wide scale. After cell treatment, the genomic DNA isolation method, as described, is ideal for high-resolution array comparative genomic hybridization (aCGH) and quantitative PCR. Guidelines for aCGH analysis and calling significant copy number variations (CNVs) for validation by qPCR are also discussed. Using this method, we describe a novel ZFN-associated chromosome 4 copy number variation (CNV) attributable to a predicted ZFN off-target cleavage site found within the CNV.
authors.affiliation: Department of Pathology and Laboratory Medicine, Raymond and Ruth Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: Cory R
authors.initials: CR
authors.lastname: Lindsay
authors.forename: David B
authors.initials: DB
authors.lastname: Roth
mesh_terms: D055028:Comparative Genomic Hybridization; D053837:DNA Cleavage; D056915:DNA Copy Number Variations; D004260:DNA Repair; D004720:Endonucleases / Q000378:metabolism*; D015870:Gene Expression; D017930:Genes, Reporter; D015894:Genome, Human; D057809:HEK293 Cells; D006801:Humans; D016335:Zinc Fingers / Q000502:physiology*
chemical_list: D004720:Endonucleases