ArticleBMC molecular biology2010
Analysis of illegitimate genomic integration mediated by zinc-finger nucleases: implications for specificity of targeted gene correction.
Article in BMC molecular biology, 2010. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 15 papers.
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Who cites it
15 citing papers in PubMed, 32 citations in OpenAlex.
- Long read sequencing reveals transgene concatemerization and vector sequences integration following AAV-driven electroporation of CRISPR RNP complexes in mouse zygotes.Frontiers in genome editing · 2025Article
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- Template plasmid integration in germline genome-edited cattle.Nature biotechnology · 2020Article
- High-frequency random DNA insertions upon co-delivery of CRISPR-Cas9 ribonucleoprotein and selectable marker plasmid in rice.Scientific reports · 2019Article
- Exploring new gene integration sites for gene knock-in by gene-trapping strategy.Transgenic research · 2015Article
- CCR5 Gene Editing of Resting CD4(+) T Cells by Transient ZFN Expression From HIV Envelope Pseudotyped Nonintegrating Lentivirus Confers HIV-1 Resistance in Humanized Mice.Molecular therapy. Nucleic acids · 2014Article
- Implications of targeted genomic disruption of β-catenin in BxPC-3 pancreatic adenocarcinoma cells.PloS one · 2014Article
- Rosa26 locus supports tissue-specific promoter driving transgene expression specifically in pig.PloS one · 2014Article
- Transcription activator like effector (TALE)-directed piggyBac transposition in human cells.Nucleic acids research · 2013Article
- The nontoxic cell cycle modulator indirubin augments transduction of adeno-associated viral vectors and zinc-finger nuclease-mediated gene targeting.Human gene therapy · 2013Article
- Zinc Finger Nucleases: Tailor-made for Gene Therapy.Drugs of the future · 2012Article
- Efficient and accurate homologous recombination in hESCs and hiPSCs using helper-dependent adenoviral vectors.Molecular therapy : the journal of the American Society of Gene Therapy · 2012Article
- Zinc-finger nucleases for somatic gene therapy: the next frontier.Human gene therapy · 2011Review
- An update on targeted gene repair in mammalian cells: methods and mechanisms.Journal of biomedical science · 2011Review
- Article
Corrections and comments
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Authors and funding
4 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundFormation of site specific genomic double strand breaks (DSBs), induced by the expression of a pair of engineered zinc-finger nucleases (ZFNs), dramatically increases the rates of homologous recombination (HR) between a specific genomic target and a donor plasmid. However, for the safe use of ZFN induced HR in practical applications, possible adverse effects of the technology such as cytotoxicity and genotoxicity need to be well understood. In this work, off-target activity of a pair of ZFNs has been examined by measuring the ratio between HR and illegitimate genomic integration in cells that are growing exponentially, and in cells that have been arrested in the G2/M phase.
resultsA reporter cell line that contained consensus ZFN binding sites in an enhanced green fluorescent protein (EGFP) reporter gene was used to measure ratios between HR and non-homologous integration of a plasmid template. Both in human cells (HEK 293) containing the consensus ZFN binding sites and in cells lacking the ZFN binding sites, a 3.5 fold increase in the level of illegitimate integration was observed upon ZFN expression. Since the reporter gene containing the consensus ZFN target sites was found to be intact in cells where illegitimate integration had occurred, increased rates of illegitimate integration most likely resulted from the formation of off-target genomic DSBs. Additionally, in a fraction of the ZFN treated cells the co-occurrence of both specific HR and illegitimate integration was observed. As a mean to minimize unspecific effects, cell cycle manipulation of the target cells by induction of a transient G2/M cell cycle arrest was shown to stimulate the activity of HR while having little effect on the levels of illegitimate integration, thus resulting in a nearly eight fold increase in the ratio between the two processes.
conclusionsThe demonstration that ZFN expression, in addition to stimulating specific gene targeting by HR, leads to increased rates of illegitimate integration emphasizes the importance of careful characterization of ZFN treated cells. In order to reduce off-target events, reversible cell cycle arrest of the target cells in the G2/M phase is an efficient way for increasing the ratio between specific HR and illegitimate integration.
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