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Retargeting Sleeping Beauty transposon insertions by engineered zinc finger DNA-binding domains

Item Type:Article
Title:Retargeting Sleeping Beauty transposon insertions by engineered zinc finger DNA-binding domains
Creators Name:Voigt, K. and Gogol-Doering, A. and Miskey, C. and Chen, W. and Cathomen, T. and Izsvak, Z. and Ivics, Z.
Abstract:The Sleeping Beauty (SB) transposon is a nonviral, integrating vector system with proven efficacy in preclinical animal models, and thus holds promise for future clinical applications. However, SB has a close-to-random insertion profile that could lead to genotoxic effects, thereby presenting a potential safety issue. We evaluated zinc finger (ZF) DNA-binding domains (DBDs) for their abilities to introduce a bias into SB's insertion profile. E2C, that binds a unique site in the erbB-2 gene, mediated locus-specific transposon insertions at low frequencies. A novel ZF targeting LINE1 repeats, ZF-B, showed specific binding to an 18-bp site represented by ~12,000 copies in the human genome. We mapped SB insertions using linear-amplification (LAM)-PCR and Illumina sequencing. Targeted insertions with ZF-B peaked at approximately fourfold enrichment of transposition around ZF-B binding sites yielding ~45% overall frequency of insertion into LINE1. A decrease in the ZF-B dataset with respect to transposon insertions in genes was found, suggesting that LINE1 repeats act as a sponge that "soak up" a fraction of SB insertions and thereby redirect them away from genes. Improvements in ZF technology and a careful choice of targeted genomic regions may improve the safety profile of SB for future clinical applications.
Keywords:Binding Sites, Computational Biology, DNA Transposable Elements, DNA-Binding Proteins, Gene Transfer Techniques, Genetic Engineering, HeLa Cells, Human Genome, Insertional Mutagenesis, Plasmids, Transfection, Transposases, Zinc Fingers
Source:Molecular Therapy
Publisher:Nature Publishing Group
Page Range:1852-1862
Date:October 2012
Official Publication:https://doi.org/10.1038/mt.2012.126
PubMed:View item in PubMed

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