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| Item Type: | Article |
|---|---|
| Title: | Retargeting Sleeping Beauty transposon insertions by engineered zinc finger DNA-binding domains |
| Creators Name: | Voigt, K., Gogol-Doering, A., Miskey, C., Chen, W., Cathomen, T., 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 |
| ISSN: | 1525-0016 |
| Publisher: | Nature Publishing Group |
| Volume: | 20 |
| Number: | 10 |
| 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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