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Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification.

Molecular cell | Jul 25, 2008

http://www.ncbi.nlm.nih.gov/pubmed/18657511

Custom-made zinc-finger nucleases (ZFNs) can induce targeted genome modifications with high efficiency in cell types including Drosophila, C. elegans, plants, and humans. A bottleneck in the application of ZFN technology has been the generation of highly specific engineered zinc-finger arrays. Here we describe OPEN (Oligomerized Pool ENgineering), a rapid, publicly available strategy for constructing multifinger arrays, which we show is more effective than the previously published modular assembly method. We used OPEN to construct 37 highly active ZFN pairs which induced targeted alterations with high efficiencies (1%-50%) at 11 different target sites located within three endogenous human genes (VEGF-A, HoxB13, and CFTR), an endogenous plant gene (tobacco SuRA), and a chromosomally integrated EGFP reporter gene. In summary, OPEN provides an "open-source" method for rapidly engineering highly active zinc-finger arrays, thereby enabling broader practice, development, and application of ZFN technology for biological research and gene therapy.

Pubmed ID: 18657511 RIS Download

Mesh terms: Base Sequence | Endonucleases | Gene Targeting | Genetic Engineering | Green Fluorescent Proteins | Humans | K562 Cells | Molecular Sequence Data | Mutagenesis | Mutation | Protein Conformation | Zinc Fingers

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Associated grants

  • Agency: NIH HHS, Id: DP1 OD006862
  • Agency: NIGMS NIH HHS, Id: R01 GM069906
  • Agency: NIGMS NIH HHS, Id: R01 GM069906-03
  • Agency: NIGMS NIH HHS, Id: R01 GM069906-04
  • Agency: NIGMS NIH HHS, Id: R01 GM069906-05
  • Agency: NHLBI NIH HHS, Id: R01 HL079295
  • Agency: NCI NIH HHS, Id: R01CA112021
  • Agency: NIGMS NIH HHS, Id: R01GM069906
  • Agency: NCRR NIH HHS, Id: R21 RR024189
  • Agency: NCRR NIH HHS, Id: R21 RR024189-01
  • Agency: NCRR NIH HHS, Id: R21RR024189
  • Agency: NIGMS NIH HHS, Id: R24 GM078369
  • Agency: NIGMS NIH HHS, Id: R24 GM078369-01A1
  • Agency: NIGMS NIH HHS, Id: R24 GM078369-02
  • Agency: NIGMS NIH HHS, Id: R24GM078369

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