Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Maeder ML

PROVIDER: S-EPMC2535758 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Maeder Morgan L ML Thibodeau-Beganny Stacey S Osiak Anna A Wright David A DA Anthony Reshma M RM Eichtinger Magdalena M Jiang Tao T Foley Jonathan E JE Winfrey Ronnie J RJ Townsend Jeffrey A JA Unger-Wallace Erica E Sander Jeffry D JD Müller-Lerch Felix F Fu Fengli F Pearlberg Joseph J Göbel Carl C Dassie Justin P JP Pruett-Miller Shondra M SM Porteus Matthew H MH Sgroi Dennis C DC Iafrate A John AJ Dobbs Drena D McCray Paul B PB Cathomen Toni T Voytas Daniel F DF Joung J Keith JK

Molecular cell 20080701 2

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 assemb ...[more]

PMID: 18657511

Similar Datasets

Project description:BackgroundCustomized zinc finger nucleases (ZFNs) form the basis of a broadly applicable tool for highly efficient genome modification. ZFNs are artificial restriction endonucleases consisting of a non-specific nuclease domain fused to a zinc finger array which can be engineered to recognize specific DNA sequences of interest. Recent proof-of-principle experiments have shown that targeted knockout mutations can be efficiently generated in endogenous zebrafish genes via non-homologous end-joining-mediated repair of ZFN-induced DNA double-stranded breaks. The Zinc Finger Consortium, a group of academic laboratories committed to the development of engineered zinc finger technology, recently described the first rapid, highly effective, and publicly available method for engineering zinc finger arrays. The Consortium has previously used this new method (known as OPEN for Oligomerized Pool ENgineering) to generate high quality ZFN pairs that function in human and plant cells.Methodology/principal findingsHere we show that OPEN can also be used to generate ZFNs that function efficiently in zebrafish. Using OPEN, we successfully engineered ZFN pairs for five endogenous zebrafish genes: tfr2, dopamine transporter, telomerase, hif1aa, and gridlock. Each of these ZFN pairs induces targeted insertions and deletions with high efficiency at its endogenous gene target in somatic zebrafish cells. In addition, these mutations are transmitted through the germline with sufficiently high frequency such that only a small number of fish need to be screened to identify founders. Finally, in silico analysis demonstrates that one or more potential OPEN ZFN sites can be found within the first three coding exons of more than 25,000 different endogenous zebrafish gene transcripts.Conclusions and significanceIn summary, our study nearly triples the total number of endogenous zebrafish genes successfully modified using ZFNs (from three to eight) and suggests that OPEN provides a reliable method for introducing targeted mutations in nearly any zebrafish gene of interest.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets