Rapid Mutation of Endogenous Zebrafish Genes Using Zinc Finger Nucleases Made by Oligomerized Pool ENgineering (OPEN) Jonathan E. Foley1., Jing-Ruey J. Yeh2,3., Morgan L. Maeder1, Deepak Reyon4, Jeffry D. Sander4, Randall T. Peterson2,3, J. Keith Joung1,5* 1 Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, United States of America, 2 Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, United States of America, 3 Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America, 4 Department of Genetics, Development and Cell Biology, Interdepartmental Graduate Program in Bioinformatics and Computational Biology, Iowa State University, Ames, Iowa, United States of America, 5 Department of Pathology, Harvard Medical School, Boston, Massachusetts, United States of America Abstract Background: Customized 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 Findings: Here 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 Significance: In 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. Citation: Foley JE, Yeh J-RJ, Maeder ML, Reyon D, Sande JD, et al. (2009) Rapid Mutation of Endogenous Zebrafish Genes Using Zinc Finger Nucleases Made by Oligomerized Pool ENgineering (OPEN). PLoS ONE 4(2): e4348. doi:10.1371/journal.pone.0004348 Editor: David W. Raible, University of Washington, United States of America Received November 19, 2008; Accepted December 15, 2008; Published February 9, 2009 Copyright: 2009 Foley et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: J.E.F., M.L.M., and J.K.J. are supported by the NIH (R01 GM069906 and R21 HL091808) and the MGH Department of Pathology. J.-R.J.Y. is supported by the National Institute of Aging (K01 AG031300) and by a Claflin Distinguished Scholar Award. J.-R.J.Y. and R.T.P. are supported by the NIH (R01 HL79267). J.D.S is supported by Pioneer-Hi-Bred International, Inc 2008 Graduate Fellowship, NIH R33-GM066387, and ISU CIAG. D.R is supported by ISU CIAG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: J.K.J. is an inventor on patent applications which describe the OPEN zinc finger engineering method. All other authors have no competing interests to declare. * E-mail: jjoung@partners.org . These authors contributed equally to this work. Introduction Engineered zinc finger nucleases (ZFNs) form the basis of a broadly applicable technology for highly efficient genome modification [1–6]. ZFNs function as dimers [7] with each monomer consisting of an engineered zinc finger array (typically composed of three or four fingers) fused to a non-specific cleavage domain from the FokI endonuclease [8,9]. Zinc finger arrays in ZFNs can be engineered to bind target DNA sequences of interest [10–17], thereby enabling the introduction of double-strand DNA breaks (DSBs) into specific genomic sequences. ZFNs can be used to alter endogenous genes in Drosophila and mammalian cells with absolute efficiencies ranging from 1%–50% [18–26]. ZFN-induced DSBs can be repaired by non-homologous end-joining (NHEJ), an imperfect process which frequently results in the creation of insertions and deletions (indels) at the site of the break. Alternatively, repair of a ZFN-induced DSB by homologous recombination (HR) with an appropriately designed exogenous PLoS ONE | www.plosone.org 1 February 2009 | Volume 4 | Issue 2 | e4348