Each zinc finger binds to approximately three base pairs (bps) of DNA and a ZFN monomer commonly utilizes three to six zinc finger motifs to bind 9–18 bp target DNA. ZFNs consist of an engineered array of zinc fingers fused to the non-specific FokI nuclease domain and function as dimers to introduce targeted DNA double-strand breaks (DSBs). Recent advances in genome engineering using Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) have facilitated the creation of targeted gene knockout mutations in zebrafish, ,, ,,. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist. is supported by National Science Foundation award DBI-0923827. is supported by an NIH Director's Pioneer Award (DP1 OD006862), NIH R01 GM088040, and the Jim and Ann Orr MGH Research Scholar Award. is supported by NIH grants K01 AR055619, RO1 CA154923, and R21 CA156056, the Alex's Lemonade Stand Foundation, the American Cancer Society, and the Harvard Stem Cell Institute. 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.įunding: D.M.L. Received: FebruAccepted: ApPublished: May 24, 2012Ĭopyright: © 2012 Moore et al. Bonkowsky, University of Utah School of Medicine, United States of America (2012) Improved Somatic Mutagenesis in Zebrafish Using Transcription Activator-Like Effector Nucleases (TALENs). Taken together, our results suggest that TALEN technology provides a robust alternative to CoDA ZFNs for inducing targeted gene-inactivation in zebrafish, making it a preferred technology for creating targeted knockout mutants in zebrafish.Ĭitation: Moore FE, Reyon D, Sander JD, Martinez SA, Blackburn JS, Khayter C, et al. TALENs exhibited similar toxicity to CoDA ZFNs, with >50% of injected animals surviving to 3 days of life. Furthermore, the broader targeting range of TALENs enabled us to induce mutations at the methionine translation start site, sequences that were not targetable using the CoDA ZFN platform. TALENs also exhibited higher mutation rates than CoDA ZFNs that had not been pre-screened using a bacterial two-hybrid assay, with DNA mutation rates ranging from 20%–76.8% compared to 1.1%–3.3%. For example, all six TALENs tested induced DNA mutations at genomic target sites while only a subset of CoDA ZFNs exhibited detectable rates of mutagenesis. We find that TALENs exhibited a higher success rate for obtaining active nucleases capable of inducing mutations than compared with CoDA ZFNs. Here, we assess the relative efficiencies of these technologies for inducing somatic DNA mutations in mosaic zebrafish.
These designer nucleases bind to and cleave DNA at particular target sites, inducing error-prone repair that can result in insertion or deletion mutations.
Zinc Finger Nucleases (ZFNs) made by Context-Dependent Assembly (CoDA) and Transcription Activator-Like Effector Nucleases (TALENs) provide robust and user-friendly technologies for efficiently inactivating genes in zebrafish.
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