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      Generation of knockout rabbits using transcription activator-like effector nucleases

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          Abstract

          Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively. This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbits using transcription activator-like effector nucleases, and a perspective of the field.

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          The online version of this article (doi:10.1186/2045-9769-3-3) contains supplementary material, which is available to authorized users.

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          Most cited references26

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          Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting

          Tomas Cermak, Erin L. Doyle, Michelle Christian … (2011)
          TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35 bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGene and a web-based version of our software is freely accessible online.
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            FLASH Assembly of TALENs Enables High-Throughput Genome Editing

            Deepak Reyon, Shengdar Tsai, Cyd Khayter … (2012)
            Engineered transcription activator-like effector nucleases (TALENs) have shown promise as facile and broadly applicable genome editing tools. However, no publicly available high-throughput method for constructing TALENs has been published and large-scale assessments of the success rate and targeting range of the technology remain lacking. Here we describe the Fast Ligation-based Automatable Solid-phase High-throughput (FLASH) platform, a rapid and cost-effective method we developed to enable large-scale assembly of TALENs. We tested 48 FLASH-assembled TALEN pairs in a human cell-based EGFP reporter system and found that all 48 possessed efficient gene modification activities. We also used FLASH to assemble TALENs for 96 endogenous human genes implicated in cancer and/or epigenetic regulation and found that 84 pairs were able to efficiently introduce targeted alterations. Our results establish the robustness of TALEN technology and demonstrate that FLASH facilitates high-throughput genome editing at a scale not currently possible with engineered zinc-finger nucleases or meganucleases.
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              Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.

              Y G Kim, J. Cha, S Chandrasegaran (1996)
              A long-term goal in the field of restriction-modification enzymes has been to generate restriction endonucleases with novel sequence specificities by mutating or engineering existing enzymes. This will avoid the increasingly arduous task of extensive screening of bacteria and other microorganisms for new enzymes. Here, we report the deliberate creation of novel site-specific endonucleases by linking two different zinc finger proteins to the cleavage domain of Fok I endonuclease. Both fusion proteins are active and under optimal conditions cleave DNA in a sequence-specific manner. Thus, the modular structure of Fok I endonuclease and the zinc finger motifs makes it possible to create "artificial" nucleases that will cut DNA near a predetermined site. This opens the way to generate many new enzymes with tailor-made sequence specificities desirable for various applications.
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                Author and article information

                Contributors
                Yu Wang: wang_yu@gibh.ac.cn
                Nana Fan: fan_nana@gibh.ac.cn
                Jun Song: jsong519@126.com
                Juan Zhong: zhong_juan@gibh.ac.cn
                Xiaogang Guo: guo_xiaogang@gibh.ac.cn
                Weihua Tian: tian_weihua@gibh.ac.cn
                Quanjun Zhang: zhang_quanjun@gibh.ac.cn
                Fenggong Cui: cui_fenggong@gibh.ac.cn
                Li Li: li_li@gibh.ac.cn
                Philip N Newsome: p.n.newsome@bham.ac.uk
                Jon Frampton: j.frampton@bham.ac.uk
                Miguel A Esteban: esteban@gibh.org
                Liangxue Lai: lai_liangxue@gibh.ac.cn
                Journal
                Journal ID (nlm-ta): Cell Regen (Lond)
                Journal ID (iso-abbrev): Cell Regen (Lond)
                Title: Cell Regeneration
                Publisher: BioMed Central (London )
                ISSN (Electronic): 2045-9769
                Publication date (Electronic): 5 February 2014
                Publication date PMC-release: 5 February 2014
                Publication date Collection: 2014
                Volume: 3
                Issue: 1
                Electronic Location Identifier: 3
                Affiliations
                [ ]Key Laboratory of Regenerative Biology of the Chinese Academy of Sciences and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, 510530 China
                [ ]NIHR Liver BRU and Centre for Liver Research, University of Birmingham, Birmingham, UK
                [ ]Liver and Hepatobiliary Unit, Queen Elizabeth Hospital Birmingham, Birmingham, UK
                [ ]Institute for Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, B15 2TT UK
                Article
                Publisher ID: 18
                DOI: 10.1186/2045-9769-3-3
                PMC ID: 4230510
                PubMed ID: 25408882
                SO-VID: ce6d531a-8755-48e6-845b-192447f9f7f0
                Copyright © © Wang et al.; licensee BioMed Central Ltd. 2014
                License:

                This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                Date received : 17 November 2013
                Date accepted : 17 November 2013
                Categories
                Subject: Methodology
                Custom metadata
                issue-copyright-statement © BioMed Central Ltd 2014

                Keywords: rabbits,animal models,zinc-finger nucleases,transcription activator-like effector nucleases,talens,genome editing,knockout
                Data availability:
                Keywords: rabbits, animal models, zinc-finger nucleases, transcription activator-like effector nucleases, talens, genome editing, knockout

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