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In vivo genome editing using a high-efficiency TALEN system

机译：使用高效TALEN系统进行体内基因组编辑

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摘要

The zebrafish (Danio rerio) is increasingly being used to study basic vertebrate biology and human disease with a rich array of in vivo genetic and molecular tools. However, the inability to readily modify the genome in a targeted fashion has been a bottleneck in the field. Here we show that improvements in artificial transcription activator-like effector nucleases (TALENs) provide a powerful new approach for targeted zebrafish genome editing and functional genomic applications. Using the GoldyTALEN modified scaffold and zebrafish delivery system, we show that this enhanced TALEN toolkit has a high efficiency in inducing locus-specific DNA breaks in somatic and germline tissues. At some loci, this efficacy approaches 100%, including biallelic conversion in somatic tissues that mimics phenotypes seen using morpholino-based targeted gene knockdowns. With this updated TALEN system, we successfully used single-stranded DNA oligonudeotides to precisely modify sequences at predefined locations in the zebrafish genome through homology-directed repair, including the introduction of a custom-designed EcoRV site and a modified loxP (mloxP) sequence into somatic tissue in vivo. We further show successful germline transmission of both EcoRV and mloxP engineered chromosomes. This combined approach offers the potential to model genetic variation as well as to generate targeted conditional alleles.
机译：斑马鱼（Danio rerio）越来越多地用于研究基本的脊椎动物生物学和人类疾病，并具有多种体内遗传和分子工具。然而，不能以靶向方式容易地修饰基因组一直是该领域的瓶颈。在这里，我们表明，人工转录激活因子样效应核酸酶（TALENs）的改进为靶向斑马鱼基因组编辑和功能基因组应用提供了强大的新方法。使用GoldyTALEN改良的支架和斑马鱼递送系统，我们证明了这种增强的TALEN工具包在诱导体细胞和种系组织中特定于基因的DNA断裂方面具有很高的效率。在某些基因座上，该功效接近100％，包括在体细胞组织中的双等位基因转化，该转化模拟了使用基于吗啉代的靶向基因敲低所观察到的表型。通过此更新的TALEN系统，我们成功地使用了单链DNA寡核苷酸，通过同源性直接修复来精确修饰斑马鱼基因组中预定位置的序列，包括将定制设计的EcoRV位点和修饰的loxP（mloxP）序列引入体内的体细胞组织。我们进一步显示了EcoRV和mloxP工程染色体的成功种系传递。这种结合的方法提供了对遗传变异进行建模以及产生靶向条件等位基因的潜力。

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  • 来源
    《Nature》 |2012年第7422期|p.114-118|共5页
  • 作者

    Victoria M. Bedell; Ying Wang; Jarryd M. Campbell; Tanya L. Poshusta; Colby G. Starker; RandallG. Krugll; Wenfang Tan; Sumedha G. Penheiter; Alvin C. Ma; Anskar Y. H. Leung; Scott C. Fahrenkrug; Daniel F. Carlson; Daniel F. Voytas; Karl J. Clark; Jeffrey J. Essner; Stephen C. Ekker;

  • 作者单位

    Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA;

    Department of Genetics, Development and Cell Biology, Iowa State University, Ames, Iowa 50011,USA;

    Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA;

    Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA;

    Department of Genetics, Cell Biology and Development, the Center for Genome Engineering, and the Department of Animal Science, University of Minnesota, Minneapolis, Minnesota 55455, USA;

    Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA;

    Department of Genetics, Cell Biology and Development, the Center for Genome Engineering, and the Department of Animal Science, University of Minnesota, Minneapolis, Minnesota 55455, USA;

    Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA;

    Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA,Departmentof Medicine, LKS Faculty of Medicine, the University of Hong Kong, Pok Fu Lam Road, Hong Kong;

    Departmentof Medicine, LKS Faculty of Medicine, the University of Hong Kong, Pok Fu Lam Road, Hong Kong;

    Department of Genetics, Cell Biology and Development, the Center for Genome Engineering, and the Department of Animal Science, University of Minnesota, Minneapolis, Minnesota 55455, USA,Recombinetics, Inc., 2575 University Ave. West, Suite 100, Saint Paul, Minnesota 55114, USA;

    Department of Genetics, Cell Biology and Development, the Center for Genome Engineering, and the Department of Animal Science, University of Minnesota, Minneapolis, Minnesota 55455, USA,Recombinetics, Inc., 2575 University Ave. West, Suite 100, Saint Paul, Minnesota 55114, USA;

    Department of Genetics, Cell Biology and Development, the Center for Genome Engineering, and the Department of Animal Science, University of Minnesota, Minneapolis, Minnesota 55455, USA;

    Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA;

    Department of Genetics, Development and Cell Biology, Iowa State University, Ames, Iowa 50011,USA;

    Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA;

  • 收录信息 美国《科学引文索引》(SCI);美国《工程索引》(EI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
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