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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Monomeric site-specific nucleases for genome editing
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Monomeric site-specific nucleases for genome editing

机译：用于基因组编辑的单体位点特异性核酸酶

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摘要

Targeted manipulation of complex genomes often requires the introduction of a double-strand break at defined locations by site-specific DNA endonucleases. Here, we describe a monomeric nuclease domain derived from GIY-YIG homing endonucleases for genome-editing applications. Fusion of the GIY-YIG nuclease domain to three-member zinc-finger DNA binding domains generated chimeric GIY-zinc finger endonucleases (GlY-ZFEs). Significantly, the l-Tevl-derived fusions (Tev-ZFEs) function in vitro as monomers to introduce a double-strand break, and discriminate in vitro and in bacterial and yeast assays against substrates lacking a preferred 5 -CNNNG-3' cleavage motif. The Tev-ZFEs function to induce recombination in a yeast-based assay with activity on par with a homodimeric Zif268 zinc-finger nuclease. We also fused the I-Tevl nuclease domain to a cata-lytically inactive LADGLIDADG homing endonuclease (LHE) scaffold. The monomeric Tev-LHEs are active in vivo and similarly discriminate against substrates lacking the 5 -CNNNG-3' motif. The monomeric Tev-ZFEs and Tev-LHEs are distinct from the Fokl-derived zinc-finger nuclease and TAL effector nuclease platforms as the GIY-YIG domain alleviates the requirement to design two nuclease fusions to target a given sequence, highlighting the diversity of nuclease domains with distinctive biochemical properties suitable for genome-editing applications.
机译：复杂基因组的靶向操作通常需要通过位点特异性DNA核酸内切酶在定义的位置引入双链断裂。在这里，我们描述了从GIY-YIG归巢核酸内切酶衍生的单体核酸酶结构域，用于基因组编辑应用。 GIY-YIG核酸酶结构域与三元锌指DNA结合结构域的融合产生了嵌合的GIY-锌指核酸内切酶（G1Y-ZFE）。重要的是，l-Tevl衍生的融合蛋白（Tev-ZFEs）在体外可作为单体引入双链断裂，并在体外以及在细菌和酵母检测中针对缺乏优选的5 -CNNNG-3'裂解基序的底物进行区分。 。 Tev-ZFE的功能是在基于酵母的分析中诱导重组，并具有与同型二聚体Zif268锌指核酸酶同等的活性。我们还将I-Tev1核酸酶结构域融合到催化失活的LADGLIDADG归巢核酸内切酶（LHE）支架上。 Tev-LHE单体在体内具有活性，并且可以与缺乏5 -CNNNG-3'基序的底物进行区分。 Tev-ZFEs和Tev-LHEs单体不同于Fokl衍生的锌指核酸酶和TAL效应子核酸酶平台，因为GIY-YIG结构域减轻了设计两个核酸酶融合以靶向给定序列的需求，从而突出了核酸酶的多样性。具有独特生化特性的区域，适合基因组编辑应用。

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    Benjamin P. Kleinstiver; Jason M. Wolfs; Tomasz Kolaczyk; Alanna K. Roberts; Sherry X. Hu; David R. Edgell;

  • 作者单位

    Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada N6A 5C1;

    Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada N6A 5C1;

    Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada N6A 5C1;

    Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada N6A 5C1;

    Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada N6A 5C1;

    Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada N6A 5C1;

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