Monomeric site-specific nucleases for genome editing
Monomeric site-specific nucleases for genome editing
复制标题
DOI：
10.1073/pnas.1117984109
复制
发表时间：
2012-05-22
影响因子：
11.1
通讯作者：
Edgell, David R.
中科院分区：
文献类型：
--
作者：
Kleinstiver, Benjamin P.;Wolfs, Jason M.;Edgell, David R.
复杂基因组的靶向操作通常需要通过特定位点的DNA内切酶在特定位置引入双链断裂。在这里，我们描述了一个从基因组编辑应用的GIY-YIG归巢内切酶衍生的单体核酸酶结构域。GIY-YIG核酸酶结构域与三成员锌指DNA结合结构域融合产生嵌合giy -锌指内切酶（GIY-ZFEs）。值得注意的是，i - tevi衍生的融合物（Tev-ZFEs）在体外作为单体发挥作用，引入双链断裂，并在体外和细菌和酵母实验中区分缺乏首选5‘-CNNNG-3’切割基序的底物。Tev-ZFEs在酵母基实验中诱导重组，其活性与同二聚体Zif268锌指核酸酶相当。我们还将I-TevI核酸酶结构域融合到催化无活性的LADGLIDADG归巢内切酶（LHE）支架上。单体Tev-LHEs在体内是活跃的，并且类似地区分缺乏5‘-CNNNG-3’基序的底物。单体Tev-ZFEs和Tev-LHEs不同于foki衍生的锌指核酸酶和TAL效应核酸酶平台，因为GIY-YIG结构域减轻了针对给定序列设计两个核酸酶融合体的要求，突出了具有独特生化特性的核酸酶结构域的多样性，适合基因组编辑应用。
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 (GIY-ZFEs). Significantly, the I-TevI-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-TevI nuclease domain to a catalytically 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 FokI-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.