Tandem repeat coupled with endonuclease cleavage (TREC): a seamless modification tool for genome engineering in yeast.
Tandem repeat coupled with endonuclease cleavage (TREC): a seamless modification tool for genome engineering in yeast.
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串联重复序列与核酸内切酶切割 (TREC) 相结合：酵母基因组工程的无缝修饰工具。
DOI：
10.1093/nar/gkq099
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发表时间：
2010-05
影响因子：
14.9
通讯作者：
Chuang, Ray-Yuan
中科院分区：
文献类型：
--
作者：
Noskov, Vladimir N.;Segall-Shapiro, Thomas H.;Chuang, Ray-Yuan
人工合成的生殖支原体全基因组(∼583kb)已在酵母中组装并克隆为环状质粒。通过涉及URA3/5-氟甲酸(5-FOA)反选择的标准遗传方法来设计克隆基因组的尝试显示了高背景的5-FOA抗性克隆，这些克隆来自于酵母中维持的细菌基因组的自发删除。在这里，我们报告了一种高效、无缝地修饰酵母中细菌基因组的方法。这种方法需要两个顺序的同源重组事件。首先，目标区域被一个突变盒取代，该盒由一个敲除核心(一个18bp的I-SCEI识别位点、GAL1启动子控制下的SCEI基因和URA3标记)和一个与靶点上游序列同源的DNA片段组成。替换产生位于核心两侧的串联重复序列。其次，半乳糖诱导I-SCEI的表达，在识别位点产生双链断裂(DSB)。这种DSB促进重复序列之间的分子内同源重组，并导致核心的切除。结果，产生了无缝修改。这种方法可以适用于各种基因组修饰，并可能为修改和设计在酵母中繁殖的天然或合成基因组提供重要工具。
The complete synthetic Mycoplasma genitalium genome (∼583 kb) has been assembled and cloned as a circular plasmid in the yeast Saccharomyces cerevisiae. Attempts to engineer the cloned genome by standard genetic methods involving the URA3/5-fluoroorotic acid (5-FOA) counter-selection have shown a high background of 5-FOA resistant clones derived from spontaneous deletions of the bacterial genome maintained in yeast. Here, we report a method that can seamlessly modify the bacterial genome in yeast with high efficiency. This method requires two sequential homologous recombination events. First, the target region is replaced with a mutagenesis cassette that consists of a knock-out CORE (an18-bp I-SceI recognition site, the SCEI gene under the control of the GAL1 promoter, and the URA3 marker) and a DNA fragment homologous to the sequence upstream of the target site. The replacement generates tandem repeat sequences flanking the CORE. Second, galactose induces the expression of I-SceI, which generates a double-strand break (DSB) at the recognition site. This DSB promotes intra-molecular homologous recombination between the repeat sequences, and leads to an excision of the CORE. As a result, a seamless modification is generated. This method can be adapted for a variety of genomic modifications and may provide an important tool to modify and design natural or synthetic genomes propagated in yeast.
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诱变的倒置重复辅助基因组工程（Mirage）。
影响因子：
14.9
作者：
Nair NU;Zhao H
通讯作者：
Zhao H
DOI：
10.1073/pnas.0535280100
发表时间：
2003-03-04
影响因子：
11.1
作者：
Gong, WJ;Golic, KG
通讯作者：
Golic, KG
影响因子：
3.3
作者：
ALANI, E;CAO, L;KLECKNER, N
通讯作者：
KLECKNER, N
影响因子：
14.9
作者：
Araki, K;Araki, M;Yamamura, KI
通讯作者：
Yamamura, KI
影响因子：
14.9
作者：
Noskov, V;Kouprina, N;Larionov, V
通讯作者：
Larionov, V