Precise manipulation of chromosomes in vivo enables genome-wide codon replacement.
Precise manipulation of chromosomes in vivo enables genome-wide codon replacement.
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DOI：
10.1126/science.1205822
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发表时间：
2011-07-15
期刊：
影响因子：
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通讯作者：
Church GM
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文献类型：
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作者：
Isaacs FJ;Carr PA;Wang HH;Lajoie MJ;Sterling B;Kraal L;Tolonen AC;Gianoulis TA;Goodman DB;Reppas NB;Emig CJ;Bang D;Hwang SJ;Jewett MC;Jacobson JM;Church GM
我们目前的基因组工程技术，能够从根本上重新设计基因组从核苷酸到兆规模。我们使用多重自动化基因组工程（法师），在32个大肠杆菌菌株中平行地用同义TAA密码子位点特异性地替换所有314个TAG终止密码子。这种方法使我们能够测量个体重组频率，确认每个修饰的可行性，并鉴定相关的表型。我们开发了分层接合组装基因组工程（CAGE），将这些密码子修饰集合并到基因组中，具有80个精确的变化，这表明这些同义密码子取代可以组合成更高阶的菌株，而不会产生合成致死效应。我们的方法将染色体视为可编辑和可进化的模板，允许探索广阔的遗传景观。
We present genome engineering technologies that are capable of fundamentally reengineering genomes from the nucleotide to the megabase scale. We used multiplex automated genome engineering (MAGE) to site-specifically replace all 314 TAG stop codons with synonymous TAA codons in parallel across 32 Escherichia coli strains. This approach allowed us to measure individual recombination frequencies, confirm viability for each modification, and identify associated phenotypes. We developed hierarchical conjugative assembly genome engineering (CAGE) to merge these sets of codon modifications into genomes with 80 precise changes, which demonstrate that these synonymous codon substitutions can be combined into higher-order strains without synthetic lethal effects. Our methods treat the chromosome as both an editable and an evolvable template, permitting the exploration of vast genetic landscapes.