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首页> 外文期刊>Nucleic acids research >Evolution of variants of yeast site-specific recombinase Flp that utilize native genomic sequences as recombination target sites
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Evolution of variants of yeast site-specific recombinase Flp that utilize native genomic sequences as recombination target sites

机译：利用天然基因组序列作为重组靶位点的酵母位点特异性重组酶Flp变体的进化

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As a tool in directed genome manipulations, site-specific recombination is a double-edged sword. Exquisite specificity, while highly desirable, makes it imperative that the target site be first inserted at the desired genomic locale before it can be manipulated. We describe a combination of computational and experimental strategies, based on the tyrosine recombinase Flp and its target site FRT, to overcome this impediment. We document the systematic evolution of Flp variants that can utilize, in a bacterial assay, two sites from the human interleukin 10 gene, IL10, as recombination substrates. Recombination competence on an end target site is acquired via chimeric sites containing mixed sequences from FRT and the genomic locus. This is the first time that a tyrosine site-specific recombinase has been coaxed successfully to perform DNA exchange within naturally occurring sequences derived from a foreign genomic context. We demonstrate the ability of an Flp variant to mediate integration of a reporter cassette in Escherichia coli via recombination at one of the IL10-derived sites.
机译：作为定向基因组操作的工具，位点特异性重组是一把双刃剑。极高的特异性，尽管非常理想，但必须先将目标位点插入所需的基因组位置，然后才能对其进行操作。我们描述了基于酪氨酸重组酶Flp及其靶位点FRT的计算和实验策略的组合，以克服这一障碍。我们记录了Flp变体的系统进化，该变体可以在细菌测定中利用人白介素10基因IL10的两个位点作为重组底物。通过包含来自FRT和基因组基因座的混合序列的嵌合位点获得在最终靶位点上的重组能力。这是第一次成功地诱使酪氨酸位点特异性重组酶在源自外源基因组环境的天然存在的序列内进行DNA交换。我们展示了一种Flp变体通过介导IL10衍生的位点之一重组来介导大肠埃希氏菌中的报告基因盒整合的能力。

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