A Tool for Multiple Targeted Genome Deletions that Is Precise, Scar-Free, and Suitable for Automation.
A Tool for Multiple Targeted Genome Deletions that Is Precise, Scar-Free, and Suitable for Automation.
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DOI：
10.1371/journal.pone.0142494
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Clare A
中科院分区：
文献类型：
--
作者：
Aubrey W;Riley MC;Young M;King RD;Oliver SG;Clare A
合成生物学的许多进展需要从基因组中去除大量的基因组元件。大多数现有的删除方法都会留下标记，并且由于标记的数量有限，因此这些方法只能应用固定的次数。回收标记的删除方法通常是不精确的（去除非靶向序列），或留下疤痕序列，这可能导致基因组不稳定和重排。没有现有的标记回收方法是自动化友好的。我们开发了一种新的公开可用的删除工具，包括：1）一种删除基因组元件的方法，该方法可以无限制地重复使用，精确，无疤痕，适合自动化;和2）设计该方法引物的软件。我们的工具是序列不可知的，可用于删除单个基因组中的大量编码序列、启动子区域、转录因子结合位点、终止子等。我们已经验证了我们的工具对删除非必需的开放阅读框架（ORF）从S。啤酒。该工具适用于任意的基因组，我们提供了引物序列的缺失：90%的ORF从S。酿酒酵母基因组中，88%的ORF来自S. pombe基因组的ORF序列，85%的ORF来自L.乳酸菌基因组。
Many advances in synthetic biology require the removal of a large number of genomic elements from a genome. Most existing deletion methods leave behind markers, and as there are a limited number of markers, such methods can only be applied a fixed number of times. Deletion methods that recycle markers generally are either imprecise (remove untargeted sequences), or leave scar sequences which can cause genome instability and rearrangements. No existing marker recycling method is automation-friendly. We have developed a novel openly available deletion tool that consists of: 1) a method for deleting genomic elements that can be repeatedly used without limit, is precise, scar-free, and suitable for automation; and 2) software to design the method’s primers. Our tool is sequence agnostic and could be used to delete large numbers of coding sequences, promoter regions, transcription factor binding sites, terminators, etc in a single genome. We have validated our tool on the deletion of non-essential open reading frames (ORFs) from S. cerevisiae. The tool is applicable to arbitrary genomes, and we provide primer sequences for the deletion of: 90% of the ORFs from the S. cerevisiae genome, 88% of the ORFs from S. pombe genome, and 85% of the ORFs from the L. lactis genome.