Genome Structure and Instability laboratory, CNRS UMR 7196, Inserm U1154, National Museum of Natural History, Life Adaptations department, 75005, Paris, France.
Sorbonne Universités, Paris, France.
Sci Rep. 2018 May 8;8(1):7127. doi: 10.1038/s41598-018-25508-3.
DNA repeats constitute a large part of genomes of multicellular eucaryotes. For a longtime considered as junk DNA, their role in genome organization and tuning of gene expression is being increasingly documented. Synthetic biology has so far largely ignored DNA repeats as regulatory elements to manipulate functions in engineered genomes. The yeast Saccharomyces cerevisiae has been a workhorse of synthetic biology, owing to its genetic tractability. Here we demonstrate the ability to synthetize, in a simple manner, tandem DNA repeats of various size by Cas9-assisted oligonucleotide in vivo assembly in this organism. We show that long tandem DNA repeats of several kilobases can be assembled in one step for different monomer size and G/C content. The combinatorial nature of the approach allows exploring a wide variety of design for building synthetic tandem repeated DNA directly at a given locus in the Saccharomyces cerevisiae genome. This approach provides a simple way to incorporate tandem DNA repeat in synthetic genome designs to implement regulatory functions.
DNA 重复序列构成了多细胞真核生物基因组的很大一部分。长期以来，它们被认为是“垃圾 DNA”，但现在越来越多的证据表明它们在基因组组织和基因表达调控中发挥着作用。合成生物学迄今为止在很大程度上忽略了 DNA 重复序列作为调控元件来操纵工程基因组中的功能。酵母酿酒酵母由于其遗传可操作性，一直是合成生物学的主要工具。在这里，我们证明了 Cas9 辅助的寡核苷酸在体内组装可以简单地合成各种大小的串联 DNA 重复序列。我们表明，对于不同的单体大小和 G/C 含量，可以一步组装几个千碱基的长串联 DNA 重复序列。该方法的组合性质允许在酿酒酵母基因组的特定基因座上直接探索构建合成串联重复 DNA 的各种设计。该方法为在合成基因组设计中引入串联 DNA 重复序列以实现调控功能提供了一种简单的方法。
