CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae
CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae
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DOI：
10.1021/acssynbio.5b00007
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发表时间：
2015-11-01
影响因子：
4.7
通讯作者：
Keasling, Jay D.
中科院分区：
文献类型：
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作者：
Jakociunas, Tadas;Rajkumar, Arun S.;Keasling, Jay D.
酿酒酵母中的同源重组（HR）已被用于质粒构建和外源DNA的染色体整合。尽管如此，天然HR机制对于现代代谢工程所需的复杂和无标记的基因组工程来说仍然不够有效。在这里，我们提出了一种无标记的多位点整合体内组装的DNA部分的方法。通过使用CRISPR/Cas9介导的单、双和三重整合位点处的一步双链断裂，我们报告了DNA部分的成功体内组装和染色体整合。我们将我们的方法称为CasEMBLR，并以两种方式验证其对基因组工程和细胞工厂开发的适用性：（i）将15个DNA部分的类胡萝卜素途径引入三个靶向基因座，以及（ii）使用10个部分创建酪氨酸生产菌株，同时敲除两个基因。该方法补充和改进了目前用于S.啤酒。
Homologous recombination (HR) in Saccharomyces cerevisiae has been harnessed for both plasmid construction and chromosomal integration of foreign DNA. Still, native HR machinery is not efficient enough for complex and marker-free genome engineering required for modern metabolic engineering. Here, we present a method for marker-free multiloci integration of in vivo assembled DNA parts. By the use of CRISPR/Cas9-mediated one-step double-strand breaks at single, double and triple integration sites we report the successful in vivo assembly and chromosomal integration of DNA parts. We call our method CasEMBLR and validate its applicability for genome engineering and cell factory development in two ways: (i) introduction of the carotenoid pathway from 15 DNA parts into three targeted loci, and (ii) creation of a tyrosine production strain using ten parts into two loci, simultaneously knocking out two genes. This method complements and improves the current set of tools available for genome engineering in S. cerevisiae.