Multiplexed refactoring provides a tool for rapid transcriptional optimization of biosynthetic gene clusters (BGCs) through simultaneous replacement of multiple native promoters with synthetic counterparts. Here, we present the mpCRISTAR, a multiple plasmid-based CRISPR/Cas9 and TAR (transformation-associated recombination), that enables a rapid and highly efficient, multiplexed refactoring of natural product BGCs in yeast. A series of CRISPR plasmids with different auxotrophic markers that could be stably maintained in yeast cells were constructed to express multiple gRNAs simultaneously. We demonstrated the multiplexing capacity of mpCRISTAR using the actinorhodin biosynthetic gene cluster as a model cluster. mpCRISTAR1, in which each CRISPR plasmid expresses one gRNA, allows for simultaneous replacement of up to four promoter sites with nearly 100% efficiency. By expressing two gRNAs from one CRISPR plasmid, termed mpCRISTAR2, we simultaneously replaced a total of six and eight promoter sites with 68% and 32% efficiency, respectively. The mpCRISTAR could be performed iteratively using two different auxotrophic markers, allowing for refactoring of any type of BGC regardless of their operon complexities. The mpCRISTAR platform we report here would become a useful tool for the discovery of new natural products from transcriptionally silent biosynthetic gene clusters present in microbial genomes.

中文翻译：

---

mpCRISTAR：CRISPR / Cas9和TAR介导的天然产物生物合成基因簇的多重重构的多重质粒方法。

多重重构提供了一种工具，可以通过用合成对应物同时替换多个天然启动子来快速优化生物合成基因簇（BGC）的转录。在这里，我们介绍了mpCRISTAR，这是一个基于多个质粒的CRISPR / Cas9和TAR（转化相关重组），可实现酵母中天然产物BGC的快速，高效，多重重构。可以稳定地维持在酵母细胞中的一系列具有不同营养缺陷型标记的CRISPR质粒被构建为同时表达多个gRNA。我们使用放线菌丝素生物合成基因簇作为模型簇证明了mpCRISTAR的多重能力。mpCRISTAR1，其中每个CRISPR质粒表达一个gRNA，允许同时替换多达四个启动子位点，效率接近100％。通过从一个称为mpCRISTAR2的CRISPR质粒表达两个gRNA，我们同时分别以68％和32％的效率替换了总共6个和8个启动子位点。mpCRISTAR可以使用两种不同的营养缺陷型标记物进行迭代操作，从而可以重构任何类型的BGC，而无需考虑其操纵子的复杂性。我们在这里报告的mpCRISTAR平台将成为从微生物基因组中存在的转录沉默生物合成基因簇中发现新的天然产物的有用工具。mpCRISTAR可以使用两种不同的营养缺陷型标记物进行迭代操作，从而可以重构任何类型的BGC，而无需考虑其操纵子的复杂性。我们在这里报告的mpCRISTAR平台将成为从微生物基因组中存在的转录沉默生物合成基因簇中发现新的天然产物的有用工具。mpCRISTAR可以使用两种不同的营养缺陷型标记物进行迭代操作，从而可以重构任何类型的BGC，而无需考虑其操纵子的复杂性。我们在这里报告的mpCRISTAR平台将成为从微生物基因组中存在的转录沉默生物合成基因簇中发现新的天然产物的有用工具。