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mpCRISTAR: Multiple Plasmid Approach for CRISPR/Cas9 and TAR-Mediated Multiplexed Refactoring of Natural Product Biosynthetic Gene Clusters.

Hiyoung KimChang-Hun JiHyun-Woo JeJong-Pyung KimHahk-Soo Kang
Published in: ACS synthetic biology (2019)
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.
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