title: Multiplexed CRISPR/Cas9- and TAR-Mediated Promoter Engineering of Natural Product Biosynthetic Gene Clusters in Yeast.
abstract: The use of DNA sequencing to guide the discovery of natural products has emerged as a new paradigm for revealing chemistries encoded in bacterial genomes. A major obstacle to implementing this approach to natural product discovery is the transcriptional silence of biosynthetic gene clusters under laboratory growth conditions. Here we describe an improved yeast-based promoter engineering platform (mCRISTAR) that combines CRISPR/Cas9 and TAR to enable single-marker multiplexed promoter engineering of large gene clusters. mCRISTAR highlights the first application of the CRISPR/Cas9 system to multiplexed promoter engineering of natural product biosynthetic gene clusters. In this method, CRISPR/Cas9 is used to induce DNA double-strand breaks in promoter regions of biosynthetic gene clusters, and the resulting operon fragments are reassembled by TAR using synthetic gene-cluster-specific promoter cassettes. mCRISTAR uses a CRISPR array to simplify the construction of a CRISPR plasmid for multiplex CRISPR and a single auxotrophic selection to improve the inefficiency of using a CRISPR array for multiplex gene cluster refactoring. mCRISTAR is a simple and generic method for multiplexed replacement of promoters in biosynthetic gene clusters that will facilitate the discovery of natural products from the rapidly growing collection of gene clusters found in microbial genome and metagenome sequencing projects.
authors.affiliation: Laboratory of Genetically Encoded Small Molecules, The Rockefeller University , 1230 York Avenue, New York, New York 10065, United States.
authors.forename: Hahk-Soo
authors.initials: HS
authors.lastname: Kang
authors.affiliation: Laboratory of Genetically Encoded Small Molecules, The Rockefeller University , 1230 York Avenue, New York, New York 10065, United States.
authors.forename: Zachary
authors.initials: Z
authors.lastname: Charlop-Powers
authors.affiliation: Laboratory of Genetically Encoded Small Molecules, The Rockefeller University , 1230 York Avenue, New York, New York 10065, United States.
authors.forename: Sean F
authors.initials: SF
authors.lastname: Brady
mesh_terms: D001688:Biological Products / Q000378:metabolism*; D064113:CRISPR-Cas Systems / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D053903:DNA Breaks, Double-Stranded; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D016680:Genome, Bacterial / Q000235:genetics; D005810:Multigene Family / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods; D015003:Yeasts / Q000235:genetics*
chemical_list: D001688:Biological Products
keywords: CRISPR/Cas9; TAR; natural products; promoter engineering