CRAGE-CRISPR Systems Facilitate Rapid Activation and Functional Characterization of Secondary Metabolite Biosynthetic Gene Clusters in Non-Model Bacteria

Abstract

Secondary metabolite biosynthetic gene clusters (BGCs) within bacterial genomes are becoming easier to predict. For subsequent characterization of their cellular functions, clustered regularly interspaced short palindromic repeats (CRISPR) systems have significantly contributed to knocking out the target genes and/or modulating their expression. However, use of CRISPR systems is limited for strains for which robust genetic tools are available. Here we present a strategy that combines CRISPR with chassis-independent recombinase-assisted genome engineering (CRAGE), which will enable implementation of CRISPR systems in diverse non-model bacteria. For demonstration, we selected 10 polyketide (PKS)/nonribosomal peptide (NRPS) BGCs created deletion and activation mutants of all 10 BGCs. Subsequent loss- and gain-of-function studies identified 22 secondary metabolites associated with the BGCs, including a product that was previously uncharacterized. These results demonstrate that the CRAGE-CRISPR system is a simple yet very powerful approach to rapidly perturb expression of defined BGCs and to profile genotype-phenotype relationships in bacteria.