With the advent of genome sequencing and mining technologies, secondary metabolite biosynthetic gene clusters (BGCs) within bacterial genomes are becoming easier to predict. For subsequent BGC characterization, clustered regularly interspaced short palindromic repeats (CRISPR) has contributed to knocking out target genes and/or modulating their expression; however, CRISPR is limited to 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 enables CRISPR systems in diverse bacteria. To demonstrate CRAGE-CRISPR, we select 10 polyketide/non-ribosomal peptide BGCs in Photorhabdus luminescens as models and create their deletion and activation mutants. Subsequent loss- and gain-of-function studies confirm 22 secondary metabolites associated with the BGCs, including a metabolite from a previously uncharacterized BGC. These results demonstrate that the CRAGE-CRISPR system is a simple yet powerful approach to rapidly perturb expression of defined BGCs and to profile genotype-phenotype relationships in bacteria.

随着基因组测序和挖掘技术的出现，细菌基因组内的次级代谢物生物合成基因簇 (BGC) 变得更容易预测。对于随后的 BGC 表征，成簇的规则间隔短回文重复序列 (CRISPR) 有助于敲除靶基因和/或调节它们的表达；然而，CRISPR 仅限于可以使用强大的遗传工具的菌株。在这里，我们提出了一种将 CRISPR 与独立于底盘的重组酶辅助基因组工程 (CRAGE) 相结合的策略，该策略使 CRISPR 系统能够应用于多种细菌。为了演示 CRAGE-CRISPR，我们在Photorhabdus luminescens中选择了 10 个聚酮化合物/非核糖体肽BGC作为模型并创建它们的删除和激活突变体。随后的功能丧失和获得研究证实了 22 种与 BGC 相关的次级代谢物，包括来自以前未表征的 BGC 的代谢物。这些结果表明，CRAGE-CRISPR 系统是一种简单而强大的方法，可以快速扰乱已定义 BGC 的表达并分析细菌中的基因型-表型关系。