Ke Jing, Robinson David, Wu Zong-Yen, Kuftin Andrea, Louie Katherine, Kosina Suzanne, Northen Trent, Cheng Jan-Fang, Yoshikuni Yasuo
US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Cell Chem Biol. 2022 Apr 21;29(4):696-710.e4. doi: 10.1016/j.chembiol.2021.08.009. Epub 2021 Sep 10.
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，细菌基因组中的次生代谢物生物合成基因簇（BGCs）越来越容易预测。对于后续的BGC表征，成簇规律间隔短回文重复序列（CRISPR）有助于敲除靶基因和/或调节其表达；然而，CRISPR仅限于拥有强大遗传工具的菌株。在此，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，该策略可在多种细菌中实现CRISPR系统。为了证明CRAGE-CRISPR，我们选择了发光杆菌中的10个聚酮/非核糖体肽BGCs作为模型，并创建了它们的缺失和激活突变体。随后的功能丧失和功能获得研究证实了与这些BGCs相关的22种次生代谢物，包括一种来自先前未表征的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，可快速干扰特定BGCs的表达并分析细菌中的基因型-表型关系。
