CRAGE-CRISPR facilitates rapid activation of secondary metabolite biosynthetic gene clusters in bacteria

Jing Ke; David Robinson; Zong-Yen Wu; Andrea Kuftin; Katherine Louie; Suzanne Kosina; Trent Northen; Jan-Fang Cheng; Yasuo Yoshikuni

doi:10.1016/j.chembiol.2021.08.009

CRAGE-CRISPR facilitates rapid activation of secondary metabolite biosynthetic gene clusters in bacteria

Cell Chem Biol. 2022 Apr 21;29(4):696-710.e4. doi: 10.1016/j.chembiol.2021.08.009. Epub 2021 Sep 10.

Authors

Jing Ke¹, David Robinson¹, Zong-Yen Wu¹, Andrea Kuftin¹, Katherine Louie¹, Suzanne Kosina¹, Trent Northen², Jan-Fang Cheng³, Yasuo Yoshikuni⁴

Affiliations

¹ US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
² 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.
³ 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. Electronic address: [email protected].
⁴ 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; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Center for Advanced Bioenergy and Bioproducts Innovation, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Global Center for Food, Land, and Water Resources, Hokkaido University, Hokkaido 060-8589, Japan. Electronic address: [email protected].

PMID: 34508657
DOI: 10.1016/j.chembiol.2021.08.009

Abstract

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.

Keywords: BGC activation; BGC deletion; BGC-to-compound characterization; CRAGE; CRISPR-Cas9; multiple sgRNA sites; secondary metabolites.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Bacteria
CRISPR-Cas Systems / genetics
Clustered Regularly Interspaced Short Palindromic Repeats* / genetics
Gene Editing
Genome, Bacterial
Multigene Family
Recombinases*

Substances

Recombinases