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Peer-reviewedCC BY 4.0mBio

Targeted, genome-scale overexpression in Proteobacteria

  • Amy B. Banta1,2
  • Ashley N. Hall1,2
  • Kevin S. Myers1,3
  • Ryan David Ward1,4,2
  • Bryce C. Davis2
  • Rodrigo A. Cuellar5
  • Michael Place1
  • Claire C. Freeh2
  • Emily E. Bacon5,2
  • Jason M. Peters6,1,7,8,2
8 affiliations
  1. 1DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA
  2. 2Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison, Madison, WI, USA
  3. 3Wisconsin Energy Institute, University of Wisconsin-Madison, Madison, WI, USA
  4. 4Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI, USA
  5. 5Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, WI, USA
  6. 6Center for Genomic Science Innovation, University of Wisconsin-Madison, Madison, WI, USA
  7. 7Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA
  8. 8Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, USA
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Abstract

Targeted, genome-scale gene perturbation screens using clustered regularly interspaced short palindromic repeats interference (CRISPRi) and activation (CRISPRa) have revolutionized eukaryotic genetics, advancing medical, industrial, and basic research. Although CRISPRi knockdowns have been broadly applied in bacteria, options for genome-scale gene overexpression face key limitations. Here, we develop a facile approach for genome-scale overexpression in bacteria we call CRISPR transposition and overexpression (CRISPRtOE). We first create a platform for comprehensive gene targeting using CRISPR-associated transposons (CASTs) and show that transposition occurs at a higher frequency in non-transcribed DNA. We then demonstrate that CRISPRtOE can upregulate gene expression in Proteobacteria with medical and industrial relevance by integrating synthetic promoters of varying strength upstream of target genes. Finally, we employ CRISPRtOE screening at the genome scale in the model bacterium Escherichia coli and the non-model biofuel producer Zymomonas mobilis, recovering known and novel antibiotic and engineering targets. We envision that CRISPRtOE will be a valuable overexpression tool for antibiotic mode of action, industrial strain optimization, and gene function discovery in bacteria.

IMPORTANCE: Systematic alteration of bacterial gene expression enables identification of genes relevant to diverse fields of study and practical applications. Although many targeted, genome-scale genetic tools exist for reducing or eliminating gene expression, there are few facile approaches for systematic gene overexpression in bacteria. Here, we develop a targeted overexpression approach for Proteobacteria of medical and industrial importance that precisely inserts strong promoters upstream of genes using clustered regularly interspaced short palindromic repeats (CRISPR)-associated transposons. We demonstrate that this approach can be used to systematically overexpress genes in both model (Escherichia coli K-12) and non-model (Zymomonas mobilis) Proteobacteria for the purpose of understanding antibiotic resistance mechanisms and improving strain resilience in biofuel production conditions, respectively.

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Cite

BibTeXRIS
BibTeX
@article{banta2026targeted,
  title = {{Targeted, genome-scale overexpression in Proteobacteria}},
  author = {Amy B. Banta and Ashley N. Hall and Kevin S. Myers and Ryan David Ward and Bryce C. Davis and Rodrigo A. Cuellar and Michael Place and Claire C. Freeh and Emily E. Bacon and Jason M. Peters},
  journal = {mBio},
  year = {2026},
  month = sep,
  doi = {10.1128/mbio.02340-25},
  url = {https://doi.org/10.1128/mbio.02340-25},
  note = {CC BY 4.0},
}
RIS
TY  - JOUR
TI  - Targeted, genome-scale overexpression in Proteobacteria
AU  - Amy B. Banta
AU  - Ashley N. Hall
AU  - Kevin S. Myers
AU  - Ryan David Ward
AU  - Bryce C. Davis
AU  - Rodrigo A. Cuellar
AU  - Michael Place
AU  - Claire C. Freeh
AU  - Emily E. Bacon
AU  - Jason M. Peters
PY  - 2026
DA  - 2026/09/21
JO  - mBio
DO  - 10.1128/mbio.02340-25
UR  - https://doi.org/10.1128/mbio.02340-25
AB  - Targeted, genome-scale gene perturbation screens using clustered regularly interspaced short palindromic repeats interference (CRISPRi) and activation (CRISPRa) have revolutionized eukaryotic genetics, advancing medical, industrial, and basic research. Although CRISPRi knockdowns have been broadly applied in bacteria, options for genome-scale gene overexpression face key limitations. Here, we develop a facile approach for genome-scale overexpression in bacteria we call CRISPR transposition and overexpression (CRISPRtOE). We first create a platform for comprehensive gene targeting using CRISPR-associated transposons (CASTs) and show that transposition occurs at a higher frequency in non-transcribed DNA. We then demonstrate that CRISPRtOE can upregulate gene expression in Proteobacteria with medical and industrial relevance by integrating synthetic promoters of varying strength upstream of target genes. Finally, we employ CRISPRtOE screening at the genome scale in the model bacterium Escherichia coli and the non-model biofuel producer Zymomonas mobilis, recovering known and novel antibiotic and engineering targets. We envision that CRISPRtOE will be a valuable overexpression tool for antibiotic mode of action, industrial strain optimization, and gene function discovery in bacteria.  IMPORTANCE: Systematic alteration of bacterial gene expression enables identification of genes relevant to diverse fields of study and practical applications. Although many targeted, genome-scale genetic tools exist for reducing or eliminating gene expression, there are few facile approaches for systematic gene overexpression in bacteria. Here, we develop a targeted overexpression approach for Proteobacteria of medical and industrial importance that precisely inserts strong promoters upstream of genes using clustered regularly interspaced short palindromic repeats (CRISPR)-associated transposons. We demonstrate that this approach can be used to systematically overexpress genes in both model (Escherichia coli K-12) and non-model (Zymomonas mobilis) Proteobacteria for the purpose of understanding antibiotic resistance mechanisms and improving strain resilience in biofuel production conditions, respectively.
SN  - 2150-7511
SN  - 2161-2129
ER  -