Gene overexpression tool for Protobacteria advances basic science and applied biodesign

Schematic overview of CRISPRtOE (CRISPR Targeted Gene OverExpression), showing VcCAST-mediated transposon insertion of a promoter upstream of a target gene to boost product output. The method is applied to medically-relevant species (Acinetobacter, Enterobacter, E. coli, Klebsiella, Pseudomonas aeruginosa) and industrially-relevant species (P. putida, Shewanella, Zymomonas mobilis), including genome-scale overexpression screens in E. coli (antibiotic resistance to trimethoprim/fosfomycin) and Z. mobilis (hy
CRISPR transposition and overexpression (CRISPRtOE) allows researchers to insert sequences of genes, known as promoters, that increase the activity of nearby genes in bacteria, providing a practical tool for identifying genes required for resistance to antibiotics or biofuel production stressors. Photo by: Courtesy of authors

Background/Objective

Targeted, genome-scale screens using clustered regularly interspaced short palindromic repeats interference (CRISPRi) and activation (CRISPRa) have revolutionized eukaryotic genetics, advancing medical, industrial, and basic research. While CRISPRi has been used for gene knockdowns in bacteria, options for genome-scale over-expression have been limited.

Approach

Scientists with the Great Lakes Bioenergy Research Center developed a facile approach for bacterial genome-scale over-expression known as CRISPR transposition and OverExpression (CRISPRtOE) and demonstrated that it can upregulate gene expression in medically and industrially relevant Protobacteria by integrating synthetic promoters of varying strength upstream of target genes.

Results

Researchers created a platform for comprehensive gene targeting using CRISPR-associated transposons (CASTs) and showed that transposition occurs at higher frequency in non-transcribed DNA. They showed CRISPRtOE can upregulate gene expression in medically and industrially relevant Proteobacteria by integrating synthetic promoters of varying strength upstream of target genes. They employed genome-scale CRISPRtOE screening in Escherichia coli K-12, recovering known and novel antibiotic targets, and in the non-model biofuel producing Zymomonas mobilis to define a potential engineering target (rpoE a.k.a. ecfZ) to overcome bioproduction stress.

Significance/Impacts

Systematic alteration of bacterial gene expression enables identification of genes relevant to diverse fields of study and practical applications, but there are few established facile approaches for systemic overexpression in bacteria. This approach can be used to systematically overexpress genes in both model and non-model Protobacteria, making it a valuable tool for both basic biology and applied biodesign, including industrial strain optimization. 

Banta, A. B., et al. Targeted, genome-scale overexpression in Proteobacteria. mBio, e02340-25. (2026). [DOI:10.1128/mbio.02340-25]

Research Theme
Sustainable Biomass Conversion
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