Targeted, genome-scale overexpression in Proteobacteria

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.