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 overexpression face key limitations. Here, we develop a facile approach for genome-scale gene overexpression in bacteria we call, "CRISPRtOE" (CRISPR transposition and OverExpression). We create a platform for comprehensive gene targeting using CRISPR-associated transposition (CAST) 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 Escherichia coli, recovering known antibiotic targets and genes with unexplored roles in antibiotic function. We envision that CRISPRtOE will be a valuable overexpression tool for antibiotic mode of action, industrial strain optimization, and gene function discovery in bacteria.