A Targeted Genome-scale Overexpression Platform for Proteobacteria.

Autor:	Banta AB; Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison, Madison, WI, USA.; DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA., Myers KS; DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA.; Wisconsin Energy Institute, University of Wisconsin-Madison, Madison, WI, USA., Ward RD; Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison, Madison, WI, USA.; DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA.; Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI, USA., Cuellar RA; Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, WI, USA., Place M; DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA., Freeh CC; Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison, Madison, WI, USA., Bacon EE; Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison, Madison, WI, USA.; Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, WI, USA., Peters JM; Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison, Madison, WI, USA.; DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, USA.; Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA.; Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, USA.; Center for Genomic Science Innovation, University of Wisconsin-Madison, Madison, WI, USA.
Jazyk:	angličtina
Zdroj:	BioRxiv : the preprint server for biology [bioRxiv] 2024 Mar 04. Date of Electronic Publication: 2024 Mar 04.
DOI:	10.1101/2024.03.01.582922
Abstrakt:	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. Competing Interests: Competing interests The authors declare no competing interests.
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu