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Nat Microbiol. 2019 Feb;4(2):244-250. doi: 10.1038/s41564-018-0327-z. Epub 2019 Jan 7.

Enabling genetic analysis of diverse bacteria with Mobile-CRISPRi.

Author information

1
Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA. jason.peters@wisc.edu.
2
Pharmaceutical Sciences Division, and Departments of Bacteriology, and of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, USA. jason.peters@wisc.edu.
3
Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA.
4
Department of Medicine, University of California, San Francisco, San Francisco, CA, USA.
5
Division of Biological Sciences, University of California, San Diego, San Diego, CA, USA.
6
Center for Microbiome Innovation, Jacobs School of Engineering, University of California, San Diego, San Diego, CA, USA.
7
Department of Clinical Laboratory, The Third People's Hospital of Shenzhen, Shenzhen, China.
8
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.
9
Department of Microbiology, Cornell University, Ithaca, NY, USA.
10
Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA. cgrossucsf@gmail.com.
11
Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA. cgrossucsf@gmail.com.
12
California Institute of Quantitative Biology, University of California, San Francisco, San Francisco, CA, USA. cgrossucsf@gmail.com.
13
Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA. oren.rosenberg@ucsf.edu.
14
Department of Medicine, University of California, San Francisco, San Francisco, CA, USA. oren.rosenberg@ucsf.edu.

Abstract

The vast majority of bacteria, including human pathogens and microbiome species, lack genetic tools needed to systematically associate genes with phenotypes. This is the major impediment to understanding the fundamental contributions of genes and gene networks to bacterial physiology and human health. Clustered regularly interspaced short palindromic repeats interference (CRISPRi), a versatile method of blocking gene expression using a catalytically inactive Cas9 protein (dCas9) and programmable single guide RNAs, has emerged as a powerful genetic tool to dissect the functions of essential and non-essential genes in species ranging from bacteria to humans1-6. However, the difficulty of establishing effective CRISPRi systems across bacteria is a major barrier to its widespread use to dissect bacterial gene function. Here, we establish 'Mobile-CRISPRi', a suite of CRISPRi systems that combines modularity, stable genomic integration and ease of transfer to diverse bacteria by conjugation. Focusing predominantly on human pathogens associated with antibiotic resistance, we demonstrate the efficacy of Mobile-CRISPRi in gammaproteobacteria and Bacillales Firmicutes at the individual gene scale, by examining drug-gene synergies, and at the library scale, by systematically phenotyping conditionally essential genes involved in amino acid biosynthesis. Mobile-CRISPRi enables genetic dissection of non-model bacteria, facilitating analyses of microbiome function, antibiotic resistances and sensitivities, and comprehensive screens for host-microorganism interactions.

Comment in

PMID:
30617347
PMCID:
PMC6424567
DOI:
10.1038/s41564-018-0327-z
[Indexed for MEDLINE]
Free PMC Article

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