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Mobile-CRISPRi: Enabling Genetic Analysis of Diverse Bacteria

Jason M. Peters, Byoung-Mo Koo, Ramiro Patino, Gary E. Heussler, Cameron C. Hearne, Yuki F. Inclan, John S. Hawkins, Candy H. S. Lu, M. Michael Harden, Hendrik Osadnik, Joseph E. Peters, Joanne N. Engel, Rachel J. Dutton, Alan D. Grossman, Carol A. Gross, View ORCID ProfileOren S. Rosenberg
doi: https://doi.org/10.1101/315499
Jason M. Peters
1Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA.
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Byoung-Mo Koo
1Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA.
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Ramiro Patino
1Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA.
2Department of Medicine, University of California, San Francisco, San Francisco, CA, USA.
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Gary E. Heussler
3Division of Biological Sciences, University of California, San Diego, San Diego, USA.
4Center for Microbiome Innovation, Jacobs School of Engineering, University of California, San Diego, San Diego, USA.
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Cameron C. Hearne
1Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA.
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Yuki F. Inclan
1Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA.
2Department of Medicine, University of California, San Francisco, San Francisco, CA, USA.
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John S. Hawkins
1Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA.
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Candy H. S. Lu
1Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA.
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M. Michael Harden
5Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.
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Hendrik Osadnik
1Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA.
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Joseph E. Peters
6Department of Microbiology, Cornell University, Ithaca, NY, USA.
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Joanne N. Engel
1Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA.
2Department of Medicine, University of California, San Francisco, San Francisco, CA, USA.
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Rachel J. Dutton
3Division of Biological Sciences, University of California, San Diego, San Diego, USA.
4Center for Microbiome Innovation, Jacobs School of Engineering, University of California, San Diego, San Diego, USA.
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Alan D. Grossman
5Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.
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Carol A. Gross
1Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA.
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Oren S. Rosenberg
1Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA.
2Department of Medicine, University of California, San Francisco, San Francisco, CA, USA.
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  • ORCID record for Oren S. Rosenberg
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Introductory paragraph

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. CRISPRi, a versatile method of blocking gene expression using a catalytically inactive Cas9 protein (dCas9) and programmable single guide RNAs (sgRNAs), has emerged as a powerful genetic tool to dissect the functions of essential and non-essential genes in species ranging from bacteria to human. However, the difficulty of establishing effective CRISPRi systems in non-model 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 combine 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 Proteobacteria and 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-microbe interactions.

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Posted May 05, 2018.
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Mobile-CRISPRi: Enabling Genetic Analysis of Diverse Bacteria
Jason M. Peters, Byoung-Mo Koo, Ramiro Patino, Gary E. Heussler, Cameron C. Hearne, Yuki F. Inclan, John S. Hawkins, Candy H. S. Lu, M. Michael Harden, Hendrik Osadnik, Joseph E. Peters, Joanne N. Engel, Rachel J. Dutton, Alan D. Grossman, Carol A. Gross, Oren S. Rosenberg
bioRxiv 315499; doi: https://doi.org/10.1101/315499
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Mobile-CRISPRi: Enabling Genetic Analysis of Diverse Bacteria
Jason M. Peters, Byoung-Mo Koo, Ramiro Patino, Gary E. Heussler, Cameron C. Hearne, Yuki F. Inclan, John S. Hawkins, Candy H. S. Lu, M. Michael Harden, Hendrik Osadnik, Joseph E. Peters, Joanne N. Engel, Rachel J. Dutton, Alan D. Grossman, Carol A. Gross, Oren S. Rosenberg
bioRxiv 315499; doi: https://doi.org/10.1101/315499

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