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Title: Development of a CRISPR/Cas9 System for Methylococcus capsulatus In Vivo Gene Editing

Abstract

Methanotrophic bacteria play a crucial role in the Earth’s biogeochemical cycle and have the potential to be employed in industrial biomanufacturing processes due to their capacity to use natural gas- and biogas-derived methane as a sole carbon and energy source. Advanced gene-editing systems have the potential to enable rapid, high-throughput methanotrophic genetics and biocatalyst development. To this end, we employed a series of broad-host-range expression plasmids to construct a conjugatable clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene-editing system in Methylococcus capsulatus (Bath). Heterologous coexpression of the Streptococcus pyogenes Cas9 endonuclease and a synthetic single guide RNA (gRNA) showed efficient Cas9 DNA targeting and double-stranded DNA (dsDNA) cleavage that resulted in cell death. We demonstrated effective in vivo editing of plasmid DNA using both Cas9 and Cas9 D10A nickase to convert green fluorescent protein (GFP)- to blue fluorescent protein (BFP)-expressing cells with 71% efficiency. Further, we successfully introduced a premature stop codon into the soluble methane monooxygenase (sMMO) hydroxylase component-encoding mmoX gene with the Cas9 D10A nickase, disrupting sMMO function. These data provide proof of concept for CRISPR/Cas9-mediated gene editing in M. capsulatus. Given the broad-host-range replicons and conjugation capability of these CRISPR/Cas9 tools, they have potential utility in othermore » methanotrophs and a wide array of Gram-negative microorganisms.« less

Authors:
 [1];  [1]; ORCiD logo [1];  [2]
  1. National Renewable Energy Lab. (NREL), Golden, CO (United States)
  2. North Carolina State Univ., Raleigh, NC (United States)
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE National Renewable Energy Laboratory (NREL), Laboratory Directed Research and Development (LDRD) Program
OSTI Identifier:
1505930
Report Number(s):
NREL/JA-5100-73621
Journal ID: ISSN 0099-2240
Grant/Contract Number:  
AC36-08GO28308
Resource Type:
Accepted Manuscript
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 85; Journal Issue: 11; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; 59 BASIC BIOLOGICAL SCIENCES; CRISPR/Cas9; methanotroph; methane monooxygenase; gene editing; Methylococcus capsulatus; methane biocatalyst

Citation Formats

Tapscott, Timothy, Guarnieri, Michael T., Henard, Calvin A., and Kelly, Robert M. Development of a CRISPR/Cas9 System for Methylococcus capsulatus In Vivo Gene Editing. United States: N. p., 2019. Web. doi:10.1128/AEM.00340-19.
Tapscott, Timothy, Guarnieri, Michael T., Henard, Calvin A., & Kelly, Robert M. Development of a CRISPR/Cas9 System for Methylococcus capsulatus In Vivo Gene Editing. United States. doi:10.1128/AEM.00340-19.
Tapscott, Timothy, Guarnieri, Michael T., Henard, Calvin A., and Kelly, Robert M. Fri . "Development of a CRISPR/Cas9 System for Methylococcus capsulatus In Vivo Gene Editing". United States. doi:10.1128/AEM.00340-19. https://www.osti.gov/servlets/purl/1505930.
@article{osti_1505930,
title = {Development of a CRISPR/Cas9 System for Methylococcus capsulatus In Vivo Gene Editing},
author = {Tapscott, Timothy and Guarnieri, Michael T. and Henard, Calvin A. and Kelly, Robert M.},
abstractNote = {Methanotrophic bacteria play a crucial role in the Earth’s biogeochemical cycle and have the potential to be employed in industrial biomanufacturing processes due to their capacity to use natural gas- and biogas-derived methane as a sole carbon and energy source. Advanced gene-editing systems have the potential to enable rapid, high-throughput methanotrophic genetics and biocatalyst development. To this end, we employed a series of broad-host-range expression plasmids to construct a conjugatable clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene-editing system in Methylococcus capsulatus (Bath). Heterologous coexpression of the Streptococcus pyogenes Cas9 endonuclease and a synthetic single guide RNA (gRNA) showed efficient Cas9 DNA targeting and double-stranded DNA (dsDNA) cleavage that resulted in cell death. We demonstrated effective in vivo editing of plasmid DNA using both Cas9 and Cas9D10A nickase to convert green fluorescent protein (GFP)- to blue fluorescent protein (BFP)-expressing cells with 71% efficiency. Further, we successfully introduced a premature stop codon into the soluble methane monooxygenase (sMMO) hydroxylase component-encoding mmoX gene with the Cas9D10A nickase, disrupting sMMO function. These data provide proof of concept for CRISPR/Cas9-mediated gene editing in M. capsulatus. Given the broad-host-range replicons and conjugation capability of these CRISPR/Cas9 tools, they have potential utility in other methanotrophs and a wide array of Gram-negative microorganisms.},
doi = {10.1128/AEM.00340-19},
journal = {Applied and Environmental Microbiology},
number = 11,
volume = 85,
place = {United States},
year = {2019},
month = {3}
}

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