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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 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 andmore » 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. https://doi.org/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. https://doi.org/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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