skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

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]
+ Show Author Affiliations
  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.
Copy to clipboard
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.
Copy to clipboard
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.
Copy to clipboard
@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}
}
Copy to clipboard
Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI:  10.1128/AEM.00340-19
Copyright Statement
Other availability
Search WorldCat Search WorldCat to find libraries that may hold this journal
Save / Share:
Export Metadata
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.

Works referenced in this record:

Engineering and characterization of a superfolder green fluorescent protein
journal, December 2005

  • Pédelacq, Jean-Denis; Cabantous, Stéphanie; Tran, Timothy
  • Nature Biotechnology, Vol. 24, Issue 1, p. 79-88
  • DOI: 10.1038/nbt1172

Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression
journal, February 2013

Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria
journal, September 2012

  • Gasiunas, G.; Barrangou, R.; Horvath, P.
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 39, p. E2579-E2586
  • DOI: 10.1073/pnas.1208507109

A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity
journal, June 2012

Metabolic engineering of Escherichia coli using CRISPR–Cas9 meditated genome editing
journal, September 2015

Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering
journal, December 2016

  • Garst, Andrew D.; Bassalo, Marcelo C.; Pines, Gur
  • Nature Biotechnology, Vol. 35, Issue 1, p. 48-55
  • DOI: 10.1038/nbt.3718

RNA-guided editing of bacterial genomes using CRISPR-Cas systems
journal, January 2013

  • Jiang, Wenyan; Bikard, David; Cox, David
  • Nature Biotechnology, Vol. 31, Issue 3, p. 233-239
  • DOI: 10.1038/nbt.2508
    [ × clear filter / sort ]

    Similar Records in DOE PAGES and OSTI.GOV collections: