Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Pivot Concepts:
Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Target Concepts:
Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Query: EC:3.1.30.2 (
endonuclease
)
18,621
document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)
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
c
lustered
r
egularly
i
nterspaced
s
hort
p
alindromic
r
epeats (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 other methanotrophs and a wide array of Gram-negative microorganisms.
IMPORTANCE
In this study, we targeted the development and evaluation of broad-host-range CRISPR/Cas9 gene-editing tools in order to enhance the genetic-engineering capabilities of an industrially relevant methanotrophic biocatalyst. The CRISPR/Cas9 system developed in this study expands the genetic tools available to define molecular mechanisms in methanotrophic bacteria and has the potential to foster advances in the generation of novel biocatalysts to produce biofuels, platform chemicals, and high-value products from natural gas- and biogas-derived methane. Further, due to the broad-host-range applicability, these genetic tools may also enable innovative approaches to overcome the barriers associated with genetically engineering diverse, industrially promising nonmodel microorganisms.
...
PMID:Development of a CRISPR/Cas9 System for Methylococcus capsulatus
In Vivo
Gene Editing. 3092 29
