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

Timothy Tapscott,Michael T Guarnieri,Calvin A Henard,Robert M Kelly

doi:10.1128/aem.00340-19

Timothy Tapscott, Michael T Guarnieri + Show 2 more

Open Access

https://doi.org/10.1128/aem.00340-19

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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 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.

Highlights

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
We employed the superfolder green fluorescent protein (GFP) [26] reporter to evaluate the functionality and strength of heterologous and native M. capsulatus promoters to be used for expression of CRISPRassociated protein 9 (Cas9) nuclease and guide RNA (gRNA) components
We tested whether pCAH01, an RK2-based broad-host-range expression plasmid that contains the inducible tetracycline promoter/operator (PtetA), previously demonstrated to function in the related gammaproteobacterial methanotroph Methylomicrobium buryatense 5GB1 [21, 23], was functional in M. capsulatus

Summary

Introduction

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. Chromosomal insertions and unmarked genetic mutations using allelic-exchange vectors and sucrose or p-chlorophenylalanine counterselection have been reported [15,16,17,18,19] These tools have served as a basis for the recent expansion of the methanotroph genetic toolbox that has enabled several proteobacterial methanotrophs to be engineered to convert methane-rich natural gas and aerobic-digestion-derived biogas into high-value products [20,21,22,23,24,25]. These tools lay the foundation for the development of advanced CRISPR genome-editing systems that facilitate multiplex or high-throughput gene-editing strategies. The CRISPR/Cas tools developed here will facilitate the development of advanced methanotrophic biocatalysts and have potential utility in an array of nonmodel, industrially promising bacteria

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