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Journal Articles Methods Year : 2020

Adaptation and application of a two-plasmid inducible CRISPR-Cas9 system in Clostridium beijerinckii

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Abstract

Recent developments in CRISPR technologies have opened new possibilities for improving genome editing tools dedicated to the Clostridium genus. In this study we adapted a two-plasmid tool based on this technology to enable scarless modification of the genome of two reference strains of Clostridium beijerinckii producing an Acetone/Butanol/Ethanol (ABE) or an Isopropanol/Butanol/Ethanol (IBE) mix of solvents. In the NCIMB 8052 ABE-producing strain, inactivation of the SpoIIE sporulation factor encoding gene resulted in sporulation-deficient mutants, and this phenotype was reverted by complementing the mutant strain with a functional spoIIE gene. Furthermore, the fungal cellulase-encoding celA gene was inserted into the C. beijerinckii NCIMB 8052 chromosome, resulting in mutants with endoglucanase activity. A similar two-plasmid approach was next used to edit the genome of the natural IBE-producing strain C. beijerinckii DSM 6423, which has never been genetically engineered before. Firstly, the catB gene conferring thiamphenicol resistance was deleted to make this strain compatible with our dual-plasmid editing system. As a proof of concept, our dual-plasmid system was then used in C. beijerinckii DSM 6423 ΔcatB to remove the endogenous pNF2 plasmid, which led to a sharp increase of transformation efficiencies.
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Dates and versions

hal-02572152 , version 1 (13-05-2020)

Licence

Attribution - NonCommercial - NoDerivatives - CC BY 4.0

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Cite

Mamou Diallo, Rémi Hocq, Florent Collas, Gwladys Chartier, François Wasels, et al.. Adaptation and application of a two-plasmid inducible CRISPR-Cas9 system in Clostridium beijerinckii. Methods, 2020, 172, pp.51-60. ⟨10.1016/j.ymeth.2019.07.022⟩. ⟨hal-02572152⟩

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