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

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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https://hal-ifp.archives-ouvertes.fr/hal-02572152
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Submitted on : Wednesday, May 13, 2020 - 2:59:06 PM
Last modification on : Saturday, October 17, 2020 - 7:16:04 PM

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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, Elsevier, 2020, 172, pp.51-60. ⟨10.1016/j.ymeth.2019.07.022⟩. ⟨hal-02572152⟩

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