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

Mamou Diallo (1) , Rémi Hocq (2) , Florent Collas (1) , Gwladys Chartier (2) , François Wasels (2) , Hani Surya Wijaya (1) , Marc W.T. Werten (1) , Emil J.H. Wolbert (1) , Servé W.M. Kengen (3) , John van Der Oost (3) , Nicolas Lopes Ferreira (2) , Ana M López-Contreras (1)

1 Wageningen Food & Biobased Research
2 IFPEN - IFP Energies nouvelles
3 WUR - Wageningen University and Research [Wageningen]

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.

Mots clés: Clostridium beijerinckii; CRISPR-Cas9; Nuclease; Genome editing

Domaines: Sciences de l'environnement

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⟩