%0 Journal Article %T Adaptation and application of a two-plasmid inducible CRISPR-Cas9 system in Clostridium beijerinckii %+ Wageningen Food & Biobased Research %+ IFP Energies nouvelles (IFPEN) %+ Wageningen University and Research [Wageningen] (WUR) %A Diallo, Mamou %A Hocq, Rémi %A Collas, Florent %A Chartier, Gwladys %A Wasels, François %A Wijaya, Hani Surya %A Werten, Marc W.T. %A Wolbert, Emil J.H. %A Kengen, Servé W.M. %A van Der Oost, John %A Ferreira, Nicolas Lopes %A López-Contreras, Ana, M %< avec comité de lecture %@ 1046-2023 %J Methods %I Elsevier %V 172 %P 51-60 %8 2020-02 %D 2020 %R 10.1016/j.ymeth.2019.07.022 %K Clostridium beijerinckii %K CRISPR-Cas9 %K Nuclease %K Genome editing %Z Environmental SciencesJournal articles %X 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. %G English %2 https://ifp.hal.science/hal-02572152/document %2 https://ifp.hal.science/hal-02572152/file/Adaptation%20and%20%20Application%20of%20Two-Plasmid.pdf %L hal-02572152 %U https://ifp.hal.science/hal-02572152 %~ SDE %~ IFP %~ OPENAIRE %~ GIP-BE