|Title||Adaptation and application of a two-plasmid inducible CRISPR-Cas9 system in Clostridium beijerinckii|
|Author(s)||Diallo, M.; Hocq, Rémi; Collas, Florent; Chartier, Gwladys; Wasels, François; Wijaya, Hani Surya; Werten, Marc W.T.; Wolbert, Emil J.H.; Kengen, Servé W.M.; Oost, John van der; Ferreira, Nicolas Lopes; López-Contreras, A.M.|
|Source||Methods : a companion to Methods in enzymology (2019). - ISSN 1046-2023 - 10 p.|
|Publication type||Refereed Article in a scientific journal|
|Keyword(s)||Clostridium beijerinckii - CRISPR-Cas9 - Genome editing - Nuclease|
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.