Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Record number 553001
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.
DOI https://doi.org/10.1016/j.ymeth.2019.07.022
Department(s) BBP Bioconversion
BacGen
WIMEK
VLAG
Publication type Refereed Article in a scientific journal
Publication year 2019
Keyword(s) Clostridium beijerinckii - CRISPR-Cas9 - Genome editing - Nuclease
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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