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

M. Diallo*, Rémi Hocq, Florent Collas, Gwladys Chartier, François Wasels, Hani Surya Wijaya, Marc W.T. Werten, Emil J.H. Wolbert, Servé W.M. Kengen, John van der Oost, Nicolas Lopes Ferreira, A.M. López-Contreras

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

2 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)51-60
Number of pages10
JournalMethods
Volume172
Early online date27 Jul 2019
DOIs
Publication statusPublished - 1 Feb 2020

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Keywords

  • Clostridium beijerinckii
  • CRISPR-Cas9
  • Genome editing
  • Nuclease

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