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

1 Citation (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
Number of pages10
JournalMethods
DOIs
Publication statusE-pub ahead of print - 27 Jul 2019

Fingerprint

Clostridium beijerinckii
Clustered Regularly Interspaced Short Palindromic Repeats
Clostridium
Butanols
Plasmids
Genes
Ethanol
Gene encoding
2-Propanol
Cellulase
Acetone
Thiamphenicol
Genome
Technology
Chromosomes
Phenotype

Keywords

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

Cite this

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. ; van der Oost, John ; Ferreira, Nicolas Lopes ; López-Contreras, A.M. / Adaptation and application of a two-plasmid inducible CRISPR-Cas9 system in Clostridium beijerinckii. In: Methods. 2019.
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Adaptation and application of a two-plasmid inducible CRISPR-Cas9 system in Clostridium beijerinckii. / 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.; van der Oost, John; Ferreira, Nicolas Lopes; López-Contreras, A.M.

In: Methods, 27.07.2019.

Research output: Contribution to journalArticleAcademicpeer-review

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T1 - Adaptation and application of a two-plasmid inducible CRISPR-Cas9 system in Clostridium beijerinckii

AU - Diallo, M.

AU - Hocq, Rémi

AU - Collas, Florent

AU - Chartier, Gwladys

AU - Wasels, François

AU - Wijaya, Hani Surya

AU - Werten, Marc W.T.

AU - Wolbert, Emil J.H.

AU - Kengen, Servé W.M.

AU - van der Oost, John

AU - Ferreira, Nicolas Lopes

AU - López-Contreras, A.M.

PY - 2019/7/27

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N2 - 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.

AB - 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.

KW - Clostridium beijerinckii

KW - CRISPR-Cas9

KW - Genome editing

KW - Nuclease

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DO - 10.1016/j.ymeth.2019.07.022

M3 - Article

JO - Methods : a companion to Methods in enzymology

JF - Methods : a companion to Methods in enzymology

SN - 1046-2023

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