Improved CRISPR/Cas9 Tools for the Rapid Metabolic Engineering of Clostridium acetobutylicum

Wilding-Steele, Tom; Ramette, Quentin; Jacottin, Paul; Soucaille, Philippe

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas (CRISPR-associated proteins)9 tools have revolutionized biology—several highly efficient tools have been constructed that have resulted in the ability to quickly engineer model bacteria, for example, Escherichia coli. However, the use of CRISPR/Cas9 tools has lagged behind in non-model bacteria, hampering engineering efforts. Here, we developed improved CRISPR/Cas9 tools to enable efficient rapid metabolic engineering of the industrially relevant bacterium Clostridium acetobutylicum. Previous efforts to implement a CRISPR/Cas9 system in C. acetobutylicum have been hampered by the lack of tightly controlled inducible systems along with large plasmids resulting in low transformation efficiencies. We successfully integrated the cas9 gene from Streptococcus pyogenes into the genome under control of the xylose inducible system from Clostridium difficile, which we then showed resulted in a tightly controlled system. We then optimized the length of the editing cassette, resulting in a small editing plasmid, which also contained the upp gene in order to rapidly lose the plasmid using the upp/5-fluorouracil counter-selection system. We used this system to perform individual and sequential deletions of ldhA and the ptb-buk operon.

• Journal Article Type: Article
• Acceptance Date: Mar 31, 2021
• Online Publication Date: Apr 2, 2021
• Publication Date: Apr 1, 2021
• Deposit Date: Apr 26, 2021
• Publicly Available Date: Apr 26, 2021
• Journal: International Journal of Molecular Sciences
• Print ISSN: 1661-6596
• Electronic ISSN: 1422-0067
• Publisher: MDPI
• Peer Reviewed: Peer Reviewed
• Volume: 22
• Issue: 7
• Article Number: 3704
• Pages: 1-11
• DOI: https://doi.org/10.3390/ijms22073704
• Public URL: https://nottingham-repository.worktribe.com/output/5460629
• Publisher URL: https://www.mdpi.com/1422-0067/22/7/3704