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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.

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