Multiplex genome engineering in Clostridium beijerinckii NCIMB 8052 using CRISPR-Cas12a

Constantinos Patinios, Stijn T. de Vries, Mamou Diallo, Lucrezia Lanza, Pepijn L.J.V.Q. Verbrugge, Ana M. López-Contreras, John van der Oost, Ruud A. Weusthuis, Servé W.M. Kengen*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review


Clostridium species are re-emerging as biotechnological workhorses for industrial acetone–butanol–ethanol production. This re-emergence is largely due to advances in fermentation technologies but also due to advances in genome engineering and re-programming of the native metabolism. Several genome engineering techniques have been developed including the development of numerous CRISPR-Cas tools. Here, we expanded the CRISPR-Cas toolbox and developed a CRISPR-Cas12a genome engineering tool in Clostridium beijerinckii NCIMB 8052. By controlling the expression of FnCas12a with the xylose-inducible promoter, we achieved efficient (25–100%) single-gene knockout of five C. beijerinckii NCIMB 8052 genes (spo0A, upp, Cbei_1291, Cbei_3238, Cbei_3832). Moreover, we achieved multiplex genome engineering by simultaneously knocking out the spo0A and upp genes in a single step with an efficiency of 18%. Finally, we showed that the spacer sequence and position in the CRISPR array can affect the editing efficiency outcome.

Original languageEnglish
Article number10153
JournalScientific Reports
Issue number1
Publication statusPublished - 22 Jun 2023


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