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

Patinios, C.; de Vries, S. T.; Diallo, M.; Lanza, L.; Verbrugge, P. L. J. V. Q.; Lopez-Contreras, A. M.; van der Oost, J.; Weusthuis, R. A.; Kengen, S. W. M.

doi:10.1101/2022.08.22.504755

bioRxiv · 10.1101/2022.08.22.504755

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

Patinios, C.·de Vries, S. T.·Diallo, M.·Lanza, L.·Verbrugge, P. L. J. V. Q.·Lopez-Contreras, A. M.·van der Oost, J.·Weusthuis, R. A.·Kengen, S. W. M.

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Abstract

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 several 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 strict 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.

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Patinios, C., de Vries, S. T., Diallo, M., Lanza, L., Verbrugge, P. L. J. V. Q., Lopez-Contreras, A. M., van der Oost, J., Weusthuis, R. A., Kengen, S. W. M.. 2022-08-23. Multiplex genome engineering in Clostridium beijerinckii NCIMB 8052 using CRISPR-Cas12a. https://doi.org/10.1101/2022.08.22.504755

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