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  • Title: Multiplex genome engineering in Clostridium beijerinckii NCIMB 8052 using CRISPR-Cas12a.
    Author: Patinios C, de Vries ST, Diallo M, Lanza L, Verbrugge PLJVQ, López-Contreras AM, van der Oost J, Weusthuis RA, Kengen SWM.
    Journal: Sci Rep; 2023 Jun 22; 13(1):10153. PubMed ID: 37349508.
    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 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.
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