Clostridiumspecies 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 inClostridium beijerinckiiNCIMB 8052. By controlling the expression of FnCas12a with the xylose-inducible promoter, we achieved efficient (25–100%) single-gene knockout of fiveC. beijerinckiiNCIMB 8052 genes (spo0A,upp,Cbei_1291,Cbei_3238,Cbei_3832). Moreover, we achieved multiplex genome engineering by simultaneously knocking out thespo0Aanduppgenes 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.