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

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.

Development of a CRISPR-Cas12a system for efficient genome engineering in clostridia

“…By deleting the uidA gene in the model microbe E. coli , we suggest that selecting targets based on pre-crRNA folding predictions may improve the efficiency of CRISPR-Cas12a systems across various species. There is a recent observation in Clostridium beijerinckii that some target sequences in the CRISPR array result in the low efficiency of their CRISPR-FnCas12a system ( 17 ).…”

“…While genome engineering in clostridia using Cas9 has been applied extensively across multiple species, Cas12a (previously known as Cpf1) is an alternative CRISPR-associated protein that has shown potential advantages over Cas9, including the recognition of T-rich PAMs for clostridia ( 13 ) and lower toxicity ( 14 ). To date, only in a few solventogenic Clostridium species has the Cas12a protein been introduced for genome engineering ( 13 , 15 – 17 ).…”

Tolerance in Solventogenic Clostridia for Enhanced Butanol Production: Genetic Mechanisms and Recent Strain Engineering Advances

“…possesses the protospacer-adjacent motif (PAM) sequence, "TTTN", which is particularly advantageous for AT-rich organisms like Clostridium [70]. This technique has been employed to make multiplex gene editing in C. beijerinckii [71]. CRISPRi employs a 'broken scissors' principle, with dead Cas9 (dCas9) being a typical example.…”