Journal of biotechnologyNov 7, 2025

Using CRISPR and anti-CRISPR tools to edit the DNA of Clostridium beijerinckii bacteria

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jun 27, 2026

Abstract

Essence

Anti-CRISPR-regulated Cas9 enabled iterative genome editing in Clostridium beijerinckii DSM 6423.

Evidence

A bacterial genome-engineering experiment combined inducible CRISPR-Cas9 with AcrIIA4 and validated edits through upp and uracil salvage pathway deletions plus 5-fluorouracil resistance.

Caveat

The strategy was demonstrated in one recalcitrant bacterial species and specific loci, so broader applicability remains inferred rather than directly tested.

Simplified

The development of CRISPR technologies has revolutionized genome editing. However, in bacteria, CRISPR-based methods can be difficult to implement due to the cytotoxicity of CRISPR-associated proteins, which often impair or entirely prevent transformation. In this work, we combine inducible expression of classical CRISPR-Cas9 components with the anti-CRISPR protein AcrIIA4 from Listeria monocytogenes to tightly regulate Cas9 activity. Using this approach, we demonstrate efficient and iterative genome editing in the genetically recalcitrant Clostridium beijerinckii DSM 6423. While deletion of upp alone was not sufficient to render the strain sensitive to 5-fluorouracil, the additional deletion of a second gene involved in the uracil salvage pathway conferred resistance to the drug and validated our gene editing strategy. Collectively, our results show that CRISPR/anti-CRISPR systems can overcome a key limitation of CRISPR-based genome editing and may offer a broadly applicable strategy for engineering otherwise intractable bacterial species.

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Using CRISPR and anti-CRISPR tools to edit the DNA of Clostridium beijerinckii bacteria

Abstract

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