CRISPR/anti-CRISPR genome editing in Clostridium beijerinckii
Year: 2025
Type: article
Abstract: 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. • A CRISPR-based gene editing method was implemented for Clostridium beijerinckii • Combining inducible CRISPR and anti-CRISPR systems reduced cytotoxicity • The system reliably yields mutants with 100% gene editing efficiency • Iterative gene editing was used to generate strains sensitive to 5-fluorouracil
Source: Journal of Biotechnology
Institution: IFP Énergies nouvelles
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $4,360
OpenAlex ID: W4415907009