Harnessing an anti-CRISPR protein for powering CRISPR/Cas9-mediated genome editing in undomesticated Bacillus strains.

backgroundWild-type Bacillus strains have significant industrial and medical value, but their effective utilization often requires strain improvement. The CRISPR/Cas9 system has become the primary tool for genome editing, allowing precise introduction of desired mutations at specific chromosomal locations. However, the practical application of CRISPR/Cas9 in most wild-type Bacillus strains remains challenging due to cellular toxicity resulting from Cas9/sgRNA activity. Therefore, controlling Cas9 toxicity is essential for the widespread application of the CRISPR/Cas9 system in wild-type Bacillus strains.

resultsWe employed AcrIIA4, an anti-CRISPR protein that inhibits the Cas9/sgRNA ribonucleoprotein complex from interacting with DNA, to mitigate Cas9/sgRNA-mediated toxicity, thereby enabling CRISPR/Cas9-based genome editing in wild-type strains. The newly constructed CRISPR/anti-CRISPR (CAC) plasmids harbor both cas9 and acrIIA4 genes controlled by the P

conclusionsIn this study, we developed a CAC system that utilizes the anti-CRISPR protein AcrIIA4 to reduce Cas9/sgRNA-mediated toxicity in Bacillus strains. This system enables precise control of AcrIIA4 and Cas9 expression through inducers, significantly enhancing the efficiency of transformation and genome editing in wild-type Bacillus strains. Therefore, the CAC system stands as a powerful tool to facilitate genome editing in diverse wild-type Bacillus species.