Development of an Efficient Gene Insertion Tool in Bacillus subtilis Based on CRISPR-associated transposase systems

Bacillus subtilis is a pivotal model organism in both industrial biotechnology and scientific research, where the efficiency of its genetic engineering is very important. However, achieving highly efficient gene insertion in this bacterium remains a significant technical challenge. To address this, we aimed to develop a novel gene insertion tool in B. subtilis . Building upon the Vibrio cholerae -derived Vch CAST system, we systematically optimized and successfully established a high-performance VchCAST system. The core components of this system include the TniQ-Cas678 complex, a guide RNA for precise targeting, and the TnsABC transposase complex responsible for DNA integration. Under antibiotic selection, screening and employing a strong promoter to drive crRNA expression increased the single-locus transposition efficiency to 41%. Subsequent genomic integration of the transposase operational unit further enhanced the efficiency to 80%. Moreover, we demonstrated that overexpressing the auxiliary factor BmrR enables simultaneous integration at two distinct genomic loci. Through protein engineering of the key transposase TnsB, we obtained optimized variants V178F and V178L with significantly enhanced activity, which improved the overall transposition efficiency by 232.6% and 178.07%, respectively. We then conducted transposition validation with the optimized system, achieving a site-specific gene insertion efficiency of approximately 95.25%. In conclusion, this study not only provides a robust gene insertion platform for B. subtilis microbial cell factory engineering, but also stands as a valuable reference for the construction of gene insertion tool in other microbial.