Development of an Efficient Genome Editing Tool in Bacillus licheniformis Using CRISPR-Cas9 Nickase.

strains are important industrial bacteria that can produce various biochemical products. However, low transformation efficiencies and a lack of effective genome editing tools have hindered its widespread application. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 techniques have been utilized in many organisms as genome editing tools because of their high efficiency and easy manipulation. In this study, an efficient genome editing method was developed for using a CRISPR-Cas9 nickase integrated into the genome of DW2 with overexpression driven by the P43 promoter. The gene was deleted using the CRISPR-Cas9n technique with homology arms of 1.0 kb as a representative example, and an efficiency of 100% was achieved. In addition, two genes were simultaneously disrupted with an efficiency of 11.6%, and the large DNA fragment (42.7 kb) was deleted with an efficiency of 79.0%. Furthermore, the heterologous reporter gene , which codes for nattokinase in , was inserted into the chromosome of with an efficiency of 76.5%. The activity of nattokinase in the DWc9nΔ7/pP43SNT-S strain reached 59.7 fibrinolytic units (FU)/ml, which was 25.7% higher than that of DWc9n/pP43SNT-S Finally, the engineered strain DWc9nΔ7 (Δ Δ Δ Δ Δ Δ Δ), with multiple disrupted genes, was constructed using the CRISPR-Cas9n technique. Taken together, we have developed an efficient genome editing tool based on CRISPR-Cas9n in This tool could be applied to strain improvement for future research. As important industrial bacteria, strains have attracted significant attention due to their production of biological products. However, genetic manipulation of these bacteria is difficult. The CRISPR-Cas9 system has been applied to genome editing in some bacteria, and CRISPR-Cas9n was proven to be an efficient and precise tool in previous reports. The significance of our research is the development of an efficient, more precise, and systematic genome editing method for single-gene deletion, multiple-gene disruption, large DNA fragment deletion, and single-gene integration in via Cas9 nickase. We also applied this method to the genetic engineering of the host strain for protein expression.