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

In this study, an efficient genome editing method was developed for Bacillus licheniformis using a CRISPR-Cas9 nickase integrated into the genome of B. licheniformis DW2 with overexpression driven by the P43 promoter. The yvmC 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 bacABC (42.7 kb) was deleted with an efficiency of 79.0%. Furthermore, the heterologous reporter gene aprN, which encodes for nattokinase in Bacillus subtilis, was inserted into the chromosome of B. licheniformis with an efficiency of 76.5%. The activity of nattokinase in the DWc9nΔ7/pP43SNT-SsacC strain reached 59.7 FU/mL, which was 25.7% higher than that of DWc9n/pP43SNT-SsacC Finally, the engineered strain DWc9nΔ7(ΔeprΔwprAΔmprΔaprEΔvprΔbprAΔbacABC) with multiple disrupted genes was construced using the CRISPR-Cas9n technique. Taken together, we have developed an efficient genome editing tool based on CRISPR/Cas9n in B. licheniformis This tool could be applied to strain improvement for future research.Importance As important industrial bacteria, Bacillus strains have attracted significant attention due to their production of biological products. However, genetic manipulation of these bacteria is difficult. The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has ...
Source: Applied and Environmental Microbiology - Category: Microbiology Authors: Tags: Appl Environ Microbiol Source Type: research