Whole genome re-sequencing and breseq analysis of ΔPvs1 and ΔpMBL43001

In our study, we developed the Cas9-NE method, which combines the CRISPR/Cas9 system with the natural excision process of mobile genetic elements (MGEs). By utilizing the Cas9-NE method, both the integrative and circular forms of MGEs can be accurately and completely eliminated through Cas9 cleavage, resulting in the generation of MGE-removed cells. We successfully applied the Cas9-NE method to remove four representative MGEs, namely plasmid pMBL43001, prophages GfP2 and Pvs1, and genomic islands VPII, from Vibrio strains.To investigate if any additional mutations occurred after the Cas9/sgRNA treatment, we conducted whole genome re-sequencing and performed breseq (version 0.37.1) analysis. Specifically, we compared an independent mutant ΔPvs1 with its corresponding wild-type strain Vibrio shilonii SCSIO 43133, as well as an independent mutant ΔpMBL43001 with its corresponding wild-type strain Vibrio coralliilyticus SCSIO 43001. The results of the analysis revealed no additional mutations in the sequenced ΔPvs1 and ΔpBML43001 mutants.This finding suggests that the Cas9-NE method specifically targets and removes the desired MGEs without inducing unintended mutations in the genome. It highlights the precision and accuracy of the Cas9-NE method in eliminating MGEs while maintaining the genomic integrity of the targeted strains.