Highly Efficient Genome Engineering in and Using the CRISPR/Cas9 System.

Genome editing is an effective tool for the functional examination of bacterial genes and for live attenuated vaccine construction. Here, we report a method to edit the genomic DNA of and using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)9 system. Using two prophages in as targets, large-fragment deletion mutants were achieved with rates of 100 or 20%. In , we successfully introduced precise point mutations into , with phenotypic assays showing that the resulting mutants lost hemolytic and phospholipase enzyme activities similar to , which is a natural mutant. Our study indicates that CRISPR/Cas9 is a powerful genetic tool for genome editing in the group, and can efficiently modify target genes without the need for residual foreign DNA such as antibiotic selection markers. This system could be developed for use in the generation of marker-free live anthrax vaccines or for safer construction of microbiological candidate-based recombinant .