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Front. Microbiol. | doi: 10.3389/fmicb.2019.01932

Highly Efficient Genome Engineering in Bacillus anthracis and Bacillus cereus using the CRISPR/Cas9 System

 Dongshu Wang1*, Chunjie Chunjie Liu1,  Xiankai Liu1, Hengliang Wang1 and  Yanchun Wang1
  • 1State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Science, China

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 Bacillus anthracis and Bacillus cereus using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)9 system. Using two prophages in B. anthracis as targets, large-fragment deletion mutants were achieved with rates of 100% or 20%. In B. cereus, we successfully introduced precise point mutations into plcR, with phenotypic assays showing that the resulting mutants lost hemolytic and phospholipase enzyme activities similar to B. anthracis, which is a natural plcR mutant. Our study indicates that CRISPR/Cas9 is a powerful genetic tool for genome editing in the Bacillus cereus 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 B. cereus.

Keywords: CRISPR/Cas9, Bacillus cereus sensu lato group, genomic Site-specific mutagenesis, Large genomic deletion, Bacillus cereus, Bacillus anthracis

Received: 23 Apr 2019; Accepted: 06 Aug 2019.

Copyright: © 2019 Wang, Chunjie Liu, Liu, Wang and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Mx. Dongshu Wang, State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Science, Beijing, Beijing Municipality, China, wangdongshu@foxmail.com