AUTHOR=Wang Yanchun , Wang Dongshu , Wang Xiaojing , Tao Haoxia , Feng Erling , Zhu Li , Pan Chao , Wang Bowen , Liu Chunjie , Liu Xiankai , Wang Hengliang 

TITLE=Highly Efficient Genome Engineering in Bacillus anthracis and Bacillus cereus Using the CRISPR/Cas9 System

JOURNAL=Frontiers in Microbiology

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2019.01932

DOI=10.3389/fmicb.2019.01932

ISSN=1664-302X

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