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

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Genome editing is crucial for bacterial gene function studies and vaccine development.
• The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system offers a precise method for genetic modification.
• Bacillus anthracis and Bacillus cereus are significant bacterial species with implications for public health and research.

Purpose of the Study:

• To establish and demonstrate the efficacy of the CRISPR/Cas9 system for genome editing in Bacillus anthracis and Bacillus cereus.
• To create large-fragment deletion mutants in B. anthracis and introduce point mutations in B. cereus.
• To assess the functional consequences of genetic modifications, specifically in relation to virulence factors.

Main Methods:

• Utilized the CRISPR/Cas9 system to target specific genomic regions in B. anthracis and B. cereus.
• Generated large-fragment deletion mutants by targeting prophages in B. anthracis.
• Introduced precise point mutations into the plcR gene in B. cereus.

Main Results:

• Achieved high efficiency (100% or 20%) in creating large-fragment deletion mutants in B. anthracis.
• Successfully introduced point mutations into the plcR gene in B. cereus, leading to loss of hemolytic and phospholipase activity.
• Demonstrated that the CRISPR/Cas9 system functions effectively without leaving residual foreign DNA, such as antibiotic resistance markers.

Conclusions:

• The CRISPR/Cas9 system is a potent and efficient tool for genome editing within the Bacillus cereus group.
• This technology enables marker-free genetic modifications, crucial for developing safer live attenuated vaccines and recombinant microbial candidates.
• The findings support the broader application of CRISPR/Cas9 for genetic manipulation in related bacterial species.