CRISPR-Cas9-mediated barcode insertion into Bacillus thuringiensis for surrogate tracking.

Area of Science:

• Microbiology
• Genetics
• Biotechnology

Background:

• Surrogate organisms are crucial for safe research on dangerous pathogens like Bacillus anthracis.
• Differentiating experimental strains from contamination is vital for accurate research outcomes.
• Genetic barcoding offers a method to track and identify specific microbial strains.

Purpose of the Study:

• To develop a straightforward method for inserting unique genetic barcodes into Bacillus thuringiensis using CRISPR-Cas9.
• To establish quantitative polymerase chain reaction (qPCR) assays for detecting these barcodes.
• To assess the stability of the barcodes and the safety of the genome engineering process.

Main Methods:

• Utilized CRISPR-Cas9 gene editing with a single plasmid for barcode insertion into Bacillus thuringiensis.
• Developed and validated qPCR assays for detecting five distinct genetic barcodes.
• Performed whole-genome sequencing and analyzed potential Cas9 off-target sites in engineered strains.

Main Results:

• Successfully inserted five distinct genetic barcodes into Bacillus thuringiensis.
• Demonstrated barcode stability through five cycles of sporulation and germination.
• Whole-genome sequencing revealed no correlation between mutations and predicted Cas9 off-target sites, indicating high specificity.

Conclusions:

• The CRISPR-Cas9 method provides a simple and effective way to create barcoded Bacillus thuringiensis strains.
• This approach facilitates the tracking of surrogate organisms in research settings.
• The genome engineering strategy is precise and does not introduce significant off-target mutations, supporting its use in various Bacillus species.