Barcode insertion into Bacillus thuringiensis and sequencing for surrogate tracking

In order to study the behavior of biothreat agents like Bacillus anthracis in non-sterile environments using surrogate strains, it is crucial to differentiate the surrogates from background contamination and distinguish between different experiments. One effective approach is to introduce unique genetic barcodes into the surrogate genome and track their presence using quantitative polymerase chain reaction (qPCR). In this report, we utilized the CRISPR-Cas9 methodology, which employs a single plasmid and transformation step, to insert five distinct barcodes into Bacillus thuringiensis, a well-established surrogate for Bacillus anthracis. We subsequently developed qPCR assays for barcode detection and successfully demonstrated the stability of the barcodes within the genome through five cycles of sporulation and germination. Additionally, we conducted whole-genome sequencing on these modified strains and analyzed 187 potential Cas9 off-target sites. We found no correlation between the mutations observed in the engineered strains and the predicted off-target sites, suggesting this genome engineering strategy did not directly result in off-target mutations in the genome. This simple approach has the potential to streamline the development Bacillus anthracis surrogate for tracking, facilitating future investigations in this field.