CRISPR Characterization and Diversity in Lactobacillus Fermentum

Lactic Acid Bacteria are gaining notoriety not only for their historical food benefits, but most recently for their rising potential in the microbiome, as probiotics, and development of CRISPR technology. Lactic Acid Bacteria are enriched for CRISPR systems, especially the Type II-A CRISPR-Cas9 system. With a growing desire to utilize CRISPR technologies for genome editing, it has become evident that the CRISPR toolbox will have to expand. Part of the Lactic Acid Bacteria complex, Lactobacillus fermentum is typically found in food products and human metagenome studies. It has recently increased in popularity due to its potential probiotic and antimicrobial effects. Due to these reasons, we investigated and characterized the CRISPR-Cas systems of Lactobacillus fermentum. Mining for CRISPR systems in 30 publicly available genomes, revealed a rich diversity of systems within this species. We determined the occurrence of CRISPR across genomes, and assigned Class and Type for each candidate. 70% of the genomes contain a putative CRISPR-Cas system, of which 81% contain a Type I CRISPR-Cas system and 52% encode a Type II CRISPR-Cas system. Our findings showed that while most systems did have a CRISPR locus, the number and type could differ greatly across genomes within this species. Next, we looked at the similarity of Cas1 and Cas2 sequences between strains. Predictably, the groupings were consistent based on the type of system, rather than the strain. Next, we examined the spacer profile. Contrary to previous studies, we found that the spacer profile of each system varied more than expected, with no conserved ancestral spacers. Actually, only two groups shared common ancestors, while the others showed unique spacer acquisition tracks. Finally, we investigated the Cas9 from the model strain ATCC 14931, a distant homolog of Sth and Spy canonical Cas9 proteins sharing only 32% AA identify, as a potential orthogonal system for the development of novel CRISPR-based molecular tools.