Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function

A recent genome-wide screen identified 300 essential or growth-supporting genes in the dental caries pathogen Streptococcus mutans. To be able to study these genes, we built a CRISPR interference tool around the Cas9 nuclease (Cas9(Smu)) encoded in the S. mutans UA159 genome. Using a xylose-inducible dead Cas9(Smu) with a constitutively active single-guide RNA (sgRNA), we observed titratable repression of GFP fluorescence that compared favorably to that of Streptococcus pyogenes dCas9 (Cas9(Spy)). We then investigated sgRNA specificity and proto-spacer adjacent motif (PAM) requirements. Interference by sgRNAs did not occur with double or triple base-pair mutations, or if single base-pair mutations were in the 3' end of the sgRNA. Bioinformatic analysis of >450 S. mutans genomes allied with in vivo assays revealed a similar PAM recognition sequence as Cas9(Spy). Next, we created a comprehensive library of sgRNA plasmids that were directed at essential and growth-supporting genes. We discovered growth defects for 77% of the CRISPRi strains expressing sgRNAs. Phenotypes of CRISPRi strains, across several biological pathways, were assessed using fluorescence microscopy. A variety of cell structure anomalies were observed, including segregational instability of the chromosome, enlarged cells, and ovococci-to-rod shape transitions. CRISPRi was also employed to observe how silencing of cell wall glycopolysaccharide biosynthesis (rhamnose-glucose polysaccharide, RGP) affected both cell division and pathogenesis in a wax worm model. The CRISPRi tool and sgRNA library are valuable resources for characterizing essential genes in S. mutans, some of which could prove to be promising therapeutic targets. Author summary Streptococcus species cause diverse infections in humans including pneumonia and meningitis. They are common inhabitants of the mouth and certain species are associated with the incredibly prevalent oral disease dental caries (tooth decay). Streptococcus mutans is the most studied and most often linked organism to the development and progression of dental caries. Recent studies have shown that the genome of this organism encodes a number of essential genes that are required for the survival of the bacteria. Many of these genes are conserved in other pathogenic streptococci, and some are unique to S. mutans. To facilitate the study of these genes in a high-throughput manner we developed a CRISPR interference gene-silencing system. This tool, which modifies the CRISPR-Cas9 system encoded by S. mutans, allowed us to study virtually all the essential genes encoded by the organism. We found that these genes are required for optimal growth of the organism. We also found that the morphological defects of these strains are diverse but also shared among genes related to similar molecular pathways. We used the CRISPRi tool to comprehensively assess the contribution of L-rhamnose glycopolymers to S. mutans viability, pathogenicity and coordination of cell division. Overall, these findings increase our understanding of Streptococcus essential genes, and may help in the development of new antimicrobial therapies.