Repurposing theStreptococcus mutansCRISPR-Cas9 System to Understand Essential Gene Function

Abstract

AbstractA recent genome-wide screen identified ∼300 essential or growth-supporting genes in the dental caries pathogenStreptococcus mutans. To be able to study these genes, we built a CRISPR interference tool around the Cas9 nuclease (Cas9Smu) encoded in theS. mutansUA159 genome. Using a xylose-inducible dead Cas9Smuwith a constitutively active single-guide RNA (sgRNA), we observed titratable repression of GFP fluorescence that compared favorably to that ofStreptococcus pyogenesdCas9 (Cas9Spy). 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 >450S. mutansgenomes allied within vivoassays revealed a similar PAM recognition sequence as the Cas9Spy. 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 inS. mutans, some of which could prove to be promising therapeutic targets.