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

摘要: A recent genome-wide screen identified close to 300 essential or growth-supporting genes in the dental caries pathogen Streptococcus mutans. To be able study these genes, we built a CRISPR interference tool around Cas9 nuclease (Cas9Smu) encoded S. mutans UA159 genome. Using xylose-inducible dead Cas9Smu with constitutively active single-guide RNA (sgRNA), observed titratable repression of GFP fluorescence that compared favorably pyogenes dCas9 (Cas9Spy). We then investigated sgRNA specificity and proto-spacer adjacent motif (PAM) requirements. Interference by sgRNAs did not occur double triple base-pair mutations, if single mutations were 39 end sgRNA. Bioinformatic analysis >450 genomes allied vivo assays revealed similar PAM recognition sequence as Cas9Spy. Next, created comprehensive library plasmids directed at genes. discovered growth defects for 77% CRISPRi strains expressing sgRNAs. Phenotypes strains, across several biological pathways, assessed using microscopy. variety cell structure anomalies observed, including segregational instability chromosome, enlarged cells, ovococci-to-rod shape transitions. was also employed observe how silencing wall glycopolysaccharide biosynthesis (rhamnose-glucose polysaccharide, RGP) affected both division pathogenesis wax worm model. The are valuable resources characterizing mutans, some which could prove promising therapeutic targets.