{"doi":"10.1371/journal.ppat.1008344","title":"Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function","abstract":"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 (Cas9Smu) encoded in the S. mutans UA159 genome. Using a xylose-inducible dead Cas9Smu with a constitutively active single-guide RNA (sgRNA), we observed titratable repression of GFP fluorescence that compared favorably to that of Streptococcus pyogenes dCas9 (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 >450 S. mutans genomes allied with in vivo assays revealed a similar PAM recognition sequence as 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 in S. mutans, some of which could prove to be promising therapeutic targets.","journal":"PLoS Pathogens","year":2020,"id":65119,"datarank":1.4879907525588432,"base_score":4.1588830833596715,"endowment":4.1588830833596715,"self_citation_contribution":0.6238324625039509,"citation_network_contribution":0.8641582900548923,"self_endowment_contribution":0.6238324625039509,"citer_contribution":0.8641582900548923,"corpus_percentile":null,"corpus_rank":null,"citation_count":63,"citer_count":51,"citers_with_citation_signal":40,"citers_with_endowment":40,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9515,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":344478,"name":"Alejandro R. Walker","orcid":"0000-0002-5839-6584","position":1,"is_corresponding":false},{"id":346337,"name":"Natalie Maricic","orcid":null,"position":2,"is_corresponding":false},{"id":344479,"name":"Brinta Chakraborty","orcid":"0000-0001-7061-3740","position":3,"is_corresponding":false},{"id":344480,"name":"Simon A. M. Underhill","orcid":"0000-0002-5214-4562","position":4,"is_corresponding":false},{"id":344481,"name":"Robert A. Burne","orcid":"0000-0002-4234-0316","position":5,"is_corresponding":false},{"id":344477,"name":"Robert C. Shields","orcid":"0000-0002-8823-9504","position":0,"is_corresponding":true}],"reference_count":65,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-18T21:13:23.327603Z","pmid":"32150575","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}