ArticlePLoS pathogens2020
Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function.
Article in PLoS pathogens, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 46 papers.
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Who cites it
46 citing papers in PubMed, 61 citations in OpenAlex.
- Technical limitations of CRISPR-Cas9 genome editing in bacteria: challenges and future directions.Archives of microbiology · 2026Review
- Cross-Strain Transferability of CRISPRi Systems and Design Rules from Laboratory to ClinicalACS synthetic biology · 2026Article
- Cross-strain transferability of CRISPRi systems and design rules from laboratory to clinicalbioRxiv : the preprint server for biology · 2026Article
- Genomics of Irish swine-derivedMicrobial genomics · 2026Article
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- tRNA Modification Landscapes in Streptococci: Shared Losses and Clade-Specific Adaptations.bioRxiv : the preprint server for biology · 2025Article
- Analysis of essential genes inJournal of bacteriology · 2025Article
- CRISPR-based genetic tools for the study of host-microbe interactions.Infection and immunity · 2025Review
- Identifying essential genes inJournal of bacteriology · 2025Article
- PAM-interacting domain turn-helix 51 motifs can improve Cas9-SpRY activity.Nucleic acids research · 2025Article
- Analysis of Essential Genes inbioRxiv : the preprint server for biology · 2025Article
- Structure and mechanism of biosynthesis of Streptococcus mutans cell wall polysaccharide.Nature communications · 2025Article
- Magnesium modulates the stress responses of oral streptococci to environmental and antibiotic challenges by altering cell envelope and nutrient transport pathways.Frontiers in microbiology · 2025Article
- Characterization of MreCD inJournal of oral microbiology · 2025Article
- MecA inmSphere · 2024Article
- Rationally designed pooled CRISPRi-seq uncovers an inhibitor of bacterial peptidyl-tRNA hydrolase.Cell reports · 2024Article
- The rise and future of CRISPR-based approaches for high-throughput genomics.FEMS microbiology reviews · 2024Review
- CRISPRi functional genomics in bacteria and its application to medical and industrial research.Microbiology and molecular biology reviews : MMBR · 2024Review
- Use of CRISPR interference for efficient and rapid gene inactivation inApplied and environmental microbiology · 2024Article
Corrections and comments
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Authors and funding
6 authors at 1 institution in 1 country.
Funding
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.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.