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Related Experiment Video

Updated: Dec 26, 2025

A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

Published on: September 2, 2021

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

Robert C Shields¹, Alejandro R Walker¹, Natalie Maricic¹

¹Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, Florida, United States of America.

|March 10, 2020

Summary

This summary is machine-generated.

Researchers developed a CRISPR interference tool to study essential genes in Streptococcus mutans, the dental caries pathogen. This tool identified numerous gene functions, revealing cell structure defects and potential therapeutic targets for treating cavities.

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Published on: April 8, 2017

Area of Science:

Microbiology
Molecular Biology
Genetics

Background:

* Streptococcus mutans is a primary pathogen responsible for dental caries.
* Understanding essential genes in S. mutans is crucial for developing targeted therapies.
* Previous methods for studying gene function in S. mutans were limited.

Purpose of the Study:

* To develop and validate a CRISPR interference (CRISPRi) tool for gene silencing in Streptococcus mutans.
* To create a comprehensive single-guide RNA (sgRNA) library targeting essential and growth-supporting genes.
* To characterize the function of essential genes and identify potential therapeutic targets.

Main Methods:

* Construction of a CRISPR interference tool using a xylose-inducible dead Cas9 nuclease (Cas9Smu) and constitutively active sgRNAs.
* Assessment of sgRNA specificity and proto-spacer adjacent motif (PAM) requirements.
* Creation of an sgRNA library targeting ~300 identified essential or growth-supporting genes.
* Analysis of CRISPRi strains using fluorescence microscopy and a wax worm infection model.

Main Results:

* The developed CRISPRi tool demonstrated titratable gene repression comparable to existing systems.
* sgRNA specificity and PAM recognition were characterized, showing similarity to Cas9Spy.
* CRISPRi screening revealed growth defects in 77% of targeted essential gene strains.
* Observed phenotypes included cell structure anomalies (e.g., enlarged cells, shape transitions) and impaired cell division.
* Silencing of rhamnose-glucose polysaccharide (RGP) biosynthesis impacted S. mutans pathogenesis.

Conclusions:

* The CRISPRi tool and sgRNA library are valuable resources for functional genomics in S. mutans.
* This system enables efficient characterization of essential genes and their roles in cell biology and pathogenesis.
* Identified essential genes represent potential targets for novel anti-dental caries therapeutics.