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Published on: June 16, 2017
CRISPR/Cas9-based efficient genome editing in Staphylococcus aureus
Abstract:
Staphylococcus aureus is an important pathogenic bacterium prevalent in nosocomial infections and associated with high morbidity and mortality rates, which arise from the significant pathogenicity and multi-drug resistance. However, the typical genetic manipulation tools used to explore the relevant molecular mechanisms of S. aureus have multiple limitations: leaving a scar in the genome, comparatively low gene-editing efficiency, and prolonged experimental period. Here, we present a single-plasmid based on the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system which allows rapid and efficient chromosomal manipulation in S. aureus. The plasmid carries the cas9 gene under the control of the constitutive promoter Pxyl/tet, a single guide RNA-encoding sequence transcribed via a strong promoter Pspac, and donor DNA used to repair the double strand breaks. The function of the CRISPR/Cas9 vector was demonstrated by deleting the tgt gene and the rocA gene, and by inserting the erm R cassette in S. aureus. This research establishes a CRISPR/Cas9 genome editing tool in S. aureus, which enables marker-free, scarless and rapid genetic manipulation, thus accelerating the study of gene function in S. aureus.
Insights
We developed a novel CRISPR/Cas9 genome editing tool for Staphylococcus aureus. This system enables rapid, scarless genetic manipulation, accelerating the study of this important pathogenic bacterium.
Area of Science:
- Microbiology
- Molecular Biology
- Bacterial Genetics
Background:
- Staphylococcus aureus is a major cause of nosocomial infections, characterized by high pathogenicity and multi-drug resistance.
- Existing genetic manipulation tools for S. aureus have limitations, including genomic scarring, low efficiency, and lengthy experimental timelines.
Purpose of the Study:
- To develop a rapid, efficient, and scarless genome editing tool for Staphylococcus aureus using a single-plasmid CRISPR/Cas9 system.
Main Methods:
- A single-plasmid CRISPR/Cas9 system was constructed, incorporating the cas9 gene, a single guide RNA, and donor DNA.
- The system utilizes a constitutive promoter (Pxyl/tet) for cas9 expression and a strong promoter (Pspac) for sgRNA transcription.
- The vector was validated by performing gene deletions (tgt, rocA) and gene insertion (ermR cassette) in S. aureus.
Main Results:
- Demonstrated successful and efficient chromosomal manipulation in S. aureus.
- Achieved marker-free and scarless genetic modifications, including gene deletion and insertion.
- The developed CRISPR/Cas9 tool significantly reduces the time required for genetic manipulation.
Conclusions:
- Established a versatile CRISPR/Cas9 genome editing platform for S. aureus.
- This tool overcomes limitations of previous methods, enabling faster and more precise genetic studies.
- Accelerates research into the molecular mechanisms and pathogenicity of Staphylococcus aureus.
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