Highly efficient base editing in Staphylococcus aureus using an engineered CRISPR RNA-guided cytidine deaminase

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Emergence of drug-resistant Staphylococcus aureus necessitates novel therapeutic strategies.
• CRISPR-based technologies offer precise genome editing capabilities.

Purpose of the Study:

• To develop a novel CRISPR-based base editor for Staphylococcus aureus.
• To enable efficient gene inactivation and point mutation generation in S. aureus.

Main Methods:

• Engineered a fusion protein pnCasSA-BEC combining a Cas9 nickase (Cas9D10A) and a cytidine deaminase (APOBEC1).
• Utilized CRISPR RNA guidance to target specific genomic loci.
• Achieved C-to-T (G-to-A) base conversions via nicking and cytidine deamination, independent of donor templates.

Main Results:

• Demonstrated highly efficient gene inactivation and point mutation generation in S. aureus.
• pnCasSA-BEC successfully introduced premature stop codons for gene inactivation.
• The system relies on endogenous DNA replication for base conversion.

Conclusions:

• The developed pnCasSA-BEC system is a powerful tool for Staphylococcus aureus research.
• This base-editing technology accelerates drug-target exploration against S. aureus infections.
• Provides insights for developing base-editing tools in other microbial systems.