Programmable Base Editing in Mycobacterium tuberculosis Using an Engineered CRISPR RNA-Guided Cytidine Deaminase

Area of Science:

• Microbiology
• Genetics
• Molecular Biology

Background:

• Multidrug-resistant tuberculosis (Mtb) poses a significant global health threat.
• Novel therapeutic strategies are urgently needed to combat drug-resistant Mtb.
• Efficient genome editing tools are crucial for understanding Mtb physiology, pathogenesis, and drug resistance.

Purpose of the Study:

• To develop a novel base editing system for gene inactivation and point mutation introduction in Mtb.
• To facilitate the study of molecular mechanisms underlying Mtb pathogenesis and drug resistance.

Main Methods:

• Development of a two-plasmid system, MtbCBE.
• The system utilizes an assistant plasmid (pRecX-NucS_E107A) to suppress DNA repair pathways.
• A base editor plasmid (pCBE) expresses a fusion protein including cytidine deaminase, nCas9, and UGI.

Main Results:

• The MtbCBE system enables efficient G:C to A:T base pair conversion at targeted genomic sites in Mtb.
• Successful gene inactivation and point mutation introduction were achieved.

Conclusions:

• The MtbCBE system represents a significant advancement in genome editing for Mtb research.
• This tool will accelerate the elucidation of Mtb pathogenesis and drug resistance mechanisms.
• The developed system provides a foundation for creating base editing tools for other microbial pathogens.