STAGE: A compact and versatile TnpB-based genome editing toolkit for Streptomyces

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Streptomyces bacteria are valuable for producing natural products and are targets for synthetic biology due to numerous uncharacterized biosynthetic gene clusters.
• Existing CRISPR-Cas systems for Streptomyces have limitations, including cytotoxicity and large effector protein size, hindering broader application.

Purpose of the Study:

• To develop a novel, efficient, and versatile genome editing platform for Streptomyces.
• To overcome the limitations of current genetic tools in Streptomyces manipulation.

Main Methods:

• Development of the Streptomyces-compatible TnpB-assisted genome editing (STAGE) toolkit using the small ISDra2 TnpB effector.
• Implementation of STAGE for site-specific gene mutation and development of STAGE-cBEST/STAGE-McBEST for C·G-to-T·A base editing.
• AI-assisted protein engineering to enhance ISDra2 TnpB efficiency and identification of a functional TnpB ortholog.

Main Results:

• STAGE demonstrated high-efficiency, site-specific gene editing with minimal off-target effects in industrial Streptomyces strains.
• STAGE-cBEST and STAGE-McBEST achieved over 75% efficiency for single and multiplexed base editing.
• Engineered TnpB variants reached nearly 100% genome editing efficiency, and a TnpB ortholog was also validated.

Conclusions:

• STAGE is a precise, programmable, and versatile genome editing platform for Streptomyces.
• This toolkit facilitates advanced genetic manipulation and synthetic biology in industrially relevant Streptomyces species.
• The development of smaller, more efficient editing tools like STAGE significantly advances the potential of Streptomyces in biotechnology.