One Plasmid Is All You Need: Genome Editing in Escherichia coli Using Endogenous TnpB and Endogenous Recombination...

Area of Science:

• Molecular Biology
• Microbial Genetics
• Biotechnology

Background:

• Escherichia coli (E. coli) is a vital microorganism in biotechnology.
• Existing CRISPR-Cas9 genome editing tools for E. coli are complex and cause metabolic stress.
• There is a need for simpler, more efficient genome editing strategies in E. coli.

Purpose of the Study:

• To investigate the potential of endogenous TnpB (EcoTnpB) in E. coli for genome editing.
• To develop a streamlined genome editing system utilizing EcoTnpB and host repair pathways.

Main Methods:

• Biochemical and cellular analyses of EcoTnpB DNA cleavage activity.
• Development of a single-plasmid editing system (SPEED) using EcoTnpB and a homologous recombination template.
• Testing editing efficiencies at multiple genomic loci in E. coli BL21 (DE3).

Main Results:

• EcoTnpB demonstrated efficient, site-specific DNA cleavage in E. coli.
• E. coli possesses endogenous homologous recombination (HR) machinery capable of repairing DNA double-strand breaks (DSBs).
• The SPEED system achieved inducible and seamless genome editing with efficiencies of 29%–56%.

Conclusions:

• Endogenous TnpB can be effectively utilized for bacterial genome editing.
• The SPEED system offers a simplified approach to E. coli genome modification.
• This work challenges previous assumptions about bacterial recombination systems and opens avenues for antimicrobial development.