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

Escherichia coli (E. coli) is a key workhorse of biotechnology. Commonly used CRISPR-Cas9 systems for E. coli genome editing are complex and impose metabolic stress on the host, creating demand for more streamlined strategies. Recent studies identified the IS605 transposon-associated TnpB as a programmable RNA-guided (ωRNA) DNA endonuclease, prompting us to explore whether endogenous TnpB in E. coli (EcoTnpB) could be harnessed for genome editing. Biochemical and cellular analyses demonstrated that EcoTnpB efficiently cleaves both chromosomal and plasmid DNA at custom-specified sites in a TAM-dependent manner. Interestingly, E. coli possesses an endogenous recombination machinery capable of repairing EcoTnpB-induced DNA double-strand breaks (DSBs), challenging the long-held view that bacteria lack efficient homologous recombination systems. Based on these findings, we established a single-plasmid editing system (SPEED) in which genome editing is achieved by simply providing ωRNA and a homologous recombination template. By utilizing endogenous EcoTnpB together with the host HR pathway, this system enabled inducible and seamless genome editing at multiple genomic loci in BL21 (DE3), with editing efficiencies ranging from approximately 29% to 56%. Our results demonstrate for the first time that endogenous TnpB can be harnessed for genome editing and may hold potential for broader applications, such as species-specific antimicrobial development.