New Plasmid Platform 'pGGTOX' Revolutionizes Genome Editing in Diverse Enterobacteriaceae
Source PublicationN/A
Primary AuthorsLiu, Tian, Cui et al.
Precise genome editing in the bacterial family Enterobacteriaceae is essential for studying gene function, pathogenesis, and antimicrobial resistance. However, many current genome editing systems face host-specific and efficiency limitations, hindering research progress in these diverse and medically important bacteria.
Addressing these challenges, researchers have developed pGGTOX, a modular plasmid platform that enables efficient homologous recombination–mediated genome editing across diverse Enterobacteriaceae. The system integrates several key features: a rhamnose-inducible toxin (MqsR) for stringent counterselection, a sfGFP reporter for visual tracking of recombination events, Golden Gate cloning for rapid assembly of homologous arms, an FRT-flanked resistance cassette for marker removal, and an oriT sequence for conjugative transfer. When used in conjunction with the companion plasmid pCP20-oriT, pGGTOX facilitates precise, marker-free genomic modifications.
The efficacy of pGGTOX was demonstrated through several applications. It achieved 100% efficiency in targeted deletions of the dapA gene in E. coli, which resulted in diaminopimelate auxotrophy, and the mrkCD gene in carbapenem-resistant K. pneumoniae, which markedly reduced biofilm formation. Beyond deletions, pGGTOX successfully deleted a 43.1-kb type IV secretion gene cluster (tra) from an IncN/FII plasmid in E. intestinihominis, which resulted in a substantial reduction in plasmid conjugation efficiency. Furthermore, it enabled the insertion of a 10-kb CRISPR–Cas9 plasmid-curing module (pCasCure) into an S. enterica IncX1 plasmid. Conjugative transfer of this engineered IncX1-pCasCure plasmid into K. pneumoniae subsequently facilitated CRISPR-mediated curing of blaKPC, sensitizing carbapenem resistance to susceptibility.
In summary, as lead author Liu notes in the paper, "pGGTOX provides a versatile, efficient, and broadly applicable platform for genome engineering and CRISPR delivery in Enterobacteriaceae, expanding the toolkit for bacterial genetics and translational antimicrobial research."