Engineered Type Six Secretion Systems Deliver Active Exogenous Effectors and Cre Recombinase.

Genetic editing has revolutionized biotechnology, but delivery of endonuclease genes as DNA can lead to aberrant integration or overexpression, leading to off-target effects. Here, we develop a mechanism to deliver Cre recombinase as a protein by engineering the bacterial type six secretion system (T6SS). Using multiple T6SS fusion proteins, Aeromonas dhakensis or attenuated Vibrio cholerae donor strains, and a gain-of-function cassette for detecting Cre recombination, we demonstrate successful delivery of active Cre directly into recipient cells. The most efficient transfer was achieved using a truncated version of PAAR2 from V. cholerae, resulting in a relatively small (118-amino-acid) delivery tag. We further demonstrate the versatility of this system by delivering an exogenous effector, TseC, enabling V. cholerae to kill Pseudomonas aeruginosa. This implies that P. aeruginosa is naturally resistant to all native effectors of V. cholerae and that the TseC chaperone protein is not required for its activity. Moreover, it demonstrates that the engineered system can improve T6SS efficacy against specific pathogens, proposing future application in microbiome manipulation or as a next-generation antimicrobial. Inexpensive and easy to produce, this protein delivery system has many potential applications, ranging from studying T6SS effectors to genetic editing. Delivery of protein-based drugs, antigens, and gene-editing agents has broad applications. The type VI protein secretion system (T6SS) can target both bacteria and eukaryotic cells and deliver proteins of diverse size and function. Here, we harness the T6SS to successfully deliver Cre recombinase to genetically edit bacteria without requiring the introduction of exogenous DNA into the recipient cells. This demonstrates a promising advantage over current genetic editing tools that require transformation or conjugation of DNA. The engineered secretion tag can also deliver a heterologous antimicrobial toxin that kills an otherwise unsusceptible pathogen, Pseudomonas aeruginosa. These results demonstrate the potential of T6SS-mediated delivery in areas including genome editing, killing drug-resistant pathogens, and studying toxin functions.