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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 (...

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Autores principales: Hersch, Steven J., Lam, Linh, Dong, Tao G.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8406194/
https://www.ncbi.nlm.nih.gov/pubmed/34281388
http://dx.doi.org/10.1128/mBio.01115-21
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author Hersch, Steven J.
Lam, Linh
Dong, Tao G.
author_facet Hersch, Steven J.
Lam, Linh
Dong, Tao G.
author_sort Hersch, Steven J.
collection PubMed
description 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.
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spelling pubmed-84061942021-09-09 Engineered Type Six Secretion Systems Deliver Active Exogenous Effectors and Cre Recombinase Hersch, Steven J. Lam, Linh Dong, Tao G. mBio Research Article 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. American Society for Microbiology 2021-07-20 /pmc/articles/PMC8406194/ /pubmed/34281388 http://dx.doi.org/10.1128/mBio.01115-21 Text en Copyright © 2021 Hersch et al. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Article
Hersch, Steven J.
Lam, Linh
Dong, Tao G.
Engineered Type Six Secretion Systems Deliver Active Exogenous Effectors and Cre Recombinase
title Engineered Type Six Secretion Systems Deliver Active Exogenous Effectors and Cre Recombinase
title_full Engineered Type Six Secretion Systems Deliver Active Exogenous Effectors and Cre Recombinase
title_fullStr Engineered Type Six Secretion Systems Deliver Active Exogenous Effectors and Cre Recombinase
title_full_unstemmed Engineered Type Six Secretion Systems Deliver Active Exogenous Effectors and Cre Recombinase
title_short Engineered Type Six Secretion Systems Deliver Active Exogenous Effectors and Cre Recombinase
title_sort engineered type six secretion systems deliver active exogenous effectors and cre recombinase
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8406194/
https://www.ncbi.nlm.nih.gov/pubmed/34281388
http://dx.doi.org/10.1128/mBio.01115-21
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