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Directed evolution of recombinase specificity by split gene reassembly
The engineering of new enzymes that efficiently and specifically modify DNA sequences is necessary for the development of enhanced gene therapies and genetic studies. To address this need, we developed a robust strategy for evolving site-specific recombinases with novel substrate specificities. In t...
Autores principales: | , , , |
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Formato: | Texto |
Lenguaje: | English |
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Oxford University Press
2010
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2896519/ https://www.ncbi.nlm.nih.gov/pubmed/20194120 http://dx.doi.org/10.1093/nar/gkq125 |
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author | Gersbach, Charles A. Gaj, Thomas Gordley, Russell M. Barbas, Carlos F. |
author_facet | Gersbach, Charles A. Gaj, Thomas Gordley, Russell M. Barbas, Carlos F. |
author_sort | Gersbach, Charles A. |
collection | PubMed |
description | The engineering of new enzymes that efficiently and specifically modify DNA sequences is necessary for the development of enhanced gene therapies and genetic studies. To address this need, we developed a robust strategy for evolving site-specific recombinases with novel substrate specificities. In this system, recombinase variants are selected for activity on new substrates based on enzyme-mediated reassembly of the gene encoding β-lactamase that confers ampicillin resistance to Escherichia coli. This stringent evolution method was used to alter the specificities of catalytic domains in the context of a modular zinc finger-recombinase fusion protein. Gene reassembly was detectable over several orders of magnitude, which allowed for tunable selectivity and exceptional sensitivity. Engineered recombinases were evolved to react with sequences from the human genome with only three rounds of selection. Many of the evolved residues, selected from a randomly-mutated library, were conserved among other members of this family of recombinases. This enhanced evolution system will translate recombinase engineering and genome editing into a practical and expedient endeavor for academic, industrial and clinical applications. |
format | Text |
id | pubmed-2896519 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2010 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-28965192010-07-06 Directed evolution of recombinase specificity by split gene reassembly Gersbach, Charles A. Gaj, Thomas Gordley, Russell M. Barbas, Carlos F. Nucleic Acids Res Synthetic Biology and Chemistry The engineering of new enzymes that efficiently and specifically modify DNA sequences is necessary for the development of enhanced gene therapies and genetic studies. To address this need, we developed a robust strategy for evolving site-specific recombinases with novel substrate specificities. In this system, recombinase variants are selected for activity on new substrates based on enzyme-mediated reassembly of the gene encoding β-lactamase that confers ampicillin resistance to Escherichia coli. This stringent evolution method was used to alter the specificities of catalytic domains in the context of a modular zinc finger-recombinase fusion protein. Gene reassembly was detectable over several orders of magnitude, which allowed for tunable selectivity and exceptional sensitivity. Engineered recombinases were evolved to react with sequences from the human genome with only three rounds of selection. Many of the evolved residues, selected from a randomly-mutated library, were conserved among other members of this family of recombinases. This enhanced evolution system will translate recombinase engineering and genome editing into a practical and expedient endeavor for academic, industrial and clinical applications. Oxford University Press 2010-07 2010-03-01 /pmc/articles/PMC2896519/ /pubmed/20194120 http://dx.doi.org/10.1093/nar/gkq125 Text en © The Author(s) 2010. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/2.5 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Synthetic Biology and Chemistry Gersbach, Charles A. Gaj, Thomas Gordley, Russell M. Barbas, Carlos F. Directed evolution of recombinase specificity by split gene reassembly |
title | Directed evolution of recombinase specificity by split gene reassembly |
title_full | Directed evolution of recombinase specificity by split gene reassembly |
title_fullStr | Directed evolution of recombinase specificity by split gene reassembly |
title_full_unstemmed | Directed evolution of recombinase specificity by split gene reassembly |
title_short | Directed evolution of recombinase specificity by split gene reassembly |
title_sort | directed evolution of recombinase specificity by split gene reassembly |
topic | Synthetic Biology and Chemistry |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2896519/ https://www.ncbi.nlm.nih.gov/pubmed/20194120 http://dx.doi.org/10.1093/nar/gkq125 |
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