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Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome

BACKGROUND: Phage-encoded serine integrases, such as φC31 integrase, are widely used for genome engineering. Fifteen such integrases have been described but their utility for genome engineering has not been compared in uniform assays. RESULTS: We have compared fifteen serine integrases for their uti...

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Autores principales: Xu, Zhengyao, Thomas, Louise, Davies, Ben, Chalmers, Ronald, Smith, Maggie, Brown, William
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4015280/
https://www.ncbi.nlm.nih.gov/pubmed/24139482
http://dx.doi.org/10.1186/1472-6750-13-87
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author Xu, Zhengyao
Thomas, Louise
Davies, Ben
Chalmers, Ronald
Smith, Maggie
Brown, William
author_facet Xu, Zhengyao
Thomas, Louise
Davies, Ben
Chalmers, Ronald
Smith, Maggie
Brown, William
author_sort Xu, Zhengyao
collection PubMed
description BACKGROUND: Phage-encoded serine integrases, such as φC31 integrase, are widely used for genome engineering. Fifteen such integrases have been described but their utility for genome engineering has not been compared in uniform assays. RESULTS: We have compared fifteen serine integrases for their utility for DNA manipulations in mammalian cells after first demonstrating that all were functional in E. coli. Chromosomal recombination reporters were used to show that seven integrases were active on chromosomally integrated DNA in human fibroblasts and mouse embryonic stem cells. Five of the remaining eight enzymes were active on extra-chromosomal substrates thereby demonstrating that the ability to mediate extra-chromosomal recombination is no guide to ability to mediate site-specific recombination on integrated DNA. All the integrases that were active on integrated DNA also promoted DNA integration reactions that were not mediated through conservative site-specific recombination or damaged the recombination sites but the extent of these aberrant reactions varied over at least an order of magnitude. Bxb1 integrase yielded approximately two-fold more recombinants and displayed about two fold less damage to the recombination sites than the next best recombinase; φC31 integrase. CONCLUSIONS: We conclude that the Bxb1 and φC31 integrases are the reagents of choice for genome engineering in vertebrate cells and that DNA damage repair is a major limitation upon the utility of this class of site-specific recombinase.
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spelling pubmed-40152802014-05-10 Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome Xu, Zhengyao Thomas, Louise Davies, Ben Chalmers, Ronald Smith, Maggie Brown, William BMC Biotechnol Research Article BACKGROUND: Phage-encoded serine integrases, such as φC31 integrase, are widely used for genome engineering. Fifteen such integrases have been described but their utility for genome engineering has not been compared in uniform assays. RESULTS: We have compared fifteen serine integrases for their utility for DNA manipulations in mammalian cells after first demonstrating that all were functional in E. coli. Chromosomal recombination reporters were used to show that seven integrases were active on chromosomally integrated DNA in human fibroblasts and mouse embryonic stem cells. Five of the remaining eight enzymes were active on extra-chromosomal substrates thereby demonstrating that the ability to mediate extra-chromosomal recombination is no guide to ability to mediate site-specific recombination on integrated DNA. All the integrases that were active on integrated DNA also promoted DNA integration reactions that were not mediated through conservative site-specific recombination or damaged the recombination sites but the extent of these aberrant reactions varied over at least an order of magnitude. Bxb1 integrase yielded approximately two-fold more recombinants and displayed about two fold less damage to the recombination sites than the next best recombinase; φC31 integrase. CONCLUSIONS: We conclude that the Bxb1 and φC31 integrases are the reagents of choice for genome engineering in vertebrate cells and that DNA damage repair is a major limitation upon the utility of this class of site-specific recombinase. BioMed Central 2013-10-20 /pmc/articles/PMC4015280/ /pubmed/24139482 http://dx.doi.org/10.1186/1472-6750-13-87 Text en Copyright © 2013 Xu et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Xu, Zhengyao
Thomas, Louise
Davies, Ben
Chalmers, Ronald
Smith, Maggie
Brown, William
Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome
title Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome
title_full Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome
title_fullStr Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome
title_full_unstemmed Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome
title_short Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome
title_sort accuracy and efficiency define bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of dna into the human genome
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4015280/
https://www.ncbi.nlm.nih.gov/pubmed/24139482
http://dx.doi.org/10.1186/1472-6750-13-87
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