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 and Thomas, Louise and Davies, Ben and Chalmers, Ronald and Smith, Maggie and Brown, William (2013) Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome. BMC Biotechnology, 13 . 87/1-87/17. ISSN 1472-6750

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Abstract

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.

Keywords: Serine recombinases, Genome manipulation, DNA damage

Item Type: Article
Additional Information: Copy of License must accompany any deposit
Schools/Departments: University of Nottingham UK Campus > Faculty of Medicine and Health Sciences > School of Life Sciences
Depositing User: de Sousa, Mrs Shona
Date Deposited: 22 Apr 2014 08:52
Last Modified: 15 Sep 2016 03:19
URI: http://eprints.nottingham.ac.uk/id/eprint/2976

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