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
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
|Journal Article Type||Article|
|Publication Date||Oct 20, 2013|
|Peer Reviewed||Peer Reviewed|
|APA6 Citation||Xu, Z., Thomas, L., Davies, B., Chalmers, R., Smith, M., & Brown, W. (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, doi:10.1186/1472-6750-13-87|
|Copyright Statement||Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0|
|Additional Information||Copy of License must accompany any deposit|
Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0