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

BackgroundPhage-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.ResultsWe 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.ConclusionsWe 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.

[1]  Daniel S. Ginsburg,et al.  Phage TP901-1 Site-Specific Integrase Functions in Human Cells , 2002, Journal of bacteriology.

[2]  W. Brown,et al.  Site-specific recombination in Schizosaccharomyces pombe and systematic assembly of a 400kb transgene array in mammalian cells using the integrase of Streptomyces phage ϕBT1 , 2007, Nucleic acids research.

[3]  Eric C. Olivares,et al.  Phage R4 integrase mediates site-specific integration in human cells. , 2001, Gene.

[4]  M. Gregory,et al.  Integration Site for Streptomyces Phage φBT1 and Development of Site-Specific Integrating Vectors , 2003, Journal of bacteriology.

[5]  Regina Z. Cer,et al.  Sequencing Bacillus anthracis Typing Phages Gamma and Cherry Reveals a Common Ancestry , 2006, Journal of bacteriology.

[6]  References , 1971 .

[7]  K. Hammer,et al.  Novel Organization of Genes Involved in Prophage Excision Identified in the Temperate Lactococcal Bacteriophage TP901-1 , 1999, Journal of bacteriology.

[8]  Graham F Hatfull,et al.  Integration and excision by the large serine recombinase φRv1 integrase , 2005, Molecular microbiology.

[9]  Julian Parkhill,et al.  Genome analysis of an inducible prophage and prophage remnants integrated in the Streptococcus pyogenes strain SF370. , 2002, Virology.

[10]  Christoph Zehe,et al.  Recombinase-mediated cassette exchange (RMCE) - a rapidly-expanding toolbox for targeted genomic modifications. , 2013, Gene.

[11]  Margaret C. M. Smith,et al.  In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[12]  J. Brookfield,et al.  Rearranging the centromere of the human Y chromosome with φC31 integrase , 2005, Nucleic acids research.

[13]  D. Ow,et al.  Site-specific cassette exchange and germline transmission with mouse ES cells expressing φC31 integrase , 2003, Nature Biotechnology.

[14]  Jason J. Hoyt,et al.  A diversity of serine phage integrases mediate site-specific recombination in mammalian cells , 2006, Molecular Genetics and Genomics.

[15]  Margaret C. M. Smith,et al.  A phage protein that binds φC31 integrase to switch its directionality , 2011, Molecular microbiology.

[16]  Ralf Kühn,et al.  Enhanced efficiency through nuclear localization signal fusion on phage PhiC31-integrase: activity comparison with Cre and FLPe recombinase in mammalian cells. , 2002, Nucleic acids research.

[17]  Margaret C. M. Smith,et al.  Switching the polarity of a bacteriophage integration system , 2004, Molecular microbiology.

[18]  W. Brown,et al.  Iterative in vivo assembly of large and complex transgenes by combining the activities of φC31 integrase and Cre recombinase , 2005, Nucleic acids research.

[19]  M. Padidam,et al.  Phage Bxb1 integrase mediates highly efficient site-specific recombination in mammalian cells. , 2006, BioTechniques.

[20]  Shigenobu Matsuzaki,et al.  A novel site-specific recombination system derived from bacteriophage phiMR11. , 2008, Biochemical and biophysical research communications.

[21]  W. Wurst,et al.  Conditional brain-specific knockdown of MAPK using Cre/loxP regulated RNA interference , 2007, Nucleic acids research.

[22]  G. Hatfull,et al.  The orientation of mycobacteriophage Bxb1 integration is solely dependent on the central dinucleotide of attP and attB. , 2003, Molecular cell.

[23]  N. Grindley,et al.  Mechanisms of site-specific recombination. , 2003, Annual review of biochemistry.

[24]  W. Brown,et al.  Serine recombinases as tools for genome engineering. , 2011, Methods.

[25]  J. Bode,et al.  Double-reciprocal crossover mediated by FLP-recombinase: a concept and an assay. , 1997, Biochemistry.

[26]  Shigeyuki Yamaguchi,et al.  A Method for Producing Transgenic Cells Using a Multi-Integrase System on a Human Artificial Chromosome Vector , 2011, PloS one.