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

Zhengyao Xu,Ben Davies,William Brown,Louise Thomas,Ronald Chalmers,Maggie Smith

doi:10.1186/1472-6750-13-87

Abstract

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.

Highlights

Phage-encoded serine integrases, such as φC31 integrase, are widely used for genome engineering
We first set out to confirm that each integrase and their cognate attachment sites were active by expression of each integrase gene in E. coli in the presence of a reporter plasmid
The major conclusion following from this work is that all of the fifteen unidirectional serine integrases for which both attachment sites have been identified are active in E.coli, only four of these; Bxb1, φC31, R4 and φBT1 are able to mediate accurate site-specific integration into genomic DNA in human cells and rank such that Bxb1 is marginally better than φC31 and both are better than R4 and φBT1 integrases

Summary

Introduction

Phage-encoded serine integrases, such as φC31 integrase, are widely used for genome engineering. Serine integrases are phage-encoded site-specific recombinases that promote conservative recombination reactions between short (40-60 bp) DNA substrates located on the phage (phage attachment site, attP) and bacterial (bacterial attachment site, attB) chromosomes [1]. In the absence of accessory factors the integrases mediate unidirectional recombination between attP and attB with greater than 80% efficiency. In the presence of a phage-encoded accessory protein, the recombination directionality factor (RDF) the attP × attB recombination is inhibited and the attL × attR recombination is carried out largely independently of one another, in different cell lines, cells of different species and using different protocols. The exceptional study was that of Yamaguchi and colleagues [11] who compared the activities of the φC31, Bxb, TP901-1 and R4 integrases in mediating site-specific recombination into a human artificial chromosome (HAC) isolated in hamster cells. It was neither possible to determine the total level of recombination promoted by these different enzymes nor to determine the fraction of recombination events that had proceeded by reciprocal and conservative site-specific recombination

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Results

Conclusion