A diversity of serine phage integrases mediate site-specific recombination in mammalian cells.
This study evaluated the ability of five serine phage integrases, from phages A118, U153, Bxb1, phiFC1, and phiRV1, to mediate recombination in mammalian cells. Two types of recombination were investigated, including the ability of an integrase to mediate recombination between its own phage att sites in the context of a mammalian cell and the ability of an integrase to perform genomic integration pairing a phage att site with an endogenous mammalian sequence. We demonstrated that the A118 integrase mediated precise intra-molecular recombination of a plasmid containing its attB and attP sites at a frequency of approximately 50% in human cells. The closely related U153 integrase also performed efficient recombination in human cells on a plasmid containing the attB and attP sites of A118. The integrases from phages Bxb1, phiFC1, and phiRV1 carried out such recombination at their attB and attP sites at frequencies ranging from 11 to 75%. Furthermore, the A118 integrase mediated recombination between its attP site on a plasmid and pseudo attB sites in the human genome, i.e. native sequences with partial identity to attB. Fifteen such A118 pseudo att sites were analyzed, and a consensus recognition site was identified. The other integrases did not mediate integration at genomic sequences at a frequency above background. These site-specific integrases represent valuable new tools for manipulating eukaryotic genomes.
Keravala
Annahita
A
Department of Genetics, M-334, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5120, USA.
Groth
Amy C
AC
Jarrahian
Sohail
S
Thyagarajan
Bhaskar
B
Hoyt
Jason J
JJ
Kirby
Patrick J
PJ
Calos
Michele P
MP
D001287:Attachment Sites, Microbiological / Q000235:genetics; D001435:Bacteriophages / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D016254:Mutagenesis, Insertional / Q000502:physiology; D011995:Recombination, Genetic / Q000502:physiology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D016662:Virus Integration / Q000502:physiology*
D014764:Viral Proteins; D019426:Integrases