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Nucleic Acids Research, 2001, Vol. 29, No. 24 5044-5051
© 2001 Oxford University Press

Directed evolution of a recombinase for improved genomic integration at a native human sequence

Christopher R. Sclimenti, Bhaskar Thyagarajan and Michele P. Calos*

Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120, USA

We previously established that a unidirectional site-specific recombinase, the phage {phi}C31 integrase, can mediate integration into mammalian chromosomes. The enzyme directs integration of plasmids bearing the phage attB recognition site into pseudo attP sites, a set of native sequences related to the phage attP recognition site. Here we use two cycles of DNA shuffling and screening in Escherichia coli to obtain evolved integrases that possess significant improvements in integration frequency and sequence specificity at a pseudo attP sequence located on human chromosome 8, when measured in the native genomic environment of living human cells. Such integrases represent custom integration tools that will be useful for modifying the genomes of higher eukaryotic cells.

* To whom correspondence should be addressed. Tel: +1 650 723 5558; Fax: +1 650 725 1534; Email: calos{at}stanford.edu


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