Journal
NUCLEIC ACIDS RESEARCH
Volume 29, Issue 24, Pages 5044-5051Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/29.24.5044
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Funding
- NCI NIH HHS [CA09302, T32 CA009302] Funding Source: Medline
- NIDDK NIH HHS [DK58187, R01 DK058187] Funding Source: Medline
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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 attBrecognition 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.
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