Integration Specificity of Phage ϕC31 Integrase in the Human Genome

The site-specific integrase from bacteriophage ϕC31 functions in mammalian cells and is being applied for genetic engineering, including gene therapy. The ϕC31 integrase catalyzes precise, unidirectional recombination between its 30–40-bp attP and attB recognition sites. In mammalian cells, the enzyme also mediates integration of plasmids bearing attB into native sequences that have partial sequence identity with attP, termed pseudo attP sites. Here, we analyzed the features of ϕC31-mediated integration into pseudo attP sites in the human genome. Sequence analysis of 196 independent integration events derived from three cell lines revealed ∼101 integration sites: 56% of the events were recurrent integrations distributed among 19 pseudo attP sequences. Bioinformatics analysis revealed a ∼30-bp palindromic consensus sequence motif shared by all of the repeat occurrences and most of the single occurrence sites, verifying that ϕC31-mediated integration into pseudo attP sites is significantly guided by DNA sequence recognition. The most favored unique sequence in these cell lines occurred at chromosome 19q13.31 and accounted for 7.5% of integration events. Other frequent integration sites were in three specific sequences in subfamilies of ERVL and L1 repetitive sequences, accounting for an additional 17.9% of integration events. Integrations could occur in either orientation at a pseudo attP site, were often accompanied by small deletions, and typically occurred in a single copy per cell. A number of aberrant events were also described, including large deletions and chromosome rearrangements. ϕC31 integrase-mediated integration only slightly favored genes and did not favor promoter regions. Gene density and expression studies suggested chromatin context effects. An analysis of the safety of integration sites in terms of proximity to cancer genes suggested minimal cancer risk. We conclude that integration systems derived from ϕC31 integrase have great potential utility.