A highly efficient site-specific integration strategy using combination of homologous recombination and the ΦC31 integrase.
The introduction of double-strand breaks (DSBs) at target sites could greatly enhance homologous recombination, and engineered nucleases, such as zinc finger and transcription activator-like effector nucleases, have been successfully developed for making such breaks. In this study, we present a highly efficient site-specific integration strategy based on homologous recombination and ΦC31 integrase. An attB sequence was introduced at the homologous arm of an insertion targeting vector. DSBs at the target locus and donor were then simultaneously generated by the ΦC31 integrase when co-transfected with the donor vector, consequently stimulating homologous recombination. The results demonstrated that our strategy is feasible and the efficiency at the BF4 target site, which we previously identified in the bovine genome, was as high as 93%. The frequency at another site (BF10) was almost two-fold greater in comparison to the vector without homologous arms. This technology requires no sophisticated nuclease design efforts, and the off-target effect is reduced by ΦC31 integrase compared to the use of engineered nucleases, thereby offering a simple and safe way to effectively express a donor gene at a desired locus. This development has great potential value, especially in transgenesis or gene therapy applications.
Shanghai Institute of Medical Genetics, Children's Hospital of Shanghai, Shanghai Jiao Tong University School of Medicine, 24/1400 West Beijing Road, Shanghai 200040, China.
Hailong
H
Ou
Ying
Y
Huang
Qingwen
Q
Ma
Zhaorui
Z
Ren
Shuzhen
S
Huang
Fanyi
F
Zeng
Yitao
Y
Zeng
D000818:Animals; D002417:Cattle; D053903:DNA Breaks, Double-Stranded; D018390:Gene Targeting; D018014:Gene Transfer Techniques; D016678:Genome; D059765:Homologous Recombination; D019426:Integrases / Q000378:metabolism*; D011995:Recombination, Genetic; D059767:Recombinational DNA Repair
D019426:Integrases
Double-strand breaks; Homologous recombination; Pseudo-attP site; Site-specific integration; ΦC31 integrase