Triplex-forming oligonucleotide directed psoralen interstrand crosslinks can induce targeted homologous recombination in mammalian cells.

摘要

Gene targeting is a strategy used to manipulate DNA sequences at a specific site in genomes, which is valuable as a molecular tool in basic scientific research and a promising application for gene therapy. To achieve efficient gene targeting, homologous recombination (HR) is often utilized. However, the frequency of HR in mammalian cells is ∼100- to 1000-fold lower than illegitimate recombination (e.g. non-homologous recombination or random integration). To increase the efficiency of targeted HR, we used chemically modified triplex-forming oligonucleotides (TFOs) to deliver a DNA damaging agent (psoralen) to the targeted site. We found that the psoralen-modified TFO (pTFO)-directed ICLs can enhance SSA frequency in mammalian cells, using a shuttle vector reporter system containing direct repeats. The SSA frequency was dependent on distance between the repeats (the longer the distance, the greater the SSA frequency), but the position of the TFO-directed ICL relative to the repeats did not affect recombination frequency or spectrum. We also observed that the gene conversion frequency was affected by the length between repeats and the relative positions of deletions within the repeats. Furthermore, we confirm that the nucleotide excision repair protein, XPA, has little effect on the SSA frequency and spectrum induced by TFO-directed psoralen ICLs. Finally, we study the intermolecular recombination induced by TFO-directed psoralen ICLs. The results indicated that TFO-directed ICLs are also efficient in inducing HR between two plasmids sharing homologous sequences. While the polarity of the TFO-directed ICL only affects the HR frequency, the deletion size in the homologous sequence can affect both recombination frequency and spectrum.