Using Nucleases to Stimulate Homologous Recombination

In essentially all organisms, double-strand breaks in chromosomal DNA stimulate repair by multiple mechanisms, including homologous recombination. It is possible to use site-specific reagents to produce a break or other recombinagenic damage at a unique site, which makes possible detailed analysis of the repair products. In addition, targeted mutagenesis and gene replacement are stimulated in the immediate vicinity of the break site. To utilize meganucleases with long recognition sequences, it is necessary to introduce the corresponding sequence prior to directed cleavage. The same is typically true of triplex-forming oligonucleotides that target polypurine-polypyrimidine tracts. Zinc-finger nucleases have the potential of being targetable to arbitrarily selected sites, owing to the flexibility of zinc finger recognition of DNA.