Chimeric nucleases stimulate gene targeting in human cells

Gene targeting is a powerful technique to introduce genetic change into the genome of eukaryotic cells. It is widely used to create defined mutations in murine embryonic stem cells and theoretically could be used to create or repair mutations in somatic cells. In this way gene targeting could be a powerful form of gene correction type gene therapy. Despite its potential, gene targeting has not been widely used in somatic cells because of its low efficiency. We report on a system based on the correction of a mutated GFP gene that allows the efficient study of gene targeting in somatic cells. Using this system we show that gene targeting is stimulated over 2000-fold by the introduction of a DNA double-stranded break in the target locus (DSB-GT). We find that the rate of DSB-GT can be increased by increasing the amount of repair substrate, the amount of homology between the gene target and repair substrate, and by increasing the frequency of double-stranded break creation. When we optimize conditions for DSB-GT we obtain targeting rates of 3-5%. Finally, we show that chimeric nucleases, protein fusions between zinc finger DNA binding domains and the endonuclease domain of the Fokl restriction enzyme, can stimulate gene targeting in the genome of human somatic cells by several-thousand fold. Our data provides a paradigm for the use of gene targeting as a form of gene therapy for monogenic diseases