Permanent modification of the human genome in vivo is impractical owing to the 
low frequency of homologous recombination in human cells, a fact that hampers 
biomedical research and progress towards safe and effective gene therapy. Here 
we report a general solution using two fundamental biological processes: DNA 
recognition by C2H2 zinc-finger proteins and homology-directed repair of DNA 
double-strand breaks. Zinc-finger proteins engineered to recognize a unique 
chromosomal site can be fused to a nuclease domain, and a double-strand break 
induced by the resulting zinc-finger nuclease can create specific sequence 
alterations by stimulating homologous recombination between the chromosome and 
an extrachromosomal DNA donor. We show that zinc-finger nucleases designed 
against an X-linked severe combined immune deficiency (SCID) mutation in the 
IL2Rgamma gene yielded more than 18% gene-modified human cells without 
selection. Remarkably, about 7% of the cells acquired the desired genetic 
modification on both X chromosomes, with cell genotype accurately reflected at 
the messenger RNA and protein levels. We observe comparably high frequencies in 
human T cells, raising the possibility of strategies based on zinc-finger 
nucleases for the treatment of disease.