Highly efficient endogenous human gene correction using designed zinc-finger nucleases

Nature. 2005 Jun 2;435(7042):646-51. doi: 10.1038/nature03556. Epub 2005 Apr 3.


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.

MeSH terms

  • Alleles
  • CD4-Positive T-Lymphocytes / metabolism
  • Cell Line
  • Cells, Cultured
  • Chromosomes, Human, X / genetics
  • DNA / genetics
  • DNA / metabolism*
  • DNA Damage / genetics
  • DNA Repair / genetics
  • Endodeoxyribonucleases / chemistry*
  • Endodeoxyribonucleases / metabolism*
  • Gene Targeting / methods*
  • Genes, Reporter / genetics
  • Genetic Linkage / genetics
  • Genetic Therapy / methods
  • Humans
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Receptors, Interleukin-2 / genetics*
  • Receptors, Interleukin-2 / metabolism
  • Recombination, Genetic / genetics
  • Sequence Homology, Nucleic Acid
  • Severe Combined Immunodeficiency / genetics*
  • Severe Combined Immunodeficiency / therapy
  • Substrate Specificity
  • Zinc Fingers*


  • RNA, Messenger
  • Receptors, Interleukin-2
  • DNA
  • Endodeoxyribonucleases