Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery

Nat Biotechnol. 2007 Nov;25(11):1298-306. doi: 10.1038/nbt1353. Epub 2007 Oct 28.


Achieving the full potential of zinc-finger nucleases (ZFNs) for genome engineering in human cells requires their efficient delivery to the relevant cell types. Here we exploited the infectivity of integrase-defective lentiviral vectors (IDLV) to express ZFNs and provide the template DNA for gene correction in different cell types. IDLV-mediated delivery supported high rates (13-39%) of editing at the IL-2 receptor common gamma-chain gene (IL2RG) across different cell types. IDLVs also mediated site-specific gene addition by a process that required ZFN cleavage and homologous template DNA, thus establishing a platform that can target the insertion of transgenes into a predetermined genomic site. Using IDLV delivery and ZFNs targeting distinct loci, we observed high levels of gene addition (up to 50%) in a panel of human cell lines, as well as human embryonic stem cells (5%), allowing rapid, selection-free isolation of clonogenic cells with the desired genetic modification.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Repair*
  • Deoxyribonucleases, Type II Site-Specific / genetics
  • Deoxyribonucleases, Type II Site-Specific / metabolism*
  • Embryonic Stem Cells / enzymology*
  • Gene Transfer Techniques
  • Genetic Engineering / methods*
  • Genetic Vectors
  • Humans
  • Integrases / genetics
  • Interleukin Receptor Common gamma Subunit / genetics
  • Lentivirus / enzymology
  • Lentivirus / genetics*
  • Point Mutation
  • Templates, Genetic
  • Transgenes
  • Virus Integration / genetics
  • Zinc Fingers*


  • IL2RG protein, human
  • Interleukin Receptor Common gamma Subunit
  • Integrases
  • Deoxyribonucleases, Type II Site-Specific