Site-specific gene correction of a point mutation in human iPS cells derived from an adult patient with sickle cell disease

Jizhong Zou, Prashant Mali, Xiaosong Huang, Sarah N. Dowey, Linzhao Cheng

Research output: Contribution to journalArticlepeer-review

245 Scopus citations


Human induced pluripotent stem cells (iPSCs) bearing monogenic mutations have great potential for modeling disease phenotypes, screening candidate drugs, and cell replacement therapy provided the underlying disease-causing mutation can be corrected. Here, we report a homologous recombination-based approach to precisely correct the sickle cell disease (SCD) mutation in patient-derived iPSCs with 2 mutated β-globin alleles (βss). Using a gene-targeting plasmid containing a loxP-flanked drug-resistant gene cassette to assist selection of rare targeted clones and zinc finger nucleases engineered to specifically stimulate homologous recombination at the βs locus, we achieved precise conversion of 1 mutated βs to the wild-type βA in SCD iPSCs. However, the resulting co-integration of the selection gene cassette into the first intron suppressed the corrected allele transcription. After Cre recombinasemediated excision of this loxP-flanked selection gene cassette, we obtained "secondary" gene-corrected βsAheterozygous iPSCs that express at 25% to 40% level of the wild-type transcript when differentiated into erythrocytes. These data demonstrate that single nucleotide substitution in the human genome is feasible using human iPSCs. This study also provides a new strategy for gene therapy of monogenic diseases using patient-specific iPSCs, even if the underlying disease-causing mutation is not expressed in iPSCs.

Original languageEnglish (US)
Pages (from-to)4599-4608
Number of pages10
Issue number17
StatePublished - Oct 27 2011
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology


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