Both TALENs and CRISPR/Cas9 Directly Target the HBB IVS2-654 (C > T) Mutation in β-thalassemia-derived iPSCs

Sci Rep. 2015 Jul 9;5:12065. doi: 10.1038/srep12065.

Abstract

β-Thalassemia is one of the most common genetic blood diseases and is caused by either point mutations or deletions in the β-globin (HBB) gene. The generation of patient-specific induced pluripotent stem cells (iPSCs) and subsequent correction of the disease-causing mutations may be a potential therapeutic strategy for this disease. Due to the low efficiency of typical homologous recombination, endonucleases, including TALENs and CRISPR/Cas9, have been widely used to enhance the gene correction efficiency in patient-derived iPSCs. Here, we designed TALENs and CRISPR/Cas9 to directly target the intron2 mutation site IVS2-654 in the globin gene. We observed different frequencies of double-strand breaks (DSBs) at IVS2-654 loci using TALENs and CRISPR/Cas9, and TALENs mediated a higher homologous gene targeting efficiency compared to CRISPR/Cas9 when combined with the piggyBac transposon donor. In addition, more obvious off-target events were observed for CRISPR/Cas9 compared to TALENs. Finally, TALENs-corrected iPSC clones were selected for erythroblast differentiation using the OP9 co-culture system and detected relatively higher transcription of HBB than the uncorrected cells. This comparison of using TALENs or CRISPR/Cas9 to correct specific HBB mutations in patient-derived iPSCs will guide future applications of TALENs- or CRISPR/Cas9-based gene therapies in monogenic diseases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • CRISPR-Cas Systems*
  • Cell Differentiation / genetics
  • Cell Self Renewal
  • Gene Order
  • Gene Targeting
  • Genetic Loci
  • Homologous Recombination
  • Humans
  • Induced Pluripotent Stem Cells / cytology*
  • Induced Pluripotent Stem Cells / metabolism*
  • Introns
  • Karyotype
  • Molecular Sequence Data
  • Mutation*
  • Sequence Alignment
  • Transcription, Genetic
  • beta-Globins / chemistry
  • beta-Globins / genetics*
  • beta-Globins / metabolism
  • beta-Thalassemia / genetics*
  • beta-Thalassemia / metabolism*

Substances

  • beta-Globins