One-Step Biallelic and Scarless Correction of a β-Thalassemia Mutation in Patient-Specific iPSCs without Drug Selection

Yali Liu, Yi Yang, Xiangjin Kang, Bin Lin, Qian Yu, Bing Song, Ge Gao, Yaoyong Chen, Xiaofang Sun, Xiaoping Li*, Lei Bu, Yong Fan

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

46 Citations (Scopus)

Abstract

Monogenic disorders (MGDs), which are caused by single gene mutations, have a serious effect on human health. Among these, β-thalassemia (β-thal) represents one of the most common hereditary hematological diseases caused by mutations in the human hemoglobin β (HBB) gene. The technologies of induced pluripotent stem cells (iPSCs) and genetic correction provide insights into the treatments for MGDs, including β-thal. However, traditional approaches for correcting mutations have a low efficiency and leave a residual footprint, which leads to some safety concerns in clinical applications. As a proof of concept, we utilized single-strand oligodeoxynucleotides (ssODNs), high-fidelity CRISPR/Cas9 nuclease, and small molecules to achieve a seamless correction of the β-41/42 (TCTT) deletion mutation in β thalassemia patient-specific iPSCs with remarkable efficiency. Additionally, off-target analysis and whole-exome sequencing results revealed that corrected cells exhibited a minimal mutational load and no off-target mutagenesis. When differentiated into hematopoietic progenitor cells (HPCs) and then further to erythroblasts, the genetically corrected cells expressed normal β-globin transcripts. Our studies provide the most efficient and safe approach for the genetic correction of the β-41/42 (TCTT) deletion in iPSCs for further potential cell therapy of β-thal, which represents a potential therapeutic avenue for the gene correction of MGD-associated mutants in patient-specific iPSCs.

Original languageEnglish
Pages (from-to)57-67
Number of pages11
JournalMolecular Therapy Nucleic Acids
Volume6
DOIs
Publication statusPublished - 17 Mar 2017
Externally publishedYes

Keywords

  • CRISPR/Cas9
  • induced pluripotent stem cells
  • ssODNs
  • β thalassemia

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Liu, Y., Yang, Y., Kang, X., Lin, B., Yu, Q., Song, B., Gao, G., Chen, Y., Sun, X., Li, X., Bu, L., & Fan, Y. (2017). One-Step Biallelic and Scarless Correction of a β-Thalassemia Mutation in Patient-Specific iPSCs without Drug Selection. Molecular Therapy Nucleic Acids, 6, 57-67. https://doi.org/10.1016/j.omtn.2016.11.010