Editing aberrant splice sites efficiently restores β-globin expression in β-thalassemia

Shuqian Xu, Kevin Luk, Qiuming Yao, Anne H. Shen, Jing Zeng, Yuxuan Wu, Hong-Yuan Luo, Christian Brendel, Luca Pinello, David H.K. Chui, Scot A. Wolfe and Daniel E. Bauer

Key points

  • Efficient aberrant splice site disruption by Cas9 and Cas12a ribonucleoproteins in β-thalassemia hematopoietic stem and progenitor cells.

  • Splice site disruption by indels results in penetrant restoration of β-globin expression.


The thalassemias are compelling targets for therapeutic genome editing in part because monoallelic correction of a subset of hematopoietic stem cells (HSCs) would be sufficient for enduring disease amelioration. A primary challenge is the development of efficient repair strategies that are effective in HSCs. Here we demonstrate that allelic disruption of aberrant splice sites, one of the major classes of thalassemia mutations, is a robust approach to restore gene function. We target the IVS1-110G>A mutation using Cas9 ribonucleoprotein (RNP) and the IVS2-654C>T mutation by Cas12a/Cpf1 RNP in primary CD34+ hematopoietic stem and progenitor cells (HSPCs) from β-thalassemia patients. Each of these nuclease complexes achieves high efficiency and penetrance of therapeutic edits. Erythroid progeny of edited patient HSPCs show reversal of aberrant splicing and restoration of β-globin expression. This strategy could enable correction of a substantial fraction of transfusion-dependent β-thalassemia genotypes with currently available gene editing technology.

  • Submitted January 3, 2019.
  • Accepted January 21, 2019.