CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells

Nature. 2016 Nov 17;539(7629):384-389. doi: 10.1038/nature20134. Epub 2016 Nov 7.


The β-haemoglobinopathies, such as sickle cell disease and β-thalassaemia, are caused by mutations in the β-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure β-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult β-globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for β-haemoglobinopathies.

Publication types

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

MeSH terms

  • Alleles
  • Anemia, Sickle Cell / genetics*
  • Anemia, Sickle Cell / pathology
  • Anemia, Sickle Cell / therapy
  • Animals
  • Antigens, CD34 / metabolism
  • CRISPR-Associated Proteins / metabolism
  • CRISPR-Cas Systems / genetics*
  • Cell Differentiation
  • Cell Lineage
  • Cell Separation
  • Dependovirus / genetics
  • Erythrocytes
  • Female
  • Flow Cytometry
  • Gene Editing / methods*
  • Gene Targeting*
  • Genes, Reporter
  • Genetic Therapy / methods*
  • Hematopoietic Stem Cells / metabolism*
  • Homologous Recombination
  • Humans
  • Magnets
  • Mice, Inbred NOD
  • Mice, SCID
  • Microspheres
  • Mutation
  • RNA, Messenger / biosynthesis
  • RNA, Messenger / genetics
  • beta-Globins / genetics*
  • beta-Thalassemia / genetics
  • beta-Thalassemia / therapy


  • Antigens, CD34
  • CRISPR-Associated Proteins
  • RNA, Messenger
  • beta-Globins