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

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

DOI

  • Daniel P Dever, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
  • ,
  • Rasmus O Bak
  • Andreas Reinisch, Department of Medicine, Division of Hematology, Cancer Institute, and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California 94305, USA.
  • ,
  • Joab Camarena, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
  • ,
  • Gabriel Washington, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
  • ,
  • Carmencita E Nicolas, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
  • ,
  • Mara Pavel-Dinu, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
  • ,
  • Nivi Saxena, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
  • ,
  • Alec B Wilkens, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
  • ,
  • Sruthi Mantri, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
  • ,
  • Nobuko Uchida, Stem Cells, Inc. 7707 Gateway Blvd., Suite 140, Newark, California 94560, USA.
  • ,
  • Ayal Hendel, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
  • ,
  • Anupama Narla, Division of Hematology/Oncology, Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94035, USA.
  • ,
  • Ravindra Majeti, Department of Medicine, Division of Hematology, Cancer Institute, and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California 94305, USA.
  • ,
  • Kenneth I Weinberg, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
  • ,
  • Matthew H Porteus, Department of Pediatrics, Stanford University, Stanford, California 94305, USA.

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.

Original languageEnglish
JournalNature
Volume539
Issue7629
Pages (from-to)384-389
Number of pages6
ISSN0028-0836
DOIs
Publication statusPublished - 17 Nov 2016
Externally publishedYes

    Research areas

  • Alleles, Anemia, Sickle Cell, Animals, Antigens, CD34, CRISPR-Associated Proteins, CRISPR-Cas Systems, Cell Differentiation, Cell Lineage, Cell Separation, Dependovirus, Erythrocytes, Female, Flow Cytometry, Gene Editing, Gene Targeting, Genes, Reporter, Genetic Therapy, Hematopoietic Stem Cells, Homologous Recombination, Humans, Magnets, Mice, Inbred NOD, Mice, SCID, Microspheres, Mutation, RNA, Messenger, beta-Globins, beta-Thalassemia, Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't

See relations at Aarhus University Citationformats

ID: 116723589