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

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

Author information

1
Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
2
Department of Medicine, Division of Hematology, Cancer Institute, and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California 94305, USA.
3
Stem Cells, Inc. 7707 Gateway Blvd., Suite 140, Newark, California 94560, USA.
4
Division of Hematology/Oncology, Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94035, USA.

Abstract

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.

PMID:
27820943
PMCID:
PMC5898607
DOI:
10.1038/nature20134
[Indexed for MEDLINE]
Free PMC Article

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