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

Daniel P. Dever, Rasmus O. Bak, Andreas Reinisch, Joab Camarena, Gabriel Washington, Carmencita E. Nicolas, Mara Pavel-Dinu, Nivi Saxena, Alec B. Wilkens, Sruthi Mantri, Nobuko Uchida, Ayal Hendel, Anupama Narla, Ravindra Majeti, Kenneth I. Weinberg, Matthew H. Porteus

Research output: Contribution to journalArticlepeer-review

627 Scopus citations

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.

Original languageEnglish
Pages (from-to)384-389
Number of pages6
JournalNature
Volume539
Issue number7629
DOIs
StatePublished - 17 Nov 2016
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2016 Macmillan Publishers Limited.

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