Nature | Article

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

(CRISPR/Cas9을 이용한 사람 조혈모 세포 유전자 교정)

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 

Nature  Volume: 539,   384–389  (17 November 2016)

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.

Subject terms: Haematopoietic stem cells  Stem-cell biotechnology