CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells.
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.
Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
Daniel P
DP
Dever
Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
Rasmus O
RO
Bak
Department of Medicine, Division of Hematology, Cancer Institute, and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California 94305, USA.
Andreas
A
Reinisch
Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
Joab
J
Camarena
Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
Gabriel
G
Washington
Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
Carmencita E
CE
Nicolas
Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
Mara
M
Pavel-Dinu
Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
Nivi
N
Saxena
Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
Alec B
AB
Wilkens
Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
Sruthi
S
Mantri
Stem Cells, Inc. 7707 Gateway Blvd., Suite 140, Newark, California 94560, USA.
Nobuko
N
Uchida
Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
Ayal
A
Hendel
Division of Hematology/Oncology, Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94035, USA.
Anupama
A
Narla
Department of Medicine, Division of Hematology, Cancer Institute, and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California 94305, USA.
Ravindra
R
Majeti
Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
Kenneth I
KI
Weinberg
Department of Pediatrics, Stanford University, Stanford, California 94305, USA.
Matthew H
MH
Porteus
D000483:Alleles; D000755:Anemia, Sickle Cell / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism; D064130:CRISPR-Associated Proteins / Q000378:metabolism; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002454:Cell Differentiation; D019070:Cell Lineage; D002469:Cell Separation; D000229:Dependovirus / Q000235:genetics; D004912:Erythrocytes; D005260:Female; D005434:Flow Cytometry; D000072669:Gene Editing / Q000379:methods*; D018390:Gene Targeting; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D059765:Homologous Recombination; D006801:Humans; D059346:Magnets; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D008863:Microspheres; D009154:Mutation; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D055544:beta-Globins / Q000235:genetics*; D017086:beta-Thalassemia / Q000235:genetics / Q000628:therapy
D018952:Antigens, CD34; D064130:CRISPR-Associated Proteins; D012333:RNA, Messenger; D055544:beta-Globins