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

Dever DP, Bak RO, Reinisch A, Camarena J, Washington G, Nicolas CE, Pavel-Dinu M, Saxena N, Wilkens AB, Mantri S, Uchida N, Hendel A, Narla A, Majeti R, Weinberg KI, Porteus MH.
Source: Nature
Publication Date: (2016)
Issue: 539(7629): 384-389
Research Area:
Immunotherapy / Hematology
Stem Cells
Cells used in publication:
CD34+ cell, human
Species: human
Tissue Origin: blood
Nucleofector™ I/II/2b
4D-Nucleofector™ X-Unit
4D-Nucleofector™ LV-Unit

CD34+ HSPCs were electroporated 1–2 days after thawing or isolation. CD34+ HSPCs were electroporated using the Lonza Nucleofector 2b (program U-014) and the Human T Cell Nucleofection Kit (VPA-1002, Lonza) as we have found this combination to be superior in optimization studies: a) 5 x10e5 cells/100µl; 30µg Cas9 protein complexed with sgRNA (1:2.5 molar ratio) b) 10µg synthetic chemically modified sgRNA with 15µg of Cas9 mRNA 15 min after nucleofection cells were transduced with AAV6 donor. Targeting experiment of freshly sorted HSCs: solution P3 (V4XP-3024); program EO-100 (4D Nucleofector X-unit; In order to target 80 x 10e6 CD34+ HSPCs: P3 solution and program DZ-100 with 4D Nucleofector LV unit. Afterwards cells were incubated in 37°C incubator.


The ß-haemoglobinopathies, such as sickle cell disease and ß-thalassaemia, are caused by mutations in the ß-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 ß-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 ß-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 ß-haemoglobinopathies.