• So Hyun Park, Ciaran M Lee, Daniel P Dever, Timothy H Davis, Joab Camarena, Waracharee Srifa, Yankai Zhang, Alireza Paikari, Alicia K Chang, Matthew H Porteus, Vivien A Sheehan, and Gang Bao.
• Department of Bioengineering, Rice University, Houston, TX 77030, USA.
• Nucleic Acids Res. 2019 Sep 5; 47 (15): 7955-7972.

AbstractSickle cell disease (SCD) is a monogenic disorder that affects millions worldwide. Allogeneic hematopoietic stem cell transplantation is the only available cure. Here, we demonstrate the use of CRISPR/Cas9 and a short single-stranded oligonucleotide template to correct the sickle mutation in the β-globin gene in hematopoietic stem and progenitor cells (HSPCs) from peripheral blood or bone marrow of patients with SCD, with 24.5 ± 7.6% efficiency without selection. Erythrocytes derived from gene-edited cells showed a marked reduction of sickle cells, with the level of normal hemoglobin (HbA) increased to 25.3 ± 13.9%. Gene-corrected SCD HSPCs retained the ability to engraft when transplanted into non-obese diabetic (NOD)-SCID-gamma (NSG) mice with detectable levels of gene correction 16-19 weeks post-transplantation. We show that, by using a high-fidelity SpyCas9 that maintained the same level of on-target gene modification, the off-target effects including chromosomal rearrangements were significantly reduced. Taken together, our results demonstrate efficient gene correction of the sickle mutation in both peripheral blood and bone marrow-derived SCD HSPCs, a significant reduction in sickling of red blood cells, engraftment of gene-edited SCD HSPCs in vivo and the importance of reducing off-target effects; all are essential for moving genome editing based SCD treatment into clinical practice.© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

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