• Megan D Hoban, Dianne Lumaquin, Caroline Y Kuo, Zulema Romero, Joseph Long, Michelle Ho, Courtney S Young, Michelle Mojadidi, Sorel Fitz-Gibbon, Aaron R Cooper, Georgia R Lill, Fabrizia Urbinati, Beatriz Campo-Fernandez, Carmen F Bjurstrom, Matteo Pellegrini, Roger P Hollis, and Donald B Kohn.
• Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California USA.
• Mol. Ther. 2016 Sep 1; 24 (9): 1561-9.

AbstractTargeted genome editing technology can correct the sickle cell disease mutation of the β-globin gene in hematopoietic stem cells. This correction supports production of red blood cells that synthesize normal hemoglobin proteins. Here, we demonstrate that Transcription Activator-Like Effector Nucleases (TALENs) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system can target DNA sequences around the sickle-cell mutation in the β-globin gene for site-specific cleavage and facilitate precise correction when a homologous donor template is codelivered. Several pairs of TALENs and multiple CRISPR guide RNAs were evaluated for both on-target and off-target cleavage rates. Delivery of the CRISPR/Cas9 components to CD34+ cells led to over 18% gene modification in vitro. Additionally, we demonstrate the correction of the sickle cell disease mutation in bone marrow derived CD34+ hematopoietic stem and progenitor cells from sickle cell disease patients, leading to the production of wild-type hemoglobin. These results demonstrate correction of the sickle mutation in patient-derived CD34+ cells using CRISPR/Cas9 technology.

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