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Mol Ther. 2016 Sep;24(9):1561-9. doi: 10.1038/mt.2016.148. Epub 2016 Jul 29.

CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ cells.

Author information

1
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California USA.
2
Division of Allergy and Immunology, Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles, California, USA.
3
Biology Department, California State University, Northridge, California, USA.
4
Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, California, USA.
5
Molecular Biology Interdepartmental PhD Program (MBIDP), University of California, Los Angeles, California, USA.
6
Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, California, USA.
7
Institute for Genomics and Proteomics, University of California, Los Angeles, California, USA.
8
Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, University of California, Los Angeles, California, USA.

Abstract

Targeted 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.

PMID:
27406980
PMCID:
PMC5113113
DOI:
10.1038/mt.2016.148
[Indexed for MEDLINE]
Free PMC Article

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