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CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ cells.
Mol Ther. 2016 09; 24(9):1561-9.MT

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.

Authors+Show Affiliations

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

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

27406980

Citation

Hoban, Megan D., et al. "CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ Cells." Molecular Therapy : the Journal of the American Society of Gene Therapy, vol. 24, no. 9, 2016, pp. 1561-9.
Hoban MD, Lumaquin D, Kuo CY, et al. CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ cells. Mol Ther. 2016;24(9):1561-9.
Hoban, M. D., Lumaquin, D., Kuo, C. Y., Romero, Z., Long, J., Ho, M., Young, C. S., Mojadidi, M., Fitz-Gibbon, S., Cooper, A. R., Lill, G. R., Urbinati, F., Campo-Fernandez, B., Bjurstrom, C. F., Pellegrini, M., Hollis, R. P., & Kohn, D. B. (2016). CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ cells. Molecular Therapy : the Journal of the American Society of Gene Therapy, 24(9), 1561-9. https://doi.org/10.1038/mt.2016.148
Hoban MD, et al. CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ Cells. Mol Ther. 2016;24(9):1561-9. PubMed PMID: 27406980.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ cells. AU - Hoban,Megan D, AU - Lumaquin,Dianne, AU - Kuo,Caroline Y, AU - Romero,Zulema, AU - Long,Joseph, AU - Ho,Michelle, AU - Young,Courtney S, AU - Mojadidi,Michelle, AU - Fitz-Gibbon,Sorel, AU - Cooper,Aaron R, AU - Lill,Georgia R, AU - Urbinati,Fabrizia, AU - Campo-Fernandez,Beatriz, AU - Bjurstrom,Carmen F, AU - Pellegrini,Matteo, AU - Hollis,Roger P, AU - Kohn,Donald B, Y1 - 2016/07/29/ PY - 2016/03/23/received PY - 2016/06/29/accepted PY - 2016/7/14/entrez PY - 2016/7/14/pubmed PY - 2017/6/7/medline SP - 1561 EP - 9 JF - Molecular therapy : the journal of the American Society of Gene Therapy JO - Mol Ther VL - 24 IS - 9 N2 - 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. SN - 1525-0024 UR - https://www.unboundmedicine.com/medline/citation/27406980/CRISPR/Cas9_Mediated_Correction_of_the_Sickle_Mutation_in_Human_CD34+_cells_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1525-0016(16)45332-3 DB - PRIME DP - Unbound Medicine ER -