title: CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ cells.
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.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California USA.
authors.forename: Megan D
authors.initials: MD
authors.lastname: Hoban
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California USA.
authors.forename: Dianne
authors.initials: D
authors.lastname: Lumaquin
authors.affiliation: Division of Allergy and Immunology, Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles, California, USA.
authors.forename: Caroline Y
authors.initials: CY
authors.lastname: Kuo
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California USA.
authors.forename: Zulema
authors.initials: Z
authors.lastname: Romero
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California USA.|Biology Department, California State University, Northridge, California, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Long
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California USA.
authors.forename: Michelle
authors.initials: M
authors.lastname: Ho
authors.affiliation: 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.
authors.forename: Courtney S
authors.initials: CS
authors.lastname: Young
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California USA.
authors.forename: Michelle
authors.initials: M
authors.lastname: Mojadidi
authors.affiliation: 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.
authors.forename: Sorel
authors.initials: S
authors.lastname: Fitz-Gibbon
authors.affiliation: Molecular Biology Interdepartmental PhD Program (MBIDP), University of California, Los Angeles, California, USA.
authors.forename: Aaron R
authors.initials: AR
authors.lastname: Cooper
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California USA.
authors.forename: Georgia R
authors.initials: GR
authors.lastname: Lill
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California USA.
authors.forename: Fabrizia
authors.initials: F
authors.lastname: Urbinati
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California USA.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Campo-Fernandez
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California USA.
authors.forename: Carmen F
authors.initials: CF
authors.lastname: Bjurstrom
authors.affiliation: 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.
authors.forename: Matteo
authors.initials: M
authors.lastname: Pellegrini
authors.affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California USA.
authors.forename: Roger P
authors.initials: RP
authors.lastname: Hollis
authors.affiliation: 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.
authors.forename: Donald B
authors.initials: DB
authors.lastname: Kohn
mesh_terms: D000755:Anemia, Sickle Cell / Q000235:genetics* / Q000628:therapy; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D002460:Cell Line; D053837:DNA Cleavage; D000072669:Gene Editing; D018390:Gene Targeting; D056426:Genetic Loci; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D011485:Protein Binding; D000094704:RNA, Guide, CRISPR-Cas Systems; D052416:Targeted Gene Repair; D000069896:Transcription Activator-Like Effector Nucleases / Q000378:metabolism; D055544:beta-Globins / Q000235:genetics*
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D055544:beta-Globins; D000069896:Transcription Activator-Like Effector Nucleases