title: Nuclease-mediated gene editing by homologous recombination of the human globin locus.
abstract: Tal-effector nucleases (TALENs) are engineered proteins that can stimulate precise genome editing through specific DNA double-strand breaks. Sickle cell disease and β-thalassemia are common genetic disorders caused by mutations in β-globin, and we engineered a pair of highly active TALENs that induce modification of 54% of human β-globin alleles near the site of the sickle mutation. These TALENS stimulate targeted integration of therapeutic, full-length beta-globin cDNA to the endogenous β-globin locus in 19% of cells prior to selection as quantified by single molecule real-time sequencing. We also developed highly active TALENs to human γ-globin, a pharmacologic target in sickle cell disease therapy. Using the β-globin and γ-globin TALENs, we generated cell lines that express GFP under the control of the endogenous β-globin promoter and tdTomato under the control of the endogenous γ-globin promoter. With these fluorescent reporter cell lines, we screened a library of small molecule compounds for their differential effect on the transcriptional activity of the endogenous β- and γ-globin genes and identified several that preferentially upregulate γ-globin expression.
authors.affiliation: Department of Pediatrics, Stanford University, 1291 Welch Rd. Stanford, CA 94305, USA and Department of Pediatrics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd. Dallas, TX 75390, USA.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Voit
authors.forename: Ayal
authors.initials: A
authors.lastname: Hendel
authors.forename: Shondra M
authors.initials: SM
authors.lastname: Pruett-Miller
authors.forename: Matthew H
authors.initials: MH
authors.lastname: Porteus
mesh_terms: D003851:Deoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes; D018390:Gene Targeting; D017930:Genes, Reporter; D056426:Genetic Loci; D049452:Green Fluorescent Proteins / Q000235:genetics; D059765:Homologous Recombination; D006801:Humans; D020014:K562 Cells; D015202:Protein Engineering; D055544:beta-Globins / Q000235:genetics*; D055545:gamma-Globins / Q000235:genetics*
chemical_list: D005456:Fluorescent Dyes; D055544:beta-Globins; D055545:gamma-Globins; D049452:Green Fluorescent Proteins; D003851:Deoxyribonucleases