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Nucleic Acids Res. 2014 Jan;42(2):1365-78. doi: 10.1093/nar/gkt947. Epub 2013 Oct 23.

Nuclease-mediated gene editing by homologous recombination of the human globin locus.

Author information

1
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.

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.

PMID:
24157834
PMCID:
PMC3902937
DOI:
10.1093/nar/gkt947
[Indexed for MEDLINE]
Free PMC Article

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