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.
Highlights
Sickle cell disease is the most common monogenic disease worldwide and is caused by a single point mutation in the b-globin gene
A recent report described Tal-effector nucleases (TALENs) designed to human b-globin and showed 5% gene correction of a mutated GFP gene, which had been disrupted by the insertion of the b-globin sequence recognized by the TALENs, but did not describe their activity at the endogenous b-globin locus [38]
The authors the used b-globin TALENs and a transposon-based targeting strategy to correct the sickle mutation in patient-derived iPS cells [39]
Summary
Sickle cell disease is the most common monogenic disease worldwide and is caused by a single point mutation in the b-globin gene. Analyses of differential expression of b- and g-globin generally have been limited to hemoglobin electrophoresis or qRT-PCR, but recent reports have described a method of using the expression of fluorescent molecules driven by the b- and g-globin promoters as a readout of differential globin regulation. In those studies, the authors integrated into the genome a bacterial artificial chromosome containing the entire 200 kb b-globin locus (which includes both b-globin and g-globin among other genes), modified such that the b- and g-globin promoters drive expression of fluorescent proteins [6,7]. Direct modification of the endogenous b- and g-globin loci eliminates those confounding variables
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