Gapped BLAST and PSI-BLAST: a new generation of protein database search programs CLUSTAL W: improvin

Richard A. Voit 1 , 2 , Ayal Hendel 1 , Shondra M. Pruett-Miller 2 and Matthew H. Porteus 1 , * 1 Department of Pediatrics, Stanford University, 1291 Welch Rd. Stanford, CA 94305, USA and 2 Department of Pediatrics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd. Dallas, TX 75390, USA *To whom correspondence should be addressed. Tel: +1 650 725 6520 ; Fax: +1 650 736 0195 ; Email: mporteus{at}stanford.edu Received August 6, 2013. Revision received September 24, 2013. Accepted September 25, 2013.
Tal-effector nucleases (TALENs) are engineered proteins that can stimulate toxel 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 toxel 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 toxel 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 toxel genes and identified several that preferentially upregulate γ-globin expression. The Author(s) 2013. Published by Oxford University Press.
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