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

Richard A. Voit, Ayal Hendel, Shondra M. Pruett-Miller, Matthew H. Porteus

Research output: Contribution to journalArticlepeer-review

78 Scopus citations


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.

Original languageEnglish
Pages (from-to)1365-1378
Number of pages14
JournalNucleic Acids Research
Issue number2
StatePublished - 1 Jan 2014
Externally publishedYes

Bibliographical note

Funding Information:
National Institutes of Health Nanomedicine Center for Nucleoprotein Machines [PN2EY018244]; U.T. Southwestern Medical Scientist Training Program [to R.A.V.]; Laurie Kraus Lacob Faculty Scholar Fund (to M.H.P.). Funding for open access charge: NIH [PN2EY018244].


Dive into the research topics of 'Nuclease-mediated gene editing by homologous recombination of the human globin locus'. Together they form a unique fingerprint.

Cite this