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Proc Natl Acad Sci U S A. 2007 Feb 27;104(9):3055-60. Epub 2007 Feb 20.

Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases.

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1
Sangamo BioSciences, Inc., Point Richmond Technology Center, 501 Canal Boulevard, Suite A100, Richmond, CA 94804, USA.

Erratum in

  • Proc Natl Acad Sci U S A. 2007 Apr 3;104(14):6090. Moehle, E A [corrected to Moehle, Erica A]; Rock, J M [corrected to Rock, Jeremy M]; Lee, Y L [corrected to Lee, Ya-Li]; Jouvenot, Y [corrected to Jouvenot, Yann]; Dekelver, R C [corrected to DeKelver, Russell C]; Gregory, P D [corrected to Gregory, Philip D]; Urnov, F D [corrected to Urnov, Fyodor D]; Holmes, M C [corrected to Holmes, Michael C].

Abstract

Efficient incorporation of novel DNA sequences into a specific site in the genome of living human cells remains a challenge despite its potential utility to genetic medicine, biotechnology, and basic research. We find that a precisely placed double-strand break induced by engineered zinc finger nucleases (ZFNs) can stimulate integration of long DNA stretches into a predetermined genomic location, resulting in high-efficiency site-specific gene addition. Using an extrachromosomal DNA donor carrying a 12-bp tag, a 900-bp ORF, or a 1.5-kb promoter-transcription unit flanked by locus-specific homology arms, we find targeted integration frequencies of 15%, 6%, and 5%, respectively, within 72 h of treatment, and with no selection for the desired event. Importantly, we find that the integration event occurs in a homology-directed manner and leads to the accurate reconstruction of the donor-specified genotype at the endogenous chromosomal locus, and hence presumably results from synthesis-dependent strand annealing repair of the break using the donor DNA as a template. This site-specific gene addition occurs with no measurable increase in the rate of random integration. Remarkably, we also find that ZFNs can drive the addition of an 8-kb sequence carrying three distinct promoter-transcription units into an endogenous locus at a frequency of 6%, also in the absence of any selection. These data reveal the surprising versatility of the specialized polymerase machinery involved in double-strand break repair, illuminate a powerful approach to mammalian cell engineering, and open the possibility of ZFN-driven gene addition therapy for human genetic disease.

PMID:
17360608
PMCID:
PMC1802009
DOI:
10.1073/pnas.0611478104
[Indexed for MEDLINE]
Free PMC Article
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