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Targeted plasmid integration into the human genome by an engineered zinc-finger recombinase.

Publication ,  Journal Article
Gersbach, CA; Gaj, T; Gordley, RM; Mercer, AC; Barbas, CF
Published in: Nucleic acids research
September 2011

The development of new methods for gene addition to mammalian genomes is necessary to overcome the limitations of conventional genetic engineering strategies. Although a variety of DNA-modifying enzymes have been used to directly catalyze the integration of plasmid DNA into mammalian genomes, there is still an unmet need for enzymes that target a single specific chromosomal site. We recently engineered zinc-finger recombinase (ZFR) fusion proteins that integrate plasmid DNA into a synthetic target site in the human genome with exceptional specificity. In this study, we present a two-step method for utilizing these enzymes in any cell type at randomly-distributed target site locations. The piggyBac transposase was used to insert recombinase target sites throughout the genomes of human and mouse cell lines. The ZFR efficiently and specifically integrated a transfected plasmid into these genomic target sites and into multiple transposons within a single cell. Plasmid integration was dependent on recombinase activity and the presence of recombinase target sites. This work demonstrates the potential for broad applicability of the ZFR technology in genome engineering, synthetic biology and gene therapy.

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Published In

Nucleic acids research

DOI

EISSN

1362-4962

ISSN

0305-1048

Publication Date

September 2011

Volume

39

Issue

17

Start / End Page

7868 / 7878

Related Subject Headings

  • Zinc Fingers
  • Transposases
  • Recombinases
  • Recombinant Fusion Proteins
  • Protein Engineering
  • Plasmids
  • Mice
  • Humans
  • Genome, Human
  • Gene Targeting
 

Citation

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Gersbach, C. A., Gaj, T., Gordley, R. M., Mercer, A. C., & Barbas, C. F. (2011). Targeted plasmid integration into the human genome by an engineered zinc-finger recombinase. Nucleic Acids Research, 39(17), 7868–7878. https://doi.org/10.1093/nar/gkr421
Gersbach, Charles A., Thomas Gaj, Russell M. Gordley, Andrew C. Mercer, and Carlos F. Barbas. “Targeted plasmid integration into the human genome by an engineered zinc-finger recombinase.Nucleic Acids Research 39, no. 17 (September 2011): 7868–78. https://doi.org/10.1093/nar/gkr421.
Gersbach CA, Gaj T, Gordley RM, Mercer AC, Barbas CF. Targeted plasmid integration into the human genome by an engineered zinc-finger recombinase. Nucleic acids research. 2011 Sep;39(17):7868–78.
Gersbach, Charles A., et al. “Targeted plasmid integration into the human genome by an engineered zinc-finger recombinase.Nucleic Acids Research, vol. 39, no. 17, Sept. 2011, pp. 7868–78. Epmc, doi:10.1093/nar/gkr421.
Gersbach CA, Gaj T, Gordley RM, Mercer AC, Barbas CF. Targeted plasmid integration into the human genome by an engineered zinc-finger recombinase. Nucleic acids research. 2011 Sep;39(17):7868–7878.
Journal cover image

Published In

Nucleic acids research

DOI

EISSN

1362-4962

ISSN

0305-1048

Publication Date

September 2011

Volume

39

Issue

17

Start / End Page

7868 / 7878

Related Subject Headings

  • Zinc Fingers
  • Transposases
  • Recombinases
  • Recombinant Fusion Proteins
  • Protein Engineering
  • Plasmids
  • Mice
  • Humans
  • Genome, Human
  • Gene Targeting