PiggyBac transposase tools for genome engineering

Xianghong Li, Erin R. Burnight, Ashley L. Cooney, Nirav Malani, Troy Brady, Jeffry D. Sander, Janice Staber, Sarah J. Wheelan, J. Keith Joung, Paul B. McCray, Frederic D. Bushman, Patrick L. Sinn, Nancy L. Craig

Research output: Contribution to journalArticle

Abstract

The transposon piggyBac is being used increasingly for genetic studies. Here, we describe modified versions of piggyBac transposase that have potentially wide-ranging applications, such as reversible transgenesis and modified targeting of insertions. piggyBac is distinguished by its ability to excise precisely, restoring the donor site to its pretransposon state. This characteristic makes piggyBac useful for reversible transgenesis,a potentially valuable feature when generating induced pluripotent stem cells without permanent alterations to genomic sequence. To avoid further genome modification following piggyBac excision by reintegration, we generated an excision competent/integration defective (Exc+Int-) transposase. Our findings also suggest the position of a target DNA-transposase interaction. Another goal of genome engineering is to develop reagents that can guide transgenes to preferred genomic regions. Others have shown that piggyBac transposase can be active when fused to a heterologous DNA-binding domain. An Exc+Int-trans-posase, the intrinsic targeting of which is defective, might also be a useful intermediate in generating a transposase whose integration activity could be rescued and redirected by fusion to a site-specific DNA-binding domain. We show that fusion to two designed zinc finger proteins rescued the Int-phenotype. Successful guided transgene integration into genomic DNA would have broad applications to gene therapy and molecular genetics. Thus, an Exc+Int-transposase is a potentially useful reagent for genome engineering and provides insight into the mechanism of transposase-target DNA interaction.

Original languageEnglish (US)
Pages (from-to)E2279-E2287
JournalProceedings of the National Academy of Sciences of the United States of America
Volume110
Issue number25
DOIs
StatePublished - Jun 18 2013

Keywords

  • Gulop
  • Induced pluripotent stem cell production
  • Protein-DNA interaction
  • ROSA26

ASJC Scopus subject areas

  • General

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  • Cite this

    Li, X., Burnight, E. R., Cooney, A. L., Malani, N., Brady, T., Sander, J. D., Staber, J., Wheelan, S. J., Joung, J. K., McCray, P. B., Bushman, F. D., Sinn, P. L., & Craig, N. L. (2013). PiggyBac transposase tools for genome engineering. Proceedings of the National Academy of Sciences of the United States of America, 110(25), E2279-E2287. https://doi.org/10.1073/pnas.1305987110