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piggyBac transposase tools for genome engineering
Journal article   Open access   Peer reviewed

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, …
Proceedings of the National Academy of Sciences - PNAS, Vol.110(25), pp.E2279-E2287
PNAS Plus
06/18/2013
DOI: 10.1073/pnas.1305987110
PMCID: PMC3690869
PMID: 23723351
url
https://doi.org/10.1073/pnas.1305987110View
Published (Version of record) Open Access

Abstract

DNA transposons that translocate by excision from a donor site and insertion into a target site are often used for genome engineering by insertional mutagenesis and transgenesis. The piggyBac element is especially useful because it can excise precisely from an insertion site, restoring the site to its pretransposon state. Precise excision is particularly useful when transient transgenesis is needed, for example, in the transient introduction of transcription factors for induced pluripotent stem cell production. We have used mutagenesis to generate an Excision + Integration − transposase that allows piggyBac excision without potentially harmful reintegration. These mutations likely lie in a target DNA-binding domain. 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 − transposase, 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.
 protein-DNA interaction Biological Sciences ROSA26 PNAS Plus GULOP induced pluripotent stem cell production

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