piggyBac transposase tools for genome engineering

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 − 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.NA "cut-and-paste" transposable elements are important tools for genome engineering, such as insertional mutagenesis and transgenesis. Research with the DNA transposon Sleeping Beauty, a "resurrected" transposon, has pioneered the use of DNA transposons in mammalian cells (1, 2). piggyBac is also a DNA transposon and a promising alternative to Sleeping Beauty. piggyBac, originally isolated from the cabbage looper moth Trichoplusia ni genome (3), has a large cargo size (4), is highly active in many cell types, and mediates long-term expression in mammalian cells in vivo (5-10). piggyBac is also distinguished by its ability to excise precisely (11), thus restoring the donor site to its pretransposon insertion sequence.Because it can excise precisely, piggyBac is especially useful if a transgene is only transiently required. Transient integration and expression of transcription factors are important approaches to generate transgene-free induced pluripotent stem cells (iPSCs) (12, 13) as well as directed differentiation of specific cell types for both research and clinical use. Removal of the transgenes is key for potential therapeutic applications of iPSCs. piggyBac has been used as a vector for reversible integration; however, reintegration of the transposon catalyzed by piggyBac (PB) transposase occurs in 40-50% of cells (14) Int− transposase whose excision frequency is five-to six-f...

Derivation and Characterization of Bovine Induced Pluripotent Stem Cells by Transposon-Mediated Reprogramming

“…Our results emphasize that the selected reprogramming factors and culture conditions play a crucial role in the efficiency of deriving and maintaining pluripotent bovine iPSCs. An attractive and advantageous feature of the PB system is that the transposon can be excised seamlessly, and the current development of excision-competent/integration-defective PB variants will facilitate the derivation of footprint-free iPSCs without permanent alterations in their genome (Li et al, 2013;Meir et al, 2013).…”

“…Briefly, the PB-reprogramming transposon contains a CAGGS promoter-driven cassette containing SOX2, OCT4, KLF4, c-MYC, NANOG, and LIN28 cDNAs, each separated by human sequences coding for selfcleaving 2A peptides and flanked by PB-inverted terminal repeats (ITR), i.e., PB-6F. A cDNA coding for a hyperactive PB is driven by the cytomegalovirus (CMV) promoter on a helper plasmid (Li et al, 2013). The transposon and transposase plasmids were mixed in a 5:1 molar ratio and used for electroporation of BFFs.…”

piggyBacTransposon

“…The mechanisms by which these mutations alter transposition activity remain elusive. In addition to the developments described above, a variant called Exc + Int − transposase that can excise a transposon but cannot integrate it back into the host genome has recently been generated (68). This variant was identified by site-directed mutagenesis of potentially DNA-interacting amino acids.…”

A stable but reversible integrated surrogate reporter for assaying CRISPR/Cas9-stimulated homology-directed repair

“…Previous studies have documented that the removal of the Piggybac DNA transposon results in clean, scar-less restoration of the genomic integration site in Ͼ99% of cases (28,(31)(32)(33)(34). This footprint-free quality of the Piggybac transposon coupled with the unique availability of both hyperactive (25) and integration defective PB transposases (30) clearly makes the Piggybac transposon a superior tool for implementation of SRIRACCHA. Alternatively, retroviral vectors are commonly used for CRISPR/Cas delivery (14, 50 -53) but are highly mutagenic because of their preferential insertion in transcriptional units (54 -58) and cannot be easily removed from the genome.…”

“…Another highly unique and practical property of the PB DNA transposon is the ability to both mobilize and remove integrated transposons using the PB transposase (PBase). With the development of an excision-only (Exc ϩ Int Ϫ ) mutant PBase (30), transposons can now be removed without the risk of reintegration. Lastly, one key advantage of PB is its "footprint-free" mobilization whereby sequence integrity around the TTAA integration site is preserved after excision of the PB transposon (28,(31)(32)(33)(34).…”