piggyBac transposase tools for genome engineering

Parts used

Key findings

• Li et al. performed scanning alanine mutagenesis of 15 conserved basic residues in the piggyBac catalytic domain and identified PB R372A/K375A as an excision-competent/integration-defective (Exc+Int−) mutant. In HEK293 cells, this mutant retained ~30–50% of wild-type excision activity while failing to produce blasticidin-resistant colonies in integration assays, indicating near-complete loss of integration activity.
• A yeast-based error-prone PCR screen of ~6,000 PB R372A/K375A variants identified M194V and D450N as excision-hyperactive suppressors. Introduced into the iPB7 background, iPB7 M194V/R372A/K375A and iPB7 R372A/K375A/D450N achieved 5–6-fold higher excision than wild-type PB while maintaining near-complete integration deficiency, yielding validated Exc+hyper Int− transposases in both HEK293 cells and yeast.
• N-terminal fusion of zinc finger proteins targeting ROSA26 or GULOP safe harbor loci to iPB7 R372A/D450N restored integration activity to 80–100% of unmodified iPB7. Genome-wide insertion site analysis revealed no enrichment near ZFP target sites, demonstrating that heterologous DNA-binding domain fusion alone is insufficient for site-directed piggyBac integration.

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.