piggyBac transposase tools for genome engineering
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.