A hyperactive piggyBac transposase for mammalian applications

Key findings

Yusa et al. generated a hyperactive piggyBac transposase (hyPBase) carrying seven combined amino acid substitutions identified by screening 10,000 error-prone PCR mutants in a yeast excision assay, then validating candidates in mouse ES cells. The combined hyPBase showed 17-fold and 9-fold increases in excision and integration activity, respectively, relative to the wild-type mammalian codon-optimized piggyBac transposase.

Yusa et al. demonstrated that hyPBase increased the efficiency of generating transgene-free mouse induced pluripotent stem cells compared to wild-type PBase. Using a piggyBac transposon-based reprogramming system, hyPBase enabled more complete excision of factor-encoding cassettes, facilitating production of iPSCs free of residual transgene sequences.

Yusa et al. evaluated whether hyPBase expression altered genomic integrity in mouse ES cells by assessing chromosomal abnormalities and footprint frequency after transposition. Footprints left by hyPBase occurred at approximately 1% frequency, comparable to wild-type PBase, and no evidence of genomic instability was detected, supporting safe use of hyPBase for mammalian gene therapy applications.