DNA transposons have been widely used for transgenesis and insertional mutagenesis in various organisms. Among the transposons active in mammalian cells, the moth-derived transposon piggyBac is most promising with its highly efficient transposition, large cargo capacity, and precise repair of the donor site. Here we report the generation of a hyperactive piggyBac transposase. The active transposition of piggyBac in multiple organisms allowed us to screen a transposase mutant library in yeast for hyperactive mutants and then to test candidates in mouse ES cells. We isolated 18 hyperactive mutants in yeast, among which five were also hyperactive in mammalian cells. By combining all mutations, a total of 7 aa substitutions, into a single reading frame, we generated a unique hyperactive piggyBac transposase with 17-fold and ninefold increases in excision and integration, respectively. We showed its applicability by demonstrating an increased efficiency of generation of transgene-free mouse induced pluripotent stem cells. We also analyzed whether this hyperactive piggyBac transposase affects the genomic integrity of the host cells. The frequency of footprints left by the hyperactive piggyBac transposase was as low as WT transposase (∼1%) and we found no evidence that the expression of the transposase affects genomic integrity. This hyperactive piggyBac transposase expands the utility of the piggyBac transposon for applications in mammalian genetics and gene therapy.reprogramming | gene correction D NA transposons are genetic elements that can mobilize from one location to an other in the host genome. These have been used as laboratory tools for transgenesis and insertional mutagenesis in a wide range of model organisms such as Drosophila (1, 2), Caenorhabditis elegans (3, 4), and plants (5). However, their application to mammalian genetics had been hampered because of the lack of active transposons in mammals. Approximately a decade ago, the first active DNA transposon in mammals, Sleeping Beauty, was reconstructed from fossilized transposon sequences found in the salmonid genome (6). This pioneer work has greatly expanded the repertoire of tools for mammalian genetics. Germline transposition has accelerated the generation of mutant mice and rats (7-11), and somatic transposition has opened up numerous possibilities to conduct forward genetic screens in vivo such as cancer gene discovery in solid tumors (12-15). Furthermore, DNA transposons hold great promise for gene therapy as nonviral vehicles (16). Since the generation of the Sleeping Beauty transposon, a number of transposons from different families have been reported to show active transposition in mammalian cells. Among them, the piggyBac transposon isolated from cabbage looper moth Trichoplusia ni is most promising because of a variety of unique characteristics, namely exhibiting the most efficient transposition in mammalian cells, the ability of the transposase to form functional protein fusions, large cargo capacity, and traceless excision, i.e., its excisi...

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