title: A hyperactive piggyBac transposase for mammalian applications.
abstract: 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.
authors.affiliation: Wellcome Trust Sanger Institute, Cambridge CB10 1SA, United Kingdom.
authors.forename: Kosuke
authors.initials: K
authors.lastname: Yusa
authors.forename: Liqin
authors.initials: L
authors.lastname: Zhou
authors.forename: Meng Amy
authors.initials: MA
authors.lastname: Li
authors.forename: Allan
authors.initials: A
authors.lastname: Bradley
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Craig
mesh_terms: D017398:Alternative Splicing / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D055028:Comparative Genomic Hybridization; D004251:DNA Transposable Elements / Q000235:genetics*; D004622:Embryo, Mammalian / Q000166:cytology; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D016678:Genome / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008957:Models, Genetic; D009036:Moths / Q000235:genetics; D016254:Mutagenesis, Insertional; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D019895:Transposases / Q000235:genetics / Q000378:metabolism*
chemical_list: D004251:DNA Transposable Elements; D029701:Saccharomyces cerevisiae Proteins; C554167:URA3 protein, S cerevisiae; D019895:Transposases