A hyperactive piggyBac transposase for mammalian applications

Key findings

• Screening approximately 10,000 error-prone-PCR-mutagenized transformants of the piggyBac transposase (PBase) in a Saccharomyces cerevisiae URA3-reversion excision assay yields 18 mutants with two- to sevenfold higher excision activity than wild-type PBase in yeast.
• Hyperactivity identified in the yeast excision assay does not reliably predict hyperactivity in mammalian cells: of the 18 yeast-hyperactive PBase mutants, 13 show excision and integration activity similar to or weaker than wild-type PBase in mouse ES cells, including Q591P — the second-strongest mutant in yeast, which shows no significant increase in ES cells — and M194V — the fourth-strongest in yeast, which shows very low activity in ES cells.
• Five PBase mutants — I30V/G165S, S103P, M282V, S509G/N570S, and N538K — each increase both excision and integration activity more than twofold in mouse ES cells relative to wild-type PBase. The fold-increases are similar between the two assays for each mutant, indicating that these mutations act primarily on the excision step of transposition rather than on integration.
• Reduced transposition activity in the PBase mutants M194V, R281G, and G316E correlates with markedly reduced steady-state protein levels by Western blot, whereas M282V — the single strongest mutation in both yeast and mouse ES cells — shows a slightly increased protein level; most other mutants retain protein levels comparable to wild-type PBase, indicating that their altered activity arises from changes to the transposition mechanism or kinetics rather than to expression level.
• Combining PBase mutations synergistically enhances transposition activity beyond any single mutation: every combination tested — pairs (I30V/G165S with M282V, M282V with N538K, and I30V/G165S with N538K), a triple (I30V/G165S, M282V, and N538K), and quadruples (adding either S103P or S509G/N570S to that triple) — shows synergistic enhancement. Combining all five mutant sets into a single mammalian codon-optimized PBase (mPBase) reading frame, a total of 7 amino acid substitutions, produces the hyperactive transposase hyPBase, which shows 17-fold higher excision activity and ninefold higher integration activity than mPBase, along with an approximately threefold increase in steady-state protein expression in mouse ES cells.
• hyPBase retains the piggyBac transposon's traceless-excision property: footprint mutations occur at the Hprt locus in mouse ES cells at a mean frequency of 0.74% for both hyPBase (18 of 2,441 excision events) and mPBase (26 of 3,478 excision events), and the large majority of footprints for both transposases — 69% for hyPBase and 81% for mPBase — are microinsertions or microdeletions rather than larger genomic rearrangements.
• In piggyBac-transposon-based reprogramming of mouse embryonic fibroblasts using a single transposon carrying a 2A-peptide-linked Oct4-Sox2-Klf4-Myc-Lin28 cassette, replacing mPBase with hyPBase increases the number of alkaline-phosphatase-positive iPS cell colonies eightfold.
• hyPBase excises the reprogramming transposon from the primary iPS cell lines iPS25 and iPS28 approximately 20-fold more efficiently than mPBase, and neither mPBase- nor hyPBase-mediated excision leaves detectable footprint mutations at the primary transposon integration sites, yielding transgene-free iPS cells with the original genomic sequence restored.
• Array comparative genomic hybridization detects genomic aberrations at a comparable frequency in hyPBase-generated transgene-free iPS cell lines derived from the iPS25 parental line (4 of 17 lines) and in untreated subclones of the same parental line (5 of 25 lines; P = 0.75, Fisher's exact test), indicating that hyPBase expression does not measurably increase genome-wide structural instability beyond the baseline variation already present in cultured mouse ES/iPS cells.
• Transfecting wild-type mouse ES cells with the original insect-derived PBase (iPBase) or mPBase produces a small number of 6-thioguanine-resistant, Hprt-deficient colonies at a rate consistent with spontaneous background mutation, whereas GFP-transfected controls and hyPBase-transfected cells produce none.
• Transient expression of iPBase, mPBase, or hyPBase in mouse ES cells by mRNA transfection does not increase double-strand-break markers relative to GFP-transfected or untreated controls: none of the three transposases raises γH2AX phosphorylation, γH2AX foci counts, or sister chromatid exchange frequency.

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.