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.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...

Transpo-mAb display: Transposition-mediated B cell display and functional screening of full-length IgG antibody libraries

“…While each of these systems has been reported to be capable of gene delivery into mammalian cells, [17][18][19] we found the PiggyBac system involving a hyperactive version of the PiggyBac transposase 20 to be most suitable for our purpose (data not shown). Hence, we designed plasmid vectors containing human antibody heavy chain (HC) or light chain (LC) expression cassettes that were flanked by PiggyBac recognition sites (inverted terminal repeats, ITRs), and thus, after delivery into host cells along with PiggyBac transposase transient expression constructs, can be "cut" from vectors and "pasted" as transposable elements (TEs) into the host cell genome by transposition ( Fig.…”

“…The amino-acid sequence of hyperactive PiggyBac transposase (hyPB) according to Yusa et al 20 was codon-optimized for murine expression, synthesized and cloned into the pcDNA3.1 transient expression vector. Vectors bearing transposable elements for antibody expression contain PiggyBac inverted terminal repeats (ITRs) up-and downstream of antibody expression cassettes, that are composed of an EF1-a promoter upstream of IgH and IgL open reading frames (ORFs) followed by an IRES sequence allowed coexpression of downstream selectable markers or reporter genes.…”

Drosophila P elements preferentially transpose to replication origins

“…The potency of DNA transposons makes them particularly attractive as tools for understanding genome evolution and as agents of genetic manipulation (2,3). The Drosophila P element, a 3-kb DNA transposon encoding a transposase, ranks among the best-understood eukaryotic transposons ( Fig.…”

A stable but reversible integrated surrogate reporter for assaying CRISPR/Cas9-stimulated homology-directed repair

“…Previous studies have documented that the removal of the Piggybac DNA transposon results in clean, scar-less restoration of the genomic integration site in Ͼ99% of cases (28,(31)(32)(33)(34). This footprint-free quality of the Piggybac transposon coupled with the unique availability of both hyperactive (25) and integration defective PB transposases (30) clearly makes the Piggybac transposon a superior tool for implementation of SRIRACCHA. Alternatively, retroviral vectors are commonly used for CRISPR/Cas delivery (14, 50 -53) but are highly mutagenic because of their preferential insertion in transcriptional units (54 -58) and cannot be easily removed from the genome.…”

“…The Piggybac (PB) transposon is a cut-and-paste mobile DNA element originally isolated from Trichoplusia ni and has undergone successive modifications through both codon optimization and directed evolution to generate one of the most highly active transposons for use in mammalian cells (25,26). Typical applications exploit PB for stable integration of foreign DNA into the genome as a safer and easier alternative to retroviral vectors in cell types that can be transfected with low or modest efficiency.…”