Contemporary Transposon Tools: A Review and Guide through Mechanisms and Applications of Sleeping Beauty, piggyBac and Tol2 for Genome Engineering

Abstract

Transposons are mobile genetic elements evolved to execute highly efficient integration of their genes into the genomes of their host cells. These natural DNA transfer vehicles have been harnessed as experimental tools for stably introducing a wide variety of foreign DNA sequences, including selectable marker genes, reporters, shRNA expression cassettes, mutagenic gene trap cassettes, and therapeutic gene constructs into the genomes of target cells in a regulated and highly efficient manner. Given that transposon components are typically supplied as naked nucleic acids (DNA and RNA) or recombinant protein, their use is simple, safe, and economically competitive. Thus, transposons enable several avenues for genome manipulations in vertebrates, including transgenesis for the generation of transgenic cells in tissue culture comprising the generation of pluripotent stem cells, the production of germline-transgenic animals for basic and applied research, forward genetic screens for functional gene annotation in model species and therapy of genetic disorders in humans. This review describes the molecular mechanisms involved in transposition reactions of the three most widely used transposon systems currently available (Sleeping Beauty, piggyBac, and Tol2), and discusses the various parameters and considerations pertinent to their experimental use, highlighting the state-of-the-art in transposon technology in diverse genetic applications.

Keywords: gene therapy; genetic screens; genome engineering; induced pluripotent stem cells; nonviral; transgenesis; transposition; transposon.

Figure 1

Schematic overview of transposase-mediated cut-and-paste transposition. A gene of interest (GOI, black bar) is mobilized by transposase (Tnpase) molecules (grey amorphous shape) from vector donor DNA (grey DNA stands) to a genomic locus (blue-red DNA strands). The transposase binds to the terminal inverted repeats (TIRs, grey arrows), induces double-stranded breaks (indicated with yellow lightning bolts), and excises the mobile element from the donor DNA leaving behind a footprint. The transposon-transposase complex finds a suitable target site (TS) and performs integration, producing a target site duplication (TSD).

Figure 2

Organization and functional domains of the Sleeping Beauty (SB), piggyBac (PB), and Tol2 autonomous transposable elements and transposases. Transposons are depicted as a double-stranded DNA helix flanked by TIRs (arrows). Transposases within each autonomous transposon appear with their respective protein domains (rectangles) after transcription and translation. (A) The SB transposon is flanked by TIRs in an inverted repeat/direct repeat (IR/DR) structure (dark grey and red arrows). The SB transposase is depicted with its domains, including a nuclear localization signal (NLS, orange circle), and the PAI and RED subdomains (blue rounded rectangles) of the DNA binding domain (DBD, green rectangle). (B) The PB transposon is flanked by its TIRs and subterminal IRs (white, blue and light grey arrows). The PB transposase is shown with its domains and NLS (orange circle). (C) The Tol2 transposon is flanked by its TIRs and subterminal regions (white and black arrows). The autonomous Tol2 transposon contains an internal Angel element (IR black arrows) and the Tol2 transposase coding sequence with its four exons coding for different protein isoforms (black bars). Tol2 is shown with its domains, as well as the typical RW-motif (light green circle) of the members of the hAT family. The structure of Tol2’s putative functional domains was matched with the coding sequence based on the general domains of the hAT family members [28], the nucleotide positions on the Tol2 DNA sequence (DDBJ/EMBL/GenBank accession no. D84375), and previous analysis of Tol2 [29,30,31]. NTD: N-terminal domain; CRD: Cysteine-rich domain; DDBD: Dimerization and DNA-binding domain; Znf-BED: BED-type zinc finger domain.

Figure 3

Overview of transposon system delivery methods. The transposon can be delivered as plasmid DNA, plasmid-free of antibiotic resistance markers (pFAR), minicircle (MC), or doggybone DNA (dbDNA) (upper left). The transposase can be delivered as plasmid DNA, pFAR, MC, dbDNA, mRNA, or recombinant protein (upper right). Additionally, hybrid delivery methods combining transposon system components with non-integrative viral vectors or nanoparticles are possible (upper middle). Combined, these delivery methods enable in vitro, ex vivo and in vivo administration (bottom).