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.

Directed evolution has emerged as one of the most powerful strategies for protein engineering, yet most platforms rely on prokaryotic hosts, creating a host mismatch when evolved proteins are intended for usage in mammalian systems. Here, we introduce Substrate-Linked Tissue culture Host Evolution via Recombination (SLiTHER), a virus-free, recombination-based platform for directed evolution entirely in mammalian cells. SLiTHER physically couples a genetic substrate to a DNA-modifying enzyme, enabling active variants to be selected and amplified in a single workflow without cell sorting, viral packaging, or antibiotic selection. Using only standard molecular biology procedures, SLiTHER supports evolution cycles as short as two days. We demonstrate the platform by evolving two site-specific recombinases and a TIGR-Tas nuclease, recovering variants with up to 25-fold improved activity within eight cycles. These results establish SLiTHER as a simple and rapid mammalian-cell platform for evolving genome engineering enzymes in their intended cellular context.