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Abstract

Transposons are DNA segments that autonomously move within and between genomes across the tree of life. Tc1/mariners in particular have frequently crossed species boundaries in nature and provide powerful broad-host-range genetic vectors. Among them, the Sleeping Beauty (SB) transposon inserts DNA in vertebrate genomes with extraordinarily high efficiency, making it a prime genetic tool with applications expanding to gene therapy clinical trials. Nevertheless, the molecular principles of SB’s distinctive activity remain elusive, greatly hampering its further development.

In the first part of this thesis, I investigated the molecular mechanisms of the SB transposon in comparison to Human mariner 1 (Hsmar1), a representative transposon of the same superfamily. Using biochemical and biophysical techniques together with fluorescence-based assays, I have characterized the initial steps of SB and Hsmar1 transposition and shown that the two transposons assemble their molecular machineries (or transpososomes) differently. By combining crystallographic data and SAXS-based modelling, I visualized the structural basis of these differences and explained how transpososome assembly is coupled to catalysis in the Hsmar1 transposon. Moreover, the data demonstrated that the unique assembly pathway of SB largely contributes to its exceptional efficiency and that it can be chemically modulated to control insertion rates in living cells. I have further reconstituted in vitro the ordered series of events comprising SB transposition, including transposon end binding, cleavage, and integration, and dissected previously unrevealed molecular features of the process. In the second part of my work, building on these mechanistic insights, I developed a novel SB transposase variant (hsSB) by employing a structure-based protein design approach. Using hsSB allowed for establishing a new genome engineering method based on the direct delivery of recombinant SB protein to cells. We showed that this new method, named SBprotAct, provides safer and more controlled genome modification of several cell types (including stem cells and human T cells), as compared to the state-of-art technology.

This work sheds first light on the molecular determinants of SB transposition and its hyper-activity, providing a unique resource for the rational design of improved genome engineering platforms for research and medicine.