DNA transposon systems such as Sleeping Beauty (SB) are useful nonviral vectors mediating efficient and long-term transgene expression. In SB transposition, stable genomic integration of a gene of interest from a DNA donor molecule is obtained through a “cut-and-paste” reaction catalysed by the SB transposase. Conditional activity of the transposase can be achieved by using tissue-specific or drug-inducible promoters and can be an advantageous feature for more precise genome engineering and safer gene therapies. Leaky expression from these promoters, however, can lead to undesired and irreversible effects, as very little amounts of transposase can be sufficient for the generation of stable integrations. Here we report the use of intein-mediated protein splicing as a way to provide additional control over SB transposition. Based on secondary structures of the functional domains of the hyperactive SB100X transposase, we introduced both cis- or trans-splicing inteins at specific amino acid positions. We demonstrate that Npu DnaE split inteins enable efficient trans-splicing of the SB transposase yielding a reconstituted transposase that preserves highly efficient transposition activity. We combined this system with either inducible (Tet-On) or hepatocyte-specific promoters for conditional expression of each of the intein-tagged transposase domains. In addition, a ligand-dependent intein that undergoes cis-splicing only in the presence of 4-hydroxytamoxifen (4HT)
provided tight post-translational control of the transposase activity with very low background in the absence of 4HT. These novel SB transposase variants permit tighter regulation of SB-mediated genome engineering and hold promise for advancing the field of gene therapy towards more targeted and controlled applications.