In a recent Nature Communications publication Weinberg et al., (2019) report developing and testing a repertoire of inducible site-specific recombinases capable of being activated by different inducers such as small molecules, light, and temperature. While developed and tested in mammalian cells and transgenic mice, they will be widely useful in insect systems.
Site-specific recombinases (SSRs) target specific regions of DNA, promoting high-fidelity DNA modifications. This technology can be combined with split recombinases that are activated by chemical or light induced dimerization, thus allowing to regulate gene expression with high spatial and temporal control. However, the number of orthogonal recombinases is limited, and the available inducible systems are mainly optimized for Cre recombinase.
Weinberg et al., (2019) describe how they developed a collection of 108 recombinases selected from the tyrosine recombinase (Cre, VCre, and Flp) and the serine integrase (ФC31, TP901, and Bxb1) families. These recombinases were split at different locations and fused to one of three different chemical-inducible dimerization domain systems (RAP, ABA, or GIB). To test their function, the recombinases were transfected into mammalian cells and activated by adding RAP, ABA, or GIB to induce chemical dimerization. Around 25 of the split recombinases induced high GFP expression and, importantly, some also showed very low levels of GFP during the inactive or basal state.
The authors selected the best responders from Cre, Flp, and ФC31 to further characterize their response dynamics by fusing them with the other dimerization domains they had not tested and measuring GFP expression in the presence of the three chemicals (RAP, ABA, and GIB). Overall, ФC31 showed the higher signal-to-noise ratio for the three chemicals, and was stable for at least 100 h.
To verify the versatility of the recombinases regarding the dimerization inducers one can potentially use, the authors also tested UV light as an induction system. To this end, they fused different Cre and Flp split recombinases to blue light-inducible dimerization domains (Magnet system). The authors report that this system also worked in mammalian cells, increasing GFP expression upon UV light stimulation. From these experiments, a serendipitous discovery was that temperatures of 22°C and 4°C also activated the recombinases.
At the end of the paper, the authors discuss possible applications of these tools. They successfully used this technology in mammalian cells and in living mice to induce cytokine and luciferase expression, respectively.
Overall, the repertoire of functional recombinases developed in Weinberg et al., (2019) proves the flexibility of the split recombinases in terms of their capacity to respond to different treatments and of having multiple functional split locations. These results show their potential as a powerful and versatile tool to study genetic circuits, model development, neuroscience, and many other different topics, in ‘non-model’ organisms.