In 2011, we reported a blue light-controlled optogenetic device for type 2 diabetes therapy1. The emergent cytotoxicity and poor penetrative capacity of blue light hampered the relevant translation of this prototype. To overcome these challenges, our group developed a far-red light (FRL)-triggered optogenetic system2, which can regulate transgene expression both in vitro and in vivo. Meanwhile, the system demonstrated negligible phototoxicity and deep tissue penetration. The new method, therefore, paves the way for a variety of applications including gene editing and immunotherapy. For examples, we have constructed a FRL-mediated CRISPR-dCas9 device (FACE)3 for epigenome engineering and a FRL–activated split-Cas9 system (FAST)4 for controllable gene editing.
Recently, we further constructed an FRL-induced split-Cre-loxP system (FISC) as a site-specific genetic manipulation tool to control DNA recombination precisely and efficiently5. By contrast, chemical- and UV light-inducible Cre-loxP systems only achieved temporal control of genome engineering and stirred up inconvenient cytotoxicity. In FISC, Cre recombinase was split into two fragments. The N-terminal Cre fragment was fused to a Coh2 domain, allowing the constitutive expression of the CreN-Coh2 fusion protein. On the other hand, the C-terminal Cre fragment was fused to a DocS domain. The expression of DocS-CreC fusion protein can be driven by the FRL-responsive promoter under FRL illumination. Consequently, Cre recombinase can be functionally restored via the heterodimerization of two fusion proteins (Fig. 1).
After meticulous and substantive optimization of promoter configurations and linkers between CreN59 and Coh2 as well as between CreC60 and DocS, we finally developed a powerful FISC which can efficiently and reliably manipulate DNA recombination. The kinetics of FISC-mediated DNA recombination demonstrate both illumination-intensity and exposure-time dependent manner and precise spatiotemporal performance in vitro. Subsequently, we evaluated the performance of FISC in vivo and further compared the findings with peers’ blue light-inducible Cre-loxP recombination systems5,6. The FISC was administrated via hydrodynamic injection (tail vein) to mice. Unsurprisingly, we found FISC worked very well in vivo. Due to the deep tissue penetration ability of FRL, FISC showed superior performance of DNA recombination in mice compared to peers’ blue-light inducible Cre-loxP system. Moreover, our recent work revealed FISC-loaded AAV can further improve its performance in vivo, which shows great translational potential towards clinical implementation in future.
Altogether, our FISC as a new tool allows precise control of genome engineering in target single individual cell or whole organisms in a spatiotemporal fashion with the capacity for deep penetration. Thus, FISC is expected to have significant impact on downstream applications, such as optical control in tissue xenografts, the creation of knock-in and knock-out organisms in a spatiotemporal fashion.
Jiali Wu, Meiyan Wang, Xueping Yang, Chengwei Yi, Jian Jiang, Yuanhuan Yu, and Haifeng Ye, A non-invasive far-red light-induced split-Cre recombinase system for controllable genome engineering in mice. Nature Communications (2020). Doi: 10.1038/s41467-020-17530-9
- Ye, H., et al., A synthetic optogenetic transcription device enhances blood-glucose homeostasis in mice. Science, 2011. 332(6037).
- Shao J, and S Xue, et al. Smartphone-controlled optogenetically engineered cells enable semiautomatic glucose homeostasis in diabetic mice. Sci Transl Med, 2017. 9(387).
- Shao, M. Wang, G. Yu, S. Zhu, Y. Yu, B. C. Heng, J. Wu, H. Ye, Synthetic far-red light-mediated CRISPR-dCas9 device for inducing functional neuronal differentiation. Proc. Natl. Acad. Sci. U.S.A. 115, E6722-e6730 (2018).
- Yu, X. Wu, N. Guan, J. Shao, H. Li, Y. Chen, Y. Ping, D. Li, H. Ye, Engineering a far-red light–activated split-Cas9 system for remote-controlled genome editing of internal organs and tumors. Sci. Adv. 6, eabb1777 (2020).
- Kawano, F., Okazaki, R., Yazawa, M. & Sato, M. A photoactivatable Cre-loxP recombination system for optogenetic genome engineering. Chem. Biol. 12, 1059-1064 (2016).
- Taslimi, A. et al. Optimized second-generation CRY2-CIB dimerizers and photoactivatable Cre recombinase. Chem. Biol. 12, 425-430 (2016).