Curated Optogenetic Publication Database

Abstract: Advances in optogenetics now allow specific modifications to the DNA of live cells with light. However, successfully using these technologies requires knowing their properties in terms of sensitivity, efficiency, kinetics and mechanism. We previously developed an optogenetic tool made of a single chimeric protein called LiCre that enables the induction of specific changes in the genome with blue light via DNA recombination between loxP sites ( Duplus-Bottin et al., 2021). Here, we used in vitro and in vivo experiments combined with kinetic modeling to provide a deeper characterization of the photoactivated LiCre-loxP recombination reaction. We find that LiCre binds DNA with high affinity in the absence of a light stimulus and that this binding is cooperative, although not as much as for the Cre recombinase from which LiCre was derived. In yeast, the addition of riboflavin to the culture medium had no effect on LiCre's efficiency, even when cells over-expressed riboflavin kinase, suggesting that the abundance of the flavin mononucleotide cofactor is not limiting for the reaction. However, LiCre's efficiency in yeast gradually increased when raising the temperature from 20°C to 37°C. The recombination kinetics observed in live cells are best explained by a model where the photoactivation of two or more DNA-bound LiCre units (happening in seconds) can produce (in several minutes) a functional recombination synapse. This model was able to capture the effect of a point mutation altering LiCre's light cycle. This deeper understanding of the LiCre-loxP system provides additional knowledge for designing experiments where specific genetic changes are induced in live cells with light.

Abstract: Optogenetics enables genome manipulations with high spatiotemporal resolution, opening exciting possibilities for fundamental and applied biological research. Here, we report the development of LiCre, a novel light-inducible Cre recombinase. LiCre is made of a single flavin-containing protein comprising the AsLOV2 photoreceptor domain of Avena sativa fused to a Cre variant carrying destabilizing mutations in its N-terminal and C-terminal domains. LiCre can be activated within minutes of illumination with blue light, without the need of additional chemicals. When compared to existing photoactivatable Cre recombinases based on two split units, LiCre displayed faster and stronger activation by light as well as a lower residual activity in the dark. LiCre was efficient both in yeast, where it allowed us to control the production of β-carotene with light, and in human cells. Given its simplicity and performances, LiCre is particularly suited for fundamental and biomedical research, as well as for controlling industrial bioprocesses.