1.
Kinetic properties of optogenetic DNA editing by LiCre-loxP.
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Dufour, A
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Duplus-Bottin, H
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Boukéké-Lesplulier, T
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Casassa, E
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Triqueneaux, G
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Darthenay-Kiennemann, C
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Dumont, A
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Moali, C
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Vittoz, F
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Jost, D
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Yvert, G
Abstract:
Previously, we developed an optogenetic tool made of a single chimeric protein called LiCre that enables the edition of specific changes in the genome of live cells 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 photo-activated LiCre-loxP recombination reaction. We find that LiCre binds DNA with high affinity in absence of light stimulus, that this binding is cooperative although not as much as for the Cre recombinase from which LiCre was derived and that increasing temperature from 20°C to 37°C gradually increased LiCre efficiency. The recombination kinetics in live cells can be explained by a model where photo-activation of two or more DNA-bound LiCre units (happening in seconds) can produce (in several minutes) a functional recombination synapse. Our conclusions provide helpful guidelines to induce specific genetic changes in live cells using light.
2.
A single-chain and fast-responding light-inducible Cre recombinase as a novel optogenetic switch.
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.
