Kinetic properties of optogenetic site-specific DNA recombination by LiCre- loxP
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.
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BibTeX
@article{dufour2026kinetic,
title = {{Kinetic properties of optogenetic site-specific DNA recombination by LiCre- loxP}},
author = {Alice Dufour and Hélène Duplus-Bottin and Thomas Boukéké-Lesplulier and Eliane Casassa and Gérard Triqueneaux and Léo Tarbouriech and Camille Darthenay-Kiennemann and Agnès Dumont and Catherine Moali and Franck Vittoz and Daniel Jost and Gaël Yvert},
journal = {Biology Open},
year = {2026},
month = may,
doi = {10.1242/bio.062381},
url = {https://hal.science/hal-05828760v1},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Kinetic properties of optogenetic site-specific DNA recombination by LiCre- loxP
AU - Alice Dufour
AU - Hélène Duplus-Bottin
AU - Thomas Boukéké-Lesplulier
AU - Eliane Casassa
AU - Gérard Triqueneaux
AU - Léo Tarbouriech
AU - Camille Darthenay-Kiennemann
AU - Agnès Dumont
AU - Catherine Moali
AU - Franck Vittoz
AU - Daniel Jost
AU - Gaël Yvert
PY - 2026
DA - 2026/05/14
JO - Biology Open
DO - 10.1242/bio.062381
UR - https://hal.science/hal-05828760v1
AB - 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.
SN - 2046-6390
ER -