Kinetic properties of optogenetic DNA editing by LiCre-loxP

Abstract

ABSTRACTPreviously, 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 betweenloxPsites (Duplus-Bottin et al., 2021). Here, we usedin vitroandin vivoexperiments combined with kinetic modeling to provide a deeper characterization of the photo-activated LiCre-loxPrecombination 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.