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 human cells. Given its simplicity and performances, LiCre is particularly suited for fundamental and biomedical research, as well as for controlling industrial bioprocesses.
Highlights
The wealth of knowledge currently available on the molecular regulations of living systems – including humans – largely results from our ability to introduce genetic changes in model organisms
We evaluated the corresponding mutants by expressing them in yeast cells, where an active Cre can excise a repressive DNA element flanked by LoxP sites and thereby switch ON the expression of a green fluorescent protein (GFP) (Figure 1c)
By performing a mutational analysis of the Cre recombinase and testing the activity of various chimeric proteins involving Cre variants and LOV domains, we have developed a novel, single-chain, lightinducible Cre recombinase (LiCre)
Summary
The wealth of knowledge currently available on the molecular regulations of living systems – including humans – largely results from our ability to introduce genetic changes in model organisms. Such manipulations have been extremely informative because they can unambiguously demonstrate causal effects of molecules on phenotypes. The vast majority of these manipulations were made by first establishing a mutant individual – or line of individuals – and studying it. When a mutation is introduced long before the phenotypic analysis, it is possible that the organism has ‘adapted’ to it either via compensatory regulations or, in case of mutant lines maintained over multiple generations, by compensatory mutations
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