Design And Application Of The Photoactivatable Dre Recombinase System

Spatiotemporal genetic engineering in specific cell types within an intact organism is challenging.Site-specific recombinases,especially tyrosine-recombinase family members,play pivotal roles in genome engineering due to their high specificity and efficiency.Among them,Cre recombinase is the most frequently used tool for mouse genetics because of its remarkable efficiency,but a single recombinase system has its technical hurdles which constrain the development of elaborate genetic models for understanding complicated biological process.Dre recombinase,with high activity and functioning orthogonally to Cre,has proven to be a very good player together with Cre in setting up dual recombinase system for sophisticated genome engineering.As with Cre recombinase,ligand-inducible Dre systems have been developed,which exhibit good ligand-responsive induction properties.However,these chemical inducible systems lack a high spatiotemporal resolution.In addition,the use of chemicals has obvious undesirable effects on living systems,such as cytotoxicity and cellular perturbation.However,light-inducible approaches are much superior in terms of high spatiotemporal precision,high tunability,and noninvasiveness.In this study,we focused on the generation of an elegant photoactivatable Dre system and a Cre-Activated Light Inducible Dre?CALID?system,and demonstrating their outstanding performance in cell labeling.Here,based on the similarity with the structure of Cre and with computational assistance,we performed a thorough screening of potential split sites in the Dre primary sequence and identified three inducible split Dre pairs(Dre N₆₀/Dre C₆₁,Dre N₁₅₀/Dre C₁₅₁ and Dre N₂₄₆/Dre C₂₄₇).Among these Dre N₂₄₆/Dre C₂₄₇ showed high efficiency and stringent regulation of gene expression.Through a series of optimization steps,we designed a highly efficient photoactivatable Dre recombinase?PA-Dre?and demonstrated its light-dependent recombination activity in mammalian cells and mice.Furthermore,to facilitate broader applications of PA-Dre,we developed a Cre-Activated Light Inducible Dre?CALID?system based on a double-floxed inverted open reading frame?DIO?strategy.Taking the advantage of well-defined cell type specific promoters or a well-established Cre transgenic strain,we demonstrated that the CALID system was able to activate endogenous reporter expression for either bulk or sparse labeling of Ca MKII?-positive excitatory neurons and parvalbumin interneurons in the brain.This flexible and tunable system could be a powerful tool for the dissection and modulation of developmental and genetic complexity in a wide range of biological systems.Taken together,we add another set of efficient tools to the genomic toolbox,which will enable more detailed investigations of different cell populations through high spatiotemporal resolution and cell-type-specific genetic manipulation.Moreover,we believe that photoactivatable Dre recombinase along with already existing Cre driver mouse lines may be useful in broad fields of biology,such as specific cellular ablation and tissue regeneration,increasing our understanding of the roles of specific cell populations in the near future.