RUBIK: A FLUORESCENT REPORTER FOR CRE AND FLP COMBINATORIAL ACTIVITY TO LABEL AND MANIPULATE SPECIFIC NEURONAL POPULATIONS

Genetic manipulation technologies largely rely on site-specific recombinases to generate conditional transgenic animal models for studying gene function with precise spatial and temporal control. However, detecting and visualizing combinatorial gene expression patterns when multiple recombinases are used is still challenging. To address this limitation, we developed Rubik, a fluorescent reporter designed to monitor the combinatorial Cre and Flp activity. Rubik enables the detection of all possible recombination outcomes: blue for Cre alone, green for Flp alone, yellow for Cre followed by Flp, and red for Flp followed by Cre. In addition, the two sequential recombination configurations co-express either an excitatory or inhibitory optogenetic tool, allowing direct manipulation of tagged cell populations. We first validated Rubik in a stable HeLa knock-in cell line, demonstrating accurate and reproducible recombination patterns with precise control over reporter expression. We then confirmed Rubik’s in vivo functionality using in-utero electroporation in mouse embryos, followed by patch-clamp recordings in acute brain slices, showing that Rubik reliably reports recombinase activity while supporting functional optogenetic modulation. To further improve temporal control over recombinase activity, we optimized a trimethoprim-inducible Flp recombinase to combine with the tamoxifen-inducible Cre recombinase. Altogether, Rubik represents a powerful tool for anatomical mapping and functional interrogation of neural circuits, with broad potential applications in neuroscience research.