A sequential event-responsive fluorescent reporter for inducible Cre and Flp combinatorial recombination

Current technologies for precise genetic manipulation of cells often utilize site-specific recombinases, enabling the creation of conditional transgenic models for studying gene functions in specific tissues and at defined time windows. The advent of drug-inducible recombinases has further enhanced this field by allowing temporal control of gene expression. However, detecting gene expression patterns when multiple recombinases are used is challenging, particularly when visualizing combinatorial maps and their temporal profile. To address this, we developed Rubik, a fluorescent genetic reporter specifically designed to monitor the action of Cre and Flp recombinases by expressing different fluorophores depending on the temporal sequence of recombination events. Rubik offers four alternative fluorescent configurations: blue for Cre recombination, green for Flp recombination, yellow for Cre followed by Flp recombination, and red for Flp followed by Cre recombination. Additionally, Rubik integrates light-gated ion channels, allowing optogenetic excitation or inhibition of electrically-excitable cells based on the temporal sequence of recombination events. Moreover, to add temporal control over Rubik recombination, we optimized a trimethoprim-inducible FlpO recombinase (FlpO-DD) to use in combination with the tamoxifen-inducible Cre recombinase (ERT2CreERT2). We validated Rubik by generating a stable knock-in HeLa cell line using CRISPR-Cas9 and single-cell sorting. Our results confirmed the effective functioning of Rubik with Cre and Flp recombinases and showed the ability to induce selective recombination via either tamoxifen or trimethoprim. This system holds potential for neuroscience research, particularly in generating knock-in mice expressing Rubik, which could be instrumental in precisely defining the roles of specific neuronal populations based on Cre and Flp activity.