Development of Fluorescent Repressor Operator System (FROS) Based on LexA for DNA Labeling in Live Cells

The spatial conformation of chromatin, namely the DNA and its associated biomolecules, plays an important role in a variety of processes, e.g. gene expression and regulation, DNA repair, nuclear transport, and more.

FROS (Fluorescent Repressor-Operator System) is a technique used to visualize DNA loci in live cells. In this method, an array of protein-binding sites (operators) is integrated at a specific locus in the genome, and is visualized through the specific binding of fluorescently tagged repressors. FROS is used in yeast to study chromatin dynamics, for example during mating-type switching process, in which the goal is to track several loci simultaneously, using three different established FROS (LacO-LacI, TetO-TetR, and LambdaO-LambdaR). The use of these different systems allows the localization of three specific loci with separate emission spectra. Developing additional FROS is highly desirable, as it will enable tagging a larger number specific loci, ultimately achieving better sampling of the genome and higher resolution of DNA dynamics. Moreover, optimization of signal intensity is crucial for the usage of advanced microscopy methods such as point spread function (PSF) engineering, for fast 3D localization microscopy.

Here, we develop a new FROS using the bacterial LexA operator -repressor system together with iRFP720 fluorescent protein. This system uses fewer operator repeats compared to the other FROS, which reduces genome perturbations. We plan to employ this new system, together with the three existing ones, to simultaneously visualize and track four different loci involved in chromatin reorganization associated with mating type switching in Saccharomyces cerevisiae.