OptoLoop: A Light-Activated System for Interrogating Genome Architecture and Gene Regulation

The genome folds inside the cell nucleus into hierarchical architectural features, such as chromatin loops and domains. While this genome organization is known, how it influences the regulation of gene expression remains only partially understood. Traditional methods for probing the structure-function relationship of genomes often involve population-wide measurements, such as mutating critical DNA elements or perturbing chromatin-associated proteins. Such approaches can lead to pleiotropic effects, affecting multiple aspects of the cell simultaneously.

To circumvent the possible pleiotropic effects of traditional approaches, OptoLoop was developed. As lead author Stortz notes in the paper, "To circumvent possible pleiotropic effects of such approaches, we have developed OptoLoop, an optogenetic system that allows direct manipulation of chromatin contacts by light in a controlled fashion." This innovative system is based on the fusion between a nuclease-dead SpCas9 protein—which can target specific DNA sequences without cutting them—and CRY2, a light-inducible oligomerizing protein. This combination allows researchers to drive the induction of contacts between genomically distant, repetitive DNA loci using light, offering a precise means of controlling genome architecture.

As a proof-of-principle application of OptoLoop, the researchers probed the functional role of DNA looping in the regulation of the human telomerase gene TERT by long-range contacts with the telomere. By analyzing both the extent of chromatin looping and nascent RNA production at individual alleles, they found evidence for looping-mediated repression of TERT.

In summary, OptoLoop represents a novel means for the interrogation of structure-function relationships within the genome at single-allele resolution. By offering precise, light-controlled manipulation, OptoLoop helps to circumvent the possible pleiotropic effects associated with previous methods, enabling more targeted investigations into genome organization and gene regulation.