Using a technique called light-activated dynamic looping, or LADL, scientists at the University of Pennsylvania are investigating the role that genome folding plays in gene expression. The team has demonstrated how LADL can be used for quickly creating specific genome folding patterns on demand, using light as a trigger. LADL combines aspects of two other biotechnological tools — CRISPR/Cas9 and optogenetics. By using CRISPR/Cas 9 to target the ends of a specific genome fold, or loop, and then using optogenetics to snap the ends together like a magnet, the researchers can temporarily create loops between exact genomic segments in a matter of hours. Once CRISPR/Cas9 was found to solve the targeting problem, the researchers turned to optogenetics for biological mechanisms that could bind the ends of the loops together. They selected proteins CIB1 and CRY2, found in Arabidopsis, a flowering plant that is a common model organism for geneticists. CIB1 and CRY2 are known to bind together when exposed to blue light. A modification of CRISPR/Cas9 allowed researchers to home in on the desired sequences of DNA on either end of the loop they wanted to form. If those sequences could be engineered to seek one another out and snap together under the other necessary conditions, the loop could be formed on demand. Courtesy of University of Pennsylvania. “Once we turn the light on, these mechanisms begin working in a matter of milliseconds and make loops within four hours,” researcher Mayuri Rege said. “And when we turn the light off, the proteins disassociate, meaning that we expect the loop to fall apart.” Researcher Ji Hun Kim said that while some DNA loops are formed slowly, many form fast, occurring within the span of a second. “If we want to study those faster looping mechanisms, we need tools that can act on a comparable timescale,” he said. The researchers tested LADL’s ability to create the desired loops using high-definition 3D genome mapping techniques, and were able to demonstrate that the newly created loops were affecting gene expression. The relationship between looping and genome function is poorly understood, and the extent to which loops are dynamic on short timescales remains an unanswered question, the researchers said. In order to conduct experiments on how genome structure configurations contribute to genome function, there is a need for techniques that can manipulate specific loops on command, and that are rapid, reversible, and able to work on the target regions with a minimum of disturbance to neighboring sequences. The ability to engineer the loops and mechanisms that determine the timing and quantity of genome expression means that researchers will be able to mimic those mechanisms in experimental conditions, making LADL a valuable tool for studying the role of genome folding on a variety of diseases and disorders. “It is critical to understand the genome structure-function relationship on short timescales because the spatiotemporal regulation of gene expression is essential to faithful human development and because the misexpression of genes often goes wrong in human disease,” professor Jennifer Phillips-Cremins said. “The engineering of genome topology with light opens up new possibilities to understanding the cause and effect of this relationship. Moreover, we anticipate that over the long term, the use of light will allow us to target specific human tissues and even to control looping in specific neuron subtypes in the brain.” The research was published in Nature Methods (https://doi.org/10.1038/s41592-019-0436-5).
