A team of scientists used a tool called CRISPR activation (CRISPRa) to find enhancer sequences that affect the development of T cells, which when not developed properly could lead to autoimmune disorders. The work appears in today's Nature from laboratories at University of California, San Francisco and University of California, Berkeley.
The scientists used an approach that would be a universal "on" switch that could target any part of the genome and, if that part included an enhancer, could activate that enhancer. The tool they used to do this is CRISPRa, which uses a "blunted" version of the DNA-cutting Cas9 protein, strapped to a chain of activating proteins. Although CRISPRa also uses guide RNA to target precise locations in the genome, instead of cutting DNA, CRISPRa can activate any enhancers in the area. By targeting the CRISPRa complex to thousands of different potential enhancer sites, they would be able to determine which had the ability to turn on a particular gene, even if that gene was far away from the enhancer on the chromosome.
The gene the team chose to study produces a protein called IL2RA, which is critical to the function of T cells. The role that IL2RA gene plays is to produce a protein that notifies T cells to trigger inflammation. If the enhancers had an error, the cells would not be able to stop inflammation, which could lead to an autoimmune disorder.
To track down locations of the enhancers that control IL2RA, the UCSF and Berkeley team produced over 20,000 different guide RNAs and put them into T cells with a modified Cas9 protein. By doing this, they were able to target some of the sequences with CRISPRa and observed an increase in IL2Ra production, providing a list of locations that might be important in regulating T cell fate.
One of the likely enhancer sequences the team identified included the site of a common genetic variant that was already known to increase the risk of irritable bowel disease, though how it did so was not understood. Alexander Marson, M.D., Ph.D., assistant professor of microbiology and immunology at UCSF, and Jacob Corn, Ph.D., assistant adjunct professor of molecular and cell biology at Berkeley, both wondered if this genetic variation might alter the switch regulating the amount of IL2RA protein present in T cells. To test out their question, they modified mouse T cells so they contained the genetic variant associated with human disease, and found that these T cells indeed produced less IL2RA.
The team next hopes to expand the method, perhaps by finding ways to search for enhancers of many different genes at once, making the search for regulators of immune disorders that much faster. They expect the method to be a widely applicable tool for untying genetic interactions in all kinds of cells.
Image: Thomas Splettstoesser (www.scistyle.com) (Own work) [CC BY-SA 4.0 (http://creativecommons.org/licenses/by-sa/4.0)], via Wikimedia Commons