Enhancers Thought to Have a Critical Role in the Development of Autoimmune Disorders

While much of the excitement around CRISPR gene editing focuses on its medical and public health applications, the ability to precisely edit virtually any DNA sequence is a revelation for basic research. In the latest demonstration, researchers at the University of California and elsewhere have used a modified version of CRISPR gene editing to identify genetic regulatory elements (enhancers) involved in autoimmune diseases.

The latest research, published this week in Nature, was led by Alexander Marson at the University of California San Francisco (UCSF) and Jacob Corn at the University of California Berkeley (UC Berkeley). The two researchers are also affiliated with the Innovative Genomics Institute (IGI), a joint UCSF-Berkeley initiative (Corn is cofounder and director), which aims to drive genome editing in medicine and agriculture.

Over the past few years, most studies utilizing CRISPR have employed a bacterial enzyme called Cas9 to engineer precise double-stranded cuts in a targeted stretch of DNA, usually in a gene. But there are many types of Cas (CRISPR-associated sequence) proteins that have different DNA cleavage and editing properties.

The new study used a method called CRISPR activation (CRISPRa), which uses a blunted version of a Cas enzyme that preserves the sequence-targeting properties but does not cleave the DNA. It was developed in 2013 by UCSF/Howard Hughes Medical Institute investigator Jonathan Weissman and colleagues. Weissman uses a musical analogy: while CRISPR/Cas9 can effectively repair a wonky key on the genomic keyboard, CRISPRa offers the possibility of composing a full score.

Using CRISPRa, Corn, Marson and colleagues have surveyed the human genome for regulatory regions called enhancers—DNA motifs that can upregulate a gene sequence and may reside many thousands of bases away from the gene sequence itself. The IGI team focused on enhancers for a gene that affects the development of T cells, a key component of the immune system. Some of these enhancers are likely to have critical roles in the aberrant pattern of gene regulation that leads to autoimmune disorders such as Crohn’s disease and inflammatory bowel disease (IBD).

“Not only can we now find these regulatory regions, but we can do it so quickly and easily that it's mind-blowing,” said Corn. “It would have taken years to find just one [enhancer] before, but now it takes a single person just a few months to find several.”

Scientists can look for potential enhancer sequences based on how they interact with proteins that bind to DNA, but figuring out which enhancers work with which genes is much more challenging. Simply cutting out an enhancer with CRISPR/Cas9 doesn't help, because it won't have a noticeable effect if the enhancer is inactive in the particular cell type used in an experiment.

If you think of the genome as a model home with 22,000 lightbulbs (the genes) and hundreds of thousands of switches (the enhancers), the challenges have been finding all of the switches and figuring out which lightbulbs they control and when. Previously, CRISPR has been used to cut out wires looking for those that would cause a bulb to go dark, giving a good idea of what that section of the circuit was doing. However, cutting out a light switch when it's off doesn't tell you anything about what it controls. So, in order to find certain light switches, it has been common to try to mimic the complicated chemical cues that activate an enhancer.

But using this method, “you can quickly go insane trying to find an enhancer,” said Benjamin Gowen, a postdoctoral fellow in Corn's lab at Berkeley and one of the study's lead authors.

A better approach 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. Fortunately, CRISPRa, recently developed by Jonathan Weissman, Ph.D., professor of cellular and molecular pharmacology at UCSF and codirector of the IGI, is just such a tool. CRISPRa 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.

While the first applications of CRISPRa involved using a single guide RNA to find promoters—sequences right next to genes that help turn them on—the UCSF/Berkeley team behind the new study realized that CRISPRa could help find enhancers too. By targeting the CRISPRa complex to thousands of different potential enhancer sites, they reasoned, 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.

“This is a fundamentally different way of looking at noncoding regulatory sequences,” said Dimitre Simeonov, a Ph.D. student in Marson's lab at UCSF and the study's other lead author.

Performing 20,000 Experiments at Once

The gene the team chose to study produces a protein called IL2RA, which is critical to the function of immune cells called T cells. Depending on conditions in the body, T cells have the ability to either trigger inflammation or suppress it. The IL2RA gene produces a protein that tells T cells that it's time to put on their anti-inflammatory hats. If the enhancers that should turn on the gene have errors, the cells fail to suppress inflammation, potentially leading to autoimmune disorders like Crohn's disease.

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. “We essentially performed 20,000 experiments in parallel to find all the sequences that turn on this gene,” Marson said.

Sure enough, targeting some of the sequences with CRISPRa increased IL2RA production, yielding a short list of locations that might be important for regulating the fate of T cells.

“Whenever you get a chance to ask a question in a totally new way, you can suddenly discover things that you would have missed with older methods,” said Gowen. “We found these enhancers without having to make any assumption of what they looked like.”

Tying Mutant Enhancers to Inflammatory Disease

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 IBD, though how it did so was not understood. Marson and Corn's teams wondered whether this genetic variation might alter the switch regulating the amount of IL2RA protein present in T cells. To test this, 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.

“This starts to unlock the fundamental circuitry of immune cell regulation, which will dramatically increase our understanding of disease,” said Marson.

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. And they expect the method to be a widely applicable tool for untying genetic interactions in all kinds of cells.

“We believe this is going to be a very generally useful method,” said Corn. “It would be easy for someone interested in neurons or any other cell type to pick it up and look for the enhancers involved in programming those cells' behavior.”

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