CRISPRa: A universal "on switch" to identify functional gene enhancers

Each one of our cells has the same 22,000 or so genes in its genome, but each uses different combinations of those same genes, turning them on and off as their role and situation demand. It is these patterns of expressed and repressed genes that determine what kind of cell -- kidney, brain, skin, heart -- each will become.

To control these shifting patterns, our genomes contain regulatory sequences that turn genes on and off in response to specific chemical cues. Among these are "enhancers," sequences that can sit tens of thousands of genetic letters away from a gene, yet still force it into overdrive when activated. Missteps in this delicate choreography can lead cells to take on the wrong role, causing debilitating diseases, but the regulatory regions involved are difficult to find and study since they only play a role in specific cells, often under very specific conditions.

Now a research team led by University of California and Chan Zuckerberg Biohub scientists has used a modified version of the gene-editing technique CRISPR to find enhancers -- not by editing them but by prompting them into action.  A team from UC San Francisco and the University of California, Berkeley, used a tool called CRISPR activation (CRISPRa), developed at UCSF in 2013, to search for enhancers of a gene that affects development of the immune cells known as T cells. The sequences they found illuminate fundamental circuitry of autoimmune disorders such as inflammatory bowel disease (IBD) and Crohn's disease.

To learn more about this research we spoke to three authors from the paper, Dr. Alex Marson, from the Chan Zuckerberg Biohub and UCSF, Dr. Benjamin Gowen, UC Berkeley and the Innovative Genomics Institute and Dimitre R. Simeonov, UCSF. 

JR: Why was there a need for a technique that can identify stimulus-responsive enhancers for a target gene, independent of exposure to the stimulus? 

Our genome contains regulatory sequences that turn genes on and off in response to specific chemical cues. Among these are “enhancers,” sequences that can sit tens of thousands of genetic letters away from a gene, yet still force it into overdrive when activated. Missteps in this delicate choreography can lead cells to take on the wrong role, causing debilitating diseases but the regulatory regions involved are difficult to find and study since they only play a role in specific cells, often under very specific conditions. 

Now we are using a modified version of the gene-editing technique CRISPR to find enhancers – not by editing them but by prompting them into action with a tool called CRISPR activation (CRISPRa), developed at UCSF in 2013. We used CRISPRa to exhaustively search for enhancers of a gene that is important for the function of immune cells known as T cells. The sequences that we have found illuminate fundamental circuitry of autoimmune disorders such as Crohn’s disease. 

JR: What problems with existing techniques does this new, CRISPRa-based approach overcome?

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. Of course, there are so many chemical cues that it is just not feasible to search for enhancers in this way.

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. CRISPRa 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. 

JR: How will this technique be utilised? What kinds of research will it support?

CRISPRa is a powerful new way to identify functional enhancers. We used it to track down locations of the enhancers that control IL2RA by producing over 20,000 different guide RNAs and putting 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. 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. 

We think that this technique will used for finding enhancers across the genome in different cell types, which will ultimately support mechanistic work on gene regulation and human disease.

JR: Apart from describing the use of this exciting new technique, what else did you find?

One of the IL2RA enhancer sequences our 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. We wondered whether this genetic variation might alter the switch regulating the amount of IL2RA protein present in T cells. To test this, we modified mouse T cells so they contained the genetic variant associated with human disease, and found that these T cells indeed produced less IL2RA. IL2RA levels help determine whether a T cell becomes pro-inflammatory or anti-inflammatory. Here we showed that the modifications in the enhancer influenced T cells to take on a more pro-inflammatory role providing one potential explanation for the increased IBD risk. 

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

JR: How are you planning to build on your findings?

The UCSF/Chan Zuckerberg Biohub team hopes to expand on 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. We expect the method to be a widely applicable tool for untying genetic interactions in all kinds of cells. We are also working towards using this technology to build functional regulatory maps of the human genome.

Reference

Simeonov, D. R., Gowen, B. G., Boontanrart, M., Roth, T. L., Gagnon, J. D., Mumbach, M. R., ... & Lituiev, D. S. (2017). Discovery of stimulation-responsive immune enhancers with CRISPR activation. Nature, 549(7670), 111-115.