NEW YORK (GenomeWeb) – A team led by researchers at the University of California, Berkeley and the University of California, San Francisco used CRISPR activation (CRISPRa) to identify enhancers of a gene that affects development of T cells, and thereby elucidate the occurrence of autoimmune disorders such as inflammatory bowel disease (IBD) and Crohn's disease.
In a study published today in Nature, Berkeley's Jacob Corn, UCSF's Alexander Marson, and their collaborators at the Broad Institute, Illumina, and elsewhere note that systematic mapping of enhancer function remains challenging "because of our limited understanding of the cellular contexts in which each enhancer contributes to gene regulation."
In order to understand the mechanisms by which non-coding genetic variation contributes to disease, the researchers developed an unbiased discovery platform to identify stimulus-responsive enhancers for a target gene independent of stimulus exposure. They used tiled CRISPRa to synthetically recruit a transcriptional activator to sites across large genomic regions surrounding two key autoimmunity risk loci: CD69 and IL2RA.
They then identified several CRISPRa Responsive Elements (CaREs) with chromatin features of stimulus-responsive enhancers, including an IL2RA enhancer that harbors an autoimmunity risk variant. And using engineered mouse models, they found that sequence perturbation of the disease-associated IL2RA enhancer delayed the timing of gene activation in response to specific extracellular signals.
"Functional enhancers can be mapped using Cas9-directed mutagenesis to disrupt genomic sequences, but this approach only identifies the subset of enhancers that are necessary in the particular cellular context being studied," the team wrote. "We hypothesized that recruitment of a strong transcriptional activator to an enhancer would be sufficient to drive target gene expression. This approach could 'fire' an enhancer that has been poised by the existing chromatin state, even if that enhancer is not currently active in the assayed cells. Compared to assays of chromatin accessibility or histone modification, this approach should be less dependent on the physiologic cellular context in which the enhancer functions."
They validated this CRISPRa method by tiling guide RNAs across the CD69 locus, and then applied their approach to the IL2RA locus by mapping functional IL2RA enhancer elements to determine how known disease risk variants affect enhancer function. IL2RA is critical to the function of T cells. The researchers produced more than 20,000 gRNAs and transduced them into T cells with a modified Cas9 protein.
The experiments yielded some sequences that increased IL2RA production when targeted with CRISPRa, and narrowed the list of enhancers that affect T cell development. One of these was also the site of a common genetic variant that was already known to increase the risk of IBD.
"The single nucleotide polymorphism rs61839660, which resides in this element, is one of only a few disease-associated variants for any common human disease that has been convincingly statistically resolved to a single non-coding variant," the authors wrote. "This individual SNP accounts for the risk of inflammatory bowel disease at the IL2RA locus 20. Consistent with a critical and complex function in immune regulation, this same SNP also paradoxically causes protection from type 1 diabetes."
The researchers are hoping to expand the method by finding ways to search for enhancers of many different genes at once. "We believe this is going to be a very generally useful method," Corn said in a statement. "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."
"While we focused on immune-related genes, we anticipate this approach will have general utility as an enhancer discovery platform and can be used for functional annotation of the vast non-coding genomic space," the authors wrote.