Introduction
Once thought to be "junk" DNA, non-coding sequences are now believed to play a significant role in the occurrence of various diseases including cancer and autoimmune disorders. Determining the biological role of these regions can be challenging and requires the development of custom in vivo models for accurate testing.
A recent publication in Nature by Simeonov and colleagues (Simeonov et al., 2017) employed CRISPR activation (CRISPRa) to understand the interplay between non-coding element variations and human immune dysfunction. CRISPRa uses modified Cas9 variants as transcription factors, allowing screening for overexpression to be done quickly and effectively within large genomic libraries (Gebre et al., 2018; Dominguez et al., 2016). By adopting the CRISPRa method, the authors were able to identify specific functional enhancers, which they studied using two mouse models engineered by JAX to determine the biological context for the enhancer.
The CRISPRa method outlined by the authors enabled enhancer discovery without prior knowledge of its biological function and revealed the function of non-coding variations. To test the application of CRISPRa, the authors examined the IL2RA locus, which encodes a subunit of the IL2 receptor (IL-2Ra), a key component in the regulation of immune activation and proliferation (Flynn and Hartley, 2017). They chose this specific locus, despite its complexity in responding to multiple signals, because non-coding variants of this locus have been implicated in at least 8 autoimmune disorders.
In vitro and in vivo studies reveal the full picture
To examine the IL2RA locus, the authors first used an in vitro platform. They used cell lines transduced with a library of guide RNAs (gRNAs) for a specific region surrounding the gene locus, including bases upstream of the transcriptional start site. As a result of the transduction, the authors identified six CRISPRa-responsive elements (CaREs). They chose to further examine CaRE3 and CaRE4 as they activated IL-2Ra to levels similar to those resulting from T cell activation.
The authors next studied the biological function of the identified CaREs using HiChIP, a method that maps active enhancers based on acetylation signatures and long-range chromatin interactions. They observed that all six CaREs exhibited a signature and interaction consistent with gene regulatory function
The authors then investigated a sequence variation in CaRE4 and assessed the effects on transactivation of IL2RA. They observed that the strongest transactivation of CaRE4 was in the highly conserved region that includes a single nucleotide polymorphism (SNP), which has been implicated in autoimmunity.
Furthermore, strong enhancer expression was only observed in response to stimulation, which was diminished following SNP introduction. Based on these collective observations, the authors concluded a link existed between the SNP and disruption of the stimulation-dependent enhancer.
Having established a link in vitro between the SNP and the enhancer, the authors needed a mouse model with the specific autoimmune-associated SNP knocked in. This model, generated for them by JAX was termed the SNP mouse model.
With this model, they observed neither immune dysregulation nor differences in T cell development and function. It did reveal, however, that the enhancer did not affect the surface expression of IL-2RA on Treg cells. The generated SNP mouse model was then used to test the in vivo effects of sequence variation within the IL2RA enhancer. To fully test the enhancer function in vivo, the authors chose another mouse model engineered by JAX, in which the entire IL2RA enhancer was deleted — the so called EDEL mouse.
The authors tested IL-2Ra surface expression on Teff and Treg cells. They had hypothesized that the cascade involving naïve CD4+ T cell response to stimulation, which results in secretion of the pro-inflammatory IL-17 in TH17 cells and the induction of Treg cells, would be skewed towards only the secretion of IL-17 with the impairment of IL2RA activation. To test this hypothesis, they first inhibited IL-2 signaling and observed that in EDEL mice, in antibody-reduced IL-2 signaling, there was an increase in IL-17 secreting cells. As noted by the authors and others, this increase in this specific cell type is associated with Crohn’s disease (Kerami Z., et al., 2014; Fujino S., et al., 2003). The authors concluded that impaired IL-2Ra receptor induction results in disrupted IL-2 signaling, which then shifts CD4+ T cells towards a pro-inflammatory state.
These experiments required not just any in vivo model, but a genetically engineered mouse model with a specific genetic background to ascertain the effects of changes within a functional enhancer. The authors leveraged the characteristics of the C57BL/6J mouse, the most widely used inbred mouse strains, to generate the SNP mouse model; whereas, the EDEL mouse was generated using NOD/ShiLtJ mouse, also known as NOD, one of the most commonly used immunodeficient mouse strains. For their study, they combined a high-throughput methodology utilizing CRISPRa with in vivo testing to determine that non-coding disease variants can alter cellular genomic responses and identified a genetic autoimmunity risk factor.
Summary
The genome is an intricate network comprised of multiple pathways that require precise manipulation to drive activation or repression. Researchers are utilizing the most up-to-date technologies, such as CRISPRa, to interrogate the genetic component of diseases as part of their in vitro testing, and in vivo models to reveal the full genetic picture.
In the publication by Simeonov and colleagues, they started their research by using in vitro methods and complimenting that research in an in vivo model. This approach not only validated their adoption of CRISPRa, but also revealed the functionality of the enhancer in vivo. By using an engineered mouse model from JAX, the researchers were able to design the model most appropriate for their research, while taking advantage of strain background characteristics.
Since 1929, JAX has built its reputation on providing the most advanced resources in the hands of researchers to empower their scientific endeavors. With tools like CRISPR/Cas9, traditional DNA microinjection, and ES cell-based techniques, researchers can leverage JAX expertise to create the best mouse model needed to advance their research. Visit the JAX model generation page to learn more about the model generation services, and how our expert mouse geneticists and therapeutic area experts will work with you to design a model that is appropriate for your study and is translationally relevant.
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References
Dominguez, AA. et al., (2016). “Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation.” Nat Rev Mol Cell Biol. 17(1):5-15. DOI: 10.1038/nrm.2015.2
Flynn, M. J. and J. A. Hartley (2017). "The emerging role of anti-CD25 directed therapies as both immune modulators and targeted agents in cancer." Br J Haematol 179(1): 20-35.
Fujino, S. et al. (2003). “Increased expression of interleukin 17 in inflammatory bowel disease.” Gut 52, 65–70.
Gebre, M., et al. (2018). “CRISPR-Cas9 Genetic Analysis of Virus-Host Interactions.” Viruses. 10(2). DOI: 10.3390/v10020055
Kerami, Z., et al. (2014). "Effect of interleukin-17 on gene expression profile of fibroblasts from Crohn's disease patients." Journal of Crohn's and Colitis 8(10): 1208-1216.
Simeonov, D. R., et al. (2017). "Discovery of stimulation-responsive immune enhancers with CRISPR activation." Nature 549(7670): 111-115.