Researchers from the University of Manchester have recently discovered an unexpected consequence of gene-editing; a particular sequence within the interleukin-1α (IL-1α) gene.
The group intended to use the CRISPR/CAS9 genetic editing technique to mutate a coding sequence in mice to alter the processing of IL-1α within the cell. By altering the nuclear localisation sequence (NLS), translocation of IL-1α to the cell nucleus is inhibited, increasing the subsequent inflammatory signalling response.
Unexpectedly however, they found that the IL-1α protein and the messenger RNA (mRNA), which codes and transcribes for the protein, were undetectable.
“We needed to generate this model so we could investigate the importance of IL-1a nuclear localisation. We were surprised that our mutations killed expression,”
said lab group lead, David Brough.
The group deduced that this unexpected finding was due to an effect on a related portion of the DNA code but within the parallel strand. IL-1α shares strand complementarity with a long-non coding RNA, important in IL-1α expression, which is also targeted by CRISPR/Cas9.
The group predicts that this off target mutation alters the secondary structure of the long-non coding region, thereby affecting its regular role as an inducer of IL-1α expression; hence the expression of IL-1a is lost when the mutations are made.
CRISPR/CAS9 is a genetic editing technique which utilises the bacterial adaptive immunity to induce precise alterations to genetic code. The widespread hype surrounding this technique is due to a mix of the effective point mutations enabled by the process and the relative simplicity to apply the technique.
The potential applications of this technique are broad, and may revolutionise healthcare and medical research, owing to the ability to effectively tune out, or in, certain genes.
This great gene-editing ability has its own ethical and technical implications. As shown by this research paper, the mutation of a NLS to produce a loss of IL-1α nuclear translocation function also had the unexpected effect of inhibiting IL-1α production.
This presents a key issue in the alteration of any genetic sequence without extensive testing to establish the full consequences of genetic editing within an organism. When asking the authors about the significance of their findings, David Brough and lead author Mike Daniels stated,
”Here we have identified a potential caveat of using CRISPR/Cas9.”
IL-1α is an inflammatory cytokine which will initiate a feedback loop which subsequently increases pro-inflammatory protein production. This cytokine has an important role in regulating inflammatory response to damage and infection, but also has a negative role in the pathogenesis of many diseases; with diabetes, Alzheimer’s disease and stroke all being heavily affected by excessive pro-inflammatory signalling.
This has led to the notion that inhibiting the inflammatory response in these diseases may result in treatment of the disease. This is especially pertinent for neurodegenerative diseases like Alzheimer’s disease, where there is currently a distinct lack of effective treatments to stop disease progression.
Research into multiple diseases can be progressed by implementing precise genetic mutations through CRISPR/CAS9. The investigation of potential ramifications which may arise from genetic alterations is crucial to avoid any detrimental off target effects and may be pivotal to establishing the effectiveness of gene-editing in a clinical setting.
Michael J. D. Daniels, Antony D. Adamson, Neil Humphreys & David Brough CRISPR/Cas9 mediated mutation of mouse IL-1α nuclear localisation sequence abolishes expression Scientific Reports. 06 December 2017. DOI:10.1038/s41598-017-17387-x
Author: Geoffrey Potjewyd; Regenerative Medicine & Neuroscience PhD student at the University of Manchester.
Image: Mike Daniels (left), and Dave Brough (right), of the School of Biological Sciences. Credit: University Of Manchester