CRISPR-Cas9 Gene Editing Reveals Potential Therapeutic Targets for ALS

Researchers have identified genes that promote amyotrophic lateral sclerosis (ALS), using the gene-editing technology CRISPR-Cas9.

The findings represent not only another piece of information to understand the molecular mechanisms triggering ALS, but also support the newly identified genes as potential targets for future therapeutics.

The study “CRISPR–Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity” was published in the journal Nature Genetics.

The accumulation of toxic protein clumps in nerve cells is the underlying cause of several neurodegenerative diseases, including ALS. But how these aggregates lead to nerve cells’ death is still an unanswered question for researchers.

“These toxic protein aggregates are what’s likely driving the pathology in the disease, but no one really knows how they cause neuronal cell death. That’s really what we wanted to probe in this study,” said Aaron Gitler, PhD, in a press release. Gitler is a professor of genetics at Stanford University School of Medicine, and co-lead author of this study.

It is known that mutations in the C9orf72 gene are the most common genetic cause for ALS. These errors expand the number of DNA repeats contained within the gene, triggering the disease.

“In a healthy person, you might see 10 to 20 of these DNA repeats,” said study co-first author Michael Haney. “But in ALS, they expand to hundreds or even thousands of repeated segments, and that’s the template for the production of these toxic proteins.”

Researchers at the Stanford University School of Medicine set out to uncover which genes could help neurons protect themselves against these toxic protein clumps, and also whether there are genes that exacerbate even further the detrimental effects of protein aggregates.

For that they used the genome-editing technology, CRISPR-Cas9, and performed a genome-wide screen by removing (“knocking-out”) each time one of the approximately 20,500 human genes that compose the entire human genome.

This strategy allows researchers to identify genes that help prevent or enhance toxicity. If, after knocking-out a particular gene, researchers see the repeats of the protein encoded by the mutated C9orf72 gene are no longer toxic, it means the absence of the gene is actually beneficial, making it a potential therapeutic target.

After knocking-out every gene in our genome, researchers ended up with a list of 200 genes either protecting or promoting cells’ death from the toxic proteins.

In the next set of experiments, they switched to primary mouse neurons (the cells of relevance for ALS) and performed the screen all over again, but this time just for the 200 genes shown to have a role in ALS.

The results confirmed a set of genes as strong protecters against the toxic proteins, but there was a particular one – Tmx2 – that caught the researchers’ attention: They saw that after knocking-out Tmx2 of the mouse neurons cells in vitro, the neurons survived almost 100 percent of the time, while normally only 10 percent survive.

“We could imagine that Tmx2 might make [a] good drug target candidate,” Haney said. “If you have a small molecule that could somehow impede the function of Tmx2, there might be a therapeutic window there.”

The Tmx2 protein resides in an intracellular organelle called the endoplasmic reticulum, but its function is still a mystery. However, some studies suggest it may work with other genes to trigger cells’ death.

“We’re still in early phases, but I think figuring out exactly what Tmx2 normally does in a cell is a good place to start — that would hint at what functions are disturbed when these toxic species kill the cell, and it could point to what pathways we should look into,” said Nicholas Kramer, another first author of the study.

This is the first study, researchers believe, to use genome-wide human CRISPR knockout screen to unveil the molecular mechanisms underlying a neurodegenerative disease, in this case ALS.

The researchers are now using their strategy to dig further into the ALS mechanisms and expand it to other neuro diseases linked to toxic protein clumps, such as Huntington’s, Parkinson’s and Alzheimer’s.

“I think it’s a really exciting application for CRISPR screens, and this is just the beginning,” said Michael Bassik, PhD, assistant professor of genetics and co-lead author of the study.