New CRISPR technology could help repair damaged neurons

When a neuron in our body gets damaged, segments of RNA produce proteins that can help repair the injury. But in neurological disorders such as ALS and spinal muscular atrophy, or following spinal cord injuries, the mechanisms for moving life-essential RNA to injured sites within the cell fail. As a result, RNA molecules can’t get to where they are needed and damage becomes permanent.

Researchers at Stanford have developed a technology for transporting RNA to specific locations within a neuron, where it can repair and even regrow parts of the cell. Their work, supported by the National Institutes of Health, forms the foundation for a new class of therapeutics that the researchers are calling “spatial RNA medicine,” which they hope will lead to treatments for neurological diseases as well as traumatic injuries.

“For the first time, we’ve harnessed the power of CRISPR technology to create a precise spatial ‘zip code’ that delivers RNA molecules exactly where they’re needed within cells,” said Stanley Qi, an associate professor of bioengineering and senior author on the paper published May 21 in Nature. “Imagine being able to specifically target damaged sites within a neuron, repairing them, and promoting their regrowth – this is what our technology achieves.”

A CRISPR-based mailman

In recent years, researchers have realized that the distribution of RNA within a cell – where specific molecules are located – may be just as important as what they are capable of doing. An individual neuron can be over a meter long, and aging, injury, and mutations can all disrupt its ability to transport the tiny RNA over such a distance.

“Therapeutic RNA can’t help if it doesn’t get to where it’s needed,” Qi said. “We wanted to create a technology that could reliably move RNA to where it needs to function.”

Qi and his colleagues used a version of the gene-editing tool CRISPR, called CRISPR-Cas13, to target individual pieces of RNA (unlike the more widely known CRISPR-Cas9, which targets DNA). Typically, CRISPR is used to slice and edit genetic code, but in this case the researchers didn’t want to make any changes. They simply wanted to move the existing RNA to a new place within the cell.

“Cas13 naturally acts like a pair of scissors, but we engineered it to act like a mailman instead,” Qi said. “Then we can tell it to carry the RNA from one precise location to another.”

The researchers paired Cas13 with specific localization signals that act as addresses, instructing the Cas13 where to deliver the RNA. Each location within the cell has its own address molecule, so the researchers can direct the RNA to various locations by adding different molecules to the cell.

Qi and his colleagues used their technology, which they are calling CRISPR-TO, to screen dozens of pieces of RNA and see if any of them would help neurons to grow. They added CRISPR-TO to mouse brain neurons in a petri dish, where it carried the RNA molecules to the tips of neurites – fingerlike protrusions that form synapses and connect to other neurons. They found several promising candidates, including one RNA molecule that increased neurite growth by as much as 50% over a 24-hour period.

“We are discovering more RNA targets that could promote neurite outgrowth and regeneration,” said Mengting Han, a postdoctoral scholar in Qi’s lab and lead author on the paper. “We’ve added a new tool to the CRISPR toolbox, using it to control RNA localization inside the cell. This has never been achieved before and, importantly, it opens new therapeutic directions for treating neurodegenerative diseases.”

Safer, more effective RNA medicine

The researchers are using CRISPR-TO to screen additional RNA molecules to determine which ones will be most effective at repairing injured neurons in the brains of mice, as well as in human neurons.

“We are at the beginning of understanding how spatial organization of RNA benefits brain repair,” Qi said. “We hope our technology will help people figure out which RNAs will be the biggest players for better therapeutics.”

Currently, the researchers are using CRISPR-TO to move endogenous RNAs – RNA molecules that are naturally produced within the cell. But it could also be used to provide precise control over RNA-based medicines, making them both safer and more efficient, Qi said.

“This potential excites us tremendously,” Qi said. “It’s not enough for a molecule to just be in the cell. We need it to be in the right location at the right time. With our precise, programmable technology, you can target any RNA in any type of cell and bring it to the site of need in the body.”

For more information

Qi is a member of Stanford Bio-X, the Stanford Cardiovascular Institute, the Maternal & Child Health Research Institute, the Stanford Cancer Institute, and the Wu Tsai Neurosciences Institute; and an institute scholar of Sarafan ChEM-H.

Additional Stanford co-authors of this research include postdoctoral scholars Yanyu Zhu, Alexander A. Choi, Emmy Li, Leanne Miles, and Yitong Ma; and graduate students Maylin L. Fu, Jon Bezney, and Sa Cai.

This work was funded by the National Science Foundation, the National Institutes of Health, the National Center for Research Resources, the Stanford School of Medicine Dean’s Postdoctoral Fellowship, and the American Heart Association Postdoctoral Fellowship.