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Chemically Induced Guide RNAs Drive CRISPR/Cas9 System for Editing Mouse Neurons In Vivo


NEW YORK (GenomeWeb) – A new CRISPR/Cas9 system designed for editing mouse neurons in vivo uses chemically induced guide RNAs (gRNAs) to help control where and when editing happens.

The virally delivered, doxycycline-inducible system will help University of Texas at Dallas researcher Jonathan Ploski study genes associated with learning and memory in the mouse model. Last month, a Ploski-led team published a study describing it in Frontiers in Molecular Neuroscience.

"If you want to do genome editing in tissues in experimental organisms, the preferred vector is AAV," Ploski told GenomeWeb. "There's been less devotion to adapting these Cas9-related technologies to AAV, because it's a smaller vector. Most of the experiments are just happening off standard plasmids transfected into cells in culture."

But the kinds of questions he wants to answer — he's a neuroscientist — require working with an animal model, necessitating a CRISPR/Cas9 system that can be delivered with AAV. In particular, Ploski developed a two-vector system, delivering the Cas9 enyzme and inducible gRNA separately. While a two-part system is more complicated, the inducible gRNA could be used separately with other CRISPR/Cas9 setups, like the Cas9 mouse, which has been engineered to express Cas9 in every cell.

"This delivery system increases the spatial specificity of this system which, due to the complexity of the brain, allows us to improve the precision by which we can target very specific cell types, which is always desirable," Ploski said.

At UT-Dallas, Ploski runs a lab where he studies the molecular and cellular basis of learning and memory, using a mouse model. Specifically, he's studying fear-based memories — where an animal learns to associate a harmless sound with a foot shock. Eventually, mice will react to the tone as if they're about to be shocked and exhibit a defensive response and the degree to which they do so can be an indication of the strength of that animal's memory, Ploski said.

Ploski reasoned that if he could manipulate a gene in a brain cell after a mouse had learned to fear the tone, he could ask questions about how that influences the animal's ability to retrieve, modify, weaken, or extinguish the memory. To do so, he would need a way to make the gene manipulation at a specific time point in the animal's life, namely, after it had made a memory, and in the right cells.

"We wanted a reliable way to manipulate genes in vivo and simply, CRISPR/Cas9 seemed like a viable way to do this," Ploski said. "For us, it would be ideal if we could localize the CRISPR/Cas9 editing in specific neurons within specific parts of the brain," he said, especially the amygdala.

While several research teams have designed inducible CRISPR systems, even using the same chemical trigger, Ploski's is the first to feature chemically induced expression of the guide RNA.

Lukas Dow, an assistant professor at Weill Cornell Medical College and a scientist who has designed a doxycycline-inducible CRISPR system in transgenic mice, said that it's important to consider that a system like Ploski's relies on a promoter that can have a basal expression level. "Systems that use inducible Cas9 are leaky," he said, meaning that sometimes the nuclease is expressed even when the system hasn't been triggered.

The decision to induce the gRNA chemically gets around that. Dow also said that the modularity of a two-component, regulatable system is a strength. "It gives you more control. Having the ability to do that is great, but how you apply it to a biological system is the key."

While Ploski's lab hasn't completed any studies yet, he said he planning to use the system to knock out candidate genes believed to be important for neuroplasticity, the ability of the brain to change over time.

Once the CRISPR system is delivered via the viral vector, the researcher can activate it by injecting doxycycline into the mouse's brain or, more simply, adding doxycycline to the mouse's food.

"If we feed [a mouse] doxycycline for 24 hours, wait a couple days, and examine it for genome editing, we see the editing was quite efficient," Ploski said.

The system also limits the amount of time during which the CRISPR system can be actively cutting DNA. "If you're constantly expressing Cas9 and the guide RNA, you may be more prone to inducing off-targets effects, due to the fact these things are hanging around longer," Dow said. "Being able to switch one component off, you can reduce the potential for collateral damage."

Not surprisingly, there's more research that can be done to optimize the system. In July, Ploski started work under a $420,750 grant from the National Institutes of Health to explore methods of localizing the tool to specific subsets of cells in the brain. He's also looking into the effects of sustained genome editing in animals. "There haven't been enough adequate studies looking at the fidelity of the CRISPR/Cas9 system over long-term use within the animal brain," he said.

Outside of his own lab, Ploski said that the system could create more opportunities to study the brains of other kinds of model organisms. While rats are often a good model for neurosciences studies, there are fewer genetically modified animals. Now, CRISPR/Cas9 could be used to get the right edits into those animals. Dow suggested that pairing Ploski's system with a Cre-lox site-specific editing system could help scientists interested in investigating two separate genetic events.

"This adds to the tool kit and I'm sure people will want to use it," he said.