Roughly five years ago, when setting up his lab to study the epigenetics of addiction and memory at The University of Alabama at Birmingham, Jeremy J. Day, PhD, bet the house on CRISPR technologies capable of manipulating chromatin modifications at specific DNA and histone loci. Day believes that CRISPR editing approaches have the potential to reveal how modulating epigenetic marks at individual chromatin sites can directly impact neuronal function and behavior.

“I did this thinking that taking specific epigenetic marks deposited or removed with experience, development, or disease states and manipulating the related chromatin modification mechanisms at desired targets would really advance our understanding of those processes by starting to establish causality,” Day says. “That’s because, up until now, we’ve seen a lot of correlational findings.”

Just a decade ago, when Day was a postdoc studying the epigenetics of neural circuits, attempts to manipulate DNA or histone marks in the brain or any biological system would materialize non-specifically throughout the genome. Day was frustrated. “It wasn’t satisfying doing epigenetic manipulation experiments knowing that you were targeting a process that was really global and happening to countless genes.”

The Missing Chromatin Link

Now an associate professor in the Department of Neurobiology at UAB, Day uses CRISPR epigenetic editing tools in which the catalytic domains of chromatin readers, writers, and erasers—DNA- or histone-modifying enzymes or chromatin-remodeling complexes—have been fused to catalytically inactive (“dead”) Cas9 endonucleases (dCas9). Chromatin modulation in this manner allows for stable, reversible manipulation of epigenetic marks at targeted cis-regulatory elements.

Day says that the most straightforward way to use CRISPR for epigenome editing in the nervous system involves distinct types of viral vectors. Lentiviruses or adeno-associated viruses (AAV) carry transgenes for either the enzymatic or targeting CRISPR-components and transfect specific projections and synaptic junctions in connected neuronal populations.

Day favors a dual lentivirus system, which means a guide RNA is expressed from one lentivirus, the CRISPR activation machinery from another. “The advantage is that you can swap out or combine the guide RNA viruses if you want to target more than one gene or different genes in series.”

This CRISPR system empowers researchers to dissect the causal links between chromatin regulation and neural responses, such as those in substance abuse and addiction.

Day’s group typically exposes animals to a drug of abuse, then uses next-generation sequencing to identify regulatory elements or promoters that are differentially regulated. “Then, we use these CRISPR epigenetic editing tools to selectively target and establish or remove modifications to see if it mimics or blocks the drug-induced changes as well as the consequences it has for the physiology of neuronal circuits and behavior of the animal.”

Epigenetic Exploration

This CRISPR-based DNA- and histone-modifying framework offers several advantages to biomedical research as well as the development of therapeutics for personalized medicine.

One major perk is that these tools are fundamentally model-free. “You can target any genome, whether it’s mouse, primate, or human,” Day says. He uses rats for their robust behavior in addiction models but has also been collaborating with researchers using prairie voles, a rodent species that forms a lifelong monogamous pair bond and is used as a model for social bonding.

These chromatin modification tools also allow for the study of genetic variants associated with brain-related diseases, mostly located in non-coding genomic regions and enriched with DNA- and histone-modifications. Now, epigenetic aberrations and chromatin alterations that have been identified in numerous neuropsychiatric conditions—such as depression, schizophrenia, autism spectrum disorders, and addiction—as well as neurodegenerative diseases can be studied on a causal basis.

Day says that these tools are enabling groundbreaking studies with human iPSC-derived neurons. “The major advantage here is that you can have the genetic background of a human with a specific gene variant that contributes to disease, pathology, or risk.” In this scenario, chromatin state modulation can be complemented with genome editing to dissect the functional and mechanistic roles of these non-coding functional variants and linked genomic regulatory regions.

In terms of personalized medicine, targeted epigenetic therapy with CRISPR can be used without genetically modifying DNA sequence to restore normal chromatin structure and correct gene expression. Crucially, this minimizes the chance to create unwanted mutations and off-target effects.

As cis-regulatory elements can interact and regulate multiple genomic loci, epigenetic therapy can provide improved therapeutic efficacy by simultaneously fine-tuning multiple chromatin states and gene activities with several guide RNAs. This is important in light that common human diseases are polygenic in origin and associated with dysregulation of chromatin state and the epigenome.

Still, this field is in its infancy and not without problems. Day says further refinements to increase the potency and heritability of epigenetic editing with improved delivery, transfection efficiency, and transgene expression stability are needed. Yet, no matter how nifty the gene-editing technology is, the brain will always be difficult to target simply for accessibility reasons, while off-target effects will be a constant concern.

These CRISPR tools are a major leap towards proving causal interplay between chromatin state and neuronal responses for cognition and behavior as well as the use of epigenetic-oriented therapeutics in the human nervous system.

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