Snapshot: The next-generation of CRISPR is prime editing – what you need to know

The CRISPR gene-editing toolbox expanded with the addition of prime editing. Prime editing has astounding potential for both basic biology research and for treating genetic diseases by theoretically correcting ~89% of known disease-causing mutations.

What is prime editing?

Prime editing is coined as a “search-and-replace” editing technique that builds on the “search-and-cut” CRISPR technology. Like CRISPR, prime editing utilizes the Cas9 enzyme targeted to a specific location in the genome by a guide RNA (gRNA). With a few ingenious modifications, including an enzyme called a reverse transcriptase (RT) fused to Cas9, prime editors can be targeted to nearly anywhere in the genome where the RT writes in new DNA letters provided by a template on the gRNA.

graphic drawing of red handled scissors
New gene-editing techniques offer more opportunities for therapy development. Each new discovery makes the techniques more and more accurate. Image courtesy of yourgenome.

 How is prime editing different from CRISPR?

Scientists are excited about prime editing because it has several advantages and overcomes many of the limitations of previous CRISPR systems. CRISPR Cas9, an endonuclease, cuts—like scissors—both DNA strands to inactivate a gene or to insert a new sequence of donor DNA. Unlike CRISPR edits, the prime editing Cas9, a nickase, cuts a single DNA strand and does not rely on the cell’s error-prone repair machinery, thereby minimizing any resulting deleterious scars left on the DNA. It has a broader range of targets because it is not limited by the location of short DNA sequences required for Cas9 binding to DNA. The versatility and flexibility of the system allows for more control to inactivate genes as well as to insert, remove, and change DNA letters, and, combine different edits simultaneously—analogous to a typewriter. Importantly, the edits are precise with relatively infrequent unwanted edits. Initial indications showed fewer off-target edits in the genome, possibly because more steps are required for a successful edit to occur. In some cases, it may be more efficient than CRISPR, depending on the targeted cell type, such as in a non-dividing cell like a neuron in the brain. However, with all these advantages, CRISPR still remains the tool of choice for making large DNA deletions and insertions because the prime editing system is limited by the RT and template RNA length.

How could prime editing help ataxia patients?

Prime editing offers enormous possibility for correcting heritable ataxia mutations accurately and safely. In dominantly inherited SCAs, like SCA1 or SCA2, prime editing could shorten the pathogenic repeat expansion allele to the normal length, or inactivate the pathogenic allele without creating unwanted, deleterious mutations. It also provides researchers with a powerful tool to study disease-causing genes in cells and animal models in new ways to advance our knowledge about the underlying mechanisms in ataxia.

What barriers are there to using prime editing as a treatment?

Prime editing will require rigorous testing in cells and animals before moving into humans in a clinical trial. Optimizing delivery and efficiency in target cells and tissues, and minimizing side-effects will be the key barriers to overcome.

To read the original Nature article describing prime editing, it can be found from the Liu lab here.

If you would like to learn more about Prime Editing, take a look at these news stories by The Broad Institute and Singularity Hub.

Snapshot written by Bryan Simpson and edited by Dr. Hayley McLoughlin.

How an ataxia gene increases the risk for Alzheimer’s disease

Written by Dr. Judit M. Perez Ortiz Edited by Dr. Marija Cvetanovic

In a tour de force study, a collaborative team of scientists led by Dr. Rudolph Tanzi (Harvard Medical School) and Dr. Huda Zhogbi (Baylor College of Medicine) found a novel relationship between the Spinocerebellar ataxia type 1 gene (ATXN1) and Alzheimer’s disease.

Alzheimer’s disease is the most common neurodegenerative disease and the most common cause of dementia. Its precise etiology remains the subject of intense investigation and debate. Alzheimer’s is a devastating disease. Persons with Alzheimer’s disease experience difficulties thinking and remembering things. As the disease worsens other symptoms begin to appear, such as getting lost easily, not recognizing loved ones, problems with language, and behavioral and psychiatric issues. In the more advanced stages, patients are completely dependent on their caregivers.

artist's sketch of a human brain, designed to look like electrical circuits
Drawing of a human brain, courtesy of Wikimedia.

Despite extensive research, a specific unifying cause of Alzheimer’s disease has not yet been identified, likely due to its complexity. There are a handful of genes responsible for a rare form of early onset Alzheimer’s disease that affects younger patients. However, the great majority of cases start late in life and have no known underlying cause (termed sporadic). While the major risk to develop Alzheimer’s disease is advanced age, scientists believe that clues to sporadic Alzheimer’s can be found in our genes. In this pursuit, hundreds of “risk genes” have been associated with Alzheimer’s disease and Ataxin-1 (ATXN1) has recently emerged as one of such risk genes. Yet how ATXN1 influence Alzheimer’s disease was not understood.

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Snapshot: How does CAG tract length affect ataxia symptom onset?

The instructions our bodies need to grow and function are contained in our genes. These instructions are made up of tiny structures called nucleobases. There are four types of nucleobases in DNA: adenine (A), cytosine (C), guanine (G), thymine (T). By putting these four nucleobases in different orders and patterns, this writes the instructions for our body.

artists drawing of a blue DNA molecule
A cartoon strand of DNA. Image by PublicDomainPictures from Pixabay

Some of the genes contain long sections of repeating ‘CAG” instructions, called CAG tracts. Everyone has repeating CAG tracts in these genes, but once they are over a certain length they can lead to disease. Some ataxias are caused by this type of mutation, including SCA1, SCA2, SCA3, SCA6, SCA7 and SCA17. These are often called polyglutamine expansion disorders. This is because “CAG” gives the body instructions to make the amino acid glutamine. You can read more about what is polyglutamine expansion in our past Snapshot about that subject.

For each disorder caused by a CAG expansion mutation, the number of times the CAG is repeated in a particular gene will determine whether someone will develop the disease. Repeat lengths under this number will not cause symptoms and repeat lengths over the threshold will usually lead to ataxia. When someone undergoes genetic testing for ataxia, doctors will be able to tell them the length of these CAG tracts and whether they have a CAG repeat number in one of these genes that is over the threshold. This table gives a summary of different CAG expansion mutations that can lead to ataxia and how the length of the repeat affects age of onset.

Affected Gene Normal
Repeat Size
Disease
Repeat Size
SCA1ATXN16-4439-88
SCA2ATXN215-3136-77
SCA3ATXN312-4055-86
SCA6CACNA1A 4-1821-33
SCA7ATXN74-3537-306
SCA12PPP2R2B4-3266-78
SCA17 TBP25-4246-63

For SCA1, SCA2, SCA3, SCA6, and SCA7; longer CAG tracts are associated with earlier onset.

For SCA12, it is hard to predict the age of onset based on repeat length as SCA12 is so rare. Some individuals with long repeats don’t develop ataxia. One study found that longer CAG tract lengths are associated with earlier onset but that it does not affect the severity of symptoms.

For SCA17, Longer CAG tracts have separately been associated with an earlier age of onset and more severe cerebellar atrophy.

In general, people with longer repeat lengths in ataxia genes are likely to present with ataxia symptoms earlier in life. However, it is important to remember that there are many other factors involved. Other genes may have mutations that either worsen the progression of ataxia or protect against more severe symptoms. Therefore, in individual people, the length of the repeat is not always enough information to determine when that person will start showing symptoms, or how severe these symptoms will be.

If you would like more information about the genetic causes of SCAs, including information about genetic testing and what CAG repeat length might mean, take a look at these resources by the National Ataxia Foundation.

Snapshot written by Anna Cook and edited by Larissa Nitschke.

Continue reading “Snapshot: How does CAG tract length affect ataxia symptom onset?”

Two or more birds with one stone: Designing a single therapeutic strategy to treat multiple types of spinocerebellar ataxia

Written by Dr. David Bushart Edited by Dr. Hayley McLoughlin

A newly-proposed treatment strategy might be effective against several forms of spinocerebellar ataxia and other CAG repeat-associated disorders

Upon receiving an initial diagnosis of spinocerebellar ataxia (SCA), a swarm of questions might enter a patient’s mind. Many of these questions will likely revolve around how to manage and treat their disease. What treatments are currently available to treat SCA? What can I do to reduce symptoms? Does SCA have a cure, and if not, are researchers close to finding one? Patients and family members who read SCASource may be able to answer some of these questions. Although scientists are aware of some of the underlying genetic causes of SCA, and patients can benefit greatly from exercise and physical therapy, there are unfortunately no current drug therapies that can effectively treat these diseases. However, this is a very exciting time in SCA research, since researchers are hard at work developing new treatment strategies for several of the most common SCAs. Many of these newly proposed therapies are specialized to treat a specific genetic subtype of SCA (e.g. SCA1, SCA3, etc.), which would allow these therapies to be very specific. However, these specialized efforts beg another question: would it be possible to treat different types of SCA with the same therapeutic strategy?

sketch of a human brain and spinal cord across a blue background
Artist’s sketch of a human brain. Image courtesy of Pixabay.

This is precisely what researchers wished to determine in a recent study, authored by Eleni Kourkouta and colleagues. This group of researchers used a technology called antisense oligonucleotides (often abbreviated ASO, or AON), to ask whether a single ASO could be used to treat multiple neurological disorders that have different underlying causes. Currently, most ASO technology depends on our ability to selectively target specific disease-causing genes, which allows the ASO to only recognize and act on the specific gene that is causing ataxia. Once recognized, these ASOs can recruit cellular machinery that lowers RNA levels of the disease-causing gene, thereby greatly limiting the amount of disease-causing protein that is produced (learn more in our What is RNA? Snapshot). This strategy has the potential to be very effective for treating SCAs that are associated with polyglutamine (polyQ) expansion (learn more in our What is Gene Therapy? Snapshot).

However, the type of ASO technology described above is not the only way to reduce levels of the disease-causing proteins in SCA. In this paper, Kourkouta and colleagues use a different type of ASO with a different mechanism of action, which also lowers levels of the disease-causing protein in two different SCAs.

Continue reading “Two or more birds with one stone: Designing a single therapeutic strategy to treat multiple types of spinocerebellar ataxia”