CRISPR-Cas3: a potential therapeutic approach for genetic disorders?
Could CRISPR-Cas3 present itself as a potential therapeutic approach for genetic disorders like transthyretin amyloidosis (ATTR)?
Researchers from the Institute of Medical Science, The University of Tokyo (IMSUT; Tokyo, Japan) have explored the use of CRISPR-Cas3 as a therapeutic strategy for treating genetic disorders such as ATTR. The findings of the study highlight the efficiency of CRISPR-Cas3, addressing some of the limitations associated with the widely known CRISPR-Cas9 system.
The development of various gene-editing techniques has enabled precise modifications to DNA, allowing for the correction of mutations or the removal of harmful genetic sequences.
“Genome editing holds the unique potential to correct the inherited disease-associated genetic abnormalities,” commented Tomoji Mashimo who co-led the study.
Among these gene-editing tools, CRISPR-Cas9 has gained significant traction for its potential clinical applications; and despite its promise, this approach has certain limitations, such as the risk of off-target DNA editing – unintended cuts in the genome, which can lead to unwanted mutations.
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To explore alternatives, the research team turned to CRISPR-Cas3. “We wanted to see if the CRISPR–Cas3 system could be developed as an efficient therapeutic genome-editing tool,” explained Mashimo.
CRISPR-Cas3 and CRISPR-Cas9 are two distinct CRISPR systems with fundamentally different structures and mechanisms of action. CRISPR-Cas9, a Class II system, uses single guide RNA (gRNA) to direct the Cas9 protein to a specific DNA sequence, where it creates precise double-strand breaks. This precision makes CRISPR-Cas9 ideal for targeted gene editing, but it carries the risk of generating in-frame mutations that may produce abnormal protein products.
On the other hand, CRISPR-Cas3, a Class I, Type I-E system, relies on a multisubunit Cascade complex to identify and bind to target DNA. Once bound, the Cascade complex recruits the Cas3 enzyme, which has helicase and nuclease activity. Cas3 unwinds and degrades DNA in a processive, unidirectional manner, shredding large regions of DNA. This long-range degradation reduces the likelihood of generating short in-frame mutations that could preserve residual gene function, potentially making CRISPR-Cas3 a safer alternative for therapeutic applications.
By exploring CRISPR-Cas3, the researchers aimed to evaluate whether it could offer a more effective or safer alternative for addressing genetic disorders, primarily ATTR.
ATTR is a systemic protein misfolding disorder caused by the deposition of amyloid fibrils derived from the TTR protein, which is primarily synthesized in the liver. It exists in two major forms: hereditary and age-associated. The age-associated form is more prevalent, affecting an estimated 500,000 individuals worldwide.
Current treatments for ATTR include TTR stabilizers, which can slow disease progression but do not address the root genetic cause. A promising therapeutic approach is NTLA-2001, a lipid nanoparticle-delivered mRNA-Cas9 therapy designed to target the TTR gene in vivo. NTLA-2001 has demonstrated sustained reductions in serum TTR levels in individuals with hereditary ATTR and is currently in Phase III clinical trials.
As mentioned earlier, CRISPR-Cas9 creates double-strand breaks. These double-strand breaks are repaired by a process called nonhomologous end joining, which can often introduce small insertions or deletions (indels) at the break site. While these indels can disrupt the gene and prevent protein production, there is a risk that the reading frame may be preserved, leading to in-frame mutations. These in-frame mutations could potentially result in the production of abnormal protein products, which may have unintended outcomes.
The researchers evaluated CRISPR-Cas3 in various models, including mouse Hepa1-6 cells, in vivo mouse liver, and human HepG2 cells. Their results showed that CRISPR-Cas3 induced reliable, extensive deletions of the TTR gene without generating indels at off-target sites – a major limitation of CRISPR-Cas9.
“Through CRISPR RNA optimization, we achieved around 59% editing at the TTR locus in our in vitro experiments. In mice models, a single LNP-based treatment helped us to achieve more than 48% hepatic editing and reduced serum TTR levels by 80%,” highlighted Mashimo.
The findings of this study highlight the therapeutic potential of CRISPR-Cas3, which may offer a safer alternative to CRISPR-Cas9 by minimizing the risk of generating unintended, harmful mutant proteins. With further optimization and safety evaluation, CRISPR-Cas3 could become a new platform for genome-editing therapies, providing patients with durable, potentially one-time treatments that directly address the root genetic causes of their conditions.
“In the coming years, this technology can lead to clinical applications not only for ATTR, but also for other currently incurable inherited diseases,” explained Mashimo.
