Researchers have found a significant drawback of the CRISPR-Cas gene editing technique. A specific molecule, intended to enhance the editing process, inadvertently damages segments of the genetic code.
The development of genome editing with various CRISPR-Cas complexes has advanced swiftly over recent years. Hundreds of laboratories globally are now striving to implement these innovations in medical applications while consistently improving the technology.
CRISPR-Cas tools enable scientists to modify specific parts of the genetic code with precision and accuracy. Current gene therapies that utilize this type of editing are already being employed to treat genetic disorders, combat cancer, and create crops that can tolerate drought and heat.
Initiating repair
The CRISPR-Cas9 complex, often referred to as “genetic scissors,” is the most commonly used tool among scientists worldwide. It cuts the double-stranded DNA at the precise location where modifications are needed, which is different from newer gene editing methods that do not cut the DNA strands.
This cut triggers two natural repair processes within the cell: a quick but less accurate method that merely reconnects the ends of the cut DNA, and a slower, more precise mechanism that is not always activated. The latter option requires a template for accurate DNA repair at the cut location.
The slower method is termed homology-directed repair (HDR). Researchers prefer this process as it facilitates the exact integration of specific DNA segments into targeted gene regions. This method is versatile and could potentially be utilized to address various genetic disorders. “In principle, it could be used to cure any disease,” states Jacob Corn, a Professor of Genome Biology at ETH Zürich.
Enhancing efficiency with a specific molecule
To encourage the use of homology-directed repair, researchers recently started employing a molecule known as AZD7648. This molecule inhibits the faster repair process, compelling the cell to utilize HDR instead. The goal was to accelerate the creation of more effective gene therapies, and initial results seemed promising—perhaps too promising, as it turned out.
A research team under Jacob Corn’s leadership has uncovered troubling side effects associated with the use of AZD7648. Their findings were published in the journal Nature Biotechnology.
Significant genetic alterations
While AZD7648 appears to enhance precise repair, it has also resulted in extensive genetic changes in a portion of the genome that was anticipated to be modified cleanly. The researchers discovered that these alterations included the deletion of thousands of genetic components, known as bases, and even the detachment of entire chromosome sections. This raises concerns about genetic stability and the unpredictable outcomes for the edited cells.
“When we examined the genome at the edited sites, it appeared correct and precise. However, when looking more broadly, we identified substantial genetic changes. These alterations do not show up when only focusing on the edited area and its immediate surroundings,” explains Grégoire Cullot, a postdoctoral fellow in Corn’s group and the primary author of the study.
Extent of the damage is considerable
The researchers were taken aback by the scale of negative effects observed. They suspect that they may not yet have a full understanding of the damage because their analysis didn’t cover the entire genome, focusing instead on specific regions.
This situation calls for new testing methods, approaches, and regulations to better understand the extent and implications of the damage.
AZD7648 is not a new discovery, as it is currently being evaluated in clinical trials for potential cancer treatments.
How did the ETH researchers become aware of this concern? Their previous studies indicated how effective and precise CRISPR-Cas9 editing could be with AZD7648, raising suspicions that prompted further investigation, according to Jacob Corn.
The ETH team analyzed the sequencing of the DNA not only at the edited sites but in the broader genomic context, leading to the discovery of these unintended catastrophic side effects caused by AZD7648.
This study marks the first acknowledgment of such side effects. Other research teams are also looking into these findings and plan to publish their results. “We are the first to state that not everything is perfect,” Corn notes. “This is a significant setback for us because, like many scientists, we hoped to use this new technique to enhance gene therapy development.”
A new beginning
Despite these challenges, Corn believes this is just the start of further advancements in CRISPR-Cas gene editing. “The pathway to any new technology is rarely smooth. A stumble does not mean we abandon the technology,” he asserts.
In the future, it may be possible to mitigate risks by using a combination of different molecules to promote HDR instead of relying solely on one. “There are numerous potential candidates. We now need to determine the right combinations that such a cocktail should contain to avoid harming the genome,” he elaborates.
CRISPR-Cas-based gene therapies are already being successfully applied in clinical settings. For instance, in recent years, around a hundred patients with the hereditary condition sickle cell anemia have been treated with CRISPR-Cas therapies—without AZD7648. “All patients are considered cured and have shown no side effects,” Corn adds. “Thus, I remain optimistic about gene therapies becoming mainstream. The challenge is to identify the right methodologies and ensure the safety of the technique for as many patients as possible.”