The AZD7648 molecule can destroy parts of the genome

Genome editing with various CRISPR-Cas molecular complexes has progressed rapidly in recent years. Hundreds of laboratories around the world are now working to put these tools to clinical use and continually advance them.

CRISPR-Cas tools allow researchers to modify individual building blocks of genetic material in a precise and targeted way. Gene therapies based on such genetic modification are already used to treat hereditary diseases, fight cancer and create crops tolerant to drought and heat.

The CRISPR-Cas9 molecular complex, also known as the genetic scissors, is the most widely used tool by scientists around the world. It cuts double-stranded DNA at the exact location where the genetic material needs to be changed. This contrasts with newer gene editing methods, which do not cut the double strand.

Cutting activates two natural repair mechanisms that the cell uses to repair such damage: a fast but imprecise mechanism that reconnects only the ends of the cut DNA, and a slow and precise mechanism that is not activated in all cases . The latter requires a copyable template for repair to accurately rejoin the DNA at the cut site.

The slow variant is called homology-directed repair. Researchers are interested in using this repair method because it allows for the precise integration of individual DNA segments into a desired genetic region. The approach is very flexible and can be used to repair different disease genes.

“In principle, it could be used to cure any disease,” says Jacob Corn, professor of genomic biology at ETH Zürich.

Increase efficiency with a single molecule

To get the cell to use homology-directed repair, researchers recently began using a molecule called AZD7648, which blocks rapid repair and forces the cell to use homology-directed repair. This approach should accelerate the development of more effective gene therapies. The first studies on these new therapies have been good. Too good to be true, it turned out.

A research group led by Corn found that the use of AZD7648 causes serious side effects. The study was published in the journal Natural biotechnology.

Massive genetic changes

Although AZD7648 promotes precise repair and therefore precise gene editing using the CRISPR-Cas9 system as hoped, in a significant proportion of cells this led to massive genetic changes in a part of the genome that was expected be modified without leaving scars.

The ETH researchers found that these changes resulted in the simple removal of thousands and thousands of DNA building blocks, called bases. Even entire chromosome arms were broken. This makes the genome unstable, with unpredictable consequences for cells modified by this technique.

“When we analyzed the genome at the sites where it had been edited, it looked correct and accurate. But when we analyzed the genome more broadly, we saw massive genetic changes. These are not visible when you only analyze the short edited section and its immediate vicinity,” explains Grégoire Cullot, a postdoctoral researcher in Corn’s group and first author of the study.

The extent of the damage is significant

The magnitude of the negative effects surprised the researchers. In fact, they suspect that they do not yet have a complete picture of the extent of the damage, because they did not examine the entire genome when analyzing the modified cells, but only partial regions.

New tests, approaches and regulations are therefore needed to clarify the extent and potential of harm.

But how did ETH researchers become aware of the problem? In other studies, researchers showed how highly efficient and precise CRISPR-Cas9 gene editing is when AZD7648 is added. “That made us suspicious, so we looked closer,” Corn says.

The ETH researchers then analyzed the sequence of DNA building blocks not only around the edited site, but also in a wider environment. They discovered these unwanted and catastrophic side effects caused by the use of AZD7648.

Their study is the first to describe these side effects. Other research groups have also studied them and support the conclusions of the ETH researchers. They also aim to publish their results. “We’re the first to say it’s not all wonderful,” Corn says.

“For us, this is a major setback because, like other scientists, we had hoped to use this new technique to accelerate the development of gene therapies.”

The start of something new

Nonetheless, Corn says this is not the end but the beginning of new advances in gene editing using CRISPR-Cas techniques. “The development of any new technology is a rocky road. One misstep does not mean we abandon the technology.”

It might be possible to avoid the danger by using not a single molecule to promote HDR in the future, but a cocktail of different substances. “There are many possible candidates. It now remains to determine which components such a cocktail should consist of so as not to damage the genome.”

Gene therapies based on the CRISPR-Cas system have already been successfully used in clinical practice. In recent years, for example, around 100 patients suffering from sickle cell disease, an inherited disease, have been treated with CRISPR-Cas-based treatments, without AZD7648.

“All patients are considered cured and have no side effects,” says Corn. “So I’m optimistic that gene therapies like this will become mainstream. The question is which approach is right and what we need to do to make this technique safe for as many patients as possible.”