CRISPR-Cas technology: balancing efficiency and safety

Genome editing with various CRISPR-Cas molecule complexes has progressed rapidly in recent years. Hundreds of labs around the world are now working to put these tools to clinical use and are continuously advancing them.

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

Starting the repair

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

The cut activates two natural repair mechanisms that the cell uses to repair such damage: a fast but imprecise one that reconnects only the ends of the cut DNA, and a slow and precise one that is not activated in every case. 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 want to use this method of repair because it allows the precise integration of individual DNA segments into a desired gene 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 Genome Biology at ETH Zürich.

Boosting efficiency with one molecule

To get the cell to use homology-directed repair, the researchers recently began using a molecule called AZD7648, which blocks fast repair and forces the cell to use homology-directed repair. This approach is expected to accelerate the development of more efficient gene therapies. Initial studies with these new therapies have been good. Too good to be true, as it turned out.

A research group led by Jacob Corn has just discovered that the use of AZD7648 has serious side effects. The study has just been published in the journal Nature Biotechnology.

Massive genetic changes

Although AZD7648 promotes precise repair and thus precise gene editing using the CRISPR-Cas9 system as hoped, in a significant proportion of cells this has led to massive genetic changes in a part of the genome that was expected to be modified without scarring. The ETH researchers found that these changes resulted in the simple deletion of thousands and thousands of DNA building blocks, known as bases. Even whole chromosome arms broke off. This makes the genome unstable, with unpredictable consequences for the cells edited by the technique.

“When we analysed the genome at the sites where it had been edited, it looked correct and precise. But when we analysed the genome more broadly, we saw massive genetic changes. These are not seen when you only analyse the short, edited section and its immediate neighbourhood,” says Grégoire Cullot, a postdoctoral fellow in Corn’s group and first author of the study.

Extent of damage is large

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

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

The molecule AZD7648 is not unknown. It is currently in clinical trials as a potential cancer treatment. 

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

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

Their study is the first to describe these side effects. Other research groups have also investigated them and support the ETH researchers’ findings. They also aim to publish their results. “We are the first to say that not everything is wonderful,” says Corn. “For us, this is a major setback because, like other scientists, we had hoped to use the new technique to accelerate the development of gene therapies.”

The beginning of something new

Nevertheless, Corn says this is not the end but the beginning of further advances in gene editing using CRISPR-Cas techniques. “The development of any new technology is a rocky road. One stumble does not mean we give up on the technology.”

It may be possible to avert the danger by using not just one molecule to promote HDR in the future but a cocktail of different substances. “There are many possible candidates. We now need to find out which components such a cocktail would have to consist of in order 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, a hundred patients suffering from the hereditary disease sickle cell anaemia have been treated with CRISPR-Cas-based therapeutics – without AZD7648. “All patients are considered cured and have no side effects,” says Corn. “So, I am optimistic that gene therapies like this will become mainstream. The question is which approach is the right one and what we need to do to make the technique safe for as many patients as possible.”