Massive sections of the targeted chromosome went missing. By Prof Michael Antoniou and Claire Robinson

CRISPR-based gene-editing tools are being developed to correct specific defective sections of the genome to cure inherited genetic diseases, with some applications already in clinical trials. However, there is a catch: under certain conditions, the repair can lead to large-scale deletions and rearrangements of DNA – as in the case of targeting the NCF1 gene in chronic granulomatous disease (CGD). This was reported by a team of researchers and physicians from the ImmuGene clinical research programme at the University of Zurich (UZH).

Their findings have important implications not just for gene editing-based therapy, but also for CRISPR-mediated gene editing of animals and plants, where the same types of large-scale genetic damage could be triggered. Indeed, because such editing is carried out with much less caution in non-human organisms, the likelihood of such large-scale damage occurring is hugely increased (see below on multiplexing).

The study also shows that attempts to avoid these problems by using adaptations of CRISPR gene editing technologies, such as prime and base editing, may not succeed.

This research on CGD is also only the latest in a series of studies that have repeatedly shown that different types of unintended mutations resulting from gene editing can affect the functioning of multiple gene systems, with potentially damaging consequences.

What is CGD?

CGD is a rare hereditary disease that affects about one in 120,000 people. The disease impairs the component of the immune system responsible for fighting off infections, which can be life-threatening to the patient. One variant of CGD is caused by the absence of two letters in the DNA base unit gene sequence which codes for the NCF1 protein. This error results in the inability of blood cells known as neutrophils to produce an enzyme complex that plays an essential role in the immune defence against bacterial, yeast, and fungal infections.

The new study’s findings

In the new study, the research team succeeded in using the CRISPR gene editing system to insert the missing DNA base unit letters in the right place in the NCF1 gene – thereby repairing the genetic defect. Initially they performed experiments in cultures of human cells containing the defective NCF1 gene. The authors then progressed to experiments using the natural cellular targets for CGD gene therapy – bone marrow stem and progenitor parent cells* from CGD patients harbouring the defect in the NCF1 gene.

However, some of the repaired cells now showed new genetic defects affecting large regions of DNA. Entire sections of the chromosome around where the gene editing repair had taken place were missing. These missing sections in some cases stretched over millions of DNA base units, resulting in the loss of many genes (17 in one instance). The reason for this is the special genetic constellation within which the NCF1 gene is located: It is present three times on the same chromosome, once as a normally functioning gene and twice in the form of defective pseudogenes (imperfect copies of the functional gene). These pseudogenes are incapable of producing normal NCF1 protein and so cannot contribute to the formation of the enzyme complex required by neutrophils to fight off infections.

The CRISPR gene-editing tool could not distinguish between the different versions of the NCF1 gene and therefore occasionally cut the DNA strand at multiple locations on the chromosome – at the normally functioning NCF1 gene as well as at the defective pseudogenes. When the sections were subsequently rejoined, in some cases, entire gene segments were misaligned or missing. The medical consequences are unpredictable and, in the worst case, can contribute to the development of leukaemia. “This calls for caution when using CRISPR technology in a clinical setting,” said lead author Janine Reichenbach.

Safer method sought

In an effort to minimise risks of inadvertently introducing large-scale DNA damage, the team tested a number of approaches using different versions of the CRISPR gene-editing tool.

First, they introduced into the cells a pre-assembled CRISPR/Cas editing complex known as an RNP (ribonucleoprotein) rather than genetic material (plasmids) encoding for this gene editing tool. Using pre-assembled CRISPR/Cas RNP complexes has become standard in the gene therapy field since once inside the targeted cells they are more short-lived than plasmid DNA, so there is less time for them to cause unintended DNA damage. The researchers found that the CRISPR/Cas RNP could successfully correct the NCF1 genetic defect in 5% or 50% of the targeted cells, depending on the cell type. However, this did not prevent the formation of large-scale DNA damage in a high proportion of these targeted cells – 25% or 35%.

Second, the researchers also tested variants of the CRISPR/Cas gene-editing tool that introduce only single DNA strand breaks rather than the more usual double-strand DNA break. This was to avoid formation of the double-strand breaks that are generally seen as the culprit for causing large-scale DNA damage. They also looked at using protective elements that reduce the likelihood of the gene-editing tool cutting the chromosome at multiple sites simultaneously. Unfortunately, none of these measures were able to completely prevent the unwanted effects, including, perhaps surprisingly, when no double-strand DNA break was caused by the CRISPR tool.

This acts as a warning to those developing CRISPR-based gene editing approaches involving processes known as prime editing and base editing, which rely on single-strand DNA breaks to undertake their editing activity. It has been commonly assumed that using such methods will minimise the risk of creating unintended large deletions and rearrangements in the DNA. But the new research shows that this may not be the case and that careful molecular genetic characterisation, such as long-read DNA sequencing, is needed to ensure that this has not taken place.

“This study highlights both the promising and challenging aspects of CRISPR-based therapies,” said co-author Martin Jinek, a professor at the UZH Department of Biochemistry. He said the study provides valuable insights for the development of gene-editing therapies for CGD and other inherited disorders. “However, further technological advances are needed to make the method safer and more effective in the future.”

Implications for gene editing of animals and plants

While this study relates to human gene therapy, it has important implications for the gene editing of animals and plants. This is because the types of large-scale damage to the genome described in this study can also arise during the gene editing of these organisms. Consequently, the functioning of many genes can be disrupted or lost. This in turn can lead to major changes in biochemical and cellular function of the organism. In animals, the unwanted effects seen in this study could impair health, including causing diseases such as cancer and developmental defects. In the case of gene-edited food plants, the result could be unexpected toxicity or allergenicity, or altered nutritional content.

Many plant genetic engineers claim that they can screen out plants with unwanted genetic mutations. However, unless a mutation causes the plant to look obviously sick or fail to grow and propagate, it could be missed, even though it may have given rise to altered biochemical composition that can harm the consumer.

Risks from multiplexing

The likelihood of the large-scale DNA damage observed in this study occurring in the gene editing of food plants is hugely increased by the aspiration of plant genetic engineers to target multiple genes simultaneously (“multiplexing”). Multiplexing involves introducing into cells multiple CRISPR/Cas tools at the same time, with the aim of targeting several different locations in the DNA of the organism. This results in the simultaneous creation of several cuts in the cells’ DNA and consequently there is a very high risk of large deletions and rearrangements taking place on repair of the DNA, as is graphically demonstrated in this study.

Careful scrutiny required

In conclusion, this comprehensive study has demonstrated how large-scale deletions and rearrangements of the genome can take place following the activity of even a single CRISPR/Cas gene-editing tool. This was found to take place when the activity of the gene-editing tool produced either a single- or double-strand DNA break at both intended and unintended editing sites. This process also mimics what takes place during CRISPR/Cas-mediated multiplex gene editing applications. The authors warn that gene editing target sites need to be carefully scrutinised to avoid such outcomes from this mechanism.

People in the agricultural gene editing field must ensure that they also do this. But so far we’ve seen no evidence that they are doing so. This omission can result in missing crucial molecular genetic characterisation and the downstream consequences of any genetic damage in a final gene-edited product that is intended for market. Those consequences can include the disruption of multiple gene functions, leading to biochemical changes resulting in unexpected toxicity or allergenicity, or compromised nutritional content. Regulations need to be in place to require that all these possibilities are examined in an in-depth risk assessment.

The new study:
Federica Raimondi et al. Gene editing of NCF1 loci is associated with homologous recombination and chromosomal rearrangements. Communications Biology. 9 October 2024. DOI: https://doi.org/10.1038/s42003-024-06959-z

* Bone marrow stem cells are the parental cells from which all types of blood cells are produced. This includes red blood cells and immune system cells (for example, T cells, B cells, and neutrophils).

For more information on the unexpected outcomes and risks of gene editing see GENE EDITING MYTHS, RISKS, & RESOURCES

Source for quotes: University of Zurich