Genome or gene editing technologies give scientists the ability to change an organism’s DNA. These technologies allow genetic material to be added, removed, or altered at particular locations in the genome. CRISPR-Cas9, short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 has generated a lot of excitement in the scientific community because it is faster, cheaper, more accurate, and more efficient than other genome editing technologies.

However, while the CRISPR-Cas9 is very effective, it is not always safe. Research has shown that in some instances cleaved chromosomes do not recover and genomic stability is compromised – which in the long run might promote cancer. [1]

This is the conclusion of a new study by researchers at Tel Aviv University (TAU) in Tel Aviv, Israel.

In the study, which was published in the June 30, 2022 edition of Nature Biotechnology, the researchers identify the risks in the use of CRISPR-Cas9, an innovative method that involves cleaving and editing DNA, and is already employed for the treatment of conditions like cancer, liver and intestinal diseases, and genetic syndromes.[1]

Using single-cell RNA sequencing to investigate the impact of this technology on T-cells (the white blood cells of the immune system), the researchers looked at genome editing outcomes in primary human T-cells transfected with CRISPR–Cas9 and guide RNAs targeting genes for TCR chains and programmed cell death protein 1 (PD-1, also known as CD279, a protein on the surface of T and B cells that has a role in regulating the immune system’s response to the cells of the human body by down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity).

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Based on the outcome of their study, the researchers warned that the loss of genetic material they observed can lead to destabilization of the genome, which, in turn, may cause cancer.

Chromosome segregation In dividing cells. Cell cytoskeleton is depicted in red, DNA is depicted in blue and a protein that marks dividing cells is depicted in green. Image courtesy: © 2022 Tom Winkler, Ben David lab. Tel Aviv University. Used with permission.

Groundbreaking technology
The study was led by Adi Barzel, Ph.D., and by Asaf Madi, Ph.D., and Uri Ben-David, Ph.D., at Tel Aviv University’s Faculty of Medicine.

The researchers explain that CRISPR-Cas9 is a groundbreaking technology for editing DNA – cleaving DNA sequences at certain locations in order to delete unwanted segments, or alternately repair or insert beneficial segments.  Developed about a decade ago, the technology has already proved impressively effective in treating a range of diseases – cancer, liver diseases, genetic syndromes, and more.

First clinical trial
The first approved clinical trial ever to use CRISPR-Cas9, was conducted in 2020 at the University of Pennsylvania, when researchers applied the method to T-cells – white blood cells of the immune system. Taking T-cells from a donor, they expressed an engineered receptor targeting cancer cells, while using CRISPR-Cas9 to destroy genes coding for the original receptor – which otherwise might have caused the T-cells to attack cells in the recipient’s body.

In the present study, the researchers sought to examine whether the potential benefits of CRISPR-Cas9 might be offset by risks resulting from the cleavage itself, assuming that broken DNA is not always able to recover.

“The genome in our cells often breaks due to natural causes, but usually it is able to repair itself, with no harm done. Still, sometimes a certain chromosome is unable to bounce back, and large sections, or even the entire chromosome, are lost. Such chromosomal disruptions can destabilize the genome, and we often see this in cancer cells. Thus, CRISPR-Cas9, in which DNA is cleaved intentionally as a means for treating cancer, might, in extreme scenarios, actually promote malignancies,” explained Uri Ben-David, Ph.D., from TAU’s Faculty of Medicine and Edmond J. Safra Center for Bioinformatics.

Extent of potential damage
To examine the extent of potential damage, the researchers repeated the 2020 Pennsylvania experiment, cleaving the T-cells’ genome in exactly the same locations – chromosomes 2, 7, and 14 (of the human genome’s 23 pairs of chromosomes). Using a state-of-the-art technology called single-cell RNA sequencing they analyzed each cell separately and measured the expression levels of each chromosome in every cell.

In this way, a significant loss of genetic material was detected in some of the cells. For example, when Chromosome 14 had been cleaved, about 5% of the cells showed little or no expression of this chromosome. When all chromosomes were cleaved simultaneously, the damage increased, with 9%, 10%, and 3% of the cells unable to repair the break in chromosomes 14, 7, and 2 respectively. The three chromosomes did differ, however, in the extent of the damage they sustained.

“Single-cell RNA sequencing and computational analyses enabled us to obtain very precise results. We found that the cause for the difference in damage was the exact place of the cleaving on each of the three chromosomes. Altogether, our findings indicate that over 9% of the T-cells genetically edited with the CRISPR-Cas9 technique had lost a significant amount of genetic material. Such loss can lead to destabilization of the genome, which might promote cancer,” said Asaf Madi, Ph.D.

Based on their findings, the researchers caution that extra care should be taken when using CRISPR therapeutics. They also propose alternative, less risky, methods, for specific medical procedures, and recommend further research into two kinds of potential solutions: reducing the production of damaged cells or identifying damaged cells and removing them before the material is administered to the patient.

“Our intention in this study was to shed light on potential risks in the use of CRISPR-Cas9. We did this even though we are aware of the technology’s substantial advantages. In fact, in other studies we have developed CRISPR-Cas9-based treatments, including a promising therapy for AIDS,” noted Adi Barzel Ph.D at the School of Neurobiology, Biochemistry and Biophysics at TAU’s Wise Faculty of Life Sciences and Dotan Center for Advanced Therapies, a collaboration between the Tel Aviv Sourasky Medical Center (Ichilov) and Tel Aviv University and also one of the lead author of the study.

“We have even established two companies – one using CRISPR-Cas9 and the other deliberately avoiding this technology. In other words, we advance this highly effective technology, while at the same time cautioning against its potential dangers. This may seem like a contradiction, but as scientists we are quite proud of our approach, because we believe that this is the very essence of science: we don’t ‘choose sides.’ We examine all aspects of an issue, both positive and negative, and look for answers,” Barzel concluded.

[1] Nahmad AD, Reuveni E, Goldschmidt E, Tenne T, Liberman M, Horovitz-Fried M, Khosravi R, Kobo H, Reinstein E, Madi A, Ben-David U, Barzel A. Frequent aneuploidy in primary human T cells after CRISPR-Cas9 cleavage. Nat Biotechnol. 2022 Jun 30. doi: 10.1038/s41587-022-01377-0. Epub ahead of print. PMID: 35773341.

Featured image: Uri Ben-David,Ph.D., Adi Barzel, Ph.D., and Asaf Madi, Ph.D. Photo courtesy: © 2022 Tel Aviv University / TAU. Used with permission

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