CRISPR-Cas9 genome editing can lead to loss of genetic material and destabilize the genome

Genome or gene editing technologies allow scientists to alter an organism’s DNA. These technologies make it possible to add, delete or modify genetic material at specific locations in the genome. CRISPR-Cas9, short for short palindromic repeats grouped regularly spaced 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, although CRISPR-Cas9 is very effective, it is not always safe. Research has shown that in some cases, cleaved chromosomes do not recover and genomic stability is compromised, which in the long term could promote cancer. [1]

That’s 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 Natural biotechnologyresearchers identify risks associated with using CRISPR-Cas9, an innovative method that involves DNA cleavage and editing, and which is already used for the treatment of diseases such as cancer, liver disease and diseases and genetic syndromes.[1]

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

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

Chromosomal segregation in dividing cells. The cell cytoskeleton is shown in red, DNA is shown in blue, and a protein that marks dividing cells is shown in green. Image Courtesy: © 2022 Tom Winkler, Ben David lab. Tel Aviv University. Used with permission.

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

The researchers explain that CRISPR-Cas9 is a breakthrough DNA editing technology – cleaving DNA sequences at certain locations in order to remove unwanted segments, or alternatively repair or insert beneficial segments. Developed a decade ago, the technology has already proven to be impressively effective in treating a whole range of diseases – cancer, liver disease, genetic syndromes, etc.

First clinical trial
The first approved clinical trial 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. By taking T cells from a donor, they expressed an altered receptor that targets cancer cells, while using CRISPR-Cas9 to destroy the genes encoding the original receptor – which otherwise could have caused the T cells to attack cells in the recipient’s body.

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

“The genome of our cells often breaks for natural reasons, but it is usually able to repair itself, without any damage. Yet, sometimes a certain chromosome is unable to rebound 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, so CRISPR-Cas9, in which DNA is intentionally cleaved as a means of treating cancer, could, in extreme scenarios, promote malignant tumors,” explained Uri Ben-David, Ph.D., of TAU School of Medicine and the 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 cell genome in exactly the same places – chromosomes 2, 7 and 14 (of the 23 pairs of chromosomes in the human genome). Using a cutting-edge technology called single-cell RNA sequencing, they analyzed each cell separately and measured the expression levels of each chromosome in each cell.

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

“Single-cell RNA sequencing and computational analyzes allowed us to obtain very precise results. We found that the cause of the difference in damage was the exact location of the cleavage on each of the three chromosomes. In total, our results indicate that more than 9% of the T cells genetically modified with the CRISPR-Cas9 technique had lost a significant amount of genetic material. Such loss can lead to destabilization of the genome, which could 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 offer alternative, less risky methods for specific medical procedures, and recommend further research into two types of potential solutions: reducing the production of damaged cells or identifying damaged cells and eliminating them before the material is administered. to the patient.

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

“We have even created two companies – one using CRISPR-Cas9 and the other deliberately avoiding this technology. In other words, we are advancing this highly effective technology, while warning of its potential dangers. seem like a contradiction, but as scientists we are quite proud of our approach, because we believe this is the essence of science: we don’t “choose sides”. We look at all aspects of a problem, both positive and negative, and seek answers,” concluded Barzel.

Reference
[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. Common aneuploidy in primary human T cells after CRISPR – Cas9 cleavage. Nat Biotechnol. June 30, 2022. 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 credit: © 2022 Tel Aviv University/TAU. Used with permission

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