Risk of cell toxicity and genomic instability with gene editing

All new technology has its risks, which must be eliminated or mitigated enough to be able to take it to its full maturity stage. Now, unexpected risks have been discovered from using the gene editing technique called CRISPR/Cas9.

CRISPR/Cas9 is highly precise and commonly used in biomedical research. Its development, led by Jennifer A. Doudna and Emmanuelle Charpentier, was recognized with the 2020 Nobel Prize in Chemistry. Commonly known as “genetic scissors”, the CRISPR technique allows the desired DNA sequence to be introduced at (virtually) any point in the genome, thereby modifying or inactivating a gene. Some therapies based on this technique are now in different phases of clinical trials for the treatment of various pathologies such as blood disorders, some types of cancer or HIV (AIDS virus), among other conditions.

Scientists at the Barcelona Institute for Research in Biomedicine (IRB Barcelona), led by ICREA researcher Dr. Fran Supek, have now revealed that, depending on where in the genome it is targeted, gene editing with CRISPR can lead to cellular toxicity and genomic instability. This unwanted effect is coordinated by the p53 protein (also known as tumor suppressor protein) and depends on the DNA sequence near the editing point and on regulatory factors in the surrounding region.

Using computational methods, researchers from the GDS (Genome Data Science) laboratory have analyzed the most popular CRISPR library designed for human cells and have detected 3,300 possible editing points in the genome that show strong toxic effects. The work also reveals that around 15% of human genes contain at least one toxic editing point.

Gene editing using CRISPR/Cas9 “gene scissors” can lead to cell-toxic effects and genomic instability. (Artistic illustration: IRB Barcelona. CC BY-NC-ND)

“Our work addresses an important and controversial question regarding TP53-associated Cas9 toxicity and also provides guidelines on how to get around the problem. Avoiding editing at these ‘risk’ points would not only make CRISPR editing more efficient but above all, safer,” explains Dr. Supek.

A specific gene can be edited at various positions. “The regions of the gene that are important for regulation or that have certain epigenetic markers are the ones that are most likely to trigger the p53 response and, therefore, should be avoided as a general recommendation,” says Dr. Miguel-Martín Álvarez, principal investigator of the study.

p53-mediated toxicity and tumorigenesis

p53 is a protein known as the guardian of the genome. It detects DNA damage, stops cells from dividing, and can direct them toward programmed cell death, thereby preventing errors in your DNA from reproducing and spreading. Therefore, p53 constitutes a natural protection mechanism against cancer and other complications related to DNA damage.

CRISPR gene editing typically requires cutting both strands of DNA. In some cases, this manipulation can trigger a p53 response, in which the edited cells can be ‘tagged’ as damaged and are then removed, thus reducing the efficiency of the gene editing process.

However, the main complication regarding p53 and gene editing is that cells that survive CRISPR editing might do so precisely because they have defects in p53. That is, these cells may not be able to detect DNA damage and/or mark cells for programmed death. As a consequence, gene editing could be favoring cell populations that have unstable genomes, meaning they are prone to accumulating more mutations, thus increasing the risk of developing malignant tumors.

“This unintended consequence could pose a risk of genomic instability, which is highly undesirable in the context of ex vivo CRISPR therapies, where a patient’s cells are edited in the lab and reintroduced back into their body. We hope that our study will provide some guidelines on how to design safer CRISPR reagents, and that it will promote further research on this question,” concludes Dr. Supek.

The study is titled “TP53-dependent toxicity of CRISPR/Cas9 cuts is differential across genomic loci and can confound genetic screening.” And it has been published in the academic journal Nature Communications. (Source: IRB Barcelona)