Study Identifies Dangerous Spots for CRISPR Editing

Researchers in Spain have identified critical spots on the genome where gene editing could cause an unwanted response—including cell toxicity and genome instability—and provided recommendations for safer approaches.

CRISPR/Cas9 is a commonly used, very precise, gene-editing technique commonly known as “genetic scissors.” The technology allows the introduction of the desired DNA sequence into virtually any spot of the genome, thus modifying or inactivating a gene. It is widely used in biomedical research and some CRISPR-based therapies are in clinical trials for the treatment of human blood disorders, some types of cancer and HIV, among other conditions.

But scientists at the Institute for Research in Biomedicine (IRB) Barcelona, led by ICREA researcher Dr. Fran Supek, report in Nature Communications that CRISPR gene editing can give rise to unwanted effects mediated by the linchpin tumor suppressor protein p53.

Known as the guardian of the genome, p53 detects DNA damage and leads the cells to stop dividing and can cause programmed death, thus preventing them from perpetuating the mistakes in their DNA. Simply put, p53 underlies a natural protection mechanism against cancer and other DNA damage-related complications.

Using computational methods, researchers in the Genome Data Science lab analyzed the most popular CRISPR library designed for human cells and detected 3,300 targeted spots that show strong toxic effects. They also report that around 15% of the human genes contain at least one toxic editing point.

"Our work addresses an important issue with TP53-associated toxicity of Cas9, which was a matter of some controversy recently, and it also provides guidelines on how to sidestep the problem. Avoiding editing in these “risky” spots would not only make CRISPR editing more efficient but, more importantly, safer," Dr. Supek says.

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

However, the main complication regarding p53 and gene editing is that cells that overcome CRISPR editing might do so precisely because of defective p53 functioning. These cells may be less able to detect DNA damage and/or tag cells for programmed death. As a result, the gene editing procedure could end up favoring cell populations that have unstable genomes, meaning they are prone to accumulating further mutations, thus increasing the risk of developing malignancies.

"This unwanted consequence might incur a risk of genomic instability, which is highly undesirable in the context of ex vivo CRISPR therapies, in which cells from a patient are edited in the lab and reintroduced back into the patient. We hope that our study provides some guidelines on how to design safer CRISPR reagents, and encourages further research into this issue," Dr. Supek says.