11 July 2022

Potential danger of CRISPR gene editing raised

 

Researchers have revealed a previously undiscovered risk of CRISPR/Cas9 gene editing which leads to large rearrangements of DNA.

A report from scientists at Boston Children’s Hospital, published in Nature Communications, warns of the potential for genetic changes called ‘retrotransposition’ in CRISPR editing.

In retrotransposition, DNA sequences known as “mobile elements” move from one location in the genome to another. Using enzymes, they replicate themselves and create a break in both strands of the DNA, where they insert themselves. While mostly harmless, mobile elements (also called “jumping genes”) have also been linked to disease, including cancer.

Dr Roberto Chiarle and Dr Jianli Tao, in the Department of Pathology, say although it is uncommon, these rearrangements occur when breaks in DNA are not repaired, which allows mismatched ends to join – such as during standard CRISPR/Cas9 gene editing.

In their study using cell lines in the lab, retrotranspositions occurred up to 5 to 6% of the time, which, say the researchers, mean it can theoretically trigger cancer.

They conclude that tests for retrotransposition should be added to safety testing for CRISPR/Cas9 editing systems. Current test technologies either sequence small stretches of DNA to ensure the desired gene has been added or deleted in the right place, or they are designed to detect small gene rearrangements. They do not look for large rearrangements caused by retrotransposition.

The researchers and colleagues also found retrotransposition is rarer during base editing, a newer and more precise technique that chemically changes just one ‘letter’ of the genetic code without causing a double-strand break in DNA. Using base editing resulted in less than 0.01% of retrotransposition events.

They were also less frequent during prime editing, an advanced technique that enables targeted insertions, deletions, and all 12 possible base changes.

Senior study investigator Dr Chiarle said: “We hope our findings will encourage investigators using CRISPR/Cas9 to include a check for insertion of mobile elements. CRISPR is really a game-changer in genetic therapy, so it’s very important to know exactly what it does to ensure its safety.”

He said while the 5 to 6% rate of retrotransposition was low, gene therapies often target millions of cells, which means in theory, just one cell with a transposition event can result in a tumour developing.

Although the study was done only in cells in the laboratory, Dr Chiarle said it is important to determine how often retrotransposition happens in clinical trials of CRISPR-based gene therapies.

In the study, Dr Tao refined a test and set up an experimental system to validate it. This demonstrated that retrotransposition events involved LINE-1, the most common mobile element.

“We think this test could help detect these events more reliably, and it may be more cost-effective than the method that’s commercially available,” Tao added.

Source: Tao J, Wang Q, Mendez-Dorantes C, Burns KH, Chiarle R. (2022) “Frequency and mechanisms of LINE-1 retrotransposon insertions at CRISPR/Cas9 sites.” Nature Communications, doi: 10.1038/s41467-022-31322-3

Link: https://www.nature.com/articles/s41467-022-31322-3

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