New Method Developed for Accurately Determining Off-Target CRISPR Edits

New Method Developed for Accurately Determining Off-Target CRISPR Edits

CRISPR has been hailed as one of the most promising methods of treating diseases, and while the gene editing technology has been shown to allow highly accurate edits to be made to DNA, there is potential for off-target edits to be made. It is the potential for harmful edits to the genome that has held back clinical trials to determine its effectiveness at treating diseases caused by defective genes and mutations.

The CRISPR system includes guide RNA (gRNA) which binds to double stranded DNA in a very precise location. The double stranded DNA is then cut by the Cas enzyme component of the system in a precise place. While the gRNA should only bind to a specific section of DNA where the edit needs to be made, it could potentially bind to other sections of DNA and cause an off-target edit to be made.

One of the problems faced by scientists is how to determine whether any off-target edits have occurred. If it is possible to accurately determine whether there have been unintentional edits, it would go a long way toward demonstrating how safe – or unsafe – the gene editing technique actually is.

There are several methods that can be employed to identify where CRISPR has made edits, but each has its limitations, including killing the cells that are being examined, producing false positives, or only being suitable for use in the cultured cells in the lab.

Researchers at the Gladstone Institutes and the Innovative Genomics Institute have been working with AstraZeneca to develop a method of accurately determining off-target effects.  Their research has focused on DNA repair factors.

When the DNA is cut, several DNA repair factors in cells spring into action and rejoin the cut DNA. The researchers have used these DNA repair factors to identify where CRISPR has cut the DNA and it has been repaired.

The researchers found that when the DNA is cut, one of the first DNA repair factors to respond to the cut is MRE11. MRE11 was discovered to be tightly distributed around the site where the Cas9 component of CRISPR makes the cuts. The researchers used this DNA repair factor to develop a new system to determine where the cut had been made and the DNA had been repaired. The method they developed, called DISCOVER-Seq, shows exactly where cuts have been made, including off-target effects of CRISPR.

“The human genome is extremely large—if you printed the entire DNA sequence, you would end up with a novel as tall as a 16-story building. When we want to cut DNA with CRISPR, it’s like we’re trying to remove one specific word on a particular page in that novel,” explained Bruce Conklin, MD, senior investigator at Gladstone. “You can think of the DNA repair factors as different types of bookmarks added to the book. While some may bookmark an entire chapter, MRE11 is a bookmark that drills down to the exact letter that has been changed.”

The team tested the method on patient cells, in mice models, and in pluripotent stem cells, and found it to be a much more reliable way of determining where edits had occurred. By using the cell’s own repair processes to identify edits, the process is less invasive than methods currently used. The system works with different CAS enzymes, not just Cas9, and multiple gRNA formats and could potentially be used in situ in patients who have undergone gene editing using CRISPR.

In addition, the system has provided new insights into the mechanisms used by CRISPR to perform edits, which has improved understanding of the gene editing technology.

The research is detailed in the paper – Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq – which was recently published in the journal Science. DOI: 10.1126/science.aav9023

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