Since 2012 when the CRISPR genome editing technology was invented, the effect has reflected great promise to treat several intractable diseases. However, it has been a real struggle for scientists to identify potential off-target impact in the therapeutically relevant cell types, which remains the main barrier to moving therapies in the clinic.
At present, a team of scientists at the Gladstone Institutes and the Innovative Genomics Institute (IGI), along with colleagues at AstraZeneca, have developed a reliable method to do just that.
CRISPR edits the genome of an individual by cutting the DNA at a specific location. The challenge is to ensure the tool doesn't also make cuts elsewhere along the DNA - damage referred to as "off-target effects," that could have unforeseen consequences.
The researchers discovered that when CRISPR cuts, the DNA is broken. To survive, the cell recruits several different DNA repair factor to that particular site in the genome to fix the break and join the cut ends back together. The researchers hope that they can find the locations of these DNA repair factors and identify the sites that have been cut by CRISPR.
The researchers tested their idea by studying a panel of different DNA repair factors. They discovered one of them, MRE11, one of the first responders to the site of the cut. With the use of MRE11, the scientists developed a new method called DISCOVER-Seq that is capable of identifying the specific location in the genome where a cut has been made by CRISPR.
There are several methods in existence to detect CRISPR off-target effects. However, researchers have come with limitations that range from producing false positive results to killing the cells they are examining. Also, the most common technique scientists use to date is currently limited to being used in cultured cells in the laboratory, excluding its use in patient-derived stem cells or animal tissue.
Scientists now use the method that relies on the cell's natural repair process to identify cuts; it has proven to be much less invasive and much more reliable. The researchers tested the new DISCOVER-Seq method in individual pluripotent stem cells, patient cells, and mice, and the outcomes indicate that this method could potentially be used in any system, rather than just in the lab.
Not only that, but DISCOVER-Seq has also revealed new insights into the mechanisms used by CRISPR to edit the genome which will lead to a better understanding of the biology of how this tool works.
