CRISPR, the gene editing tool developed several years ago, has already helped advance biomedical research and holds a lot of promise as a potential therapeutic for human disease. There are still challenges to the clinical applications of the tool, however. One of those is the possibility that the gene editing machinery will make an edit in places where they are not intended. A team of researchers has now created a way to find those off-target effects in the relevant cell types. The work, by scientists from the Gladstone Institutes, the Innovative Genomics Institute (IGI), and AstraZeneca, has been reported in Science.
"When CRISPR makes a cut, the DNA is broken," said the co-first author of the report Beeke Wienert, Ph.D. "So, in order to survive, the cell recruits many different DNA repair factors to that particular site in the genome to fix the break and join the cut ends back together. We thought that if we could find the locations of these DNA repair factors, we could identify the sites that have been cut by CRISPR." Learn more about the off-target effects caused by CRISPR from the video above by IGI.
In this work, the team focused on several of those DNA repair factors; they determined that one, MRE11, is one of the first to rush to the scene of a cut. The researchers developed DISCOVER-Seq, which is based on MRE11 and can show exactly where in the genome CRISPR has made a cut.
"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," noted Bruce R. Conklin, MD, a senior investigator at Gladstone and deputy director at IGI.
"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. 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," explained Conklin.
While there are other ways to find the off-target effects produced by CRISPR, they can produce false positives and kill the cells under study. Those methods aren’t useful in the clinic, either.
"Because our method relies on the cell's natural repair process to identify cuts, it has proven to be much less invasive and much more reliable," said Jacob E. Corn, Ph.D. of ETH Zurich. "We were able to test our new DISCOVER-Seq method in induced pluripotent stem cells, patient cells, and mice, and our findings indicate that this method could potentially be used in any system, rather than just in the lab. The new method greatly simplifies the process of identifying off-target effects while also increasing the accuracy of the results," said Conklin.
"This could allow us to better predict how genome editing would work in a clinical setting. As a result, it represents an essential step in improving pre-clinical studies and bringing CRISPR-based therapies closer to the patients in need.”