The possibility of altering the genome by altering the DNA sequence inside a living cell is powerful for research and holds great promise for the treatment of disease. However, existing genome editing technologies frequently lead to unwanted mutations or may not introduce changes at all. These issues have kept the field from reaching its full potential.
Now, new research from the lab of Princeton University researcher Britt Adamson, conducted with collaborators from the lab of Jonathan Weissman, fellow of the Whitehead Institute and professor of biology at the Massachusetts Institute of Technology and researcher at the Howard Hughes Medical Institute, and Cecilia Cotta-Ramusino, formerly at Editas Medicine, details a new method called Repair-seq that reveals in detail how genome editing tools work.
“We have long known that the mechanisms involved in the repair of broken DNA are essential for genome editing, because in order to change the DNA sequence, it must first be broken,” said Britt Adamson, lead author of the study and assistant professor in the Department of Molecular Biology at Princeton and the Lewis-Sigler Institute for Integrative Genomics. “But these processes are incredibly complex and therefore often difficult to disentangle.”
To repair DNA, cells use many different mechanisms, each involving sets of genes working together in separate pathways. Repair-seq allows researchers to probe the contribution of these pathways to repairing specific DNA damage by simultaneously profiling how hundreds of individual genes affect mutations produced at damaged sites. Researchers can then generate mechanistic models of DNA repair and learn how these mechanisms impact genome editing. Adamson and his colleagues applied their method to one of the most commonly used genome editing approaches, CRISPR-Cas9, which uses the bacterial Cas9 nuclease to cut both strands of the double-stranded DNA molecule, creating lesions called double-strand breaks.
âEditing with double-stranded breaks has been the bread and butter of genome editing for a long time, but making desired changes without unwanted mutations has been a huge challenge,â said the study‘s first author, Jeffrey Hussmann, who conducted the work during a postdoctoral fellowship. researcher in Jonathan Weissman’s laboratory. “We set out to understand the mechanisms behind as many induced mutations as possible, believing that this could help us optimize the system.”
Repair-seq experiments generate a huge amount of data. Analysis of this data, led by Hussmann, produced a map of how different DNA repair pathways relate to particular types of Cas9-induced mutations. Drawing on a rich history of research in the field, Hussmann’s analysis shed light on already known pathways and identified new ones, which together highlight the enormous complexity and myriad of systems involved. in repairing double strand breaks. All of the data unearthed in this work is now posted on an online portal that others can use to query genes and DNA repair pathways.
Separately, a team led by David Liu of the Broad Institute at MIT and Harvard developed a genome editing system called âmaster editingâ that doesn’t rely on creating double-stranded breaks. Primary editing efficiencies vary widely depending on cell type and target site, but the researchers suspected that identifying the DNA repair pathways involved could help identify avenues for improvement. With that in mind, Adamson and Hussmann teamed up with Liu and their colleagues to study the main cut using Repair-seq.
âWorking together has been a huge advantage,â said Adamson. “For us, it was a fantastic team-oriented, collaborative science experience.”
The collaborating researchers found that the ability to achieve the intended edits with the main edition was affected by proteins in the DNA mismatch repair pathway. They went on to show that inhibiting or bypassing this pathway dramatically improved the efficiency and accuracy of major editing results – positioning major editing to become a more widely applicable genome editing technology.
âWorking with Britt, Jonathan and their labs has been a beautiful integration of basic science, tool application and technological development – a real testament to the power of multidisciplinary collaboration,â said Liu.
Importantly, this work also demonstrates how Repair-seq can be used to enhance other genome editing technologies. In fact, the collaborating researchers have already applied it to a third genome editing system, which was also developed by scientists working under Liu. The results of this study were recently published in the journal Natural biotechnology.
âRepair-seq is a beautiful marriage of technological knowledge and biological knowledge,â said John Doench, director of research and development for the Broad Institute’s genetic disruption program, who was not involved in the work.
“And for the work on the main edition, what a wonderful example of collaboration! The main editors have often proved difficult to work with, and this article is starting to understand why, while launching new solutions,” he added. .
Going forward, the team will continue to improve the platform and apply it to other genome editing technologies.
âWe see Repair-seq as a tool that allows you to take a detailed picture of what genome editors are doing inside cells and then assess very quickly: ‘Is this a landscape that I can find design principles that will help improve the tool? ‘”said Adamson.” We are really excited to explore future applications. “
The studies were funded by grants from the National Institutes of Health, Howard Hughes Medical Institute, Searle Scholars Program, National Science Foundation, Damon Runyon Cancer Research Foundation, China Scholarship Council, and National Cancer Institute.