Genome editing has become a widely accepted technology to modify DNA in cells, allowing scientists to study diseases in the laboratory and develop therapies that repair disease-causing mutations. However, with the current approach it is only possible to edit cells in one location at a time.
Now, a team of scientists from Gladstone Institutes has developed a new method that allows them to perform precise operations at multiple locations within a cell – all at once. Using molecules called retrons, they created a tool that can efficiently modify DNA in bacteria, yeast and human cells.
“We wanted to push the boundaries of genomic technologies by providing engineering tools to help us study the true complexity of biology and disease,” said Associate Investigator Seth Shipman, PhD, senior author of a new study published in Nature Chemical Biology.
Overcoming limitations
Shipman is a leader in the emerging and rapidly growing field of retrons, molecular components of a bacterial immune system that can produce large amounts of DNA. By combining retrons with CRISPR-Cas9 genome editing, his lab pioneered a system in 2022 to edit human cells quickly and efficiently.
With the new study, the researchers wanted to use their system to overcome a limitation of current genome editing methods.
“If you wanted to edit a cell at multiple locations in the genome that are not close to each other, the standard approach until now has been to make the changes one after the other,” explains Alejandro González-Delgado, PhD, one of the first authors . of the study and a postdoctoral researcher in Shipman’s laboratory. “It was a laborious cycle: first you made an edit, then you used the edited cells to make a new edit, and so on.”
Instead, the team found a way to encode a retron so that it can generate different stretches of DNA. When delivered to a cell, these specially designed retrons, called multitrons, can perform multiple operations simultaneously.
Another advantage of multitrons is their ability to delete large parts of the genome.
“With multitrons, we can make sequential deletions to cut and collapse middle parts of the genome region we are targeting, bringing the widely separated ends closer together until the entire region is completely deleted,” says González- Delgado.
Many potential applications
As part of their research, Shipman and his team demonstrated immediate applications for their new method in the fields of molecular recording and metabolic engineering.
They have previously shown that retrons can be used to record molecular events in a cell, creating a detailed log of the cell’s activity and changes in its environment. With multitrons, the researchers have expanded this approach and can now record with greater sensitivity.
“Multitrons allows us to record very weak and very strong signals at the same time, increasing the dynamic range of our recordings,” says González-Delgado. “Ultimately, we can imagine implementing these types of tools into the gut microbiome to register a signal such as inflammation.”
In terms of metabolic engineering, the scientists have shown that multitrons can be used to simultaneously edit multiple genes in a metabolic pathway to quickly increase the production of a targeted substance in a cell. They tested their approach on a powerful antioxidant called lycopene and successfully increased the production of this compound by a factor of three.
“To start modeling complex genetic diseases and ultimately find treatments or cures, we need to make many different mutations in cells at once,” says Shipman, who is also an associate professor in the Department of Bioengineering and Therapeutic Sciences at UC San Francisco . , as well as a Chan Zuckerberg Biohub researcher. “Our new approach is a step in that direction.”
