all A single mutation in the human genome can be the difference between health and disease, and base editing allows researchers to replicate these mutations in the lab to study how genetic changes mess up cells. But the genetic basis of complex diseases involves multiple mutations, and current base editing strategies fall short.
“We think it’s really important to functionally characterize specific combinations of mutations,” he said. Alexis ComorChemist at the University of California, San Diego. “We need tools that can multiplex.”
Unfortunately, multiplexing isn’t as easy as throwing a bunch of base editors at them all at once, especially when researchers want to make multiple types of edits at once—for example, changing cytosine to thymine at one location and adenine to guanine at another.
This machinery is complex, and includes both an enzyme that makes the desired modification to a base and a guide RNA that points the complex to the correct site in the genome. But when multiple base editors are working simultaneously, the guide RNA for one base editor can interact with components of another base editor, directing the wrong enzyme complex to the target site and creating the wrong type of edit at that site.
To solve this problem, Komor’s team developed four new things. Multiplex orthogonal based editor (MOBE) system allows researchers to perform multiple types of editing simultaneously without worrying about interference between the machines required for each editing task.One The system is posted on: Nature BiotechnologyIt opens the door to building models to study complex genetic diseases caused by multiple mutations.
The researchers used complementary pairs of RNA and protein molecules, called aptamers and coat proteins, that selectively bind to each other. By incorporating RNA aptamers into guide RNAs and coat proteins into their corresponding base-editing protein complexes, they forced the correct guide RNA and base-editing pairs to work together, minimizing incorrect pairings.
“When we came up with it, it was a very simple idea in theory,” Comor said. “It was a Herculean effort to actually make it work and do all the protein engineering.”
Queen CowanBiochemists leading the research in Komor’s lab tested hundreds of different combinations of RNA and protein components to find the optimal approach that maximizes editing specificity and minimizes crosstalk. Crosstalk is when one base editor edits at the target site of another editor. Typically, when combining base editors that target adenine and cytosine, the crosstalk rate is 30%. But with MOBE, crosstalk dropped to 5%. About a quarter of cells ended up with the correct editing pair.
“I see a lot of utility in this platform,” he said. Krishnanu SahaA biomedical engineer at the University of Wisconsin-Madison who was not involved in the study. “What’s interesting about the strategy that this team has developed is how modular it is.” The system can be used for many different types of base editors, and can also be fine-tuned by changing how the different components are connected. Saha also notes that this modularity could make it easier to deliver to cells than other, larger, multiplexed editors.
While some members of the Comor team are improving the performance of base-editing enzymes, others are using the system to edit multiple underlying mutations into cell lines to create genetic models of complex diseases. For example, in this study, the researchers used MOBE to edit a pair of mutations that cause the hormonal disorder Kallmann syndrome and the neurological disorder anencephaly into cell lines. They were then able to study how these mutations affect the characteristics of the cells, such as their transcriptional profile or morphology.
Saha is optimistic about the potential applications of MOBE in his work creating cell therapies. For example, certain mutations can make T cells more potent in immunotherapy, and MOBE allows researchers to screen multiple combinations of mutations at scale to see which ones produce the most therapeutically promising T cells.
“Expanding the scope of disease modeling and cell engineering is very exciting to me,” Saha said. “This will take the application of these genome-writing tools to another level.”
reference
1. Cowan QT, etc. Development of a Multiplexed Orthogonal-Based Editor (MOBE) System. Nat Biotechnology. 2024.
