a Single mutations in the human genome can make the difference between health and disease, and base editing allows researchers to recreate these mutations in the lab and study how genetic changes wreak havoc on cells. However, the genetic underpinnings of complex diseases involve multiple mutations, making current base editing strategies inadequate.
“We think it’s really important to functionally characterize specific combinations of mutations.” Alexis Komol“You need tools that allow you to do multiplexing,” says the University of California, San Diego chemist.
Unfortunately, multiplexing is not as simple as putting together a set of multiple base editors, especially if researchers want to make different types of edits simultaneously, such as converting a cytosine to a thymine at one position and an adenine to a guanine at another.
This machinery is complex and involves both enzymes that make the desired changes to the bases and guide RNAs that direct the complexes to the correct site in the genome. However, when multiple base editors are functioning simultaneously, it is possible that the guide RNA for one base editor can interact with components of another base editor, directing the incorrect enzyme complex to its target site and creating the wrong type of edit at that site.
To solve this problem, Komor’s team developed four new Multi-orthogonal basis editor The (MOBE) system allows researchers to make multiple types of edits simultaneously without worrying about crosstalk between the machines for each change.1 the system, Nature BiotechnologyIt opens the door to building models to study complex genetic diseases caused by multiple mutations.
The researchers took advantage of complementary pairs of RNA and protein molecules, called aptamers and coat proteins, that selectively bind to each other. By incorporating an RNA aptamer into the guide RNA and a coat protein into the corresponding base-editing protein complex, they ensured that the correct pair of guide RNA and base-editing protein worked together, minimizing incorrect pairing.
“When we came up with it, it was a very simple idea in theory,” Komor says, “but it was a huge effort to actually make it work and do all the protein engineering.”
Quinn CowanBiochemists in the Komol lab who led the study tested hundreds of different configurations of RNA and protein components to find the best approach that maximized editing specificity and minimized crosstalk, which is when one base editor edits the target position of the other. Typically, pairing adenine- and cytosine-targeting base editors results in a 30 percent crosstalk rate. But with MOBE, crosstalk dropped to 5 percent. A quarter of cells had the correct edit pair.
“I think this platform has a lot of utility,” he said. Krishnan Saha“The beautiful thing about the strategy they developed is how modular it is,” says Saha, a biomedical engineer at the University of Wisconsin-Madison, who was not involved in the work. The system can be used for many different types of base-editing programs, and it can be further fine-tuned by varying how the different components are linked together. Saha also notes that this modularity may make it easier to deliver into cells than other bulky multiple-editing programs.
While some members of Komol’s team are improving the performance of base-editing enzymes, others are using the system to create genetic models of complex diseases by editing multiple underlying mutations in cell lines. For example, in this study, the researchers used MOBE to edit a pair of mutations that cause Kallmann syndrome, a hormonal disorder, and anencephaly, a neurological disorder, into cell lines. They could then study how these mutations affect cellular features such as transcriptional profiles and morphology.
Saha is optimistic about MOBE’s potential applications in his work to generate cell therapies: For example, certain mutations may make T cells more powerful in immunotherapy, and MOBE could allow researchers to screen many combinations of mutations at scale to see which ones produce the most therapeutic T cells.
“The expanded scope of disease modeling and cell engineering is very exciting to me,” Saha says, “and will push the application of genome-writing tools to a new level.”
reference
1. Cowan QT, et al. Development of the Multiple Orthogonal Basis Editor (MOBE) system. National Biotechnology2024.
