When researchers first reported three years ago that they had created base editors, a version of the powerful CRISPR genome-editing tool, the excitement revolved around their distinct powers to subtly alter DNA compared to CRISPR itself . But the weaknesses of the base editors have become increasingly apparent, and a new study shows that they can also accidentally transform the RNA strands that help build proteins or accomplish other important cellular tasks. Researchers say this can complicate the development of safe therapies with technology and hamper other research applications.
Sickle cell human diseases to Tay-Sachs disease are caused by a single mutation in one of the four DNA bases adenine, guanine, cytosine and thymine, and CRISPR has often had difficulty changing the bad actors. This is partly because CRISPR cuts double-stranded DNA at specific sites and then relies on mystical cell repair mechanisms to do the heavy lifting of inserting a corrected DNA sequence into a mutation. Base editors, in contrast, chemically alter one base of DNA to another with enzymes called deaminases, which do not require a cut or help from the cell.
Base editors, who adapt key components of CRISPR to target sites in the genome, have been shown to have many off-target effects on DNA. But so far, its effects on RNA, which contain three of the same bases as DNA, have escaped scrutiny. Then, J. Keith Joung, a pathologist and molecular biologist at Massachusetts General Hospital in Boston, led a team that placed the base editors in human liver and kidney cells. Its discovery: the deaminases can also alter the RNA, informs the group today in Nature.
Joung, a pioneer developer of base editors, was surprised by changes in RNA, which had cytosines being converted to uracil, an RNA base that is related to thymine. "When a postdoc showed me the results and we saw tens of thousands of RNA cytosines being edited, I was like, wait a minute, what are we seeing here?"
Jia Chen, who does genomic editing research at ShanghaiTech University in China and was not involved in the new work, was not so surprised, noting that deamines were originally described as having the ability to change RNA. But he says the new job will push the field to solve the problem. "Discovery [lead to] developing new base editors with greater editing accuracy, "says Chen.
Joung says that his recent discovery of ancient literature deaminase is what led his laboratory to do these experiments. And they have already created deamines that substantially reduce the number of inadvertent RNA issues. "This was very encouraging for us," says Joung. "We are ultimately protein engineers and we want to find out if we can design the system to make the mutations disappear."
David Liu, a Harvard chemist who created the first base editor and co-founded two technology-based companies with Joung, notes that deaminases naturally edit cellular RNA, emphasizing that the biological consequences of such editing are unclear. He adds that studies from his own laboratory on base editors have also found out-of-target RNA editions, but at much lower levels. The differences between the results, Liu says, probably have less to do with the amount of off-target RNA editing than the different way in which the Joung group separated their cells and analyzed the results.
Both Liu and Joung emphasize that their labs have found DNA or RNA-only deaminases, which makes them confident that they can decouple the off-target effects observed with current base editors. "Base editors are still incredibly powerful tools," says Joung. "This is just another parameter we need to understand."