Powerful CRISPR Cousin accidentally mutates RNA during DNA target editing | Science

By Jon CohenApril 17, 2019, 4:10 pm

When researchers first announced, 3 years ago, that they had created basic editors, a version of the powerful CRISPR genome editing tool, enthusiasm swirled around their distinct powers to modify DNA more subtly in relation to the CRISPR itself. But the weaknesses of the core publishers have become increasingly obvious, and a new study shows that they can also accidentally mutate RNA strands that help build proteins or other key cellular tasks. Researchers say this could complicate the development of safe therapies with technology and hinder other applications of research.

Human diseases ranging from sickle cell disease to Tay-Sachs are caused by a single mutation of one of the four bases of DNA (adenine, guanine, cytosine and thymine) and CRISPR has often had difficulty exchanging the bad actors . This is partly because CRISPR cuts double-stranded DNA at targeted locations, and then relies on difficult cell repair mechanisms to perform the heavy task of inserting a corrected DNA sequence for a specific target. mutation. Baseline writers, on the other hand, chemically modify one DNA base in another with enzymes called deaminases, which do not require cleavage or help from the cell.

Basic editors, which adapt key components of CRISPR to reach targeted areas of the genome, have many non-targeted effects on DNA. But until now, its effects on RNA, which contains three identical bases to those of DNA, had escaped examination. For example, J. Keith Joung, a pathologist and molecular biologist at Massachusetts General Hospital in Boston, led a team that placed editors in human liver and kidney cells. Their discovery: deaminases can also alter RNA, reports the group today in Nature.

Joung, a pioneering developer of database editors, was surprised by the changes in RNA that turned cytosines into uacil, a thymine-related RNA base. "When a postdoc showed me the results for the first time and we saw tens of thousands of RNA cytosines being modified, I thought to myself: 'Wait a minute, that's right. are we looking here? "

Jia Chen, who is doing research on genome modification at ShanghaiTech University in China and did not take part in the new work, was not so surprised, noting that deaminases had to Originally been described as having the ability to modify RNA. But he says the new job will push the field to solve the problem. "The result will be [lead to] develop new basic editors with superior editing accuracy, "says Chen.

Joung explains that it is his recent discovery of the old literature on deaminase that led his laboratory to do these experiments. And they have already developed disinfection solutions that dramatically reduce the number of RNA changes inadvertently. "It has been very encouraging for us," says Joung. "We are finally protein engineers, and we want to know if we can design the system to remove the mutations."

David Liu, a chemist at Harvard University who created the first basic editor and co-founded two companies based on Joung technology, notes that deaminases naturally modify cellular RNA, pointing out that the biological consequences of such a modification are not clear. He adds that studies from his own lab on core editors have also revealed untargeted changes in RNA, but at much lower levels. The differences in their results, says Liu, probably have less to do with the amount of non-target RNA editing that takes place than the different way in which the Joung group sorted its cells and analyzed the results.

Liu and Joung both point out that their labs have found deaminases that only work on DNA or RNA, which makes them confident in the ability to decouple off-target effects seen with basic editors. current. "Basic editors are still incredibly powerful tools," says Joung. "That's just another parameter we need to understand."