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Base Editors Cause Off-Target Mutations in RNA

A new study indicates that the modified CRISPR-Cas9 technology will need to be further refined before it can safely be used for research and therapeutic applications.

Apr 18, 2019
Catherine Offord

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Base editors designed to convert one DNA nucleotide to another may also perform large numbers of unwanted edits to RNA, according to a study published earlier this week (April 17) in Nature. Although base editing was touted as a more precise genome-editing approach than more traditional methods, researchers in the US found that the technique appears to cause widespread changes to the transcriptome of human cells, suggesting that the technology needs more work before it can be used reliably in research and therapeutics.

“Most investigation of off-target base editing has focused on DNA, but we have found that this technology can induce large numbers of RNA alterations as well,” study coauthor J. Keith Joung of Massachusetts General Hospital and Harvard Medical School says in a statement. “This surprising finding suggests the need to look at more than just genetic alterations when considering unintended off-target effects of base editors in cells.”

The most widely used form of base editing was developed by Harvard University’s David Liu, and relies on a modified form of CRISPR-Cas9 technology. Unlike standard CRISPR-guided genome editing, which results in a double-strand break in DNA, base editing allows researchers to precisely swap one DNA nucleotide for another. 

See “Advances in Genome Editing

Scientists have raised questions about the approach’s off-target effects before. Earlier this year, two studies—one in mouse embryos and one in rice—found that CRISPR-guided base editors can cause hundreds of unwanted mutations in other parts of the genome. But this study was one of the first to thoroughly scan the transcriptome for off-target effects on RNA. 

The team specifically tested a version of base editor that targets cytosine. They found that the technology induced tens of thousands of base changes—switching cytosine bases for uracil—across human cells’ RNA, leading to mutations in both protein-coding and noncoding sequences.

The extent of the off-target effects came as a surprise, Joung tells Science. “When a postdoc first showed me the results and we saw tens of thousands of RNA cytosines being edited, I was like, ‘Wait a minute, what are we looking at here?’”

Liu, who was not involved in the work, tells Science that his own lab has carried out analyses of base editors’ off-target effects in RNA and found lower levels than the ones report in Joung’s study. He suggests some of the differences might have more to do with the way the results were analyzed than true differences in the level of RNA editing.

He also notes that it will be possible to design base editors that only work on RNA or DNA, rather than both. Joung’s lab also investigated this possibility in its study, engineering new variants of the cytosine base editor that had substantially fewer effects on RNA, while preserving the same level of on-target editing in DNA.

“Base editors are still incredibly powerful tools,” Joung tells Science. “This is just another parameter we need to understand.”

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