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CRISPR DNA Base Editors Cause Tens of Thousands of Off-Target RNA Mutations

NEW YORK (GenomeWeb) – A team of Chinese researchers has found that CRISPR-based DNA base editors can induce tens of thousands of off-target mutations in RNA.

Adenine base editors (ABEs) and cytosine base editors (CBEs) are known to efficiently and precisely edit point mutations in DNA with minimal off-target DNA editing. However, recent studies have also shown that despite their reputations for generating low levels of off-target DNA edits, base editors may create unexpected problems.

In March, an international team reported that the cytosine base editor 3 (BE3) induced SNVs with frequencies more than 20-fold higher than the spontaneous mutation rate. In April, a team of researchers from Massachusetts General Hospital led by Keith Joung reported that both ABEs and CBEs could cause extensive transcriptome-wide off-target RNA editing in human cells. And in May, a Broad Institute team led by David Liu reported that they had found ABEs to generate low but detectable levels of widespread adenosine-to-inosine editing in cellular RNAs

In their new study, published yesterday in Nature, the Chinese researchers reported that they quantitatively evaluated the RNA SNVs induced by CBEs and by ABEs and found that both BE3 and the adenine base editor ABE7.10 generated tens of thousands of off-target RNA SNVs.

In order to evaluate the off-target effect of base editors at the RNA level, the investigators transfected BE3 or ABE7.10 along with a green fluorescent protein, with or without single guide RNAs into HEK293T cultured cells. They validated the high on-target efficiency of DNA editing by both BE3 and ABE7.10 in the cells using Sanger sequencing and then performed RNA sequencing at an average depth of 125X.

The team observed between five- and 40-fold higher levels of RNA SNVs in cells transfected with various combinations of base editors and sgRNAs compared to the cells transfected with GFP only. Moreover, they found that the number of off-target RNA SNVs was increased when expressing higher level of CBEs or ABEs.

"Notably, nearly 100 percent of the RNA SNVs identified in the BE3-treated cells were mutations from G to A or C to U, significantly higher than that of the GFP-alone control cells," the authors wrote. "This mutation bias was the same as that of APOBEC1 itself, indicating that these mutations were not spontaneous but rather were induced by BE3 or [the APOBEC1 protein]. Similarly, 95 percent of the ABE7.10-induced mutations were A to G or U to C, consistent with the action of [the TadA protein]."

Further, the researchers observed that ABE7.10 induced 56 and 12 non-synonymous RNA SNVs in oncogenes and tumor suppressor genes, respectively. Many of these genes showed an editing rate higher than 40 percent, raising concerns about possible oncogenic risks of DNA base editing.

In order to explore approaches that could eliminate the RNA off-target activity of base editors, the researchers looked at the effects of destabilizing the RNA binding capacity of APOBEC1 and TadA.

Specifically, they introduced a point mutation called W90A to a predicted hydrophobic region in APOBEC1, but they found that although the BE3W90A mutant no longer caused RNA off-target effects, its on-target DNA editing activity was also essentially muted. Further experiments, however, showed that double mutations to BE3 (W90Y and R126E) reduced RNA off-target effects to base level but maintained BE3-like DNA on-target efficiencies.

They also tested whether replacing APOBEC1 with human APOBEC3A could eliminate the RNA off-target activity of BE3 and found that BE3 (hA3A)-transfected HEK293T cells showed significantly lowered off-target RNA SNVs, as compared to that of BE3 (APOBEC1)-transfected cells. To further reduce off-target effects, they introduced a point mutation into the predicted RNA and single-strand DNA binding domain of hA3A, respectively, and found that the number of off-target RNA SNVs in both variants was decreased to the base level.

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