Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis.

Recently developed DNA base editing methods enable direct generation of desired point mutation in genomic DNA without generating any double-strand breaks (DSBs)1–3, yet the issue of off-target edits have limited the application of these methods. While several previous studies have evaluated off-target mutations in genomic DNA4–8, it is now appreciated that the deaminases integral to commonly used DNA base editors often exhibit RNA binding activity9–13. For example, the cytosine deaminase APOBEC1 used in cytosine base editors (CBEs) was found to target both DNA and RNA12, and the adenine deaminase TadA used in adenine base editors (ABEs) was found to induce site-specific inosine formation on RNA9,11. However, any potential RNA mutations caused by DNA base editors has not been evaluated. Adeno-associated viruses are the most common delivery system for DNA editing gene therapies; these viruses can sustain long-term gene expression in vivo, so the extent of potential RNA mutation induced by DNA base editors is of great concern14–16[REMOVED HYPERLINK FIELD]. Here, we quantitatively evaluated the RNA single nucleotide variations (SNVs) induced by CBEs and by ABEs. We found that both the cytosine base editor BE3 and the adenine base editor ABE7.10 generate tens of thousands of off-target RNA SNVs. Subsequently, by engineering deaminases, we found that three CBE variants and one ABE variant reduced off-target RNA SNVs to the baseline while maintaining their efficient DNA on-target activity. This study reveals a previously overlooked aspect of off-target effects in DNA editing and also demonstrates that such effects can be eliminated by engineering deaminases.