Non-target RNA mutation induced by the editing of DNA bases and its elimination by mutagenesis

Recently developed DNA database editing methods allow direct generation of the desired point mutation in genomic DNA without generating double-strand breakage (DSB)^1-3However, the problem of off-target controls has limited the application of these methods. Although several previous studies have evaluated non-targeted mutations of genomic DNA^4-8It is now accepted that deaminases incorporated into commonly used DNA database editors often exhibit RNA binding activity^9-13. For example, the cytosine deaminase APOBEC1 used in basic cytosine editors (CBE) has been shown to target both DNA and RNA¹²and adenine deaminase TadA used in adenine base editors (ABE) induce site-specific inosine formation on RNA^9.11. However, no potential mutation of RNA caused by DNA-based editors was evaluated. Adeno-associated viruses are the most common delivery system for gene editing gene therapy; these viruses can support long-term gene expression in vivo, so the magnitude of the potential mutation of RNA-induced DNA database editors is a big concern^14-16[REMOVED HYPERLINK FIELD]. Here, we quantitatively evaluated CBE and ABE-induced single nucleotide RNA (SNV) variations. We found that the basic BE3 cytosine editor and the ABE7.10 adenine core editor generated tens of thousands of non-targeted SNV SNVs. Subsequently, in developing deaminases, we found that three CBE variants and one ABE variant reduced non-targeted SNV RNAs to the baseline while maintaining their effective DNA activity on the target. This study reveals a previously neglected aspect of non-targeted effects in DNA editing and also demonstrates that such effects can be eliminated by engineering deaminases.