DddA homolog search and engineering expand sequence compatibility of mitochondrial base editing.

Expanding mitochondrial base editing tools with broad sequence compatibility is of high need for both research and therapeutic purposes. In this study, we identify a DddA homolog from Simiaoa sunii (Ddd_Ss) which can efficiently deaminate cytosine in DC context in double-stranded DNA (dsDNA). We successfully develop Ddd_Ss-derived cytosine base editors (DdCBE_Ss) and introduce mutations at multiple mitochondrial DNA (mtDNA) loci including disease-associated mtDNA mutations in previously inaccessible GC context. Finally, by introducing a single amino acid substitution from Ddd_Ss, we successfully improve the activity and sequence compatibility of DdCBE derived from DddA of Burkholderia cenocepacia (DdCBE_Bc). Our study expands mtDNA editing tool boxes and provides resources for further screening and engineering dsDNA base editors for biological and therapeutic applications.