Engineered ADARs enable precision A-to-G base editing of DNA

Adenine base editors (ABEs), which enable A•T-to-G•C base editing, have emerged as a powerful tool with potential therapeutic applications. However, conventional ABEs suffer from bystander nucleotide conversions, limiting their utility for precise editing. Here we present a single-nucleotide resolution ABE (snuABE) created by fusing a nickase Cas9, nCas9-H840A, with the deaminase domain of ADAR (adenosine deaminase acting on RNA), which acts on DNA:RNA hybrids, instead of TadA, which acts on single-stranded DNA in conventional ABEs. snuABE requires a target-adenine guide RNA (tagRNA) that introduces a mismatch at the target adenine, enabling highly specific A-to-G editing by ADAR. Engineering ADAR from Pediculus humanus using the in silico protein evolution algorithm EvolvePro, along with 3′-end protection of the tagRNA, enhanced snuABE activity, yielding a median efficiency of 5.4% and a maximum efficiency of 50.0% across 32 targets in HEK293T cells. snuABE exhibits no detectable DNA off-target editing at predicted off-target or orthogonal R-loop sites, highlighting its potential as a precise and safe base-editing technology. Bystander edits are minimized by engineering an adenosine deaminase acting on RNA for DNA base editing.