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bioRxiv · 10.1101/2025.07.19.664718

Engineered ADARs enable single-nucleotide resolution DNA A-to-G editing without bystander effects

Im, H.·Jeong, B.·Lee, Y.·Oh, Y. E.·Jung, C.·Kim, Y.-W.·Uhm, H.·Bae, S.
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Abstract

The adenine base editor (ABE), which enables A*T-to-G*C base conversion, has emerged as a powerful tool for 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, which acts on DNA:RNA hybrids, instead of TadA, which acts on single-stranded DNA in conventional ABEs. snuABE requires a specially designed 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, further enhances the editing activity of snuABE in human cells. Moreover, snuABE exhibits significantly reduced DNA off-target activity, highlighting its potential as a safer and more precise base editing technology for therapeutic applications.

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Im, H., Jeong, B., Lee, Y., Oh, Y. E., Jung, C., Kim, Y.-W., Uhm, H., Bae, S.. 2025-07-19. Engineered ADARs enable single-nucleotide resolution DNA A-to-G editing without bystander effects. https://doi.org/10.1101/2025.07.19.664718

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