A streamlined base editor engineering strategy to reduce bystander editing

Izabella Valdez, Ian O’Connor, Divesh Patel, Katherine Gierer, Jan Harrington, Ethan Ellis, Stephen A. Caponetti, Robert P. Sebra, Hillary C. Valley, Kevin Coote, Martin Mense, Samuele G. Marro and Tingting Jiang ()
Additional contact information
Izabella Valdez: Icahn School of Medicine at Mount Sinai
Ian O’Connor: Icahn School of Medicine at Mount Sinai
Divesh Patel: CFFT Lab
Katherine Gierer: CFFT Lab
Jan Harrington: CFFT Lab
Ethan Ellis: Icahn School of Medicine at Mount Sinai
Stephen A. Caponetti: Icahn School of Medicine at Mount Sinai
Robert P. Sebra: Icahn School of Medicine at Mount Sinai
Hillary C. Valley: CFFT Lab
Kevin Coote: CFFT Lab
Martin Mense: CFFT Lab
Samuele G. Marro: Icahn School of Medicine at Mount Sinai
Tingting Jiang: Icahn School of Medicine at Mount Sinai

Nature Communications, 2025, vol. 16, issue 1, 1-14

Abstract: Abstract Base editing (BE) can permanently correct over half of known human pathogenic genetic variants without requiring a repair template, thus serving as a promising therapeutic tool to treat a broad spectrum of genetic diseases. However, the broad activity windows of current base editors pose a major challenge to their therapeutic application. Here, we show that integrating a naturally occurring oligonucleotide binding module into the deaminase active center of TadA-8e, a highly active deoxyadenosine deaminase, enhances its editing specificity. When conjugated with a Cas9 nickase or alternative PAM Cas9 variants, the engineered TadA variant—TadA-NW1—consistently achieves robust A-to-G editing efficiencies within an editing window consisting of four nucleotides, substantially narrower than the 10-bp editing window of the TadA-8e-derived ABEs. Moreover, compared to ABE8e, ABE-NW1 shows significantly decreased Cas9-dependent and -independent off-target activity while maintaining similar on-target editing efficiency. Further, TadA-NW1 can be reprogrammed to perform desired cytidine deamination and adenine transversion within a restricted editing window. Finally, in a cystic fibrosis (CF) cell model, ABE-NW1 outperforms existing ABEs in accurately and efficiently correcting the CFTR W1282X variant, one of the most common CF-causing mutations. In all, we engineered a suite of base editors with refined activity windows, enabling more precise base editing. Importantly, this study presents a streamlined genome editor re-engineering strategy to accelerate the development of therapeutic base editing.

Date: 2025
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DOI: 10.1038/s41467-025-63609-6

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