An adenine base editor variant expands context compatibility

ABSTRACT Adenine base editors (ABEs) are precise gene-editing agents that convert A:T pairs into G:C through a deoxyinosine intermediate. Existing ABEs function most effectively when the

target A is in a TA context. Here we evolve the _Escherichia coli_ transfer RNA-specific adenosine deaminase (TadA) to generate TadA8r, which extends potent deoxyadenosine deamination to RA

(R = A or G) and is faster in processing GA than TadA8.20 and TadA8e, the two most active TadA variants reported so far. ABE8r, comprising TadA8r and a _Streptococcus pyogenes_ Cas9 nickase,

expands the editing window at the protospacer adjacent motif-distal end and outperforms ABE7.10, ABE8.20 and ABE8e in correcting disease-associated G:C-to-A:T transitions in the human

genome, with a controlled off-target profile. We show ABE8r-mediated editing of clinically relevant sites that are poorly accessed by existing editors, including sites in _PCSK9_, whose

disruption reduces low-density lipoprotein cholesterol, and _ABCA4-p.Gly1961Glu_, the most frequent mutation in Stargardt disease. Access through your institution Buy or subscribe This is a

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* Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS ENGINEERING A PRECISE ADENINE BASE EDITOR WITH MINIMAL

BYSTANDER EDITING Article 13 October 2022 RE-ENGINEERING THE ADENINE DEAMINASE TADA-8E FOR EFFICIENT AND SPECIFIC CRISPR-BASED CYTOSINE BASE EDITING Article 10 November 2022 ADENINE BASE

EDITOR ENGINEERING REDUCES EDITING OF BYSTANDER CYTOSINES Article 01 July 2021 DATA AVAILABILITY All next-generation sequencing data have been deposited to the NCBI’s Gene Expression Omnibus

and can be accessed through accession no. GSE243181 (ref. 65). Amplicon sequencing data have been deposited to the NCBI Sequence Read Archive under BioProject no. PRJNA925224. REFERENCES *

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Download references ACKNOWLEDGEMENTS We thank H. Yan for optimizing the transfection workflow and K. M. Watters for scientific editing of the paper. This work was completed in part with

computing resources provided by the University of Chicago Research Computing Center. We thank the Single Cell Immunophenotyping Core Facility at the University of Chicago for sequencing

support. W.T. is supported by the Searle Scholars Program (grant no. SSP-2021-113), the Cancer Research Foundation Young Investigator Program, the American Cancer Society (grant no.

RSG-22-043-01-ET) and the David & Lucile Packard Foundation (grant no. 2022-74685). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Chemistry, The University of Chicago,

Chicago, IL, USA Yu-Lan Xiao, Yuan Wu & Weixin Tang * Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA Yu-Lan Xiao, Yuan Wu & Weixin Tang Authors *

Yu-Lan Xiao View author publications You can also search for this author inPubMed Google Scholar * Yuan Wu View author publications You can also search for this author inPubMed Google

Scholar * Weixin Tang View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Y.-L.X. and W.T. conceived and designed the study. Y.-L.X. carried

out directed evolution, purified and characterized TadA8r, constructed the paired sgRNA-target library and evaluated TadA8r in human cells. Y.W. assisted with deaminase purification and

characterization. Y.W. analyzed transcriptome-wide off-target effects for all ABEs and editing data generated using the paired sgRNA-target library. W.T. supervised the study. Y.-L.X., Y.W.

and W.T. wrote the paper. CORRESPONDING AUTHOR Correspondence to Weixin Tang. ETHICS DECLARATIONS COMPETING INTERESTS A patent has been filed for TadA8r and its applications in gene editing

by the University of Chicago. PEER REVIEW PEER REVIEW INFORMATION _Nature Biotechnology_ thanks the anonymous reviewers for their contribution to the peer review of this work. ADDITIONAL

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INFORMATION Supplementary Notes 1–4, Tables 1–10 and Figs. 1–39. REPORTING SUMMARY SUPPLEMENTARY TABLE 1 Evaluated ABE targets of clinical relevance_full table. RIGHTS AND PERMISSIONS

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CITE THIS ARTICLE Xiao, YL., Wu, Y. & Tang, W. An adenine base editor variant expands context compatibility. _Nat Biotechnol_ 42, 1442–1453 (2024).

https://doi.org/10.1038/s41587-023-01994-3 Download citation * Received: 14 January 2023 * Accepted: 15 September 2023 * Published: 02 January 2024 * Issue Date: September 2024 * DOI:

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