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Adenine base editor engineering reduces editing of bystander cytosines

Nature Biotechnology volume 39, pages 1426–1433 (2021)Cite this article

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Abstract

Adenine base editors (ABEs) catalyze specific A-to-G conversions at genomic sites of interest. However, ABEs also induce cytosine deamination at the target site. To reduce the cytosine editing activity, we engineered a commonly used adenosine deaminase, TadA7.10, and found that ABE7.10 with a D108Q mutation in TadA7.10 exhibited tenfold reduced cytosine deamination activity. The D108Q mutation also reduces cytosine deamination activity in two recently developed high-activity versions of ABE, ABE8e and ABE8s, and is compatible with V106W, a mutation that reduces off-target RNA editing. ABE7.10 containing a P48R mutation displayed increased cytosine deamination activity and a substantially reduced adenine editing rate, yielding a TC-specific base editing tool for TC-to-TT or TC-to-TG conversions that broadens the utility of base editors.

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Fig. 1: Rational design to reduce cytosine deamination activity of adenine base editors.
Fig. 2: Screening for decreased ABE-induced cytosine deamination.
Fig. 3: D108Q is a key mutation for reducing the frequency of cytosine deamination.
Fig. 4: The P48R mutation promotes specific cytosine conversion.
Fig. 5: ABE-P48R and ABE-P48R-UGI can be used for TC-specific base editing.

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Data availability

High-throughput sequencing data have been deposited in the NCBI Sequence Read Archive database (https://www.ncbi.nlm.nih.gov/sra) under accession number PRJNA661167. RNA-seq data have been deposited in the Gene Expression Omnibus under accession GSE165164. Plasmids encoding ABE8eWQ (Addgene no. 161815), ABE-P48R (Addgene no. 161816) and ABE-P48R-UGI (Addgene no. 161817) engineered in this work are available on Addgene at https://www.addgene.org/Sangsu_Bae/. Any other additional relevant data are available from the authors upon reasonable request. Source data are provided with this paper.

Code availability

The authors declare that all unreported custom python code used in this study are available within the article or from the corresponding author upon reasonable request.

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Acknowledgements

This research was supported by grants from the National Research Foundation of Korea no. 2020M3A9I4036072, no. 2020R1A6A1A06046728, no. 2021R1A2C3012908 and no. 2021M3A9H3015389 to S.B., and no. 2018R1C1B6004447 to J.-S.W.

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Author notes

  1. These authors contributed equally: You Kyeong Jeong, SeokHoon Lee.

Authors and Affiliations

  1. Department of Chemistry and Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul, South Korea

    You Kyeong Jeong, SeokHoon Lee, Gue-Ho Hwang, Sung-Ah Hong, Se-eun Park & Sangsu Bae

  2. Center for Genome Engineering, Institute for Basic Science, Daejeon, South Korea

    Jin-Soo Kim

  3. Department of Chemistry, Seoul National University, Seoul, South Korea

    Jin-Soo Kim

  4. Department of Life Sciences, Korea University, Seoul, South Korea

    Jae-Sung Woo

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  1. You Kyeong Jeong
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Contributions

S.B. and J.-S.W. conceived this project; Y.K.J., S.H.L., S.-A.H. and S.-e.P. performed the experiments; Y.K.J. and G.-H.H. performed bioinformatics analyses; J.-S.K. gave critical comments; Y.K.J., J.-S.W. and S.B. wrote the manuscript with the approval of all other authors.

Corresponding authors

Correspondence to Jae-Sung Woo or Sangsu Bae.

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Competing interests

Y.K.J., S.H.L, J.-S.W. and S.B. have filed a patent application based on this work. The remaining authors declare no competing interests.

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Peer review information Nature Biotechnology thanks Nozomu Yachie, Hui Yang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary Figs. 1–10, Tables 1–4 and References.

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Jeong, Y.K., Lee, S., Hwang, GH. et al. Adenine base editor engineering reduces editing of bystander cytosines. Nat Biotechnol 39, 1426–1433 (2021). https://doi.org/10.1038/s41587-021-00943-2

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