Rationally Designed APOBEC3B Cytosine Base Editors with Improved Specificity

Shuai Jin, Hongyuan Fei, Zixu Zhu, Yingfeng Luo, Jinxing Liu, Shenghan Gao, Feng Zhang, Yu Hang Chen, Yanpeng Wang, Caixia Gao

Research output: Contribution to journalArticlepeer-review

98 Scopus citations

Abstract

Cytosine base editors (CBEs) generate C-to-T nucleotide substitutions in genomic target sites without inducing double-strand breaks. However, CBEs such as BE3 can cause genome-wide off-target changes via sgRNA-independent DNA deamination. By leveraging the orthogonal R-loops generated by SaCas9 nickase to mimic actively transcribed genomic loci that are more susceptible to cytidine deaminase, we set up a high-throughput assay for assessing sgRNA-independent off-target effects of CBEs in rice protoplasts. The reliability of this assay was confirmed by the whole-genome sequencing (WGS) of 10 base editors in regenerated rice plants. The R-loop assay was used to screen a series of rationally designed A3Bctd-BE3 variants for improved specificity. We obtained 2 efficient CBE variants, A3Bctd-VHM-BE3 and A3Bctd-KKR-BE3, and the WGS analysis revealed that these new CBEs eliminated sgRNA-independent DNA off-target edits in rice plants. Moreover, these 2 base editor variants were more precise at their target sites by producing fewer multiple C edits.

Original languageEnglish (US)
Pages (from-to)728-740.e6
JournalMolecular Cell
Volume79
Issue number5
DOIs
StatePublished - Sep 3 2020

Bibliographical note

Funding Information:
We thank Dr. Fei Lu (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences [IGDB, CAS]) for advice in the statistics analysis. This work was supported by grants from the National Transgenic Science and Technology Program (2019ZX08010-003 and 2019ZX08010-001), the Strategic Priority Research Program of the CAS (Precision Seed Design and Breeding, XDA24020102), the National Natural Science Foundation of China (31788103), the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2020000003), and by a grant from the United Soybean Board (1920-152-0131-B) to F.Z. Y.W. and C.G. designed the experiments. S.J. H.F. and Z.Z. performed most of the experiments. Y.L. S.J. and S.G. performed the WGS analysis. J.L. performed the rice transformation experiments. Y.-H.C. analyzed the structure of the A3Bctd protein. Y.W. and C.G. supervised the project. S.J. H.F. Z.Z. Y.W. F.Z. and C.G. wrote the manuscript. The authors have submitted two patent applications based on the results reported in this article.

Funding Information:
We thank Dr. Fei Lu (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences [IGDB, CAS]) for advice in the statistics analysis. This work was supported by grants from the National Transgenic Science and Technology Program ( 2019ZX08010-003 and 2019ZX08010-001 ), the Strategic Priority Research Program of the CAS (Precision Seed Design and Breeding, XDA24020102 ), the National Natural Science Foundation of China ( 31788103 ), the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2020000003), and by a grant from the United Soybean Board ( 1920-152-0131-B ) to F.Z.

Publisher Copyright:
© 2020 Elsevier Inc.

Keywords

  • A3Bctd deaminase
  • cytosine base editors
  • nSaCas9
  • off-target editing
  • rationally designing
  • specificity
  • whole-genome sequencing
  • Reproducibility of Results
  • Genes, Plant
  • Humans
  • Oryza/genetics
  • Minor Histocompatibility Antigens/genetics
  • Cytidine Deaminase/genetics
  • Gene Editing/methods
  • Cytosine/chemistry
  • RNA, Plant/chemistry
  • RNA, Guide/chemistry
  • Mutation

PubMed: MeSH publication types

  • Research Support, Non-U.S. Gov't
  • Journal Article
  • Validation Study

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