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

doi:10.1016/j.molcel.2020.07.005

Rationally Designed APOBEC3B Cytosine Base Editors with Improved Specificity

Mol Cell. 2020 Sep 3;79(5):728-740.e6. doi: 10.1016/j.molcel.2020.07.005. Epub 2020 Jul 27.

Authors

Shuai Jin¹, Hongyuan Fei¹, Zixu Zhu¹, Yingfeng Luo², Jinxing Liu³, Shenghan Gao², Feng Zhang⁴, Yu-Hang Chen⁵, Yanpeng Wang⁶, Caixia Gao⁷

Affiliations

¹ State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China; College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China.
² State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
³ State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China.
⁴ Department of Plant and Microbial Biology, Center for Plant Precision Genomics, The Microbial and Plant Genomics Institute, University of Minnesota, Minneapolis, MN 55108, USA.
⁵ State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China.
⁶ State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China; College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China. Electronic address: yanpengwang@genetics.ac.cn.
⁷ State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China; College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China. Electronic address: cxgao@genetics.ac.cn.

PMID: 32721385
DOI: 10.1016/j.molcel.2020.07.005

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.

Keywords: A3Bctd deaminase; cytosine base editors; nSaCas9; off-target editing; rationally designing; specificity; whole-genome sequencing.

Publication types

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

MeSH terms

Cytidine Deaminase / genetics*
Cytosine* / chemistry
Gene Editing / methods*
Genes, Plant
Humans
Minor Histocompatibility Antigens / genetics*
Mutation
Oryza / genetics*
RNA, Guide, CRISPR-Cas Systems
RNA, Plant / chemistry
Reproducibility of Results

Substances

Minor Histocompatibility Antigens
RNA, Plant
Cytosine
APOBEC3B protein, human
Cytidine Deaminase