Rationally Designed APOBEC3B Cytosine Base Editors with Improved Specificity

被引：116

作者：

Jin, Shuai ^{[1
,2
]}

Fei, Hongyuan ^{[1
,2
]}

Zhu, Zixu ^{[1
,2
]}

Luo, Yingfeng ^{[3
]}

Liu, Jinxing ^{[1
]}

Gao, Shenghan ^{[3
]}

Zhang, Feng ^{[4
]}

Chen, Yu-Hang ^{[5
]}

Wang, Yanpeng ^{[1
,2
]}

Gao, Caixia ^{[1
,2
]}

机构：

[1] Chinese Acad Sci, State Key Lab Plant Cell & Chromosome Engn, Ctr Genome Editing, Inst Genet & Dev Biol,Innovat Acad Seed Design, Beijing, Peoples R China

[2] Univ Chinese Acad Sci, Coll Adv Agr Sci, Beijing, Peoples R China

[3] Chinese Acad Sci, Inst Microbiol, State Key Lab Microbial Resources, Beijing, Peoples R China

[4] Univ Minnesota, Microbial & Plant Genom Inst, Ctr Plant Precis Genom, Dept Plant & Microbial Biol, Minneapolis, MN 55108 USA

[5] Chinese Acad Sci, Inst Genet & Dev Biol, Innovat Acad Seed Design, State Key Lab Mol Dev Biol, Beijing, Peoples R China

来源：

MOLECULAR CELL | 2020年 / 79卷 / 05期

基金：

中国国家自然科学基金;

关键词：

CATALYTIC DOMAIN; GENOMIC DNA; OFF-TARGET; DEAMINASE;

D O I：

10.1016/j.molcel.2020.07.005

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：728 / +

页数：19

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[1] Nuclear Magnetic Resonance Structure of the APOBEC3B Catalytic Domain: Structural Basis for Substrate Binding and DNA Deaminase Activity [J].