A rationally engineered cytosine base editor retains high on-target activity while reducing both DNA and RNA off-target effects

Erwei Zuo; Yidi Sun; Tanglong Yuan; Bingbing He; Changyang Zhou; Wenqin Ying; Jing Liu; Wu Wei; Rong Zeng; Yixue Li; Hui Yang

doi:10.1038/s41592-020-0832-x

A rationally engineered cytosine base editor retains high on-target activity while reducing both DNA and RNA off-target effects

Nat Methods. 2020 Jun;17(6):600-604. doi: 10.1038/s41592-020-0832-x. Epub 2020 May 18.

Authors

Erwei Zuo^#^{1

2}, Yidi Sun^#^{3

4}, Tanglong Yuan^#⁵, Bingbing He^#⁶, Changyang Zhou^#⁶, Wenqin Ying⁶, Jing Liu⁵, Wu Wei^{4

7}, Rong Zeng^{3

8}, Yixue Li^{9

10

11

12}, Hui Yang^{13

14}

Affiliations

¹ Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China. erweizuo@163.com.
² Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. erweizuo@163.com.
³ CAS Key Laboratory of Systems Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China.
⁴ Bio-Med Big Data Center, Key Laboratory of Computational Biology, CAS-MPG Partner Institute for Computational Biology, Shanghai Institute of Nutrition and Health, Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China.
⁵ Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China.
⁶ Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
⁷ Center for Biomedical Informatics, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai, China.
⁸ Department of Life Sciences, Shanghai Tech University, 100 Haike Road, Shanghai, China.
⁹ Bio-Med Big Data Center, Key Laboratory of Computational Biology, CAS-MPG Partner Institute for Computational Biology, Shanghai Institute of Nutrition and Health, Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China. yxli@sibs.ac.cn.
¹⁰ Department of Life Sciences, Shanghai Tech University, 100 Haike Road, Shanghai, China. yxli@sibs.ac.cn.
¹¹ School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China. yxli@sibs.ac.cn.
¹² Collaborative Innovation Center for Genetics and Development, Fudan University, Shanghai, China. yxli@sibs.ac.cn.
¹³ Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. huiyang@ion.ac.cn.
¹⁴ Shanghai Center for Bioinformation Technology, Shanghai Academy of Science & Technology, Shanghai, China. huiyang@ion.ac.cn.

^# Contributed equally.

PMID: 32424272
DOI: 10.1038/s41592-020-0832-x

Abstract

Cytosine base editors (CBEs) offer a powerful tool for correcting point mutations, yet their DNA and RNA off-target activities have caused concerns in biomedical applications. We describe screens of 23 rationally engineered CBE variants, which reveal mutation residues in the predicted DNA-binding site can dramatically decrease the Cas9-independent off-target effects. Furthermore, we obtained a CBE variant-YE1-BE3-FNLS-that retains high on-target editing efficiency while causing extremely low off-target edits and bystander edits.

Publication types

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

MeSH terms

Base Sequence
CRISPR-Associated Protein 9 / genetics*
CRISPR-Cas Systems / genetics
Cytosine / metabolism*
DNA / genetics*
Gene Editing / methods*
HEK293 Cells
Humans
Mutation
Point Mutation
RNA / genetics*

Substances

RNA
Cytosine
DNA
CRISPR-Associated Protein 9