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Nature. 2019 Jun 10. doi: 10.1038/s41586-019-1314-0. [Epub ahead of print]

Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis.

Zhou C1,2, Sun Y2,3,4, Yan R5, Liu Y2,6, Zuo E1,7, Gu C5, Han L1, Wei Y1, Hu X1,2, Zeng R3,6, Li Y8,9,10, Zhou H11, Guo F12, Yang H13.

Author information

1
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, 200031, China.
2
College of Life Sciences, University of Chinese Academy of Sciences, Beijing, 100049, China.
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, Shanghai, 200031, 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, Shanghai, 200031, China.
5
Center for Translational Medicine, Ministry of Education Key Laboratory of Birth Defects and Related Diseases of Women and Children, Department of Obstetrics and Gynecology, West China Second University Hospital, College of Life Sciences, Sichuan University, Chengdu, Sichuan, 610041, China.
6
School of Life Science and Technology, Shanghai Tech University, Shanghai, China.
7
Center for Animal Genomics, Agricultural Genome Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, 518124, China.
8
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, Shanghai, 200031, China. yxli@sibs.ac.cn.
9
School of Life Science and Technology, Shanghai Tech University, Shanghai, China. yxli@sibs.ac.cn.
10
Shanghai Jiao Tong University, Fudan University, Shanghai Academy of Science & Technology, Shanghai, 200240, China. yxli@sibs.ac.cn.
11
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, 200031, China. hbzhou@ion.ac.cn.
12
Center for Translational Medicine, Ministry of Education Key Laboratory of Birth Defects and Related Diseases of Women and Children, Department of Obstetrics and Gynecology, West China Second University Hospital, College of Life Sciences, Sichuan University, Chengdu, Sichuan, 610041, China. guofan@scu.edu.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, 200031, China. huiyang@ion.ac.cn.

Abstract

Recently developed DNA base editing methods enable direct generation of desired point mutation in genomic DNA without generating any double-strand breaks (DSBs)1-3, yet the issue of off-target edits have limited the application of these methods. While several previous studies have evaluated off-target mutations in genomic DNA4-8, it is now appreciated that the deaminases integral to commonly used DNA base editors often exhibit RNA binding activity9-13. For example, the cytosine deaminase APOBEC1 used in cytosine base editors (CBEs) was found to target both DNA and RNA12, and the adenine deaminase TadA used in adenine base editors (ABEs) was found to induce site-specific inosine formation on RNA9,11. However, any potential RNA mutations caused by DNA base editors has not been evaluated. Adeno-associated viruses are the most common delivery system for DNA editing gene therapies; these viruses can sustain long-term gene expression in vivo, so the extent of potential RNA mutation induced by DNA base editors is of great concern14-16[REMOVED HYPERLINK FIELD]. Here, we quantitatively evaluated the RNA single nucleotide variations (SNVs) induced by CBEs and by ABEs. We found that both the cytosine base editor BE3 and the adenine base editor ABE7.10 generate tens of thousands of off-target RNA SNVs. Subsequently, by engineering deaminases, we found that three CBE variants and one ABE variant reduced off-target RNA SNVs to the baseline while maintaining their efficient DNA on-target activity. This study reveals a previously overlooked aspect of off-target effects in DNA editing and also demonstrates that such effects can be eliminated by engineering deaminases.

PMID:
31181567
DOI:
10.1038/s41586-019-1314-0

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