Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos PMID: 30819928

GLP-1 and Obesity Research

2026-08-18

Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos

Science. 2019 Apr 19;364(6437):289-292. doi: 10.1126/science.aav9973.

Erwei Zuo ^# ¹ ² , Yidi Sun ^# ³ , Wu Wei ^# ⁴ ⁵ ⁶ , Tanglong Yuan ^# ² , Wenqin Ying ¹ , Hao Sun ⁷ , Liyun Yuan ⁴ , Lars M Steinmetz ⁸ ⁹ ¹⁰ , Yixue Li ¹¹ ¹² ¹³ , Hui Yang ¹⁴

Affiliations

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. Center for Animal Genomics, Agricultural Genome Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518124, 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, University of Chinese Academy of Sciences, Shanghai, 200031, China.
4. Key Lab of Computational Biology, CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
5. Stanford Genome Technology Center, Stanford University, Palo Alto, CA 94304, USA.
6. Center for Biomedical Informatics, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai 200040, China.
7. University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0726, USA.
8. Stanford Genome Technology Center, Stanford University, Palo Alto, CA 94304, USA. [email protected] [email protected] [email protected].
9. Department of Genetics, School of Medicine, Stanford University, Stanford, CA 94305, USA.
10. Genome Biology Unit, European Molecular Biology Laboratory (EMBL), 69117 Heidelberg, Germany.
11. Key Lab of Computational Biology, CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China. [email protected] [email protected] [email protected].
12. Collaborative Innovation Center for Genetics and Development, Fudan University, Shanghai 200438, China.
13. Shanghai Center for Bioinformation Technology, Shanghai Industrial Technology Institute, Shanghai 201203, China.
14. 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. [email protected] [email protected] [email protected].
# Contributed equally.

Abstract

Genome editing holds promise for correcting pathogenic mutations. However, it is difficult to determine off-target effects of editing due to single-nucleotide polymorphism in individuals. Here we developed a method named GOTI (genome-wide off-target analysis by two-cell embryo injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. Comparison of the whole-genome sequences of progeny cells of edited and nonedited blastomeres at embryonic day 14.5 showed that off-target single-nucleotide variants (SNVs) were rare in embryos edited by CRISPR-Cas9 or adenine base editor, with a frequency close to the spontaneous mutation rate. By contrast, cytosine base editing induced SNVs at more than 20-fold higher frequencies, requiring a solution to address its fidelity.