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

  • Nature. 2019 Jul;571(7764):275-278. doi: 10.1038/s41586-019-1314-0.
Changyang Zhou  #  1  2 ,  Yidi Sun  #  2  3  4 ,  Rui Yan  #  5 ,  Yajing Liu  #  2  6 ,  Erwei Zuo  #  1  7 ,  Chan Gu  5 ,  Linxiao Han  1 ,  Yu Wei  1 ,  Xinde Hu  1  2 ,  Rong Zeng  3  6 ,  Yixue Li  8  9  10 ,  Haibo Zhou  11 ,  Fan Guo  12 ,  Hui Yang  13
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, China.
  • 2. College of Life Sciences, University of Chinese Academy of Sciences, Beijing, 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, 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. 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, 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, China.
  • 8. 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, China. [email protected].
  • 9. School of Life Science and Technology, Shanghai Tech University, Shanghai, China. [email protected].
  • 10. Shanghai Jiao Tong University, Fudan University, Shanghai Academy of Science & Technology, Shanghai, China. [email protected].
  • 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, China. [email protected].
  • 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, China. [email protected].
  • 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. [email protected].
  • # Contributed equally.
PMID: 31181567 DOI: 10.1038/s41586-019-1314-0
Abstract

Recently developed DNA base editing methods enable the direct generation of desired point mutations in genomic DNA without generating any double-strand breaks1-3, but the issue of off-target edits has limited the application of these methods. Although several previous studies have evaluated off-target mutations in genomic DNA4-8, it is now clear that the deaminases that are integral to commonly used DNA base editors often bind to RNA9-13. For example, the Cytosine deaminase APOBEC1-which is used in Cytosine base editors (CBEs)-targets both DNA and RNA12, and the Adenine deaminase TadA-which is used in Adenine base editors (ABEs)-induces site-specific Inosine formation on RNA9,11. However, any potential RNA mutations caused by DNA base editors have not been evaluated. Adeno-associated viruses are the most common delivery system for gene therapies that involve DNA editing; these viruses can sustain long-term gene expression in vivo, so the extent of potential RNA mutations induced by DNA base editors is of great concern14-16. Here we quantitatively evaluated RNA single nucleotide variations (SNVs) that were induced by CBEs or ABEs. Both the Cytosine base editor BE3 and the Adenine base editor ABE7.10 generated tens of thousands of off-target RNA SNVs. Subsequently, by engineering deaminases, we found that three CBE variants and one ABE variant showed a reduction in off-target RNA SNVs to the baseline while maintaining 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.