Article

APOBEC3 induces mutations during repair of CRISPR–Cas9-generated DNA breaks

  • Nature Structural & Molecular Biology 254552 (2018)
  • doi:10.1038/s41594-017-0004-6
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Abstract

The APOBEC-AID family of cytidine deaminase prefers single-stranded nucleic acids for cytidine-to-uridine deamination. Single-stranded nucleic acids are commonly involved in the DNA repair system for breaks generated by CRISPR–Cas9. Here, we show in human cells that APOBEC3 can trigger cytidine deamination of single-stranded oligodeoxynucleotides, which ultimately results in base substitution mutations in genomic DNA through homology-directed repair (HDR) of Cas9-generated double-strand breaks. In addition, the APOBEC3-catalyzed deamination in genomic single-stranded DNA formed during the repair of Cas9 nickase-generated single-strand breaks in human cells can be further processed to yield mutations mainly involving insertions or deletions (indels). Both APOBEC3-mediated deamination and DNA-repair proteins play important roles in the generation of these indels. Therefore, optimizing conditions for the repair of CRISPR–Cas9-generated DNA breaks, such as using double-stranded donors in HDR or temporarily suppressing endogenous APOBEC3s, can repress these unwanted mutations in genomic DNA.

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Acknowledgements

We are grateful to A. Furano, H. Lin and H. Wang for discussing and commenting on this manuscript, L.-L. Chen and N. Jing for technical support, X. Li and Y. Pan for participating in the examination of APOBEC expression, J. Wu for maintaining cell lines and H. Fang for participating in deep-sequencing library preparation. Next-generation deep sequencing was performed at the CAS-MPG PICB Omics Core, Shanghai, China. This work is supported by a MOST grant (2014CB910600 to L. Yang), NSFC grants (91540115 to L. Yang, 31571372 to B.S., 31471241 to L. Yang, 31600619 to B.Y. and 31600654 to J.C.), the Shanghai Pujiang program (16PJ1407000 to J.C. and 16PJ1407500 to B.Y.) and CAS Key Laboratory of Computational Biology grants (2015KLCB01 and 2016KLCB01 to L. Yang and J.C.).

Author information

Author notes

  1. Liqun Lei, Hongquan Chen, Wei Xue, Bei Yang and Bian Hu contributed equally to this work.

Affiliations

  1. School of Life Science and Technology, ShanghaiTech University, Shanghai, China

    • Liqun Lei
    • , Bian Hu
    • , Lijie Wang
    • , Wanjing Shang
    • , Min Zhuang
    • , Xingxu Huang
    •  & Jia Chen
  2. Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China

    • Liqun Lei
    • , Lijie Wang
    • , Lei Yan
    •  & Wanjing Shang
  3. University of Chinese Academy of Sciences, Beijing, China

    • Liqun Lei
    • , Lijie Wang
    • , Lei Yan
    •  & Wanjing Shang
  4. State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, China

    • Hongquan Chen
    • , Yiqiang Cui
    • , Wei Li
    • , Jianying Wang
    • , Jiahao Sha
    •  & Bin Shen
  5. School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, China

    • Hongquan Chen
    •  & Jimin Gao
  6. Key Laboratory of Computational Biology, CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai, China

    • Wei Xue
    • , Jia Wei
    •  & Li Yang
  7. Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, China

    • Bei Yang
    •  & Lei Yan
  8. MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University, National Resource Center for Mutant Mice, Nanjing, China

    • Bian Hu

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Contributions

J.C., L. Yang and B.S. conceived, designed and supervised the project. L.L., H.C., B.Y. and B.H. performed most of the experiments with the help of L.W., Y.C., W.L. and J. Wang on RT–qPCR, plasmid construction and in vitro transcription and W.S. and L. Yan on Cas9 protein purification. J. Wei prepared samples for deep sequencing, and W.X. performed the deep-sequencing data analyses and bioinformatics analysis, supervised by L. Yang. J.G., J.S., M.Z. and X.H. provided critical technical assistance. B.Y., J.C., L. Yang and B.S. wrote the paper with inputs from all authors. J.C. managed the project.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Bin Shen or Li Yang or Jia Chen.

Integrated Supplementary Information

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–10, Supplementary Tables 1–4 and Supplementary Note 1.

  2. Life Sciences Reporting Summary

  3. Supplementary Dataset 1

    Uncropped images

  4. Supplementary Dataset 2

    Base substitution frequency determined by deep sequencing

  5. Supplementary Dataset 3

    Indel frequency determined by deep sequencing

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