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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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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  1. 1.

    Harris, R. S. & Liddament, M. T. Retroviral restriction by APOBEC proteins. Nat. Rev. Immunol. 4, 868–877 (2004).

  2. 2.

    Henderson, S. & Fenton, T. APOBEC3 genes: retroviral restriction factors to cancer drivers. Trends Mol. Med. 21, 274–284 (2015).

  3. 3.

    Salter, J. D., Bennett, R. P. & Smith, H. C. The APOBEC protein family: united by structure, divergent in function. Trends Biochem. Sci. 41, 578–594 (2016).

  4. 4.

    Yang, B., Li, X., Lei, L. & Chen, J. APOBEC: From mutator to editor. J. Genet. Genomics 44, 423–437 (2017).

  5. 5.

    Chen, J., Miller, B. F. & Furano, A. V. Repair of naturally occurring mismatches can induce mutations in flanking DNA. eLife 3, e02001 (2014).

  6. 6.

    Chen, J. & Furano, A. V. Breaking bad: the mutagenic effect of DNA repair. DNA Repair (Amst.) 32, 43–51 (2015).

  7. 7.

    Roberts, S. A. et al. Clustered mutations in yeast and in human cancers can arise from damaged long single-strand DNA regions. Mol. Cell 46, 424–435 (2012).

  8. 8.

    Taylor, B. J. et al. DNA deaminases induce break-associated mutation showers with implication of APOBEC3B and 3 A in breast cancer kataegis. eLife 2, e00534 (2013).

  9. 9.

    Nik-Zainal, S. et al. Mutational processes molding the genomes of 21 breast cancers. Cell 149, 979–993 (2012).

  10. 10.

    Burns, M. B., Temiz, N. A. & Harris, R. S. Evidence for APOBEC3B mutagenesis in multiple human cancers. Nat. Genet. 45, 977–983 (2013).

  11. 11.

    Roberts, S. A. et al. An APOBEC cytidine deaminase mutagenesis pattern is widespread in human cancers. Nat. Genet. 45, 970–976 (2013).

  12. 12.

    Helleday, T., Eshtad, S. & Nik-Zainal, S. Mechanisms underlying mutational signatures in human cancers. Nat. Rev. Genet. 15, 585–598 (2014).

  13. 13.

    Chan, K. & Gordenin, D. A. Clusters of multiple mutations: incidence and molecular mechanisms. Annu. Rev. Genet. 49, 243–267 (2015).

  14. 14.

    Mali, P., Esvelt, K. M. & Church, G. M. Cas9 as a versatile tool for engineering biology. Nat. Methods 10, 957–963 (2013).

  15. 15.

    Doudna, J. A. & Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096 (2014).

  16. 16.

    Sander, J. D. & Joung, J. K. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347–355 (2014).

  17. 17.

    Cox, D. B., Platt, R. J. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121–131 (2015).

  18. 18.

    Komor, A. C., Badran, A. H. & Liu, D. R. CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell 168, 20–36 (2017).

  19. 19.

    Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

  20. 20.

    Wu, Y. et al. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell 13, 659–662 (2013).

  21. 21.

    Yin, H. et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 32, 551–553 (2014).

  22. 22.

    Symington, L. S. & Gautier, J. Double-strand break end resection and repair pathway choice. Annu. Rev. Genet. 45, 247–271 (2011).

  23. 23.

    Myler, L. R. et al. Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins. Proc. Natl. Acad. Sci. USA 113, E1170–E1179 (2016).

  24. 24.

    Burns, M. B. et al. APOBEC3B is an enzymatic source of mutation in breast cancer. Nature 494, 366–370 (2013).

  25. 25.

    Refsland, E. W. et al. Quantitative profiling of the full APOBEC3 mRNA repertoire in lymphocytes and tissues: implications for HIV-1 restriction. Nucleic Acids Res. 38, 4274–4284 (2010).

  26. 26.

    Harris, R. S. et al. DNA deamination mediates innate immunity to retroviral infection. Cell 113, 803–809 (2003).

  27. 27.

    Anand, R., Beach, A., Li, K. & Haber, J. Rad51-mediated double-strand break repair and mismatch correction of divergent substrates. Nature 544, 377–380 (2017).

  28. 28.

    Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

  29. 29.

    Cho, S. W. et al. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res. 24, 132–141 (2014).

  30. 30.

    Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M. & Joung, J. K. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32, 279–284 (2014).

  31. 31.

    Tsai, S. Q. et al. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat. Biotechnol. 32, 569–576 (2014).

  32. 32.

    Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

  33. 33.

    Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, aaf8729 (2016).

  34. 34.

    Kim, K. et al. Highly efficient RNA-guided base editing in mouse embryos. Nat. Biotechnol. 35, 435–437 (2017).

  35. 35.

    Zhou, C. et al. Highly efficient base editing in human tripronuclear zygotes. Protein Cell (2017).

  36. 36.

    Li, G. et al. Highly efficient and precise base editing in discarded human tripronuclear embryos. Protein Cell 8, 772–775 (2017).

  37. 37.

    Paquet, D. et al. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533, 125–129 (2016).

  38. 38.

    Carter, R. J. & Parsons, J. L. Base excision repair, a pathway regulated by posttranslational modifications. Mol. Cell. Biol. 36, 1426–1437 (2016).

  39. 39.

    Daley, J. M., Niu, H., Miller, A. S. & Sung, P. Biochemical mechanism of DSB end resection and its regulation. DNA Repair (Amst.) 32, 66–74 (2015).

  40. 40.

    Starrett, G. J. et al. The DNA cytosine deaminase APOBEC3H haplotype I likely contributes to breast and lung cancer mutagenesis. Nat. Commun. 7, 12918 (2016).

  41. 41.

    Long, C. et al. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science 351, 400–403 (2016).

  42. 42.

    Nelson, C. E. et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science 351, 403–407 (2016).

  43. 43.

    Tabebordbar, M. et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science 351, 407–411 (2016).

  44. 44.

    Bonvin, M. et al. Interferon-inducible expression of APOBEC3 editing enzymes in human hepatocytes and inhibition of hepatitis B virus replication. Hepatology 43, 1364–1374 (2006).

  45. 45.

    Kim, Y. B. et al. Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nat. Biotechnol. 35, 371–376 (2017).

  46. 46.

    Li, J., Sun, Y., Du, J., Zhao, Y. & Xia, L. Generation of targeted point mutations in rice by a modified CRISPR/Cas9 system. Mol. Plant 10, 526–529 (2017).

  47. 47.

    Liang, P. et al. Correction of β-thalassemia mutant by base editor in human embryos. Protein Cell 8, 811–822 (2017).

  48. 48.

    Lu, Y. & Zhu, J. K. Precise editing of a target base in the rice genome using a modified CRISPR/Cas9 system. Mol. Plant 10, 523–525 (2017).

  49. 49.

    Rees, H. A. et al. Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat. Commun. 8, 15790 (2017).

  50. 50.

    Shimatani, Z. et al. Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. Nat. Biotechnol. 35, 441–443 (2017).

  51. 51.

    Zhang, Y. et al. Programmable base editing of zebrafish genome using a modified CRISPR-Cas9 system. Nat. Commun. 8, 118 (2017).

  52. 52.

    Zong, Y. et al. Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. Nat. Biotechnol. 35, 438–440 (2017).

  53. 53.

    Hess, G. T., Tycko, J., Yao, D. & Bassik, M. C. Methods and applications of CRISPR-mediated base editing in eukaryotic genomes. Mol. Cell 68, 26–43 (2017).

  54. 54.

    Mitsunobu, H., Teramoto, J., Nishida, K. & Kondo, A. Beyond native Cas9: manipulating genomic information and function. Trends Biotechnol. 35, 983–996 (2017).

  55. 55.

    Komor, A. C. et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T: Abase editors with higher efficiency and product purity. Sci. Adv. 3, eaao4774 (2017).

  56. 56.

    Wang, L. et al. Enhanced base editing by co-expression of free uracil DNA glycosylase inhibitor. Cell Res. 27, 1289–1292 (2017).

  57. 57.

    Shen, B. et al. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat. Methods. 11, 399–402 (2014).

  58. 58.

    Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013).

  59. 59.

    Bogerd, H. P., Wiegand, H. L., Doehle, B. P. & Cullen, B. R. The intrinsic antiretroviral factor APOBEC3B contains two enzymatically active cytidine deaminase domains. Virology 364, 486–493 (2007).

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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.


  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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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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