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Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects

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Abstract

Bacterial RNA–directed Cas9 endonuclease is a versatile tool for site-specific genome modification in eukaryotes. Co-microinjection of mouse embryos with Cas9 mRNA and single guide RNAs induces on-target and off-target mutations that are transmissible to offspring. However, Cas9 nickase can be used to efficiently mutate genes without detectable damage at known off-target sites. This method is applicable for genome editing of any model organism and minimizes confounding problems of off-target mutations.

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Figure 1: NHEJ-mediated mutations induced in mice by Cas9 nickases.
Figure 2: Optimal orientation and distance of paired sgRNA for efficient Cas9 nickase–induced modification.

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Acknowledgements

We thank members of the entire Huang laboratory for their support and advice, G. Tischler for the C++ alignment code, and J. Liu and C. Gao for help with deep sequencing. This work was supported by grants from the 973 program (2010CB945101 and 2011CB944301) and a core grant from the Wellcome Trust.

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Author notes

  1. Bin Shen, Wensheng Zhang and Jun Zhang: These authors contributed equally to this work.

Authors and Affiliations

  1. Ministry of Education Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University, Nanjing, China

    Bin Shen, Jun Zhang, Jiankui Zhou, Jianying Wang, Li Chen & Xingxu Huang

  2. Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK

    Wensheng Zhang, Alex Hodgkins, Vivek Iyer & William C Skarnes

  3. Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China

    Lu Wang

Authors
  1. Bin Shen
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  2. Wensheng Zhang
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  3. Jun Zhang
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  4. Jiankui Zhou
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  5. Jianying Wang
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  6. Li Chen
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  7. Lu Wang
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  8. Alex Hodgkins
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  9. Vivek Iyer
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  10. Xingxu Huang
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Contributions

B.S., W.Z., J. Zhang, X.H. and W.C.S. designed the experiments and wrote the manuscript. B.S., W.Z., J. Zhang, J. Zhou, J.W. and L.C. performed the experiments. L.W., A.H. and V.I. performed the computational analysis of off-target sites.

Corresponding authors

Correspondence to Xingxu Huang or William C Skarnes.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10, Supplementary Tables 1–14 and Supplementary Note (PDF 3275 kb)

Supplementary Data

Paired off-target sites for AR-A and AR-B sgRNAs. Ten closest paired sites in the mouse genome with similar sequence to each combination of AR-A and AR-B Cas9 target sites. (XLS 92 kb)

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Shen, B., Zhang, W., Zhang, J. et al. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat Methods 11, 399–402 (2014). https://doi.org/10.1038/nmeth.2857

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  • DOI: https://doi.org/10.1038/nmeth.2857

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