Letter | Published:

Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements

Nature Biotechnology volume 36, pages 765771 (2018) | Download Citation

  • An Erratum to this article was published on 06 September 2018

This article has been updated

Abstract

CRISPR–Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR–Cas9 was reasonably specific. Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR–Cas9 editing may have pathogenic consequences.

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Change history

  • 31 July 2018

    In the version of this article initially published online, four figure citations were incorrect on p.2: left-hand column, after "complex rearrangements," "Supplementary Fig. 2a,b" should have been "Fig. 2a,b"; right-hand column, in three places, the citation for "Supplementary Fig. 3..." should have been for "Supplementary Fig. 2." The errors have been corrected for the print, PDF and HTML versions of this article.

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European Nucleotide Archive

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Acknowledgements

We wish to thank M. Friedrich for sharing his gRNA expression construct and technical advice, E. Metzakopian for technical advice and critical reading of the manuscript, G. Rutledge for critical reading of the early manuscript, A. Ferguson-Smith for the CAST/B6 hybrid ES cells, P. Liu and X. Gao for mCherry/GFP reporter cells, S. Jackson's group for the Cas9-expressing RPE1 cell line and the Cytometry Core Facility for assistance with cell sorting. This work was supported by the Wellcome Trust Grant number 098051.

Author information

Affiliations

  1. Wellcome Sanger Institute, Hinxton, UK.

    • Michael Kosicki
    • , Kärt Tomberg
    •  & Allan Bradley

Authors

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Contributions

M.K. performed most of the experiments and analyzed the data. K.T. performed the primary cell work. A.B. supervised the project. All authors contributed to writing of the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Allan Bradley.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–7

  2. 2.

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Note

    Supplementary Note 1

Excel files

  1. 1.

    Supplementary Table 1

    Flow cytometry results and gRNA sequences.

  2. 2.

    Supplementary Table 2

    Automatic annotation of all PigA alleles in the study

  3. 3.

    Supplementary Table 3

    Detailed description of PigA alleles recovered from single cell clones.

  4. 4.

    Supplementary Table 4

    Diagnostic PCRs at the PigA locus.

  5. 5.

    Supplementary Table 5

    Summary of PCR genotyping experiments

  6. 6.

    Supplementary Table 6

    PCR primers.

Text files

  1. 1.

    Supplementary Data 1

    PigA alleles

Zip files

  1. 1.

    Supplementary Data 2

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DOI

https://doi.org/10.1038/nbt.4192

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