Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Correspondence
  • Published:

Off-target mutations are rare in Cas9-modified mice

Nature Methods volume 12, page 479 (2015)Cite this article

Subjects

Access through your institution
Buy or subscribe

To the Editor:

The clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas system is a highly efficient technology for genome editing of mouse zygotes1. Previously, we reported cotransmission of a Cas9-induced mutation in the X-linked Ar gene and an off-target mutation to offspring of founder animals from pronuclear injection of Cas9 mRNA and two single guide RNAs (sgRNAs)2. No off-target damage is observed in offspring derived from founder animals injected with Cas9 nickase mRNA and a pair of sgRNAs, showing that CRISPR-Cas specificity is improved by employing nickases2,3. Here, we used whole-genome sequencing to more thoroughly assess any damage induced by injection of Cas9 versus Cas9 nickase.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

References

  1. Wang, H. et al. Cell 153, 910–918 (2013).

    Article  CAS  Google Scholar 

  2. Shen, B. et al. Nat. Methods 11, 399–402 (2014).

    Article  CAS  Google Scholar 

  3. Ran, F.A. et al. Cell 154, 1380–1389 (2013).

    Article  CAS  Google Scholar 

  4. Smith, C. et al. Cell Stem Cell 15, 12–13 (2014).

    Article  CAS  Google Scholar 

  5. Veres, A. et al. Cell Stem Cell 15, 27–30 (2014).

    Article  CAS  Google Scholar 

  6. Suzuki, K. et al. Cell Stem Cell 15, 31–36 (2014).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the Sanger Institute sequencing pipeline for whole-genome sequencing. This work was supported by the 973 program (2011CB944301) to X.H. and a core grant from the Wellcome Trust to W.C.S.

Author information

Author notes

  1. Vivek Iyer and Bin Shen: These authors contributed equally to this work.

Authors and Affiliations

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

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

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

    Bin Shen & Xingxu Huang

  3. Cambridge-Suda Genome Research Center, Soochow University, Suzhou, China

    Wensheng Zhang

Authors
  1. Vivek Iyer
    View author publications

    Search author on:PubMed Google Scholar

  2. Bin Shen
    View author publications

    Search author on:PubMed Google Scholar

  3. Wensheng Zhang
    View author publications

    Search author on:PubMed Google Scholar

  4. Alex Hodgkins
    View author publications

    Search author on:PubMed Google Scholar

  5. Thomas Keane
    View author publications

    Search author on:PubMed Google Scholar

  6. Xingxu Huang
    View author publications

    Search author on:PubMed Google Scholar

  7. William C Skarnes
    View author publications

    Search author on:PubMed Google Scholar

Corresponding authors

Correspondence to Xingxu Huang or William C Skarnes.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Confirmation of the Ar mutation from the analysis of whole-genome sequencing

The raw PINDEL output (top panel) and pileup of supporting sequence reads (lower panel) for F1 animal F18-9 shows a heterozygous deletion of 58 bp in exon 1 of Ar, previously characterized in founder animal F18 by PCR sequencing2. Note the fall in read coverage across the region and the mapping of soft-clipped reads.

Supplementary Figure 2 Cas9-induced damage at a bona fide off-target site, OTS3

The raw PINDEL output (top panel) and pile up of supporting reads (lower panel) for F1 animal F18-9 shows a heterozygous deletion of 2 bp in OTS3 (Chr8;:121017179-121017180), detected previously by PCR sequencing2.

Supplementary Figure 3 Alignment of Ar sgRNAs to high-quality variants do not correspond to a bona fide off-target site

The PAM site in the genomic sequence is highlighted in red text. (a) Ungapped alignment showing 7 mismatches between the sgRNA and genome sequence. The variant sequence is located 9 bp upstream of the predicted Cas9 cleavage site (arrowhead). The 2bp deletion detected at this site (Chr10:39516743) in animal F18-6 is underlined. (b) Gapped alignment showing 4 mismatches. The variant lies outside of the target sequence, 8 bp downstream of the predicted Cas9 cut site (arrowhead). The 6 bp deletion detected at this site (Chr3:141956378) in animals F25-5 and F25-6 is underlined.

Supplementary information

Supplementary Text, Figures and Tables

Supplementary Figures 1–3, Supplementary Tables 1 and 2 and Supplementary Methods (PDF 348 kb)

Supplementary Data

List of high quality variant sites and sequences (XLSX 2837 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Iyer, V., Shen, B., Zhang, W. et al. Off-target mutations are rare in Cas9-modified mice. Nat Methods 12, 479 (2015). https://doi.org/10.1038/nmeth.3408

Download citation

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1038/nmeth.3408

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research