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.

Mutation frequency is not increased in CRISPR–Cas9-edited mice

Nature Methods volume 15, pages 756–758 (2018)Cite this article

Subjects

We microinjected Cas9 mRNA, a single guide RNA and one donor oligonucleotide into fertilized eggs (Supplementary Methods). Detailed descriptions of the insertions at the target locus can be found in Supplementary Notes 1 and 2. We observed a high frequency of complex modified alleles, as well as more than two mutant alleles in several of the mice, which is evidence that CRISPR-induced mutagenesis continues beyond the one-cell stage. We applied GATK best practices with joint genotyping3,4,5 and Lumpy6 to identify single-nucleotide polymorphisms (SNPs), simple insertions and deletions (indels) and larger/complex indels. The parent–progeny design allowed us to explicitly identify putative de novo mutations (Fig. 1b, Supplementary Methods).

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

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

Fig. 1: Parent–progeny study.

Data Availability

The data are available at SRA under project number PRJNA470569.

References

  1. Schaefer, K. A. et al. Nat. Methods 14, 547–548 (2017).

    Article  CAS  Google Scholar 

  2. Anonymous. Nat. Methods 15, 229 (2018).

  3. McKenna, A. et al. Genome Res. 20, 1297–1303 (2010).

    Article  CAS  Google Scholar 

  4. Van der Auwera, G. A. et al. Curr. Protoc. Bioinformatics 43, 11.10.1–11.10. 33 (2013).

    Google Scholar 

  5. DePristo, M. A. et al. Nat. Genet. 43, 491–498 (2011).

    Article  CAS  Google Scholar 

  6. Layer, R. M., Chiang, C., Quinlan, A. R. & Hall, I. M. Genome Biol. 15, R84 (2014).

    Article  Google Scholar 

  7. Shin, H. Y. et al. Nat. Commun. 8, 15464 (2017).

    Article  CAS  Google Scholar 

  8. Iyer, V. et al. PLoS Genet. 14, e1007503 (2018).

    Article  Google Scholar 

  9. Luo, X. et al. bioRxiv Preprint at https://www.biorxiv.org/content/early/2018/06/05/339143 (2018).

Download references

Acknowledgements

This study was funded through the IRPs of NIDDK and NHLBI. Research support was received from the NIH Director’s Challenge Innovation Award program (to L.H.). We also thank the team of the NIH HPC Biowulf cluster, especially W. Resch, for their advice, as well as their continued maintenance of hardware and software. We acknowledge the Genomics Platform at The Broad Institute for their support and services provided for the generation of the WGS data.

Author information

Author notes

  1. These authors contributed equally: Michaela Willi and Harold E. Smith.

Authors and Affiliations

  1. Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health, Bethesda, MD, USA

    Michaela Willi, Chaochen Wang & Lothar Hennighausen

  2. Genomics Core, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health, Bethesda, MD, USA

    Harold E. Smith

  3. Transgenic Core, National Heart, Lung, and Blood Institute, US National Institutes of Health, Bethesda, MD, USA

    Chengyu Liu

Authors
  1. Michaela Willi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Harold E. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chaochen Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chengyu Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lothar Hennighausen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.W., H.E.S., C.L., C.W. and L.H. designed the study. C.L. and C.W. conducted mouse experiments. M.W. and H.E.S. carried out computational analyses. M.W., H.E.S. and L.H. wrote the manuscript.

Corresponding authors

Correspondence to Michaela Willi or Lothar Hennighausen.

Ethics declarations

Competing interests

The authors declare no competing interests.

Integrated supplementary information

Supplementary Figure 1 Equivalence test.

(a) The equivalence test for the de novo SNPs is significant, which verifies that the number of de novo SNPs is equivalent. (CRISPR and control group: n = 6; δ = +/- 1.35) (b) The equivalence test for the de novo indels is inconclusive, as the 90% CI reaches over zero and the lower boundary (CRISPR and control group: n = 6; δ = +/- 0.82).

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1, Supplementary Methods, Supplementary Notes 1–4 and Supplementary Tables 1–7

Reporting Summary

Supplementary Data

De novo mutations in CRISPR–Cas9-edited and control mice

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Willi, M., Smith, H.E., Wang, C. et al. Mutation frequency is not increased in CRISPR–Cas9-edited mice. Nat Methods 15, 756–758 (2018). https://doi.org/10.1038/s41592-018-0148-2

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41592-018-0148-2

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