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

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

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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).

The SNP analysis unveiled between 10,004 and 13,731 SNPs in each of the 36 mice, demonstrating that all samples had a comparable number of SNPs relative to that in the reference genome (Supplementary Table 3). The parent population carried between 10,004 and 12,501 SNPs (Supplementary Note 3, Supplementary Table 4), whereas 11,186–13,024 were present in the noninjected control mice and 11,236–13,731 were present in the CRISPR–Cas9-edited mice. Thus, the number of SNPs present in the CRISPR–Cas9-edited mice was in the same range as that identified in the controls and the parents. After subtracting the mutations present in the parent population from those identified in the offspring, we obtained ~100 de novo SNPs (Supplementary Data) in each progeny. Consistent with the similar number of overall detected SNPs, CRISPR–Cas9-edited mice did not have an increased frequency of mutations compared with that in control mice (Fig. 1c, Supplementary Table 3).

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Fig. 1: Parent–progeny study.

Data Availability

The data are available at SRA under project number PRJNA470569.

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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.

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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
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  2. Harold E. Smith
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  5. Lothar Hennighausen
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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.

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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

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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

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