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Response to “Unexpected mutations after CRISPR–Cas9 editing in vivo”

Nature Methods volume 15, pages 238–239 (2018)Cite this article

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To the Editor:

Schaefer et al.1 recently stated that CRISPR–Cas9 nuclease can induce off-target alterations at genomic loci that do not resemble the intended on-target site. This new proposed CRISPR–Cas9 off-target activity runs contrary to previously published work (performed mostly in cells, but also in mice)2,3,4,5,6 and, if the authors are correct, could have profound implications for research and therapeutic applications. However, we here demonstrate that the simplest interpretation of data in Schaefer et al.1 is that the two CRISPR–Cas9-treated mice are genetically more closely related to each other than to the control mouse. This strongly suggests that the so-called 'unexpected mutations' simply represent shared single-nucleotide polymorphisms (SNPs) and indels that existed before nuclease treatment.

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Figure 1: Measures of genetic relatedness in the F03, F05, and FVB mice.

References

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Acknowledgements

This work was supported by the National Science Foundation (DGE1144152 to C.A.L.), National Institutes of Health (R35 GM118158 to J.K.J. and R00 HG008399 to L.P.), the Defense Advanced Research Projects Agency (HR0011-17-2-0042 to J.K.J. and L.P.), and the Desmond and Ann Heathwood Massachusetts General Hospital Research Scholar Award (J.K.J.). We thank B. Kleinstiver, J. Lopez, and A. Anthony for helpful discussions.

Author information

Author notes

  1. Caleb A Lareau, Kendell Clement and Jonathan Y Hsu: These authors contributed equally to this work.

Authors and Affiliations

  1. Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA

    Caleb A Lareau, Kendell Clement, Jonathan Y Hsu, Vikram Pattanayak, J Keith Joung, Martin J Aryee & Luca Pinello

  2. Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA

    Caleb A Lareau & Martin J Aryee

  3. Cell Circuits and Epigenomics Program, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

    Caleb A Lareau, Kendell Clement, Martin J Aryee & Luca Pinello

  4. Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA

    Kendell Clement, Vikram Pattanayak, J Keith Joung, Martin J Aryee & Luca Pinello

  5. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA

    Kendell Clement

  6. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Jonathan Y Hsu

Authors
  1. Caleb A Lareau
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  2. Kendell Clement
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  3. Jonathan Y Hsu
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  4. Vikram Pattanayak
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  5. J Keith Joung
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Corresponding authors

Correspondence to J Keith Joung, Martin J Aryee or Luca Pinello.

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Competing interests

J.K.J. has financial interests in Beam Therapeutics, Editas Medicine, Monitor Biotechnologies, Pairwise Plants, Poseida Therapeutics, and Transposagen Biopharmaceuticals. M.J.A. has financial interests in Monitor Biotechnologies. J.K.J.'s and M.J.A.'s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies.

Integrated supplementary information

Supplementary Figure 1 Reproduction of reported variant calls.

Variants were called using the intersection of three somatic cancer mutation calling software frameworks for each mouse using FVB as a “normal” tissue. We were able to reproduce 91.1% and 93.5% of the previously reported variants (green; left) though our analysis framework differed slightly as we employed GATK best practices. The number of mutations called and concordant with the previously reported variants was similar for an identical iteration of the pipeline run on downsampled data for the F03 and F05 mice (right), indicating that the differential coverage reported by the authors did not have a substantial impact on the analysis.

Supplementary Figure 2 Measures of genetic relatedness between the three mice.

Panel E shows the computed genetic distance between the three mice under each of the two models (Panels A, C) and observed variants (Panels B, D).

Supplementary Figure 3 Distance distributions of Schaefer et al.-reported SNVs to closest Cas-OFFinder predictions.

Log10 minimum distances were plotted for all Schaefer et al.-reported SNVs to the closest Cas-OFFinder in-silico off-target prediction (mismatch up to 6bp, DNA/RNA bulges up to 2bp). Three independent randomly sampled common dbSNPs were also plotted to assess the background of the analysis.

Supplementary Figure 4 Motif enrichment at variants identified through the Schaefer et al. pipeline.

Motif sequences were inferred using HOMER. Motifs were inferred using sequences of 100bp centered at the SNPs obtained with the pipeline proposed by Schaefer et al. (mm10 reference genome) for the groups F03 vs FVB and F05 vs FVB as target sets. No enriched motifs were present for both SNP sets, leaving no clear mechanism for potential shared DNA sequence recognition by Cas9.

Supplementary Figure 5 Empirical evaluation of indel concordance in F03 and F05 mice.

We sought to quantify the empirical probability that 118 indels shared between F03 and F05 mice would all be the same length (left). From empirical indel distributions described in van Overbeek et al., we simulated paired indel occurrences (right) to highlight the heterogeneity of the indel outcome process. With the least heterogeneous indel size distribution, we use the binomial test to assess the significance of observing 118 paired indel size matches (p = 1.5 e-42).

Supplementary Figure 6 SNP substitution comparison between F03 and F05.

Heatmaps showing the overlap of the nucleotide observed at a SNV in F03, and the nucleotide observed in F05 at the same location. In each heatmap, only SNVs that are homozygous reference, and then show a variant in both F03 and F05 are shown. Notably, the density along the diagonal shows that a vast majority (> 99%) of variant loci shared in F03 and F05 also share the same nucleotide base.

Supplementary Figure 7 Characterization of mutations called from variant calling pipelines.

Left: Pie chart showing the fraction of dbSNP variants called by the GATK pipeline that have the same genotype in F03 and F05 (but not in FVB). Right: Variants called by the Schaefer et al. pipeline were mapped to the closest dbSNP variant. This pie chart shows the fraction of these dbSNP variants that had the same genotype in F03 and F05. The variants that are closest to calls from the Schaefer et al. pipeline are significantly enriched for variants whose genotype is shared between F03 and F05.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Notes 1–6 and Supplementary Table 1 (PDF 2437 kb)

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Lareau, C., Clement, K., Hsu, J. et al. Response to “Unexpected mutations after CRISPR–Cas9 editing in vivo”. Nat Methods 15, 238–239 (2018). https://doi.org/10.1038/nmeth.4541

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