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Unexpected mutations after CRISPR–Cas9 editing in vivo

A Retraction to this article was published on 27 April 2018

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Figure 1: CRISPR gene correction introduces an unexpectedly high number of mutations in a mouse model of gene therapy.

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Change history

  • 14 June 2017

    Editorial Note: readers are alerted that the conclusions of this paper are subject to criticisms that are being considered by editors. A further editorial response will follow the resolution of these issues.

  • 25 July 2017

    Editorial Expression of Concern: The editors of Nature Methods are issuing an editorial expression of concern regarding this paper to alert our readers to concerns about interpretation of the data. Multiple groups have questioned the interpretation that single nucleotide changes seen in whole-genome sequences of two CRISPR–Cas9-treated mice are due to the CRISPR treatment. Since the background genetic variation between the control mouse and the CRISPR-treated animals is not known, an alternative proposed interpretation is that the observed changes are due to normal genetic variation. We are in contact with the critics and with the authors to examine this matter further. We will update our readers once these investigations are complete. All the authors do not agree with the journal's decision to issue an editorial expression of concern.

  • 30 March 2018

    This paper is being retracted because the genomic variants observed by the authors in two CRISPR-treated mice cannot be conclusively attributed to CRISPR–Cas9. The paper was a peer-reviewed Correspondence in the journal. The authors made their observation as part of their work on correction of a gene involved in blindness. The authors used mice of the inbred FVB/NJ strain from the JAX genetic quality control program that were purchased within months of each other and that were not bred in the authors' laboratory. The assumption was that this design was sufficient to control for genetic variation in an inbred strain. Since publication of the work, however, it has been brought to the journal's and the authors' attention that without parental controls or more analysis of genetic background, it is not certain that the variants reported are due to CRISPR treatment (https://doi.org/10.1038/nmeth.4559, https://doi.org/10.1038/nmeth.4552, https://doi.org/10.1038/nmeth.4553, https://doi.org/10.1038/nmeth.4541, https://doi.org/10.1038/nmeth.4554). The study is therefore being retracted to maintain the accuracy of the scientific record. S.H.T. and W.-H.W. agree with the retraction. K.A.S., D.F.C., A.G.B. and V.B.M. do not agree with the retraction. All authors note that there is very little whole-genome sequencing data on the effects of CRISPR treatment in vivo. The question of whether CRISPR has effects on the in vivo genome will require further study; the authors are carrying out follow-up studies using whole-genome sequencing.

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Acknowledgements

We acknowledge the efforts and expertise of the New York Genome Center. S. Wu assisted with data analysis. K.A.S. is supported by NIH grant F31EY026789. V.B.M. and A.G.B. are supported by NIH grants (R01EY026682, R01EY024665, R01EY025225, R01EY024698, R01NS098590 and R21AG050437) and Research to Prevent Blindness (RPB), New York, New York. The Bernard & Shirlee Brown Glaucoma Laboratory is supported by NIH grants (5P30EY019007, R01EY018213). The National Cancer Institute Core is supported by an NIH grant (5P30CA013696), the RPB Physician-Scientist Award, and unrestricted funds from RPB, New York, New York, USA. S.H.T. is a member of the RD-CURE Consortium and is supported by the Tistou and Charlotte Kerstan Foundation, the Schneeweiss Stem Cell Fund, New York State (grant C029572), the Crowley Family Fund, and the Gebroe Family Foundation.

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Correspondence to Alexander G Bassuk or Vinit B Mahajan.

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Integrated supplementary information

Supplementary Figure 1 CRISPR-dependent mutations favor transitions over transversions.

Heat maps represent the percentage of specific point mutation nucleotide changes detected in CRISPR-treated animals. For both animals, most mutations were G to A and C to T mutations.

Supplementary Figure 2 Sanger sequencing confirms CRISPR-induced mutations detected by WGS.

a. Example of a coding SNV confirmed to be heterozygous in F03. F05 is wildtype as is reported in Supplementary Tables 1 and 2. b. Example of an intronic SNV confirmed in both mice as reported in Supplementary Table 5. c. Example of an SNV in a snoRNA confirmed in both mice as reported in Supplementary Table 5.

Supplementary Figure 3 Sequence alignment of guide RNA to actual off-target regions does not show substantial homology.

a. The top-10 predicted, off-target regions aligned to the gRNA. Sequences are 80 to 95% homologous to the gRNA. b. Regions surrounding 10 selected, experimentally-observed SNVs in coding regions aligned to the gRNA. All regions were observed in both CRISPR-treated mice. Sequences are 15 to 45% homologous to the gRNA. c. Regions surrounding 10 selected, experimentallyobservedSNVs in non-coding regions aligned to the gRNA. All regions were observed in both CRISPR-treated mice. Sequences are 5-65% homologous to the gRNA. d. Regions surrounding 10 selected, experimentally-observed indels in both coding and non-coding regions aligned to the gRNA. All regions were observed in both CRISPR-treated mice. Sequences are 25 to 65% homologous to the gRNA.

Supplementary Figure 4 Off-target CRISPR mutations could cause unwanted phenotypes.

Pie charts show the SNVs and indels detected in WGS of CRISPR-treated animals based on assigned biotype. Biotypes were assigned by SNPEff software (Cingolani P, et al. Fly, 2012.). Intragenic regions were not assigned a biotype.

Supplementary information

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Supplementary Figures 1–4, Supplementary Methods and Supplementary Tables 1–5 (PDF 3893 kb)

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Schaefer, K., Wu, WH., Colgan, D. et al. Unexpected mutations after CRISPR–Cas9 editing in vivo. Nat Methods 14, 547–548 (2017). https://doi.org/10.1038/nmeth.4293

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