Article

Correction of a pathogenic gene mutation in human embryos

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Abstract

Genome editing has potential for the targeted correction of germline mutations. Here we describe the correction of the heterozygous MYBPC3 mutation in human preimplantation embryos with precise CRISPR–Cas9-based targeting accuracy and high homology-directed repair efficiency by activating an endogenous, germline-specific DNA repair response. Induced double-strand breaks (DSBs) at the mutant paternal allele were predominantly repaired using the homologous wild-type maternal gene instead of a synthetic DNA template. By modulating the cell cycle stage at which the DSB was induced, we were able to avoid mosaicism in cleaving embryos and achieve a high yield of homozygous embryos carrying the wild-type MYBPC3 gene without evidence of off-target mutations. The efficiency, accuracy and safety of the approach presented suggest that it has potential to be used for the correction of heritable mutations in human embryos by complementing preimplantation genetic diagnosis. However, much remains to be considered before clinical applications, including the reproducibility of the technique with other heterozygous mutations.

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Change history

  • Updated online 02 October 2017

    Editorial Note: Readers are alerted that some of the conclusions of this paper are subject to critiques that are being considered by editors. Some of these critiques have been publicly deposited in preprint form. A further editorial response will follow the resolution of these issues.

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Acknowledgements

We acknowledge the OHSU Institutional Review Board (IRB), Innovative Research Advisory Panel (IRAP), Scientific Review Committee (SRC) and Data Safety Monitoring Committee (DSMC) for oversight and guidance on this study. We thank all study participants for tissue donations; the Women’s Health Research Unit staff, IVF laboratory staff, University Fertility Consultants and the Reproductive Endocrinology and Infertility Division in the Department of Obstetrics and Gynecology, OHSU for support and procurement of human gametes; S. Olson and Research Cytogenetics Laboratory at OHSU for cytogenetic analysis of ES cells; S. Cooper from the Wallace Division of Smiths Medical for donating ICSI and gamete manipulation micropipettes; Y. Wang, T. Wu and Y. Shen from BGI-Shenzhen for help with sample preparation, sequencing and data analyses; M. Ku from the H. A. and Mary K. Chapman Charitable Foundations Genomic Sequencing Core of the Salk Institute for next generation sequencing; and E. Aizawa and R. Hernandez-Benitez from the laboratory of JCIB for assistance. Studies conducted at OHSU were supported by OHSU institutional funds. Work in the laboratory of J.-S.K. was supported by the Institute for Basic Science (IBS-R021-D1). Work in the laboratory of J.C.I.B. was supported by the G. Harold and Leila Y. Mathers Charitable Foundation, the Moxie Foundation and the Leona M. and Harry B. Helmsley Charitable Trust. Work at BGI was supported by the Shenzhen Municipal Government of China (DRC-SZ [2016] 884).

Author information

Author notes

    • Hong Ma
    • , Nuria Marti-Gutierrez
    • , Sang-Wook Park
    •  & Jun Wu

    These authors contributed equally to this work.

    • Juan Carlos Izpisua Belmonte
    • , Paula Amato
    • , Jin-Soo Kim
    • , Sanjiv Kaul
    •  & Shoukhrat Mitalipov

    These authors jointly supervised this work.

Affiliations

  1. Center for Embryonic Cell and Gene Therapy, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, Oregon 97239, USA

    • Hong Ma
    • , Nuria Marti-Gutierrez
    • , Yeonmi Lee
    • , Amy Koski
    • , Dongmei Ji
    • , Tomonari Hayama
    • , Riffat Ahmed
    • , Hayley Darby
    • , Crystal Van Dyken
    • , Ying Li
    • , Eunju Kang
    • , David Battaglia
    • , Don P. Wolf
    • , Paula Amato
    •  & Shoukhrat Mitalipov
  2. Center for Genome Engineering, Institute for Basic Science, 70, Yuseong-daero 1689-gil, Yuseong-gu, Daejeon, 34047, Republic of Korea

    • Sang-Wook Park
    • , A.-Reum Park
    • , Sang-Tae Kim
    •  & Jin-Soo Kim
  3. Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, USA

    • Jun Wu
    • , Keiichiro Suzuki
    •  & Juan Carlos Izpisua Belmonte
  4. Department of Chemistry, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul, 151-747, Republic of Korea

    • Daesik Kim
    •  & Jin-Soo Kim
  5. BGI-Shenzhen, Build 11, Beishan Industrial Zone, Yantian District, Shenzhen, 518083, China

    • Jianhui Gong
    • , Ying Gu
    •  & Xun Xu
  6. China National GeneBank, BGI-Shenzhen, Jinsha Road, Dapeng District, Shenzhen, 518210, China

    • Jianhui Gong
    • , Ying Gu
    •  & Xun Xu
  7. BGI-Qingdao, 2877 Tuanjie Road, Sino-German Ecopark, Qingdao, 266000, China

    • Jianhui Gong
    • , Ying Gu
    •  & Xun Xu
  8. Shenzhen Engineering Laboratory for Innovative Molecular Diagnostics, BGI-Shenzhen, Build 11, Beishan Industrial Zone, Yantian District, Shenzhen, 518083, China

    • Jianhui Gong
  9. Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Oregon Health & Science University, 3303 Southwest, Bond Avenue, Portland, Oregon 97239, USA

    • David Battaglia
    • , Sacha A. Krieg
    • , David M. Lee
    • , Diana H. Wu
    •  & Paula Amato
  10. Knight Cardiovascular Institute, Oregon Health & Science University, 3181 Southwest, Sam Jackson Park Road, Portland, Oregon 97239, USA

    • Stephen B. Heitner
    • , Sanjiv Kaul
    •  & Shoukhrat Mitalipov

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Contributions

S.M., S.K., Y.Le., N.M.-G. and H.M. conceived the study and designed the experiments. S.-W.P., J.-S.K. and K.S. designed CRISPR–Cas9 constructs. A.K., S.B.H. and S.K. coordinated recruitment of gamete donors. Y.Le. and R.A. derived patient iPSCs and S.-W.P., J.W.,.K.S and J.C.I.B. tested the specificity of the CRISPR–Cas9 systems in patient iPSCs. P.A., D.B., D.M.L., S.A.K. and D.H.W. conducted ovarian stimulation and oocyte retrieval. N.M.-G., Y.Le., D.J. and T.H. performed CRISPR–Cas9 injections, fertilizations, embryo culture and blastomere isolation experiments. N.M.-G., D.J., H.D., C.V.D., Y.Li and E.K. performed DNA extraction, PCR and Sanger sequencing. S.-W.P., A.-R.P., D.K., S.-T.K. and J.-S.K. performed Digenome-seq, WGS and analyses and independently corroborated embryo on-target editing by deep sequencing. J.G., Y.G., X.X. and J.W. performed whole-exome sequencing and analyses. H.M., N.M.-G., S.-W.P., J.W., A.K., D.P.W., S.B.H., J.C.I.B., P.A., J.-S.K., S.K. and S.M. analysed the data and wrote the manuscript.

Competing interests

J.-S.K. is a co-founder and shareholder of ToolGen, Inc, and S.M. is a co-founder and shareholder of Mitogenome Therapeutics, Inc and Health and Science Center “M1” Inc and declare competing financial interests. The other authors declare no competing financial interests.

Corresponding authors

Correspondence to Juan Carlos Izpisua Belmonte or Paula Amato or Jin-Soo Kim or Sanjiv Kaul or Shoukhrat Mitalipov.

Reviewer Information Nature thanks F. Lanner and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Reporting summary

Excel files

  1. 1.

    Supplementary Table 1

    Genotypes of individual iPSC clones

  2. 2.

    Supplementary Table 2

    Genotypes of individual blastomeres in control embryos

  3. 3.

    Supplementary Table 3

    Genotypes of individual blastomeres in zygote injected embryos

  4. 4.

    Supplementary Table 4

    Genotypes of individual blastomeres in M-phase injected embryos

  5. 5.

    Supplementary Table 5

    Mutagenic indel frequencies at potential off-target sites captured by Digenome-sequencing

  6. 6.

    Supplementary Table 6

    Whole exome sequencing (WES) analyses of variants identified using Hg19 as reference genome

  7. 7.

    Supplementary Table 7

    List of predicted off-target sites

  8. 8.

    Supplementary Table 8

    WES analyses of non-inherited variants

Videos

  1. 1.

    Video: CRISPR/Cas9 injection into human oocytes and zygotes

    CRISPR/Cas9 injection into human oocytes and zygotes. S-phase CRISPR/Cas9 injection was performed 18 hours after ICSI. M-phase injections were co-injected with sperm during ICSI.

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