• An Erratum to this article was published on 04 October 2017

This article has been updated


Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR–Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR–Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.

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  • 22 September 2017

    In the AOP version of this Letter, the received date was corrected on 22 September 2017.


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Gene Expression Omnibus


  1. 1.

    et al. Optimal timing of inner cell mass isolation increases the efficiency of human embryonic stem cell derivation and allows generation of sibling cell lines. Cell Stem Cell 4, 103–106 (2009)

  2. 2.

    & Analysis of human embryos from zygote to blastocyst reveals distinct gene expression patterns relative to the mouse. Dev. Biol. 375, 54–64 (2013)

  3. 3.

    et al. Defining the three cell lineages of the human blastocyst by single-cell RNA-seq. Development 142, 3613 (2015)

  4. 4.

    et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012)

  5. 5.

    et al. Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing. J. Assist. Reprod. Genet. 33, 581–588 (2016)

  6. 6.

    et al. CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein. Mol. Genet. Genomics 292, 525–533 (2017)

  7. 7.

    et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell 6, 363–372 (2015)

  8. 8.

    et al. Correction of a pathogenic gene mutation in human embryos. Nature 548, 413–419 (2017)

  9. 9.

    et al. Oct4 cell-autonomously promotes primitive endoderm development in the mouse blastocyst. Dev. Cell 25, 610–622 (2013)

  10. 10.

    et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95, 379–391 (1998)

  11. 11.

    et al. Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs. Development 143, 4405–4418 (2016)

  12. 12.

    et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013)

  13. 13.

    , , , & Phenotypic complementation establishes requirements for specific POU domain and generic transactivation function of Oct-3/4 in embryonic stem cells. Mol. Cell. Biol. 22, 1526–1536 (2002)

  14. 14.

    , , , & New type of POU domain in germ line-specific protein Oct-4. Nature 344, 435–439 (1990)

  15. 15.

    et al. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat. Methods 12, 237–243 (2015)

  16. 16.

    , , , & Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq. Genome Res. 26, 406–415 (2016)

  17. 17.

    et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013)

  18. 18.

    et al. Oct4 is required for lineage priming in the developing inner cell mass of the mouse blastocyst. Development 141, 1001–1010 (2014)

  19. 19.

    et al. Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes. Dev. Biol. 393, 3–9 (2014)

  20. 20.

    , , , & Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins. Genetics 195, 1177–1180 (2013)

  21. 21.

    et al. CRISPR/Cas9 targeting events cause complex deletions and insertions at 17 sites in the mouse genome. Nat. Commun. 8, 15464 (2017)

  22. 22.

    , , & The timing of pronuclear formation, DNA synthesis and cleavage in the human 1-cell embryo. Mol. Hum. Reprod. 2, 299–306 (1996)

  23. 23.

    , & Characterization of the first cell cycle in human zygotes: implications for cryopreservation. Fertil. Steril. 59, 359–365 (1993)

  24. 24.

    , , , & Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 (2014)

  25. 25.

    , , , & Prediction model for aneuploidy in early human embryo development revealed by single-cell analysis. Nat. Commun. 6, 7601 (2015)

  26. 26.

    et al. Clinical utilisation of a rapid low-pass whole genome sequencing technique for the diagnosis of aneuploidy in human embryos prior to implantation. J. Med. Genet. 51, 553–562 (2014)

  27. 27.

    et al. Chromosomal aneuploidies and early embryonic developmental arrest. Int. J. Fertil. Steril. 9, 346–353 (2015)

  28. 28.

    et al. Non-invasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage. Nat. Biotechnol. 28, 1115–1121 (2010)

  29. 29.

    et al. G&T-seq: parallel sequencing of single-cell genomes and transcriptomes. Nat. Methods 12, 519–522 (2015)

  30. 30.

    et al. Single-cell RNA-seq profiling of human preimplantation embryos and embryonic stem cells. Nat. Struct. Mol. Biol. 20, 1131–1139 (2013)

  31. 31.

    et al. Human hypoblast formation is not dependent on FGF signalling. Dev. Biol. 361, 358–363 (2012)

  32. 32.

    et al. The roles of FGF and MAP kinase signaling in the segregation of the epiblast and hypoblast cell lineages in bovine and human embryos. Development 139, 871–882 (2012)

  33. 33.

    , & Asymmetric parental genome engineering by Cas9 during mouse meiotic exit. Sci. Rep. 4, 7621 (2014)

  34. 34.

    , , , , & Inter-homologue repair in fertilized human eggs? Preprint at (2017)

  35. 35.

    et al. A global reference for human genetic variation. Nature 526, 68–74 (2015)

  36. 36.

    et al. Genome engineering using the CRISPR–Cas9 system. Nat. Protocols 8, 2281–2308 (2013)

  37. 37.

    et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013)

  38. 38.

    , , & Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 42, e168 (2014)

  39. 39.

    , & Genome editing assessment using CRISPR Genome Analyzer (CRISPR-GA). Bioinformatics 30, 2968–2970 (2014)

  40. 40.

    , , & Cas-analyzer: an online tool for assessing genome editing results using NGS data. Bioinformatics 33, 286–288 (2017)

  41. 41.

    et al. Analysis of implantation and ongoing pregnancy rates following the transfer of mosaic diploid-aneuploid blastocysts. Hum. Genet. 136, 805–819 (2017)

  42. 42.

    , , , & A rapid and efficient 2D/3D nuclear segmentation method for analysis of early mouse embryo and stem cell image data. Stem Cell Reports 2, 382–397 (2014)

  43. 43.

    Serum-free and feeder-free culture conditions for human embryonic stem cells. Methods Mol. Biol. 690, 57–66 (2011)

  44. 44.

    et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013)

  45. 45.

    , & featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014)

  46. 46.

    et al. SCnorm: robust normalization of single-cell RNA-seq data. Nat. Methods 14, 584–586 (2017)

  47. 47.

    , , & Normalization of RNA-seq data using factor analysis of control genes or samples. Nat. Biotechnol. 32, 896–902 (2014)

  48. 48.

    , & HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015)

  49. 49.

    , & Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014)

  50. 50.

    , , , & Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods 5, 621–628 (2008)

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We thank the generous donors whose contributions have enabled this research; M. Macnamee, P. Snell and L. Christie at Bourn Hall Clinic for their support and assistance with the donation of embryos; T. Hiroda, P. Singh and J. Schimenti for the DMC1 sgRNA sequence and product; R. Lovell-Badge, I. Henderson, J. Haber, J. Rossant and A. Handyside for discussions and advice; the Wellcome Trust policy advisers, especially K. Littler and S. Rappaport, as well as J. Lawford-Davies and M. Chatfield for advice and support; and the Francis Crick Institute’s Biological Resources, Advanced Light Microscopy, High Throughput Sequencing, Research Illustration (Fig. 2a) and Bioinformatics facilities. D.W. was supported by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme. N.K. was supported by the University of Oxford Clarendon Fund and Brasenose College Joint Scholarship. A.B. was supported by a British Heart Foundation PhD Studentship (FS/11/77/39327). K.E.S. was supported by the NIHR Cambridge BRC. L.V. was supported by core grant funding from the Wellcome Trust and Medical Research Council (PSAG028). J.-S.K. was supported by the Institute for Basic Science (IBS-R021-D1). Work in the K.K.N. and J.M.A.T. labs was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK, the UK Medical Research Council, and the Wellcome Trust (FC001120 and FC001193). Work in the K.K.N. laboratory was also supported by the Rosa Beddington Fund.

Author information

Author notes

    • Alessandro Bertero

    Present address: Department of Pathology, University of Washington, Seattle, Washington 98109, USA.


  1. Human Embryo and Stem Cell Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK

    • Norah M. E. Fogarty
    • , Afshan McCarthy
    • , Paul Blakeley
    • , Rebecca Lea
    • , Sissy E. Wamaitha
    •  & Kathy K. Niakan
  2. NIHR Cambridge Biomedical Research Centre hIPSC Core Facility, Department of Surgery, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0SZ, UK

    • Kirsten E. Snijders
    •  & Ludovic Vallier
  3. Sex Chromosome Biology Laboratory, The Francis Crick Institute, London NW1 1AT, UK

    • Benjamin E. Powell
    • , Valdone Maciulyte
    •  & James M. A. Turner
  4. Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK

    • Nada Kubikova
    •  & Dagan Wells
  5. Bourn Hall Clinic, Bourn, Cambridge CB23 2TN, UK

    • Kay Elder
  6. Department of Chemistry, Seoul National University, Seoul 151-747, South Korea

    • Daesik Kim
    •  & Jin-Soo Kim
  7. Bioinformatics Facility, The Francis Crick Institute, London NW1 1AT, UK

    • Jens Kleinjung
  8. Center for Genome Engineering, Institute for Basic Science, Daejeon 34047, South Korea

    • Jin-Soo Kim
  9. Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SA, UK

    • Ludovic Vallier
  10. Wellcome Trust and MRC Cambridge Stem Cell Institute and Biomedical Research Centre, Anne McLaren Laboratory, Department of Surgery, University of Cambridge, Cambridge CB2 0SZ, UK

    • Ludovic Vallier
    •  & Alessandro Bertero


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K.K.N. conceived the project, designed and performed experiments, microinjected embryos and analysed data. N.M.E.F. performed single-cell analysis, human ES cell experiments, human and mouse embryo phenotyping and genotyping. A.M. performed genotyping of human ES cells, stem cell derivation, mouse embryo phenotyping and generated the sgRNAs. K.E.S. generated the inducible human ES cells, independently performed human ES cell phenotyping and performed flow cytometry analysis. A.B. designed and assisted with human ES cell experiments and L.V. and A.B. supervised the experiments. N.K. and D.W. performed cytogenetic analysis and independently confirmed human embryo genotyping analysis. K.E. coordinated donation of embryos to the research project. B.E.P. generated some of the sgRNAs used in mice and supplied sgRNA sequences. P.B. and J.K. performed the RNA-seq analysis. R.L. and S.E.W. assisted with phenotyping. D.K. and J.-S.K. performed Digenome-seq analysis. V.M. assisted with genotyping. K.K.N., J.M.A.T. and N.M.E.F. wrote the manuscript with help from all of the authors. All authors assisted with experimental design, generated figures and/or commented on the manuscript.

Competing interests

J.-S.K. is a co-founder of and holds stocks in ToolGen, Inc., a company focused on genome editing. All other authors declare no competing financial interests.

Corresponding author

Correspondence to Kathy K. Niakan.

Reviewer Information Nature thanks D. Egli, J. Kimmelman 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

    DESeq analysis of genes that are differentially expressed. RNA-seq dataset generated from human single cell samples at the blastocyst stage following microinjection of sgRNA2b/Cas9 ribonucleoprotein complex compared to Cas9 injected controls.

CSV files

  1. 1.

    Supplementary Table 2

    This file contains the read depth and alignment rate for each single-cell RNA-seq sample from the Cas9 microinjected controls and the sgRNA2b/Cas9 ribonucleoprotein complex microinjected embryos collected at the blastocyst stage.


  1. 1.

    Human pronuclear stage zygote microinjected with sgRNA2b/Cas9 ribonucleoprotein complex

    Video of human pronuclear stage zygote microinjected with sgRNA2b/Cas9 ribonucleoprotein complex.

  2. 2.

    Development of a human embryo following microinjection of the sgRNA2b/Cas9 ribonucleoprotein complex. AVI format

    Development of a human embryo following microinjection of the sgRNA2b/Cas9 ribonucleoprotein complex.

  3. 3.

    Development of a human embryo following microinjection of Cas9 protein

    Development of a human embryo following microinjection of Cas9 protein.

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