Genome editing reveals a role for OCT4 in human embryogenesis

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


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Screening sgRNAs targeting OCT4 in optimized inducible CRISPR–Cas9 knockout human ES cells and mouse embryos.
Figure 2: The developmental potential of human embryos following CRISPR–Cas9-mediated genome editing.
Figure 3: Genotypic characterization of OCT4-targeted human embryos.
Figure 4: Phenotypic characterization of OCT4-targeted human embryos.

Accession codes

Primary accessions

Gene Expression Omnibus

Change history

  • 22 September 2017

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


  1. 1

    Chen, A. E. 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

    Niakan, K. K. & Eggan, K. 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

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

  4. 4

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

  5. 5

    Kang, X. 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

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

  7. 7

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

  8. 8

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

  9. 9

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

  10. 10

    Nichols, J. 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

    Bertero, A. 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

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

  13. 13

    Niwa, H., Masui, S., Chambers, I., Smith, A. G. & Miyazaki, J. 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

    Schöler, H. R., Ruppert, S., Suzuki, N., Chowdhury, K. & Gruss, P. New type of POU domain in germ line-specific protein Oct-4. Nature 344, 435–439 (1990)

  15. 15

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

  16. 16

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

  17. 17

    Wang, H. 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

    Le Bin, G. C. 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

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

  20. 20

    Cho, S. W., Lee, J., Carroll, D., Kim, J. S. & Lee, J. Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins. Genetics 195, 1177–1180 (2013)

  21. 21

    Shin, H. Y. 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

    Capmany, G., Taylor, A., Braude, P. R. & Bolton, V. N. The timing of pronuclear formation, DNA synthesis and cleavage in the human 1-cell embryo. Mol. Hum. Reprod. 2, 299–306 (1996)

  23. 23

    Balakier, H., MacLusky, N. J. & Casper, R. F. Characterization of the first cell cycle in human zygotes: implications for cryopreservation. Fertil. Steril. 59, 359–365 (1993)

  24. 24

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

  25. 25

    Vera-Rodriguez, M., Chavez, S. L., Rubio, C., Reijo Pera, R. A. & Simon, C. Prediction model for aneuploidy in early human embryo development revealed by single-cell analysis. Nat. Commun. 6, 7601 (2015)

  26. 26

    Wells, D. 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

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

  28. 28

    Wong, C. C. 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

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

  30. 30

    Yan, L. 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

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

  32. 32

    Kuijk, E. W. 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

    Suzuki, T., Asami, M. & Perry, A. C. Asymmetric parental genome engineering by Cas9 during mouse meiotic exit. Sci. Rep. 4, 7621 (2014)

  34. 34

    Egli, D., Zuccaro, M. V., Kosicki, M., Church, G. M., Bradley, A. & Jasin, M. Inter-homologue repair in fertilized human eggs? Preprint at (2017)

  35. 35

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

  36. 36

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

  37. 37

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

  38. 38

    Brinkman, E. K., Chen, T., Amendola, M. & van Steensel, B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 42, e168 (2014)

  39. 39

    Güell, M., Yang, L. & Church, G. M. Genome editing assessment using CRISPR Genome Analyzer (CRISPR-GA). Bioinformatics 30, 2968–2970 (2014)

  40. 40

    Park, J., Lim, K., Kim, J. S. & Bae, S. Cas-analyzer: an online tool for assessing genome editing results using NGS data. Bioinformatics 33, 286–288 (2017)

  41. 41

    Fragouli, E. 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

    Lou, X., Kang, M., Xenopoulos, P., Muñoz-Descalzo, S. & Hadjantonakis, A. K. 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

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

  44. 44

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

  45. 45

    Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014)

  46. 46

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

  47. 47

    Risso, D., Ngai, J., Speed, T. P. & Dudoit, S. Normalization of RNA-seq data using factor analysis of control genes or samples. Nat. Biotechnol. 32, 896–902 (2014)

  48. 48

    Anders, S., Pyl, P. T. & Huber, W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015)

  49. 49

    Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014)

  50. 50

    Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L. & Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods 5, 621–628 (2008)

Download references


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

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.

Correspondence to Kathy K. Niakan.

Ethics declarations

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.

Additional information

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 figures and tables

Extended Data Figure 1 POU5F1 targeting and comparison of sgRNAs.

a, Schematic representation of the human POU5F1 locus and sgRNA targeting sites. The location (not to scale) and sequences of the sgRNAs tested are shown and the PAM sequences are underlined and in red font. Sequences within the exons are in uppercase and introns are in lowercase. The mouse sgRNA sequences are shown below. The exons encoding the N-terminal domain (NTD), POU DNA-binding domain or the C-terminal domain (CTD) are indicated. b, Representative flow cytometry analysis quantifying OCT4 expression in human ES cells induced to express each sgRNA over 5 days compared to uninduced controls. The percentage of OCT4 protein expression is shown. c, qRT–PCR analysis after 4 days of sgRNA induction in mTeSR medium. Relative expression reflected as fold difference over uninduced cells normalized to GAPDH. Data points and mean for all samples are shown: n = 2 sgRNA1-1 clones; n = 3, sgRNA 1-2, 2b or 4 clones, representative of two independent experiments and ± s.e.m. where there are three samples. Two-way ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. d, Heat maps of selected genes showing unsupervised hierarchical clustering of uninduced and sgRNA2b-induced human ES cells. Normalized RNA-seq expression levels are plotted on a high-to-low scale (purple–white–green). Source data

Extended Data Figure 2 Further characterization of sgRNA2b-induced human ES cells.

a, Human ES cells induced to express sgRNA2b for 4 days (+Tet) in chemically defined medium with activin A and FGF2 (CDM/AF) compared to uninduced controls (No Tet). Immunofluorescence analysis for the pluripotency markers OCT4, NANOG and SOX2 or markers associated with differentiation to early derivatives of the germ layers (SOX1-expressing ectoderm cells or SOX17-expressing endoderm cells). DAPI nuclear staining (blue) is shown. Scale bars, 400 μm. b, qRT–PCR analysis for selected genes associated with either pluripotency or differentiation to derivatives of the germ layers in human ES cells induced to express each of the sgRNAs for 4 days. Relative expression reflected as fold difference over wild-type human ES cells and normalized to PBGD. Data points and mean ± s.e.m. are shown: n = 3 wild-type H9 and sgRNA2b, representative of two independent experiments. Two-way ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, ns, not significant. Source data

Extended Data Figure 3 On-target mutation spectrum in human ES cells induced to express sgRNA1-1, sgRNA1-2, sgRNA2b or sgRNA4.

Shown are frequent types of indel mutations and corresponding sequences observed in human ES cells induced to express sgRNA1-1, sgRNA1-2, sgRNA2b or sgRNA4. The cells were induced to express each sgRNA for 4 days and the data shown are representative of the types of indel mutations observed in other clonal lines (n = 2 sgRNA1-1 clones; n = 3, sgRNA 1-2, 2b or 4 clones) and across time (from 1 to 4 days following induction of each sgRNA).

Extended Data Figure 4 Off-target analysis of sgRNA2b-induced human ES cells.

a, The POU5F1 sgRNA2b 12-bp seed sequence is highlighted in green and the NGG PAM sequence in red. In black are the nucleotide sequences 5′ to the sgRNA seed sequence. Seven putative off-target sequences and associated genes are shown including POU5F1 pseudogenes. In orange are the nucleotides that differ from the sgRNA2b sequence. b, Percentage of indel mutations detected at putative off-target sites in human ES cells 4 days after tetracycline induction of sgRNA2b compared to uninduced controls. Data are percentages of indel mutations detected by targeted deep sequencing in the cell lines at each of the sites indicated. Comparisons made between three clonal human ES cell lines induced to express sgRNA2b versus uninduced controls. The percentage of indel mutations induced at the on-target site were significantly different while all other sites were not significantly different. Two-way ANOVA. ***P < 0.001. c, Digenome-seq results displayed as a genome-wide circos plot. The height of the peak corresponds to the DNA cleavage score. The red arrow points to the POU5F1 locus on chromosome 6. d, Percentage of indel mutations observed in sgRNA2b-induced human ES cells and in wild-type H9 control cells at each locus following targeted deep sequencing of putative off-target sites identified by Digenome-seq. e, Off-target candidate nucleotides displayed as sequence logos using the WebLogo program. f, Percentage of indel mutations observed in sgRNA2b-induced human ES cells and in wild-type H9 control cells following targeted deep sequencing of putative off-target sites determined by WebLogo sequence homology. Source data

Extended Data Figure 5 Assessing a range of Cas9 and sgRNA combinations for microinjection into mouse pronuclear zygotes.

a, b, Additional conditions were tested in mouse embryos microinjected with the sgRNA2b either with Cas9 mRNA (a) or as a complex with the Cas9 protein (b) at the ratios indicated. Quantification was performed on the proportion of mouse embryos at the blastocyst stage that are phenotypically null (loss of OCT4 and SOX17 protein expression), mosaic or heterozygous (partial OCT4 and/or SOX17 expression) or uninjected (strong OCT4 and SOX17 expression). Data are mean ± s.d. from three independent experiments. Comparisons were made between the percentage of OCT4-null embryos observed versus wild-type uninjected control embryos. Chi-squared test. *P < 0.05; ***P < 0.001; ****P < 0.0001. c, The types of indel mutations detected in mouse embryos microinjected with the sgRNA2b–Cas9 complex. The sgRNA sequence is boxed and the NGG PAM site underlined. Dash, deletion position. d, Further characterization of mouse embryos microinjected with sgRNA2b–Cas9 compared to uninjected control blastocysts. Immunofluorescence analysis for markers of the trophectoderm (CDX2) or primitive endoderm (GATA4, GATA6, PDGFRA and SOX7) lineages together with DAPI nuclear staining. Confocal z-section. Scale bars, 100 μm. e, Quantification of blastocyst inner cell mass (ICM) or trophoblast outgrowths in mouse embryonic stem cell derivation conditions. Uninjected, Cas9-injected or Cas9 plus Dmc1 sgRNA-injected cells (targeting a gene not essential for preimplantation development) were used as controls. Comparisons were made between the percentage of ICM outgrowths observed in blastocysts that developed following sgRNA2b–Cas9 microinjection. Two-tailed t-test. *P < 0.05. Source data

Extended Data Figure 6 Further assessing human embryo quality.

a, Karyotype analysis following whole-genome sequencing of either single blastomeres, a clump of three cells from a cleavage stage embryo or a clump of 3–5 cells from trophectoderm biopsies. Multiple biopies were analysed from embryos C8, C12 and C16. Analysis was also performed on blastocysts that developed following microinjection of Cas9 protein. The type of chromosome gains and losses are indicated. b, Representative karyotype analysis by whole-genome sequencing of human blastocysts. A representative graph indicating aneuploidy in embryos following Cas9 protein and sgRNA2b–Cas9 ribonucleoprotein complex microinjection. c, Phase-contrast images of starting blastocysts and blastocysts that developed following microinjection of the sgRNA2b–Cas9 complex compared to Cas9 protein-injected controls. White arrows point to the presumptive inner cell mass and a black arrow to a representative zona pellucida.

Extended Data Figure 7 Evaluating on-target and putative off-target mutations in human embryo cells.

a, The type and relative proportion of indel mutations observed compared to all observable indel mutations within each human embryo. b, Quantification of indels by TIDE analysis. Representative plots and Sanger sequencing chromatograms are shown from OCT4-null, heterozygous and wild-type human cells. c, Percentage of indel mutations detected at the sgRNA2b on-target site and putative off-target sites in single cells microdissected from Cas9 protein-microinjected control blastocysts or blastocysts that developed following sgRNA2b–Cas9 complex microinjection. Putative off-target sites were evaluated in cells that were previously determined to be OCT4-null (green), heterozygous (orange) or wild-type (blue) along with samples from Cas9 protein-microinjected embryos (red). Three representative examples of wild-type and edited cells are shown. d, Sanger sequencing chromatograms from OCT4-null single cells collected from human blastocysts that developed following sgRNA2b–Cas9 microinjection. The chromatograms exemplify the sequence detected in all of the other samples analysed. Underlined is the sequence of the putative off-target site. Source data

Extended Data Figure 8 Phenotypic characterization of OCT4-targeted embryos.

a, Immunofluorescence analysis for OCT4 (green) and DAPI nuclear staining (blue) in human cleavage stage embryos following sgRNA2b–Cas9 complex microinjection (n = 5). Confocal z-section. Arrow, OCT4-expressing cell. Scale bars, 100 μm. b, Immunofluorescence analysis for OCT4 (green), SOX17 (red) and DAPI nuclear staining (blue) in an uninjected control blastocyst (n = 3) or a human blastocyst that developed following sgRNA2b–Cas9 complex microinjection (n = 3). Confocal z-section. Scale bars, 100 μm. c, d, Immunofluorescence analysis for OCT4 (green), NANOG (red) and DAPI nuclear staining (blue) in a human blastocyst that developed following sgRNA2b–Cas9 complex microinjection (c, n = 3) or in a mouse uninjected control blastocyst or in blastocysts that developed following sgRNA2b–Cas9 complex microinjection (d, n = 7). Confocal z-section. Scale bars, 100 μm. e, Quantification of NANOG and OCT4 expression in mouse uninjected control blastocysts (n = 5) or in blastocysts that developed following sgRNA2b–Cas9 complex microinjection (n = 7). One-tailed t-test. **P < 0.01. f, Immunofluorescence analysis for GATA2 (green) and DAPI nuclear staining (blue) in a human blastocyst that developed following sgRNA2b–Cas9 complex microinjection (n = 3). Confocal projection. Scale bar, 100 μm. Source data

Extended Data Figure 9 Transcriptome analysis of OCT4-targeted embryos.

a, Hierarchical clustering and heat map of a selection of genes following single-cell RNA-seq analysis of human embryos. Embryos C8, C9, C12 and C16 (samples denoted in orange font) were targeted with the sgRNA2b–Cas9 complex. Embryos 2, 5, 7 and 8 were microinjected with Cas9 protein as a control. An uninjected control reference dataset labelled PE (primitive endoderm cells), EPI (epiblast cells) or TE (trophectoderm cells) is included3. Control cells clustered according to lineage and are indicated with the coloured bars: red, primitive endoderm; green, epiblast; and blue, trophectoderm. Grey bar highlights the samples that have low expression of markers of each of the lineages shown. The genotypes of the samples are noted as POU5F1 wild-type (WT), heterozygous (Het) or knockout (KO). Five samples failed repeated genotyping but the RNA quality is good and these are listed as X. Normalized expression levels are plotted on a high–low scale (purple–white–green). b, c, Principal component analysis of a previously published human single-cell RNA-seq dataset30 integrated with the data from the Cas9 protein control and the sgRNA2b–Cas9 ribonucleoprotein (RNP) complex-microinjected embryos. Each point represents a single cell. Data were plotted along the second and third (b) or the first and third (c) principal components.

Extended Data Table 1 Reagent list

Supplementary information

Reporting Summary (PDF 74 kb)

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. (XLSX 4955 kb)

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. (CSV 6 kb)

Human pronuclear stage zygote microinjected with sgRNA2b/Cas9 ribonucleoprotein complex

Video of human pronuclear stage zygote microinjected with sgRNA2b/Cas9 ribonucleoprotein complex. (MP4 22466 kb)

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. (AVI 29627 kb)

Development of a human embryo following microinjection of Cas9 protein

Development of a human embryo following microinjection of Cas9 protein. (AVI 30180 kb)

PowerPoint slides

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Fogarty, N., McCarthy, A., Snijders, K. et al. Genome editing reveals a role for OCT4 in human embryogenesis. Nature 550, 67–73 (2017).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.