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CRISPR/Cas9 Embryo Gene-Editing Reveals Species-specific OCT4 Function

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Review of “Genome editing reveals a role for OCT4 in human embryogenesis” from Nature by Stuart P. Atkinson

Initial studies of the gene editing in abnormally fertilized tripronuclear human zygotes and a limited number of normally fertilized human zygotes [1-4] have concentrated on the correction of disease-related mutations. However, a team of researchers from the laboratory of Kathy K. Niakan (Francis Crick Institute, London, UK) sought to follow a different approach.

In their new Nature article, Fogarty et al. targeted the POU5F1 locus (better known as OCT4) in mouse and human embryos via CRISPR/Cas9 gene editing to fully appreciate the role of this pluripotency factor during the development the one-cell zygote through to the blastocyst stage embryo [5]. The potent combination of high-efficiency on-target editing and undetectable off-target editing makes CRISPR/Cas9 the most efficient strategy for embryo manipulation and this approach permitted the authors to discover some significant differences between OCT4 function across these two species.

The team first screened for OCT4-specific single-guide RNAs (sgRNAs) using human embryonic stem cells (hESCs) engineered to constitutively express Cas9 and inducibly express an sgRNA cassette. Excitingly, one sgRNA prompted a 2-bp deletion that generated a frameshift mutation and a premature stop codon, leading to the desired downregulation of OCT4 expression. Of note, targeted deep sequencing revealed no unwanted editing at putative off-target sites

Before moving to human embryos, the authors first confirmed the suitability of their gene-editing strategy via microinjection of gene-editing components into mouse fertilized zygotes. Live embryo imaging demonstrated that a lack of Oct4 still permitted blastocyst formation, but not the outgrowth of the inner cell mass when cultured under mouse ESC derivation conditions. At the transcriptional level, Oct4 loss reduced the expression of some differentiation-associated genes but did not affect pluripotency genes, so suggesting a role for Oct4 in embryo maintenance in mouse.

Finally, CRISPR/Cas9 genome editing of the OCT4 locus in human embryos employed microinjection of an sgRNA–Cas9 ribonucleoprotein complex into in vitro fertilized (IVF) zygotes donated by patients undergoing infertility treatment. Encouragingly, the authors observed high on-target editing efficiency and low off-target activity; however, in contrast to the mouse study, OCT4 loss reduced both viability and quality of human blastocysts, suggesting a role for OCT4 in blastocyst establishment. Indeed, loss of OCT4 in human embryos not only downregulated differentiation-associated genes but also inhibited pluripotency gene expression, thus disrupting embryonic development.

While this research has relevance to both IVF treatments and stem cell therapeutics, the author´s findings also highlight the limitations of relying heavily on animal models in basic research. However, future studies will take encouragement from the observed efficiency of CRISPR/Cas9 genome editing in human zygotes, although the team proposes that higher gene-editing efficiency may be attainable through the co-injection of gene-editing components with sperm during the creation of the fertilized zygote [4].

For all the news on CRISPR/Cas9 gene editing of the human embryo, stay tuned to the Stem Cells Portal.

References

  1. Kang X, He W, Huang Y, et al., Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing. J Assist Reprod Genet 2016;33:581-588.
  2. Tang L, Zeng Y, Du H, et al., CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein. Mol Genet Genomics 2017;292:525-533.
  3. Liang P, Xu Y, Zhang X, et al., CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein & cell 2015;6:363-72.
  4. Ma H, Marti-Gutierrez N, Park S-W, et al., Correction of a pathogenic gene mutation in human embryos. Nature 2017;548:413-419.
  5. Fogarty NME, McCarthy A, Snijders KE, et al., Genome editing reveals a role for OCT4 in human embryogenesis. Nature 2017;550:67.