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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.

Early mammalian embryogenesis is controlled by mechanisms that govern the balance between pluripotency and differentiation. Expression of early lineage-specific genes varies substantially between species1-3, with implications for developmental control and stem cell derivation. However, the mechanisms that pattern the human embryo are unclear, because of a lack of methods to efficiently perturb gene expression of early lineage specifiers in this species.

Recent advances in genome editing using the CRISPR (clustered regularly interspaced, short palindromic repeat)-Cas (CRISPRassociated) system have greatly increased the efficiency of genetic modification. The Streptococcus pyogenes Cas9 endonuclease is guided to homologous DNA sequences via a single-guide RNA (sgRNA) whereby it induces double strand breaks (DSBs) at the target site4. Endogenous DNA repair mechanisms function to resolve the DSBs, including error-prone non-homologous or micro-homology- mediated end joining, which can lead to insertions or deletions (indels) of nucleotides that can result in the null mutation of the target gene. CRISPR-Cas9-mediated editing has been attempted in abnormally fertilized tripronuclear human zygotes and a limited number of normally fertilized human zygotes, with variable success5-8. To determine whether CRISPR-Cas9 can be used to understand gene function in human preimplantation development, we chose to target POU5F1, a gene encoding the developmental regulator OCT4, as a proof-of-principle. Zygotic POU5F1 is thought to be first transcribed at the four- to eight-cell...

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