Unravelling the molecular mechanisms governing early embryonic development is of great interest for both assisted reproductive technology (ART) and for the fundamental research field. CRISPR/Cas9 is a versatile gene editing tool, able to efficiently edit targeted DNA sequences in the germline genome. In this research proposal, I will make use of CRISPR/Cas9 germline genome editing to study the mechanisms involved in both trophectoderm (TE) and inner cell mass (ICM) formation, the two cell types formed during first lineage segregation. In a first set of experiments, I will study the function of two important TE markers (Gata2/GATA2, Gata3/GATA3), by generating gene knockouts (KO) in both mouse and human, as important interspecies differences might exist. Analysis of the KO embryos on DNA, RNA and protein level will yield more information about their function following gene KO. Besides, I will also explore the link between these key transcription factors and cytoskeletal components/regulators in determining cell fate during first lineage segregation, using time-lapse confocal imaging. Secondly, I will make use of the same methodology to unravel the function of important ICM markers (Sox2/SOX2, Nanog/NANOG), in both mouse and human embryos. This way, I aim to gain a comprehensive understanding of the mechanisms governing the first lineage segregation, besides unravelling previously unknown interspecies differences.