CRISPR/Cas9 is a versatile tool that allows efficient editing of targeted DNA sequences, providing an unique opportunity to establish reliable models to study gene function during early embryogenesis. In this proposal, I will use this technique to specifically investigate trophectoderm (TE) development, focusing on mouse/human inter-species differences. The TE is crucial for the implantation process, therefore, unraveling its fundamental function could provide insights into the causes of implantation failure after assisted reproductive technology. I will focus on TFAP2C and GATA3, two master regulators of TE, and will document their expression and localization during different stages of mouse/human embryonic development, and assess the consequences of their knock-out by CRISPR/Cas9. The analysis entails both evaluation of lineage-specific markers and known interactors with the two transcription factors and the involved pathways, as well as an unbiased assessment via single-cell transcriptomic approaches. Assessment will take place at the blastocyst level, as well beyond the blastocysts stage, by the establishment of a peri-implantation model, which will be extremely relevant for studying trophectoderm development and maintenance. Lastly, depending on the outcome of Tfap2c/TFAP2C and Gata3/GATA3 knock-out, I will also knock-out highly similar protein isoforms (TFAP2a/c and GATA2/3) to unravel potential functional redundancy in human, as has been shown previously in mouse.