Differential identification of genes related to implantation process via use of CRISPR/Cas9

Implantation failure occurs when chorionic gonadotropin secreted by trophoectoderm cells is not produced during the invasion of the embryo into the endometrium, or if it is produced, it cannot form the gestational sac. Even in spontaneous pregnancies where assisted reproductive techniques are not applied, the probability of implantation failure on the first attempt varies between 25-40%. This project aims to elucidate the activity, interactions, temporal and cell type-dependent variability of genes affecting implantation, changes in in vitro conditions, and their effects on implantation using the efficient CRISPR/Cas9 genome editing method. To achieve this goal, samples were collected at 24 and 72 hours from blastocysts incubated in in vitro three-dimensional culture environment in different experimental groups. RNA sequence analysis was performed on these samples and active genes were revealed. For each identified active gene, silencing was performed separately on endometrial epithelial cells and endometrial stroma cells using the CRISPR/Cas9 method. In vitro implantation parameters were examined for each group. The study focused on Hmga2 in endometrial epithelial cells, Uxs1 genes in stromal cells, and Gm15452 genes (a pseudogene) in both epithelial and stromal cells. Knockout of these genes using the CRISPR/Cas9 method caused significant changes in the relevant genes identified as adhesion, invasion, and developmental and functional implantation parameters. Knockout of Hmga2 in epithelial cells revealed significant changes in E-Cadherin, Fibronectin, LIF, Mmp9, and Wnt4 genes at different time groups. Knocking out of Gm15452 in epithelial cells led to significant changes in E-Cadherin and LIF expressions. Uxs1 knockout in stroma cells affected the expressions of E-Cadherin, LIF, and Mmp9, while Gm15452 knockout in stroma cells significantly affected the expressions of E-Cadherin, LIF, and Mmp9.