The Beginnings of Difference: Using Prime Editing to Investigate Early Embryonic Development, Yonit Krebs, UG '24 (A3432925)

Following fusion of the egg and sperm during fertilization, the embryo divides to form a structure called a blastocyst. Differences between cells in the embryo emerge by the formation of the early blastocyst. At this point the outer layer of cells has differentiated and will form the trophectoderm, the future placenta, while the inner cluster of cells will become the rest of the embryo. In mice, this five-day transition from one-cell to blastocyst is followed by implantation, and although timescales differ among mammalian species, much of preimplantation development is conserved. Therefore, studying this process is crucial for understanding early mammalian development. Prime editing, a new gene editing approach developed by the Posfai and Adamson labs at Princeton University to precisely edit early embryos holds much promise. However, tools for predicting the efficiency of prime editing guide RNA (pegRNA) molecules have not been evaluated in embryos. Evaluation of two tools found that while screening for pegRNA sequences in human cell lines was an effective approach, DeepPrime, a computational model was less successful. Use of prime editing to interfere with gene function along with live imaging of embryo development further demonstrated the power of this approach. Loss of CDH1, a protein responsible for forming contacts between cells, severely disrupted the embryo's ability to form the typical blastocyst structure and resulted in changes in regulation of the first cell differentiation event. This study demonstrates the feasibility of this technique, opening up avenues for further research into the critical events of preimplantation development.