Understanding the intricate interplay between host factors and viral pathogens remains a focal point of research in molecular virology. Here, we describe a new role for Y-box binding protein 1 (YBX1) during Human Cytomegalovirus (HCMV) infection in regulating the translation of mRNAs encoding the immediate early proteins IE1 and IE2. In this study, we introduce a novel technique for investigating RNA-protein interactions, leveraging the strengths of CRISPR-Cas13, allowing for precise and sensitive exploration of RNA-protein interactions during infection in a temporal manner. Through this approach, we explored the role of YBX1 in HCMV replication, providing a deeper understanding of the molecular host-pathogen interactions. This research not only advances our knowledge of YBX1’s role in viral pathogenesis, but also introduces a powerful tool for unraveling RNA-protein dynamics that can be used in a wide range of biological processes.