Investigations of the programmed DNA elimination (PDE) process of the unicellular ciliate Tetrahymena have provided important insights into how eukaryotes use small noncoding RNAs to regulate transposable elements (TEs), which impact genome organization and integrity. Despite the two decades of efforts to clarify the small RNA-directed PDE mechanism, previous studies using candidate-driven reverse genetics by gene knockout and biochemical protein complex purifications have failed to identify many of the molecular factors involved in this process. To further understand the molecular mechanism of small RNA-directed PDE in Tetrahymena, in this proposed project, we aim to optimize the CRISPR-Cas13-based gene knock-down method in Tetrahymena and establish sorting methods isolating cells and nuclei that are defective in different steps of PDE. Using these methods, we will perform genome-wide forward genetic screening to identify and characterize previously undescribed genes involved in PDE. In parallel, we also aim to use proximity labeling to identify and characterize novel proteins associated with the factors known to be involved in PDE. We expect that the proposed study will provide important insights into the evolution of small RNA-directed TE silencing as well as advanced understandings about the conserved molecular principles for small RNA-mediated epigenetic chromatin regulation. Moreover, we believe that the establishment of a cells sorting-based high-throughput forward genetics protocol will revolutionize Tetrahymena genetics to understand the various fundamental processes of eukaryotes.