Genome editing is an essential tool for life sciences. Recent ground-breaking discovery in microbiology drew our attention to the genome editing ability of bacteria (CRISPR). Since its discovery, CRISPR has revolutionized the way of editing a genome. Despite its wide use, CRISPR-genome editing has limitations, especially in the use for medical applications. Numerous studies have shown that it suffers from the off-target effect. Its use is also restricted by its particular sequence requirement and its poor accessibility to a structured genome. Furthermore, recent studies suggested that it might act as a virulence factor within human cells. These limitations demand new genome editing tools. This proposal sets out to understand the molecular mechanism of Tetrahymena DNA elimination. This naturally occuring genome editing is mediated by a eukaryotic RNA system (Twi1). This system uses an entirely different mechanism from CRISPR and has potential to perform more effectively. I will first investigate how small RNA-loaded Twi1 (“target searcher”) recognizes its target and whether its performance exceeds other target searchers including CRISPR/Cas9. I will use single-molecule fluorescence for high resolution observations and develop a high-throughput single-molecule method for transcriptome-wide understanding. Second, I aim to identify a Twi1-related DNA nuclease(s) that carries out DNA elimination. I will use cutting-edge tools of single-molecule pull-down and multi-color FRET together with mass spectrometry. The nanoscopic understanding of a searcher (Twi1) and the identification of a nuclease will help create a new genome editing tool (e.g. a fusion of Twi1 and the nuclease) that potentially perform better than Cas9. Thereby, this fundamental study on “mighty RNA” will make a long-term impact for applications in science and technology. To realize this ambitious project, I will utilize my experience of studying small RNAs (funded by ERC Starting Grant).