Epitranscriptomic Markers in Microbiome-Derived Cell-Free RNA from Plasma for Colorectal Cancer Diagnosis - PROJECT SUMMARY Circulating cell-free DNA (cfDNA) and RNA (cfRNA) offer enormous potential as a new class of biomarkers for developing non-invasive liquid biopsies applicable to numerous diseases and conditions. Recent years have seen intense study of cfDNA and cfRNA as tools for non-invasive prenatal testing, solid organ transplantation, cancer screening, and tumor monitoring. However, early cancer detection has been hindered by the low concentration of tumor-derived cfDNA in plasma, resulting in decreased sensitivity and specificity. While epigenetic-based cfDNA sequencing approaches have been developed; however, their effectiveness remains limited by the low abundance of target analyte at early stages. Cell-free RNA (cfRNA) presents a promising alternative, yet its practical application of cfRNA faces hurdles: rapid degradation of cell-free mRNA, insufficient quantities in clinical samples, and cellular contamination altering mRNA levels, thus compromising the detection specificity. Moreover, the study of epitranscriptomic markers from cfRNA remains challenging due to the lack of robust sequencing methods. To address these critical knowledge gaps, this project aims to (i) profile the transcriptomics and epitranscriptomics of plasma cfRNA, (ii) develop epitranscriptomic patterns for microbiome- derivative cfRNA as biomarkers for colorectal cancer (CRC) diagnosis. As described in the preliminary data in this application, I have developed an innovative method called Low-Input Multiple MEthylation Sequencing (LIME-seq) to monitor abundance and site-specific modifications across multiple RNA species from ultra-low- input cfRNA (sourced from only 600 μL plasma). Applying this method to human plasma samples, I have successfully uncovered the presence of tRNAs and specific ncRNAs as major cfRNA components originating from both human and microbiome sources. Importantly, I discovered that methylation patterns in microbiome- derived cfRNA represent an effective class of non-invasive biomarkers with unprecedented potential for early CRC diagnosis, offering a distinct advantage over microbial abundance profiles obtained from cfRNA/cfDNA sequences. These methylation patterns accurately reflect microbiota activity, surpassing traditional abundance- based methods and providing a more prescise approach to study human microbiota. Leveraging the LIME-seq technique, I will conduct a comparative analysis between the transcriptomic and epitranscriptomic landscapes of specific microbiomes and that of plasma cfRNA. This will validate the presence of certain microbiome-derived cfRNA and confirm the RNA methylation sites. I will also demonstrate the utility of microbiome-derived cfRNA profiles sourced from plasmain CRC diagnosis, with a particular focus on adenoma and stage I. The projected outcomes of this study will generate both diagnostic and predictive biomarkers from methylation sites of microbiome-derived cfRNA and provide a non-invasive method for assessing microbiota. Furthermore, a more ambitious vision for this research could be to identify novel microbiome targets, which could become potential therapeutic strategies for CRC prevention and treatment.