Project Summary/Abstract RNA editing is a widespread strategy employed by cells to post-transcriptionally alter protein sequence and gene expression levels. High-throughput techniques such as RNA-seq have enabled the identification of putative editing sites and can provide information on the average editing frequency at each of these sites. However, these methods can only measure average RNA editing frequencies on whole cell populations and lack the ability to probe the epitranscriptomic contributions of individual cells. In contrast, the ability to probe RNA editing fre- quency at single-cell resolution can dramatically improve our understanding of the molecular pathogenesis of cancer and other diseases, enabling advances in diagnostic and therapeutic interventions. Here we propose the development and validation of novel multi-sequence capture beads that can be employed with the estab- lished Drop-seq single-cell sequencing pipeline in order to quantitatively assess A-to-I editing frequency across ~104 individual cells. Future application of this technology will enable biomedical researchers to address im- portant questions, including: (1) What is the heterogeneity of editing frequency at each site across a cell popu- lation? (2) Is there interplay of editing frequency between different transcripts within a cell? (3) How does editing frequency at specific sites correlate with expression of other genes in the cell? For our initial validation, we will benchmark the performance of our method using acute myeloid leukemia (AML) cells. However, this technology will be broadly applicable to a wide variety of contexts including multiple cancer types, stem cell differentiation, development, immune function, and mental and neurological disorders.