Base Editing–Enabled Technologies and Multiplex Genome Editing

Abstract

Base editing is a precision genome editing technology that enables targeted single-nucleotide changes without requiring double-strand DNA breaks. This dissertation presents several distinct advances in multiplex base editing and base editing–enabled technologies in mammalian systems. First, we demonstrate large-scale editing of transposable elements, which can be targeted with a single guide RNA due to their repetitive nature. Using catalytically inactive Cas9 base editors, which minimize editing-associated cytotoxicity, we achieve several thousand edits per cell. Second, we develop Genomic Sequence Encryption (GSE), a cryptographic framework that uses multiplex base editing and pooled guide RNAs to encode information across more than one hundred distinct genomic loci. We implement GSE in mammalian cell lines and stem cells, establishing a robust method for introducing a high number of edits in both bulk populations and individual stem cells. We devise an enrichment strategy that enables the isolation of stem cells carrying more than two dozen distinct precision edits across a single diploid genome with minimal screening. This represents a significant advancement in the scale of simultaneous precision editing achievable in primary or stem cells, and, in the context of GSE, paves the way for encrypted genomic signatures in living animals. Lastly, we develop reprogrammable ADAR sensors, a programmable RNA-sensing platform that links endogenous transcript detection to protein translation through A-to-I RNA editing. Together, these contributions expand the scope of base editing by enabling large-scale genome modification, secure biological information encoding, and transcript-responsive regulation in mammalian cells.