Project Summary/Abstract Gene editing technologies enable the efficient targeted introduction of desired genetic alterations of interest in a wide range of organisms and have broad applications for biological research and human therapeutics. First- generation gene editing platforms use site-specific endonucleases (e.g., RNA-guided CRISPR-Cas nucleases) to induce targeted DNA double-stranded breaks, using cellular DNA repair mechanisms to create mutations. More recently developed gene editing platforms include the CRISPR cytosine or adenine base editors, which use Cas9-directed single-stranded DNA deaminases to induce targeted C-to-T or A-to-G alterations, respectively, and CRISPR-guided prime editors (which use a reverse transcriptase-dependent process that can induce any nucleotide substitution or small indel mutations). Although gene editing technology development has accelerated over the past decade, many important limitations of existing platforms have yet to be addressed and opportunities remain to create multiple new impactful technologies enabled by recent advances. For the next cycle of this MIRA, the focus will be on four broadly important Challenges: 1) expand the sensitivities and scope of gene editor off-target detection technologies; 2) engineer next-generation prime editors with enhanced and optimized architectures; 3) create new epigenetic editing platforms for targeted heritable activation of human genes; and 4) develop new universal modalities for delivery of all gene editors to human cells.