MitoScribe single-cell molecular recorder logs graded signaling dynamics into mitochondrial DNA

Genetically encoded DNA recorders convert transient biological events into stable genomic mutations, offering a means to reconstruct past cellular states. However, current approaches to log historical events by modifying genomic DNA have limited capacity to record the magnitude of biological signals within individual cells. Here, we introduce MitoScribe, a mitochondrial DNA (mtDNA)-based recording platform that uses mtDNA base editors (DdCBEs) to write graded biological signals into mtDNA as neutral, single-nucleotide substitutions at a defined site. Taking advantage of the hundreds to thousands of mitochondrial genome copies per cell, we demonstrate MitoScribe enables reproducible, highly sensitive, non-destructive, durable, and high-throughput measurements of molecular signals, including hypoxia, NF-{kappa}B activity, BMP and Wnt signaling. We show multiple modes of operation, including multiplexed recordings of two independent signals, and coincidence detection of temporally overlapping signals. Coupling MitoScribe with single-cell RNA sequencing and mitochondrial transcript enrichment, we further reconstruct signaling dynamics at the single-cell transcriptome level. Applying this approach during the directed differentiation of human induced pluripotent stem cells (iPSCs) toward mesoderm, we show that early heterogeneity in response to a differentiation cue predicts the later cell state. Together, MitoScribe provides a scalable platform for high-resolution molecular recording in complex cellular contexts.