High-Resolution Phylogenetic Lineage Recording with CRISPR Base Editors

Dividing and differentiating cells form exquisitely organized structures across every facet of multicellular life. If we could measure the complete history of cells as they divide, change transcriptional state, and move spatially, we could address critical questions about stem cell differentiation, development, and the onset of disease. However, determining cellular ontologies is challenging except in rare cases where continual optical access is possible. Base editing technology enables the generation of stochastic, heritable mutations into genomic DNA while cells grow and divide. Comparing mutation patterns between cells allows inference of their lineage relationships in a manner analogous to evolutionary phylogenetic reconstruction. Here, we present two phylogenetic recording systems that enable high resolution lineage reconstruction over long time scales. In the first system, termed baseMEMOIR, we introduce a multiplexed, genomically dispersed set of editable targets that can be read out by imaging in situ. This system preserves spatial organization of cells and is compatible with downstream transcriptional measurements. In the second system, which we term the hypercascade, we take advantage of the predictability of A-to-G base editing to create a system in which edits not only alter bases but also generate new editable target sites in synthetic sequences. This behavior linearizes the rate at which mutations accumulate, improving lineage reconstruction. These methods enable analysis of temporal dynamics in diverse biological contexts.