Genome Editing by Dual Nucleases Records Deep Lineage Hierarchy with High Fidelity

Single-cell lineage tracing based on CRISPR/Cas9 gene editing tracks both cell states and genetic lineages at a single-cell resolution. Despite accurately dissecting cell fate determination, current implementations of this technology are hindered by limited recording intervals and considerable dropout of recording information. Here, we introduce a new method, DuTracer, which utilizes two inducible gene editing systems to record deep cell lineage history at single-cell resolution, minimizing target dropouts. Application of DuTracer in mouse embryoid bodies demonstrates the transition pattern of the lineage potency hierarchy across different developmental stages. Moreover, we have developed an entropy-based approach to quantify the lineage recording ability of DuTracer in cell differentiation models. Together, aided with entropy-based quantification of the barcoding process, DuTracer facilitates the elaborate interrogation of cell lineages.