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. We developed DuTracer, a single-cell lineage tracing method, to avoid rapid target exhaustion and barcode information dropout. Firstly, we introduced this system into HEK293T cells and initiated DuTracer for tracing. We collected single-cell transcriptome and barcode amplicon, demonstrating that this system could record more hierarchical information with minimal inter-site deletions. Subsequently, we applied DuTracer to a mouse embryoid body differentiation model to track lineage transitions of different cell types. We revealed cell potency hierarchy during the stochastic differentiation process of mES cells.