Dual nuclease single-cell lineage tracing by Cas9 and Cas12a

Single-cell lineage tracing based on CRISPR/Cas9 gene editing enables the simultaneous linkage of cell states and lineage history at a high resolution. Despite its immense potential in resolving the cell fate determination and genealogy within an organism, existing implementations of this technology suffers from the limitations in recording capabilities and considerable information dropout. Here, we introduce a versatile tool, DuTracer, which utilizes two orthogonal gene editing systems to record deep cell lineage history at single-cell resolution in an inducible manner. DuTracer shows the ability of enhanced lineage recording with minimized target dropouts and deepened tree depth. Application of DuTracer in mouse embryoid bodies and neuromesodermal organoids illustrates the transition pattern of the lineage relationship of different cell types and proposes potential lineage-biased molecular drivers. Moreover, we have developed an entropy-based approach to quantify the lineage recording ability of DuTracer in cell differentiation models. Together, DuTracer facilitates the precise and regulatory interrogation of cellular lineages of complex biological processes. Overall design: All samples have undergone DuTracer-based lineage tracing. For neuromesodermal organoids (NMO), two biological batches of samples were collected. Batch 1 only contained NMO D30, while batch 2 included both neuromesodermal progenitor D3 and NMO D15. To increase the harvest rate, NMO D15 were sampled for three times from the same organoid. To generate 10 neuromesodermal organoid libraries, the samples were prepared halfway through the DNBelab C4 protocol and the cDNA pool was split. One quarter the pool is used to generate the RNA-seq libraries and one quarter is used to make amplicon libraries (Note: target site amplicon samples). For mouse embryonic stem cells and HEK293T samples, cells were collected every two days from D0-D16. Genomic DNAs were extracted and subsequently subjected to amplicon sequencing.