scRIPT: a rapid, high-coverage single-cell lineage-tracing method applied to the hematoendothelial differentiation of embryonic stem cells

Single-cell lineage tracing, which often involves CRISPR-Cas-based barcode insertion followed by novel barcode creation, links a living cell's transcriptome to its clonal history. It is often less suitable for short cell differentiation protocols: the barcoding time window does not always align well with that of differentiation, barcode dropout rates can be too high, library preparation remains laborious, and many studies lack appropriate lineage-specific quality control. We developed scRIPT (single-cell Rapid Integrated Platform for Tracing), which combines rapid ribonucleoprotein (RNP)-based barcoding with fluorescent reporter coding sequence insertion, reporter-based enrichment of informative cells by cell sorting, streamlined library preparation, and dual short-read sequencing, coupled to dedicated quality-control metrics for lineage data. scRIPT achieves a low cell dropout rate (90% of cells). We applied scRIPT to document mouse embryonic stem cell (ESC) differentiation into early hematoendothelial cells and revealed clonal relationships and fate coupling among the emerging cell (sub)types. A subset of cell clones was biased towards single fates, and bifurcating progenitors already express mRNA of canonical regulators of their eventual fate. The results also suggest a heritable, fate-independent clonal memory enriched with imprinted genes. Taken together, scRIPT provides a convenient, rapid, reliable, high-coverage, and quality-controlled platform that combines cell lineage and transcriptome readouts.