Temporal recording of mammalian development and precancer [rna-seq]

Insights into many biological phenomena requires knowing the temporal order of cellular events, which is traditionally achieved through continuous direct observations [1, 2]. An alternative solution leverages irreversible genetic changes, such as naturally occurring mutations, to create indelible markers that enables retrospective temporal ordering [3-8]. Using Native sgRNA Capture and sequencing (NSC-seq), a newly devised and validated multi-purpose single-cell CRISPR platform, we developed a molecular clock approach to record the timing of cellular events and clonality in vivo while incorporating cell state and lineage information. Using this approach, we uncovered precise timing of tissue-specific cell expansion during murine embryonic development, unconventional developmental relationships between cell types, and new epithelial progenitor states by their unique genetic histories. NSC-seq analysis of murine adenomas coupled to multi-omic and single-cell profiling of human precancers, with clonal analysis of 418 human polyps, demonstrated the occurrence of polyancestral initiation in 15-30% of colonic precancers, revealing their origins from multiple normal founders. Our study presents a multimodal framework that lays the foundation for in vivo recording, integrating synthetic or natural indelible genetic changes with single-cell analyses to explore the origins and timing of development and tumorigenesis in mammalian systems.