Temporal recording of mammalian development and precancer

Key to understanding many biological phenomena is knowing the temporal ordering of cellular events, which often require continuous direct observations [[1][1], [2][2]]. An alternative solution involves the utilization of irreversible genetic changes, such as naturally occurring mutations, to create indelible markers that enables retrospective temporal ordering [[3][3]-[8][4]]. Using NSC-seq, a newly designed 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 assigned cell state and lineage information. Using this approach, we uncovered precise timing of tissue-specific cell expansion during murine embryonic development and identified new intestinal epithelial progenitor states by their unique genetic histories. NSC-seq analysis of murine adenomas and single-cell multi-omic profiling of human precancers as part of the Human Tumor Atlas Network (HTAN), including 116 scRNA-seq datasets and 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. Thus, our multimodal framework augments existing single-cell analyses and lays the foundation for in vivo multimodal recording, enabling the tracking of lineage and temporal events during development and tumorigenesis. ### Competing Interest Statement M.J.S. received funding from Janssen; G.M.C.'s competing financial interests can be found at ; L.T.T is currently an employee of Genentech. All other authors declare no competing interests. [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: #ref-8