Development and optimization of a cell cycle molecular clock based on the ABE-CRISPR system.

Clustered regularly interspaced short palindromic repeats (CRISPR)-guided multiplex mutagenesis has emerged as a tool for lineage tracing; however, existing systems often exhibit disordered mutation patterns and cannot reliably record cell division history. To overcome these limitations, we develop the adenine base editor template number (ATN) system, which implements ordered, stepwise editing of double-stranded DNA through a programmable cascade of modifications. The ATN system incrementally marks each cell cycle, providing a quantitative representation of proliferation events. Once a predetermined number of divisions is reached, the system triggers activation of a downstream gene through frame restoration, effectively serving as a cellular "molecular clock" that links cell age to inducible genetic control. This approach therefore offers a systematic framework for studying cell proliferation dynamics and enables temporally regulated manipulation of cell fate.