Using CRISPR barcoding as a molecular clock to capture dynamic processes at single-cell resolution

Biological processes are fundamentally dynamic, yet existing methods for capturing these temporal changes are limited. We present scDynaBar, a novel approach that integrates CRISPR-Cas9 dynamic barcoding with single-cell sequencing to enable the recording of temporal cellular events. In this system, genetic barcodes accumulate mutations over a 25-day period and then are sequenced together with the transcriptome of each single cell. We propose that this gradual accumulation of genetic diversity can be exploited to create an ordered record of a cellular event. We apply this approach to track the transition from a pluripotent state to a two-cell (2C)–like state in mouse embryonic stem cells (mESCs). The results provide compelling evidence for the transient nature of the 2C-like state. Additionally, our system shows consistent mutation rates across diverse cell types in a mouse gastruloid model, underscoring its robustness and versatility across various biological contexts. This technique not only improves our ability to study single-cell dynamics but also creates new opportunities for recording other temporal signals—in other words, using dynamic barcoding as a molecular clock in individual cells.