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

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 4-week timeframe 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 a time-ordered record of cellular events. To demonstrate this, we apply the system 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 cellular dynamics but also creates exciting new opportunities for future applications based on recording temporal signals at the single-cell level—in other words, using dynamic barcoding as a molecular clock. Overall design: We applied scDynabar in mESCs using different gRNA sequences and two versions of Cas9 (BE3 and canonical Cas9). Data was collected for bulk processing across different time points.