Summary
Genetically encoded DNA recorders noninvasively convert transient biological events into durable mutations in a cell’s genome, allowing for the later reconstruction of cellular experiences using high-throughput DNA sequencing1. Existing DNA recorders have achieved high-information recording2–14, durable recording3,5–10,13,15–18, prolonged recording over multiple timescales3,5,8,10, multiplexed recording of several user-selected signals5–8,18, and temporally resolved signal recording5–8,18, but not all at the same time. We present a DNA recorder called peCHYRON (prime editing19 Cell HistorY Recording by Ordered iNsertion) that does. In peCHYRON, prime editor guide RNAs19 (pegRNAs) insert a variable triplet DNA sequence alongside a constant propagation sequence that deactivates the previous and activates the next step of insertion. This process results in the sequential accumulation of regularly spaced insertion mutations at a synthetic locus. Accumulated insertions are permanent throughout editing because peCHYRON uses a prime editor that avoids cutting both DNA strands, which risks deletions. Editing continues indefinitely because each insertion adds the complete sequence needed to initiate the next step. Constitutively expressed pegRNAs generate insertion patterns that support straightforward reconstruction of cell lineage relationships. Pulsed expression of different pegRNAs enables the reconstruction of pulse sequences, which may be coupled to biological stimuli for temporally-resolved multiplexed event recording.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
↵* e-mail: ccl{at}uci.edu; theresa.berens.loveless{at}gmail.com