Open-ended molecular recording of sequential cellular events into DNA

Abstract

SummaryGenetically 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.

Emergent evolutionary forces in spatial models of luminal growth and their application to the human gut microbiota

“…4 . Even stronger tests are possible if the strain labeling events could be coupled to local environmental conditions (e.g., pH and mucin) ( 78 – 80 ). By tracking the frequencies of these labeled lineages as they spread out across the colon—and potentially into the intestinal walls ( 32 )—one could directly measure the weighting function u ( x ) and its associated mixing timescales.…”

Predicting efficiency of writing short sequences into the genome using prime editing

“…Communicated, but as yet unpublished examples include insertion of recombinase sites using prime editing to enable directed insertion of large DNA cargo of up to 36 kb 1,2 , as well as clever utilization of short sequence insertion to generate a molecular recorder for sequential cellular events [40][41][42] . Better understanding of how cellular determinants 17 and pegRNA features affect prime editing rates 14,18 provides a foundation for these advances.…”

Chromatin context-dependent regulation and epigenetic manipulation of prime editing

“…The high programmability and specificity of prime editing, together with its avoidance of double-stranded breaks, offers considerable advantages over alternatives in the context of therapeutic genome editing 5 . Its programmability and specificity have also facilitated other goals, such as sequential genome editing for molecular recording [6][7][8] and the high-throughput functional characterization of genetic variants 9 .…”