Carlson, C. K. | Search bioRxiv

bioRxiv2021

Molecular recording of sequential cellular events into DNA

Genetically encoded DNA recorders noninvasively convert transient biological events into durable mutations in a cells genome, allowing for the later reconstruction of cellular experiences using high-throughput DNA sequencing1. Existing DNA recorders have achieved high-information recording2-15, durable recording3,5-10,13,15-19, multiplexed recording of several cellular signals5-8,19,20, and temporally resolved signal recording5-8,19,20, but not all at the same time in mammalian cells. We present a DNA recorder called peCHYRON (prime editing21 Cell HistorY Recording by Ordered iNsertion) that does. In peCHYRON, mammalian cells are engineered to express prime editor and a collection of prime editing guide RNAs21 (pegRNAs) that facilitate iterative rounds of prime editing. In each round of editing, prime editor inserts a variable triplet DNA sequence alongside a constant propagator sequence that deactivates the previous and activates the next step of insertion. Editing can continue indefinitely because each insertion adds the complete sequence needed to initiate the next step. Because only one active target site is present at any given time, insertions accumulate sequentially, in a unidirectional order. Thus, temporal information is preserved in the order of insertions. Durability is achieved through the use of a prime editor that only nicks a single DNA strand, effectively avoiding deletion mutations that could otherwise corrupt the information stored at the recording locus. High-information content is established by co-expressing a variety of pegRNAs, each harboring unique triplet DNA sequences. We demonstrate that constitutive expression of such a library of pegRNAs generates insertion patterns that support straightforward reconstruction of cell lineage relationships. In an alternative pegRNA expression scheme, we also achieve multiplexed recording by manually pulsing expression of different pegRNAs, then reconstructing pulse sequences from the peCHYRON records. Additionally, we coupled the expression of specific pegRNAs to specific biological stimuli, which allowed temporally resolved, multiplexed recording of chemical exposures in populations of mammalian cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/467507v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@3dcbb2org.highwire.dtl.DTLVardef@1081b72org.highwire.dtl.DTLVardef@141fed3org.highwire.dtl.DTLVardef@1c0e8ef_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2019

Ordered insertional mutagenesis at a single genomic site enables lineage tracing and analog recording in mammalian cells

The study of intricate cellular and developmental processes in the context of complex multicellular organisms is difficult because it can require the non-destructive observation of thousands, millions, or even billions of cells deep within an animal. To address this difficulty, several groups have recently reported CRISPR-based DNA recorders that convert transient cellular experiences and processes into changes in the genome, which can then be read by sequencing in high-throughput. However, existing DNA recorders act primarily by erasing DNA: they use the random accumulation of CRISPR-induced deletions to record information. This is problematic because in the limit of progressive deletion, no record remains. Here, we present a new type of DNA recorder that acts primarily by writing new DNA. Our system, called CHYRON (Cell HistorY Recording by Ordered iNsertion), inserts random nucleotides at a single locus in temporal order in vivo and can be applied as an evolving lineage tracer as well as a recorder of user-selected cellular stimuli. As a lineage tracer, CHYRON allowed us to perfectly reconstruct the population lineage relationships among 16 groups of human cells descended from four starting groups that were subject to a series of splitting steps. In this experiment, CHYRON progressively wrote and retained base insertions in 20% percent of cells where the average amount written was 8.4 bp (~14.5 bits), reflecting high information content and density. As a stimulus recorder, we showed that when the CHYRON machinery was placed under the control of a stress-responsive promoter, the frequency and length of writing reflected the dose and duration of the stress. We believe CHYRON represents a conceptual advance in DNA recording technologies where writing rather than erasing becomes the primary mode of information accumulation. With further engineering of CHYRONs components to increase writing efficiency, CHYRON should lead to single-cell-resolution recording of lineage and other information through long periods of time in complex animals or tumors, advancing the pursuit of a full picture of mammalian development.

synthetic biology↗