NEW YORK – Researchers in Jay Shendure's lab at the University of Washington have incorporated prime editing technology into strategies that write the history of cellular molecular events, such as gene network transcription, into DNA.
The approach offers advantages in the level of detail that can be stored in and retrieved from the genome, according to Wei Chen, a postdoc at UW who did his graduate work under Shendure and is the first author on a paper in Nature last month describing the method, called enhancer-driven genomic recording of transcriptional activity in multiplex (ENGRAM).
In the paper, the researchers described how they were able to incorporate a specific DNA barcode, or "symbol," into a predetermined genomic location in response to a corresponding molecular signal. "We preassign a symbol to a signal," Chen said. "We can represent, to some extent, the signal activity, but we still cannot record what gene got transcribed."
Specifically, signals are coupled to the production of guide RNAs that help prime editors mark up a stretch of the genome comprising otherwise inactive CRISPR target sites. In proof-of-concept studies, the researchers were able to record cell-type specific activities of dozens of regulatory elements.
The approach enables better cellular lineage tracing and makes it easier to read out a cell's molecular history.
"This new work from the Shendure lab creatively and cleverly uses prime editing to record information flow in cells and tissues, equipping living systems with signal recorders that write representations of events into the genome," David Liu, a genome editing researcher at the Broad Institute whose lab invented the prime editing approach, said in an email. "It's the latest beautiful study from an innovative, multidisciplinary lab."
Shendure has been pursuing genome editing-based cellular recording technologies since at least 2016. His lab's Genome Editing of Synthetic Target Arrays for Lineage Tracing (GESTALT) used CRISPR-Cas9 for cell lineage tracing, one of several such approaches proposed at the time.
"We thought, 'OK, we can report lineage; it would be fun to record all these [transcription] signals. as well,'" Chen said. "In 2019, when prime editing came out, we realized it's the tech that's perfect for reporting." Prime editing offers genomic search-and-replace functionality without the use of double-strand breaks in DNA.
They combined prime editing with DNA Typewriter, another DNA-based technology developed in the Shendure lab, published in Nature in 2022. DNA Typewriter uses an array of genome editing target sites that make up a DNA tape, which can record as many as 20 events in sequence. "The acronym ENGRAM is inspired by the use of 'engram' in neuroscience to refer to the physical manifestation of a unit of memory," the authors noted.
Shendure and Chen are inventors on a patent application that is partially based on the new paper; however, Chen declined to talk about potential plans to commercialize ENGRAM. Shendure plans to use ENGRAM and DNA Typewriter in the recently launched Seattle Hub for Synthetic Biology.
Previous approaches to CRISPR-based cell history recording looked genome-wide for random edits made by Cas9. The edits weren't truly random, though, and cells with no relation to each other could end up with similar mutation profiles, adding noise to cell lineage tracing.
By recording the cellular activity in a predetermined spacer, one can be more certain that cells with similar DNA tapes are related, Chen said. The tape also makes data retrieval easier. Rather than doing genome-wide sequencing, where the mutational profile is the barcode, "we just need to sequence the tape," he said. The tape can be selected for sequencing with PCR; alternatively, for a non-sequencing approach, a fluorescence in situ hybridization (FISH) probe could be used to read out the tape.
Targeted sequencing is more cost-efficient, but the overall sequencing costs are low, "and there are other advantages that are more important than sequencing costs," Chen said.
The researchers demonstrated use of ENGRAM and DNA Typewriter in stem cells and gastruloids, a three-dimensional cell aggregate that mimics aspects of early mammalian development.
Though ENGRAM can record transcription events in general, it does not offer gene-level resolution. "We can represent, to some extent, the signal activity, but we still cannot record what gene got transcribed," Chen said. "We put a probe into cells to see how active the signal is, but we're not necessarily recording all the genes regulated by this signal." The authors noted that ENGRAM "is not well suited to biological signals or states that are not readily coupled to cis-regulatory elements, nor to recording at fast timescales."
The ability to record lineage and certain transcription programs means that ENGRAM could help further demystify embryonic development. "With lineage tracing, we can see where cells come from, but we don't know why," Chen said. "ENGRAM will provide the why and what signal they received."