A Prime-Editing Based Approach Records Cellular Genetic History

Memories can live as remarkably cogent substances in the brain. It’s the definition of the word engram, which describes the physical content of how a memory is stored. But memories extend beyond the organism level; cells also retain memories of the variety of genes they express over time. As cis-regulatory elements (CREs) regulate gene expression, measuring the activity of these CREs could provide insights into how genes are expressed in cells. However, existing techniques can only record cellular gene regulation at a single point in time.¹

Motivated to find a way to record the activity of these CREs over time, Jay Shendure, a geneticist at the University of Washington, and his team developed a technique called enhancer driven genomic recording of transcriptional activity in multiplex, or ENGRAM.² By leveraging prime editing, which can insert a short, unique DNA sequence into a specific location, Shendure and his team could track and write gene regulation information into a genomic location. Their approach, published in Nature, could potentially provide insights into how, through time, certain genes shape a cell’s identity.²

A CRE is a stretch of DNA that regulates the expression of a nearby gene. When the gene is about to be turned on, transcription factors bind to a section of the CRE and begin the process of gene expression. For the ENGRAM system, the team used CREs to drive prime editing guide RNAs (pegRNAs) to insert small DNA sequences (barcodes) into a specified location in the genome. When a CRE was turned on, prime editing ensued, and the corresponding barcode was written. By assigning each CRE a unique barcode, the scientists could track the activity of many different CREs.

By using the same spacer sequence on each pegRNA, the scientists ensured that all barcodes were written into the same place in the genome to create a “list” of barcodes. “This is the key to the multiplexity,” said Wei (Will) Chen, who is a coauthor of the paper and was previously a graduate student in Shendure’s lab. Chen is now a postdoctoral researcher at the Institute for Protein Design.

The scientists could simply sequence the cellular DNA, review which barcodes were written, and unravel the cell’s regulatory history. When the scientists tested the system in live cells to see if the number of recorded barcodes matched the amount of RNA produced by gene expression, they found that the two correlated with one another, validating that the CREs quantitatively captured the extent of expression.

The scientists then tested if the ENGRAM recorder system could correctly capture the effects of multiple signals by incorporating three CREs that would activate in response to different stimuli, such as application of a drug. They found that the recorded barcodes reflected the amount of applied external signal—more signal, for example, resulted in more barcodes.

Finally, when the team tested ENGRAM in mouse embryonic cells, they were able to measure the activity of 98 synthetic CREs as the embryonic cells differentiated over time. “At the end, we can map, in a time series, the dynamics of different signals,” Chen said.

“[The study] addresses an important limitation in the field, which is that we need to have the ability to record expression levels directly from genes,” said Harris Wang, a biologist at Columbia University who was not involved in the study.

Some might worry that the very process of inserting synthetic DNA into the genome to record the activity of regulatory elements could disrupt the normal state of the cell. But according to Reza Kalhor, a bioengineer at Johns Hopkins University who was not involved in the study, it’s an inevitable risk. “The Heisenberg principle tells us that we cannot measure anything without changing it,” he said.

For Shendure, it’s about uncovering the cell’s journey, not just looking at its final destination. “The vision is that we can get to a world where we’re routinely making measurements across all cells in a system that are reflective of not only the final dynamics of that system, but also everything that happened along the way,” he said.