BALTIMORE – Researchers from the Gladstone Institute of Data Science and Biotechnology and their collaborators have engineered a CRISPR-based DNA recorder that can directly log the order of transcriptional events within living cells.
Described in a Nature study in July, the genetic machinery, named Retro-Cascorder, deploys CRISPR-Cas integrases to incorporate barcoded DNA sequence produced by retrons — DNA segments commonly found in bacterial genomes that help defend the cell against infection — into the genome of a living cell. By integrating the molecular receipts into a CRISPR array in a unidirectional fashion, Retro-Cascorder offers researchers a simple and physical way to track the temporal orders of transcriptional events inside a cell.
"When we look at biological processes, we basically have to destroy a sample to look at what's going on inside," said Santi Bhattarai-Kline, the study's first author and a former research associate in Seth Shipman's lab at Gladstone. "The objective of [molecular recording] is to build systems that allow you to record biological events as they occur over time, so that you only need to take a destructive measurement at the end of the process."
According to Bhattarai-Kline, scientists have previously designed ways to record cellular events using different DNA-modifying schemes such as site-specific recombinases and CRISPR-Cas nucleases. However, one bottleneck with recombinase-based recordings is that, because there is only a limited number of well-characterized DNA recombinases, the approach is not easily scalable.
Meanwhile, CRISPR-Cas nuclease-based recorders, which exploit the fact that DNA repair is imperfect and work by accumulating DNA mutations throughout the cell lineage, typically require computational reconstruction of the mutational data to infer the order of the biological events, Bhattarai-Kline said.
"This work leverages a piece of the CRISPR system that I think gets a lot less attention … that is the integrase portion of the CRISPR system," Bhattarai-Kline said. "Rather than inferring how events took place in time using statistical methods, we are attempting to make a physical record of how these events occurred."
Retro-Cascorder is built upon previous work by the team of Shipman, he added, who is also the senior author of this study, which demonstrated the information storage capabilities of CRISPR arrays. "The CRISPR array has a very unique structure that lends itself well to data storage applications," he said, adding that, mechanistically, Retro-Cascorder makes use of the function of CRISPR arrays as immune memories in bacterial cells.
During infection, the CRISPR Cas1 and Cas2 proteins can incorporate a foreign DNA piece into the CRISPR array, which consists of a leader sequence followed by unique spacer sequences that are derived from foreign DNA fragments and are interspersed by identical sequences called repeats. Because a CRISPR array extends linearly, when a new spacer is integrated, it can only be added next to the leader sequence, shifting the previous spacers away from the leader. Therefore, the distance between spacers and the leader sequence becomes indicative of the order of the related biological events.
To record transcriptional events using CRISPR arrays inside cells, Bhattarai-Kline's team engineered custom retrons, which can produce compact and distinct molecular tags, and placed them under the control of various gene promoters of interest. When a target gene is turned on, the tag sequence of the retron is transcribed into RNA and subsequently reverse-transcribed by the retron reverse transcriptase into single-stranded DNA, serving as a DNA receipt of the transcriptional event. That DNA receipt is then incorporated into the CRISPR array, creating a physical record of transcription. Similarly, when another gene is later turned on, a correlating molecular tag will be produced and added to the CRISPR array in a linear fashion.
The team tested out Retro-Cascorder in Escherichia coli cells. They engineered the cells to contain two types of barcoded retrons, which are placed behind chemically inducible gene promoters responsive to the presence of anhydrotetracycline or choline chloride, respectively. In short, the results showed that, after culturing these cells in liquid containing either anhydrotetracycline or choline chloride for 24 hours and then switching to the other solution for another 24 hours, Retro-Cascorder could accurately record the order of gene expression, revealing the correct sequence of the chemicals the cells were exposed to. According to Bhattarai-Kline, so far, the team is able to sequence and analyze CRISPR arrays that record up to three separate integration events.
To gauge the limits of retron recording in conditions that are currently difficult to create in the laboratory, Bhattarai-Kline and his team developed a computational model of Retro-Cascorder based on data from the experiment and the current understanding of biology. Using the model, the researchers extrapolated four variables "critical to the design of these recording experiments," including signal strength, length of recording, number of reads, and number of replicates. Boosting any of these four parameters can enhance the fidelity of the temporal recording outcomes, they concluded.
"We fundamentally know that time matters in biology, but we don't have a good way of reading that right now," said Timothy Lu, a bioengineering professor at the Massachusetts Institute of Technology who also studies DNA recording. "I'm really excited about these sorts of systems going and helping us to decipher the biological networks and events that happen in disease models."
According to Lu, because previous CRISPR-Cas nuclease-based recorders mostly track how mutated the guide RNA sequence is in order to computationally reconstruct the biological events that happened in a cell, it is harder for these recorders to trace the exact order of these events. As such, one advantage of the retron recording machinery is that it offers a direct way to record the timing of cellular events, offering a higher-fidelity readout.
However, since retrons only occur in bacteria, Lu said more engineering is still needed to make the technology work effectively in mammalian cells. "Whenever you move a bacterial system into mammalian cells, you have to think about localization or other cofactors," he noted. "So, there's a bit more engineering that needs to be done." He also pointed out that there have been recent efforts by researchers to transfer the retron system into mammalian cells.
Mirroring Lu's point, Nozomu Yachie, a biomedical engineering professor at the University of British Columbia, said one current drawback of the Retro-Cascorder is that it is limited to bacteria at this point. "In our field, people have been working so hard to make the Cas1/Cas2 system work in mammalian cells, but I believe we haven't been successful yet," he said.
Still, he thinks that Retro-Cascorder, with its linear recording capability, can be advantageous to trace temporal transcriptional events, even if they are from the same gene. The CRISPR-Cas9 recorders typically document gene expression activity by altering the DNA tape in response to targeted transcriptional events, but the machinery tends not to effectively capture situations where the same gene is turned on multiple times, he explained. For example, if a gene fires at three different time points in a cell, "you never know if the second or third event happened because your DNA is already edited by the first event."
Commenting on the DNA recording research field in general, Yachie said it is also important to harness these early technologies for high-resolution DNA event recording systems in order to be practically useful for biomedical research. In an article published in Science in July, he and his colleagues identified four "substantial limitations" for the field to overcome: information recording capacity, available molecular sensors to capture diverse biological events, number of cells that can be analyzed, and computational capacity to reconstruct high-content biological history.
In terms of applications, Bhattarai-Kline said that Retro-Cascorder could be useful in the short term as a live-recording biosensor for environments that might otherwise be difficult to monitor, such as wastewater or the human gut.
In the long run, he said the hope is to deploy Retro-Cascorder to record complicated biological processes, such as the development of cells and degenerative diseases. However, he acknowledged that the current system, which is still a proof of concept, requires further engineering for that.
"It's worth saying that this work was done in bacteria, and there are still real questions about how portable these elements are that we're working with, [and whether they] can be moved to more complex cell systems," he said. "That's an open question, but it's something that we're working on."