What If Cells Kept Receipts of Their Gene Expression?

At first glance, an Escherichia Coli ( E. Coli ) bacterium looks a bit like a Cheeto, with the same puffy cylindrical shape. But it is a Cheeto lookalike with incredible immune defenses.

Behind the bacteria’s unassuming exterior are complex systems that help protect it from attacks by foreign invaders. For Seth Shipman, a bioengineer at the University of California, San Francisco, leveraging these defenses has opened new technological possibilities for recording gene expression in cells. “We’re taking a bunch of bacterial parts and repurposing them for biotechnology that they weren’t intended to be used for,” he says.

Shipman’s laboratory has developed a system that, when implemented in bacteria like E. Coli , can act as a recorder to track when certain genes are turned on or off. This system relies on molecular parts that the bacteria normally use for immunity, now slightly modified to serve new functions.

Named the Retro-Cascorder and recently described in Nature , the technology creates DNA “receipts” that store a record of gene expression. The scientists think that equipping cells with this recording capability can allow them to serve as tiny biological sentinels, providing precise insights into gene expression patterns during disease and development. Previously, to figure out which individual genes were expressed in cells—as well as when and where—scientists had to remove RNA at certain points in time, which meant killing the cells.

“Generally, the way that we measure things in biology requires destroying your biological sample,” says Santi Bhattarai-Kline, a coauthor on the paper and a student in Shipman’s lab. “Either you can look at all the genes in the cell, or you can let the cell continue to live and do what it’s going to do in the future, but not both,” agrees Theresa Loveless, a biologist at the University of California, Irvine, who was unaffiliated with the study. To circumvent this problem, the UCSF team and others have wondered how one might store molecular data over time without having to halt the cell’s activities.

Imagine the cell as a kind of reality TV star, with a log of its transcriptional lifetime preserved for scientists to probe and analyze for posterity. Bhattarai-Kline says this would be useful for tracking something like gene expression by being “able to record multiple different kinds of events, and the order in which they occur, and then at a final time point, being able to determine what happened in the past. ” The scientists’ wish to look back at what has happened within cells served as inspiration for the Retro-Cascorder.

It uses two main components: a retron (a little bacterial gene sequence) and Crispr-Cas, a genome editing system that bacteria use as part of their immune response. Scientists aren’t entirely sure what function retrons normally serve for bacteria—though recent studies have shown them to be useful in host defense against foreign invaders. But they have a very convenient power: They create proteins that can turn RNA into DNA.

(As a reminder, DNA is double stranded and used to store genetic information, while RNA is single stranded and codes for proteins. ) This RNA turned DNA can then be stored away in a bacteria’s genome as a “receipt” of gene expression. DNA is a good storage medium for something like a receipt because, unlike RNA, which degrades faster, it is stable over long periods of time.

“It’s compact, it’s flexible, it’s got a nice code we can work with, it’s stable,” says Shipman. “It’s not something that you ever have to worry about falling apart, even over really long timescales. ” Shipman and other scientists have found that retrons also generate a noncoding RNA sequence, or a string of code that does not produce proteins.

Shipman’s team realized they could modify these sequences so that they contained a unique “barcode”—a short set of bases within the RNA string. This subset of the string would serve as a marker of gene expression, kind of like sticking a tracking number on a mailed package. By creating a different barcode for each gene they wanted to track, the scientists could check these receipts to see whether the gene was being expressed.

In order to match each gene to the right barcode, the scientists placed the retron under control of the promoter from the gene they were interested in tracking. That way every time the gene was expressed, the retron was also activated to generate the noncoding RNA sequence with its barcode marker. Then, the retron would reverse transcribe the RNA sequence, including the gene-specific barcode.

This produced the final DNA receipt, complementary to the original noncoding RNA, along with the barcode. Next, the scientists needed to figure out a way to store those receipts within the bacteria’s genome so that they could be read in the future. To do that they used Crispr arrays: sections of the genome that hold a series of DNA chunks.

(Normally, bacteria use these arrays to store viral genomic information as part of their immune defenses—this helps them remember which viruses they have previously encountered so they can fight them off in the future. ) These arrays are created by Cas proteins, which pick up pieces of DNA and stockpile them inside the array. Critically, the scientists had noticed that a Cas protein doesn’t just add the DNA pieces randomly.

“It adds them directionally,” Shipman says. “It’s not just logging them, it’s logging them in order. ” That’s important because it creates a chronological record.

To co-opt the Crispr arrays for storing DNA receipts rather than viral information, the scientists engineered the noncoding RNA strings (and their subsequent DNA receipts) to also contain a “spacer” sequence that could be recognized by Cas proteins. The proteins would pick up the receipts by binding to the spacer and stick them into the Crispr array in chronological order. A gene that was expressed first would have its DNA receipt logged before a gene that was expressed later.

After running the cell’s Crispr array through a sequencing machine and reading the DNA receipts, the scientists could determine not only which genes were expressed, but also the order in which it happened—unfurling a living history of the cell’s gene activity. To test whether the Retro-Cascorder actually worked, the team decided to track the activity of two genes in E. Coli that would be turned on in the presence of specific chemicals.

Each gene drove the expression of a retron that created a DNA receipt with a unique barcode. To make things simpler, the scientists dubbed these barcodes A and B. They added the chemical that triggered the first gene (corresponding to barcode A) for 24 hours, followed by the one for the second gene (corresponding to barcode B) for the next 24.

“In theory we should have all the recording proteins turned on throughout the process, but only the RNA for signal A in the first half and signal B in the second half,” Bhattarai-Kline says. When the scientists sequenced the E. Coli ’s genomes, that’s exactly what they found: The DNA receipts for barcode A were integrated into the Crispr array first, followed by those of barcode B.

To double-check their work, they reversed the conditions, adding the chemical for barcode B before that of A. Once again, the Crispr array read out the expected pattern. This indicated that the Retro-Cascorder recorded the expression of both genes in the correct order.

While other recording systems have been developed that store information in DNA , the one made by Shipman’s group has an additional degree of specificity—the gene-specific barcodes—coupled with the ability to view gene expression in order. “It’s a really cool demonstration and optimization of cell recording,” says Timothy Lu, a synthetic biologist at the Massachusetts Institute of Technology who was unaffiliated with the study. Harris Wang, a biologist at Columbia University who has developed molecular recording systems, agrees.

This work “pushes us into a new area in terms of how we are able to glean information about the inner workings of the cell,” he says, adding that “you have much better control of what signals you can record. ” Wang, who was unaffiliated with the study, is curious to see whether these recording systems can one day keep track of the degree to which a gene is turned on or off, since gene expression does not always operate on a binary scale. For example, something like epigenetic regulation (chemical changes to DNA) can easily modulate genes to be expressed at varying levels, rather than simply on or off.

Lu is interested in seeing this system, and other cell recording systems, one day implemented in mammalian cells—an interest shared by Shipman and his team. “Our long-term goal is recording really complex events that play out over weeks and months in mammalian development and disease states,” Shipman says. Then, for something like cancer or Parkinson’s, scientists might be able to better understand how different genes are turned on and off as the disease progresses.

In the immediate future, the scientists envision the Retro-Cascorder as a bit of additional gear that could turn a bacterium into a biosensor. These bacteria could be unleashed to track chemical exposure in wastewater or study the human gut. Bacteria “interact with their environment, and they sense a lot of things that we would normally care about on a very sensitive level,” says Shipman.

“If we could just get them to store that information, then we can put them to work in some environment that is difficult to monitor. ” Since substances like pollutants and metabolites often elicit changes in gene expression, the bacterium’s DNA receipt book can be used to identify which molecules are present and when. For now, Shipman is grateful that the Retro-Cascorder works.

It shows that cell parts can be jerry-rigged for newer purposes. “We let evolution get us to something useful, and then we cherry-pick it,” he says with a laugh. .