Using a sophisticated transcriptional recording method called Record-seq in a study conducted on mice, scientists have engineered E.coli sentinel cells so that they can generate a historical record of changes in their gene expression as they travel through the gut, by integrating DNA snippets into spacer sequences of engineered CRISPR arrays in their genomes. These arrays can then be bioinformatically analyzed to uncover physiological details of the intestinal tract and diverse microbial communities that colonize it.
“The gut and its microbiome are central to health. There is a paucity of options for investigating host and microbe physiology in the human intestine, making the human gut a black box. Fecal and blood measurements are indirect. Endoscopy requires fasting and purging,” said Randall Platt, PhD, senior author of the study and a professor of biological engineering at ETH Zurich. “We developed transcriptional recording sentinel cells with the idea that they could safely traverse the gastrointestinal tract and reveal critical features of health and disease within the gut noninvasively.”
The study was published in the journal Science, in an article titled, “Noninvasive assessment of gut function using transcriptional recording sentinel cells.” It opens new avenues for developing noninvasive, diagnostic, microbial sensors that could uncover early symptoms of intestinal disease or assess the effect of diet or therapies on health.
The CRISPR-Cas mechanism, a kind of bacterial immune memory, enables bacteria to incorporate RNA or DNA snippets from attacking viruses into a region of their genome called the CRISPR array, allowing them to recall past viruses to stave off future attacks expeditiously.
The researchers of the current study exploited the CRISPR-Cas mechanism to enable E. coli to incorporate snippets of their own mRNA. Here, bacterial mRNAs serve as blueprints that reveal which genes are being deployed to execute cellular functions as the bacteria traverses the length of the intestinal tract.
The scientists introduced the CRISPR array of the bacterial species Fusicatenibacter saccharivorans into an E. coli strain. This included a reverse transcriptase—an enzyme that transcribes RNA into DNA—along with CRISPR-associated proteins necessary for incorporating the DNA fragment into the CRISPR array. The basic molecular method used in this study was developed in earlier work by Platt’s team published in 2018 and 2020.
“In the current work, we further develop and expand the method to enable applications in different types of mouse models, including germ-free gnotobiotic mice and mice harboring a model microbiota. Each mouse model requires a different protocol in terms of how much bacteria we gavage, how we extract the recorded information from feces, and how we process the data,” explained Platt.
Collaborating teams led by Andrew Macpherson, PhD, a professor of gastroenterology at the University Hospital of Bern, administered the sentinel E. coli in mice and analyzed bacterial DNA in fecal samples to determine how often the gut bacteria manufactured a given mRNA molecule during their passage through the gut, and which genes were activated.
“This new method lets us obtain information directly from the gut, without having to disturb intestinal functions,” said Macpherson.
Platt said, “Transcriptional recording sentinel cells open avenues in basic research and medicine. We now have a tool that can reveal host and microbial physiology within the intact and unperturbed intestine.”
Harris Wang, PhD, an associate professor of systems biology, pathology, and cell biology at Columbia University said, “The study is a wonderful example of the power of temporal recording to detect and capture cellular environments and responses using cutting-edge CRISPR molecular machinery. The authors showed an impressive array of uses for biological recording inside the gut of an animal. I expect there will be many emerging applications as the technology is further refined in terms of sensitivity, temporal resolution, and resilience in open environments.”
Through the analysis of bacteria isolated from fecal samples of mice on different diets, the researchers showed how the sentinel bacteria adapted their metabolism to the nutrient supply.
“This work is really exciting and a great breakthrough in the field. Capturing transcriptional events that are not realized by traditional RNAseq in a noninvasive manner is outstanding,” said Joseph Bondy-Denomy, PhD, associate professor of microbiology and immunology at the University of California, San Francisco. “Extending this technology to non-E. coli microbes is an exciting next step that the field can now begin to tackle. I specifically am interested in understanding what these data in E. coli can tell us about the factors that dictate the success or failure of phage infection in vivo.”
“This work nicely extends Record-seq to assess the physiological state of the digestive tract. The authors provide a number of important demonstrations, including detection of nutrient content, inflammation, and microbial interactions. These examples nicely highlight how Record-seq can be uniquely applied, compared to existing cell-based biosensing technologies by providing genome-wide expression signatures, and it offers opportunities in place of otherwise highly invasive medical procedures,” said Chase Beisel, PhD, professor of medicine at the Julius Maximilian University of Würzburg, Germany, and head of the Institute for RNA-based Infection Research.
Beisel added, “The limitations of the technology are that Record-req remains limited to E. coli, which may not be the best choice when thinking about applying this approach in humans. E. coli also may not be best equipped to sense particular conditions or effectively inhabit different parts of the digestive tract. Deciphering the signature is also complicated, and time will tell how well the signature can be decoded to provide an accurate diagnosis. These mostly represent next steps in developing the technology.”
Wang, Bondy-Denomy, and Beisel were not involved in the current study and provided their independent opinions.
Having demonstrated that the system works in mice, in future work Platt and his team intend to translate the technology to humans. To bring this about, Platt’s team is conducting experiments to ensure that the engineered bacteria are safe for humans and do not survive in an open environment.