Abstract
Writing in Science, Randall Platt, Andrew Macpherson, and colleagues use a clever transcriptional recording technique (RECORD-seq) using CRISPR spacer acquisition in sentinel bacteria to provide noninvasive information on the gut microbiome.
The past decade has seen an impressive number of studies unraveling links between our gut environment and our health. Imbalances in the composition of microbial communities, referred to as dysbiosis, are commonly associated with an inflamed gut environment and loss of barrier function.1,2 A perturbed gut environment has been found in many pathologies where it might drive or promote disease, including inflammatory bowel disease, type 1 diabetes, colorectal cancer, autism, and more. 3
To better understand these pathologies and the role that microbes play, we need to obtain a detailed picture of the gut environment and how it responds to various stimuli. The ideal method should enable capturing the dynamics of the gut response along time in live animals and possibly in humans. It should also provide information across different sections of the gut while being noninvasive. This might sound like a pipe dream, but teams from ETH Zurich and the University of Bern in Switzerland just took a major step in that direction in a study recently published in Science. 4
The strategy is based on the use of sentinel bacterial cells that are able to record their physiological state into DNA as they pass through the gut (Fig. 1). This feat is achieved by harnessing a CRISPR-Cas system, but not the usual Cas9/Cas12 RNA-guided nuclease. In a clever twist, Platt, Macpherson, and colleagues employed the machinery used by the CRISPR-Cas immune systems of Fusicatenibacter saccharivorans to capture novel DNA sequences from invading genetic elements. This bacterium carries a type III system, a type of CRISPR-Cas system known to detect target RNA molecules rather than DNA. F. saccharivorans carries a peculiar adaptation machinery where the cas1 gene is fused to a reverse transcriptase (RT), which enables this immune system to capture sequences that match the invader's transcripts.
An Escherichia coli strain carrying the Record-seq device can generate cDNAs from pieces of transcripts and integrate them into a CRISPR array. Sequencing the captured cDNAs from bacteria recovered in feces sheds light on the physiological state of the bacteria as they pass through the gut and face different conditions.
In a previous article published in Nature in 2018, 5 Platt's group at ETH Zurich showed that when overexpressed in Escherichia coli, the RT-Cas1-Cas2 from F. saccharivorans could capture sequences from the E. coli's messenger RNAs into the CRISPR array. Sequencing the captured sequences at the population level provides quantitative information about the highly expressed genes in this bacterial population. As the information is stably recorded at the DNA level, the strategy can even provide information on historical states of the population.
In the new study, the team first fed E. coli MG1655 carrying their Record-seq device to mice and showed how the repertoire of captured sequences enabled the simple classification of mice according to the diet they received. The method even helped distinguish mice up to 2 weeks after they had returned to a common diet. Interestingly, although both RNA-seq and Record-seq on feces material could inform on the metabolic pathways used by E. coli under different dietary intakes, part of the signal was unique to the Record-seq approach.
The transcripts uniquely captured by Record-seq could in part be traced back to genes expressed only in the cecum and proximal parts of the colon, a signal that is lost from the transcriptome by the time bacteria end up in feces pellets. Genes enriched in Record-seq but not in RNA-seq could thus inform on conditions transiently faced by the bacteria during their passage throughout the intestinal tract.
In mice treated with dextran sodium sulfate (DSS) to induce colitis, the Record-seq approach captured signals consistent with decreased anaerobic respiration and increased oxidative stress, which persisted for >10 days after the end of the treatment. Increased oxygenation is known to occur during colitis and likely contributes to the expansion of aerotolerant pathobionts. 6 Record-seq could thus become a useful method to probe such altered gut states in patients.
With all the previous experiments performed in monocolonized animals, the authors then sought to demonstrate that the approach would still work in the presence of other bacteria and could even shed light on how the E. coli niche is modified in the presence of other bugs. In mice cocolonized with Bacteroides thetaiotaomicron, Record-seq revealed interesting cross-feeding behaviors. In the presence of a complex community consisting of a dozen species, the approach still performed well to differentiate various dietary intakes. This opens the path for the use of the technology in more complex models of the microbiome's impact on health.
Finally, modifying the sequence of the CRISPR repeats enabled the construction of orthogonal Record-seq devices that can be used simultaneously in an animal and help record the transcription profile of two different E. coli strains that only differ by a single mutation, something that would be impossible to achieve using RNA-seq.
Altogether, this approach provides exciting opportunities for measuring rich phenotypes of bacteria across the gut environment while only requiring access to fecal samples. This opens several avenues to better understand the interactions between sentinel bacteria, the host, and the rest of the microbiota. The phenomena that can be measured by the technique are currently restricted by what E. coli can sense and metabolize, but we can anticipate that Record-seq will be adapted in the future to other members of the gut ecosystem that will help capture different and more complex phenomena.
Beyond the application of Record-seq as a research tool, we hope that the approach could provide rich data for diagnostic purposes, enabling researchers to access information that cannot be captured in any other way, be it in the blood, breath, or feces. In addition, the ability to keep an historical record of transiently altered conditions without requiring longitudinal sampling and invasive methods such as endoscopies could be particularly valuable for patients of chronic disease.