Transcriptional recording enables noninvasive measurement of the intestinal tract with potential for biomedical research and future biomedical diagnostic applications

Bacteria within the gut continuously adapt their gene expression to environmental conditions that are associated with diet, health, and disease. Noninvasive measurements of bacterial gene expression patterns throughout the intestine are important to understand in vivo microbiota physiology and pathophysiology. Current methods do not offer sufficient information about transient or proximal events within the intestine without using indirect or invasive approaches that disturb normal physiology and are inapplicable to clinical practice.

Transcriptional recording by CRISPR spacer acquisition from RNA (Record-seq) enables engineered bacteria to continuously record the history of gene expression in a population of bacteria. Over time, snippets of intracellular RNA are converted into DNA and integrated as a historical record of spacer sequences within CRISPR arrays through the action of an integration complex that contains a reverse transcriptase Cas1 fusion protein (RT-Cas1) and Cas2. Here, using a refined Record-seq methodology, ETH Zurich researchers used transcriptional recording Escherichia coli sentinel cells to reveal intestinal and microbiota physiology under different dietary and disease contexts along the length of the unmanipulated mouse intestine.

The researchers used transcriptional recording sentinel cell technology in the gastrointestinal tract of germ-free and gnotobiotic mice to assess how the DNA record of spacer sequences in fecal samples uncovered distinct transcriptional records during passage from the proximal to the distal intestine, which depended on diet, inflammation, and microbe-microbe interactions. Record-seq was able to capture diet-specific signatures in mice colonized with a 12-organism model microbiota. Moreover, by using barcoded CRISPR arrays, the researchers could show that Record-seq can be multiplexed in several strains of the same bacterial species that cocolonize the intestine, thus elucidating the compensatory response of a single-gene mutant to competition with the WT strain.

Transcriptional recording sentinel cells noninvasively report interactions
with diet, host, other microbes, and pathological environments

Throughout intestinal transit, sentinel cells capture information about transient mRNA expression into plasmid-encoded CRISPR arrays through the action of a reverse transcriptase Cas1-Cas2 complex. This information is retrieved by means of fecal sampling and deep sequencing followed by computational analyses. Barcoded CRISPR arrays enable transcriptional profiling of isogenic bacteria coinhabiting the intestine.