The CRISPRhistory project focused on operationalizing transcriptional recording bacteria as non-invasive living microbial diagnostics of the gastrointestinal tract. The project had three main aims, including improving the efficiency of transcriptional recording (WP1), combining transcriptional recording with biosensing gene circuits (WP2), and demonstrating that transcriptional recording sentinel cells facilitate interrogation of the mammalian gut environment and can be applied as a living diagnostic (WP3). We succsesfully completed each of these work packages.
For WP1, using a newly developed selection assay, we completed an E. coli host factor screen enabling us to identify E. coli proteins that are involved with transcriptional recording. The logic here is that transcriptional recording requires known and unknown host factors, which we can leverage to improve the efficiency of our technology. The hits that emerged from the screen helped us reveal fundamental insights into the mechanism of CRISPR spacer acquisition from RNA and prioritize methods to increase the efficiency of sentinel cell recording technology. Specifically, we engineered second generation sentinel cells to express E. coli host factors as open reading frames, which resulted in sentinel cells with enhanced recording efficiency both in vitro in batch culture and in vivo in the mouse intestine. To diseminate this work, we are in the process of preparing a manuscript for submission to a scientific journal. Also for WP1, we leveraged the same selection assay to perform a large-scale RT-Cas1 and Cas2 deep mutational scanning (DMS) experiment with the goal of both understanding critical biochemical bottlenecks in CRISPR spacer acquisition from RNA and identifying genetic variants that increase the efficiency of the sentinel cell technology. We expect to conclude these efforts this year and submit a manuscript describing the work for publication in a peer reviewed journal.
For WP2, we set out to engineer sentinel cells with multiplexed, barcoded biosensors. The underlying concept is that transcriptional recording relies on E. coli gene expression to interpret the extracellular world. We can therefore only observe what E. coli can sense. With biosensors we can gain specific information on important molecules in addition to recording the general cellular response. For proof of concept, we evolved novel biosensors biosensors for the short chain fatty acids propionate and butyrate. We are now working to validate these biosensors in vivo in mice as well as barcode the outputs of these biosensors and integrate them with transcriptional recording. We expect to conclude these efforts this year and submit a manuscript describing the work for publication in a peer reviewed journal.
For WP3, we have made substantial progress in demonstrating the value of sentinel cell technology to reveal fundamental knowledge on host and microbial physiology in the intestine. In a published article directly resulting from the ERC project (Schmidt, Science, 2022), we described the development of a non-invasive microbial-cell based in vivo molecular recording system to report on characteristic signatures of health and disease. The underlying concept is that engineered E. coli harboring an RNA CRISPR spacer acquisition complex ‘record’ gene expression information while passing through the gastrointestinal tract. In doing so, they pick up characteristic signatures of the physiological or pathological state of the intestine, which can be reconstructed through Record-seq performed on fecal samples. We have shown that our technology can capture a wide range of dynamic conditions present in vivo in the intestine, including quantitative shifts in molecular and chemical features resulting from alterations in inflammation, nutrition, and presence of other bacteria. In the future, our technology may be applicable in humans as non-invasive diagnostics.
