Periodic Reporting for period 4 - CRISPRhistory (Reconstructing cellular histories with transcriptional recording)
Période du rapport: 2024-06-01 au 2024-11-30
What are the main differences between a photograph and a video? Photographs record a single point in time and videos continuously record a sequence of events. While the content and interpretation of a photograph is heavily reliant on a single moment in time, a video is not. The tools that scientists have to understand the molecular and cellular world around them are most like film cameras – producing single snapshots to describe dynamic processes. Towards the goal of continuously recording molecular events within cells, my laboratory recently developed ‘transcriptional recording’, an approach that employs CRISPR spacer acquisition from RNA to capture and convert intracellular RNAs into DNA, permanently storing transcriptional information in the DNA of living cells. The newly acquired sequences serve as transcriptional records, which are retrievable via deep sequencing and can be leverage to reconstruct cellular histories. This technology provides an entirely new mode of interrogating dynamic biological and physiological processes and opens up numerous avenues for future work.
The overall goal of this proposal was to develop transcriptional recording sentinel cells with the capacity to continuously monitor biological and pathological processes within the mammalian gut. To achieve this goal, we will employed a variety of methods that included molecular and cellular biology, synthetic biology, protein engineering, bioinformatics, and mouse models. We focused our efforts on improving the efficiency of transcriptional recording, combining transcriptional recording with biosensing gene circuits, and demonstrating that transcriptional recording sentinel cells facilitate interrogation of the mammalian gut environment and can be applied as a living diagnostic. In summary, this study developed a fundamentally new approach to interrogating dynamic biological and pathological processes within the intestine.
The overall goal of this proposal was to develop transcriptional recording sentinel cells with the capacity to continuously monitor biological and pathological processes within the mammalian gut. To achieve this goal, we will employed a variety of methods that included molecular and cellular biology, synthetic biology, protein engineering, bioinformatics, and mouse models. We focused our efforts on improving the efficiency of transcriptional recording, combining transcriptional recording with biosensing gene circuits, and demonstrating that transcriptional recording sentinel cells facilitate interrogation of the mammalian gut environment and can be applied as a living diagnostic. In summary, this study developed a fundamentally new approach to interrogating dynamic biological and pathological processes within the intestine.
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.
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.
Intestinal physiology is central to the absorption of nutrients and can be disturbed either by intestinal disease or by malnutrition from a diet that contains insufficient and/or excess nutrients. Over 800 million people globally suffer from calorie or micronutrient insecurity, which particularly affects growth, development, ageing, and immunity. Approaches to assess the composition of the intestine are thus central to health and disease diagnosis. Current objective clinical metrics for assessing nutrition are indirect and insufficient.
To overcome this challenge, we developed transcriptional recording. Based on experiments in mice, we have shown that our technology can captures a wide range of dynamic conditions present in vivo in the intestine. Our new technology has the potential to open up numerous avenues for promoting human health and wellbeing. These areas include diagnostics, clinical decision support, biomarker discovery, precision nutrition, guiding healthy lifestyles, and nutritional and microbiome-directed therapeutics.
Our ERC project empowered these efforts by providing sentinel cells with increased efficiency, capacity to integrate information from biosensors, and proof of concept demonstrations of sentinel cell diagnostics in mouse models.
To overcome this challenge, we developed transcriptional recording. Based on experiments in mice, we have shown that our technology can captures a wide range of dynamic conditions present in vivo in the intestine. Our new technology has the potential to open up numerous avenues for promoting human health and wellbeing. These areas include diagnostics, clinical decision support, biomarker discovery, precision nutrition, guiding healthy lifestyles, and nutritional and microbiome-directed therapeutics.
Our ERC project empowered these efforts by providing sentinel cells with increased efficiency, capacity to integrate information from biosensors, and proof of concept demonstrations of sentinel cell diagnostics in mouse models.