Bacterial CRISPR Recording Cells for Non-Invasive Gut Assessment
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Solution Overview
Problem
Current molecular recording technologies for monitoring gastrointestinal health are invasive, fail to capture the complexity of the mammalian gastrointestinal tract, and cannot preserve transient signals effectively.
Innovation Solution
A composition of two bacterial cell populations, each expressing a Cas1 RT fusion protein and Cas2 protein, with distinct CRISPR direct repeat sequences, records transcriptional events in the gut and distinguishes them by sequencing the integrated spacers in CRISPR arrays.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If current molecular recording technologies (recombinases, CRISPR-Cas9) are used to encode biological features into DNA, then cellular histories can be converted into heritable nucleotide sequence archives, but these technologies have largely only been deployed in vitro and cannot non-invasively sample the gastrointestinal tract
Solution Approach 1:
The patent introduces engineered bacterial cells as intermediary carriers that traverse the gastrointestinal tract and deliver molecular recording tools to host cells. These sentinel cells act as mediators between the external environment and the host's biological systems, enabling non-invasive sampling while maintaining the complexity capture capability of molecular recording technologies.
Solution Approach 2:
The patent replaces invasive mechanical sampling methods with a biological delivery system. Instead of using physical instruments to access the gastrointestinal tract, the system uses orally-administered bacterial cells that naturally traverse the gut, substituting mechanical intervention with biological transport to achieve non-invasive access.
2Measurement precision
If biosensors are used to report on single biomolecules in sentinel cells, then molecular recording can be achieved, but the complexity of the mammalian gastrointestinal tract cannot be captured
Solution Approach 1:
The patent creates universal sentinel cells equipped with multiplexed molecular recording tools that can simultaneously capture multiple biological features including gene expression, metabolic states, and environmental metabolite concentrations. This multi-functional recording system enables comprehensive profiling of the gastrointestinal tract's complexity rather than limited single-biomolecule detection.
Solution Approach 2:
The patent transitions from single-biomolecule detection to transcriptome-scale analysis by recording RNA sequences. This dimensional expansion from molecular level to genomic level enables capture of the gastrointestinal tract's biological complexity through transcriptomic profiles that integrate multiple cellular processes and environmental interactions.
3Quantity of substance
If omics-based measurements of transient stimuli are performed, then snapshot data is obtained, but transient signals are lost because metabolites and RNA are short-lived
Solution Approach 1:
The patent performs preliminary action by capturing transient RNA signals while they are still present in the bacterial cells during their transit through the gastrointestinal tract. The molecular recording system immediately converts these transient RNA molecules into stable DNA sequences in the CRISPR arrays, preserving the signal before it degrades or is diluted in the complex gut environment.
Solution Approach 2:
The patent creates a permanent copy of transient biological signals by recording RNA sequences into the CRISPR array as stable DNA spacers. This copying mechanism transforms the ephemeral RNA molecules into durable genetic records that can be stored and analyzed long after the original transient signals have disappeared from the gastrointestinal tract.
4Ease of operation
If invasive procedures or devices are used to sample the gastrointestinal tract, then direct access to the lumen is achieved, but the sampling process is disruptive and cannot preserve transient signals
Solution Approach 1:
The patent employs self-service by utilizing the body's own physiological processes to deliver the sampling system. The orally-administered bacterial cells naturally traverse the gastrointestinal tract through peristalsis and digestive movements, using the host's own mechanisms for transport rather than requiring external invasive devices that disrupt normal intestinal function.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables non-invasive, comprehensive monitoring of intestinal and microbial health by capturing transcriptome-scale information on microbial-host interactions, providing insights into intestinal function and disease states.
Implementation Method 1
a fusion protein of a Cas1 polypeptide and a reverse transcriptase
Implementation Method 2
the CRISPR spacer adaptation complex of Fusicatenibacter saccharivorans (FsRT-Cas1-Cas2) to acquire snippets of cellular RNAs as DNA within CRISPR arrays
Data Source
AI summary
The invention relates to a bacterial cell comprising a Cas1 RT fusion protein, a Cas2 protein and a CRISPR direct repeat (DR) sequence, wherein an RNA polymerase promoter in addition to the leader sequence is associated with the DR sequence. The invention further relates to a composition comprising two bacterial cell populations, each comprising a Cas1 RT fusion protein and Cas2 protein. The two cell types contain different versions of a CRISPR direct repeat (DR) sequence. The invention further relates to methods for analysis of transcription recording events of bacteria having passed through a subject's intestine, to assign a probability to the subject having a condition, such as malnutrition or inflammation of the intestine.